ACS Publications. Most Trusted. Most Cited. Most Read
Quick View

OR SEARCH CITATIONS

My Activity
Publications
CONTENT TYPES

Figure 1Loading Img
Download Hi-Res ImageDownload to MS-PowerPointCite This:J. Phys. Chem. B 2022, 126, 48, 10000-10017
RETURN TO ISSUEPREVB: Biophysical and B...B: Biophysical and Biochemical Systems and ProcessesNEXT

Galectin-3 Binding to α5β1 Integrin in Pore Suspended Biomembranes

  • Nirod Kumar Sarangi
    Nirod Kumar Sarangi
    School of Chemical Sciences and National Centre for Sensor Research, Dublin City University, DCU Glasnevin Campus, D09 V209Dublin 9, Ireland
    More by Nirod Kumar Sarangi
  • , 
  • Massiullah Shafaq-Zadah
    Massiullah Shafaq-Zadah
    Institut Curie, PSL Research University, U1143 INSERM, UMR3666 CNRS, Cellular and Chemical Biology Unit, 75248Paris Cedex 05, France
    More by Massiullah Shafaq-Zadah
    Orcidhttps://orcid.org/0000-0002-7582-8131
  • , 
  • Guilherme B. Berselli
    Guilherme B. Berselli
    School of Chemical Sciences and National Centre for Sensor Research, Dublin City University, DCU Glasnevin Campus, D09 V209Dublin 9, Ireland
    More by Guilherme B. Berselli
  • , 
  • Jack Robinson
    Jack Robinson
    School of Chemical Sciences and National Centre for Sensor Research, Dublin City University, DCU Glasnevin Campus, D09 V209Dublin 9, Ireland
    More by Jack Robinson
  • , 
  • Estelle Dransart
    Estelle Dransart
    Institut Curie, PSL Research University, U1143 INSERM, UMR3666 CNRS, Cellular and Chemical Biology Unit, 75248Paris Cedex 05, France
    More by Estelle Dransart
  • , 
  • Aurélie Di Cicco
    Aurélie Di Cicco
    Institut Curie, PSL Research University, UMR 168 CNRS, 75248Paris Cedex 05, France
    More by Aurélie Di Cicco
  • , 
  • Daniel Lévy
    Daniel Lévy
    Institut Curie, PSL Research University, UMR 168 CNRS, 75248Paris Cedex 05, France
    More by Daniel Lévy
  • , 
  • Ludger Johannes*
    Ludger Johannes
    Institut Curie, PSL Research University, U1143 INSERM, UMR3666 CNRS, Cellular and Chemical Biology Unit, 75248Paris Cedex 05, France
    *Email: [email protected]
    More by Ludger Johannes
  • , and 
  • Tia E. Keyes*
    Tia E. Keyes
    School of Chemical Sciences and National Centre for Sensor Research, Dublin City University, DCU Glasnevin Campus, D09 V209Dublin 9, Ireland
    *Email: [email protected]
    More by Tia E. Keyes
    Orcidhttps://orcid.org/0000-0002-4604-5533
Cite this: J. Phys. Chem. B 2022, 126, 48, 10000–10017
Publication Date (Web):November 22, 2022
https://doi.org/10.1021/acs.jpcb.2c05717

Copyright © 2022 The Authors. Published by American Chemical Society. This publication is licensed under

CC-BY 4.0.
  • Open Access

Article Views

1614

Altmetric

-

Citations

-
LEARN ABOUT THESE METRICS
PDF (10 MB)
Get e-Alerts
Supporting Info (1)»
SUBJECTS:

Abstract

High Resolution Image
Download MS PowerPoint Slide

Galectin-3 (Gal3) is a β-galactoside binding lectin that mediates many physiological functions, including the binding of cells to the extracellular matrix for which the glycoprotein α5β1 integrin is of critical importance. The mechanisms by which Gal3 interacts with membranes have not been widely explored to date due to the complexity of cell membranes and the difficulty of integrin reconstitution within model membranes. Herein, to study their interaction, Gal3 and α5β1 were purified, and the latter reconstituted into pore-suspended lipid bilayers comprised eggPC:eggPA. Using electrochemical impedance and fluorescence lifetime correlation spectroscopy, we found that on incubation with low nanomolar concentrations of wild-type Gal3, the membrane’s admittance and fluidity, as well as integrin’s lateral diffusivity, were enhanced. These effects were diminished in the following conditions: (i) absence of integrin, (ii) presence of lactose as a competitive inhibitor of glycan–Gal3 interaction, and (iii) use of a Gal3 mutant that lacked the N-terminal oligomerization domain (Gal3ΔNter). These findings indicated that WTGal3 oligomerized on α5β1 integrin in a glycan-dependent manner and that the N-terminal domain interacted directly with membranes in a way that is yet to be fully understood. At concentrations above 10 nM of WTGal3, membrane capacitance started to decrease and very slowly diffusing molecular species appeared, which indicated the formation of protein clusters made from WTGal3−α5β1 integrin assemblies. Overall, our study demonstrates the capacity of WTGal3 to oligomerize in a cargo protein-dependent manner at low nanomolar concentrations. Of note, these WTGal3 oligomers appeared to have membrane active properties that could only be revealed using our sensitive methods. At slightly higher WTGal3 concentrations, the capacity to generate lateral assemblies between cargo proteins was observed. In cells, this could lead to the construction of tubular endocytic pits according to the glycolipid–lectin (GL–Lect) hypothesis or to the formation of galectin lattices, depending on cargo glycoprotein stability at the membrane, the local Gal3 concentration, or plasma membrane intrinsic parameters. The study also demonstrates the utility of microcavity array-suspended lipid bilayers to address the biophysics of transmembrane proteins.

This publication is licensed under

CC-BY 4.0.
  • cc licence
  • by licence

Introduction

ARTICLE SECTIONS
Jump To
Integrins are transmembrane heterodimeric proteins comprised of an α and a β subunit that mediate signals between the extra- and intracellular spaces via binding to different extracellular matrix ligands and numerous binding partners present at the cytosolic side of the plasma membrane. (1) Twenty-four integrins are known in vertebrates that all have common structural motifs including in the α domain, a 7-bladed β propeller connected to a thigh and two calf domains. A metal ion-dependent adherent site is also conserved and is crucial for ligand binding. (2) Integrins regulate various biological functions such as cell adhesion, migration, proliferation, differentiation, and spreading, and the remodeling of the extracellular matrix. (3) This ability to regulate crosstalk between the cell and the surrounding environment positions integrins as key players in the process of tumor progression. (4) Overexpression of integrins leads to therapy resistance, tumor reoccurrence, and survival issues. α5β1 integrin, also known as the fibronectin receptor, has been identified as a potential therapeutic target on certain solid tumors. For example, in breast cancer, it has been observed that the ligation of β1 integrins, such as α5β1 and α2β1, with extracellular matrix components significantly reduces drug-induced apoptosis from chemotherapeutic agents. (5)
Galectins are a family of 15 proteins that contain highly conserved carbohydrate-recognition domains (CRDs) that allow binding to glycosylated proteins and lipids at the surface of cells or in the extracellular matrix, including integrins. Galectins access the extracellular milieu by unconventional secretion. (6,7) They have been classified into three subtypes, (i) prototype, (ii) tandem repeat, and (iii) chimera. (3,8−11) A single CRD is found on prototype galectins, and two CRDs separated by a linker sequence on tandem repeat galectins. (12)
Galectin-3 (Gal3) is the sole representative of the chimera galectin subtype. This lectin has an important role, in part mediated by integrins, in physiological and pathological phenomena (13) and is the most documented galectin family member. Gal3 is widely distributed in many tissues. It carries two specific domains, one CRD as for all galectin members, but also an intriguing N-terminal domain comprising proline/glycine/tyrosine-rich repeats. This N-terminal domain strongly determines the oligomerization capacity of Gal3 upon its binding to surface glycoproteins, (14,15) possibly involving liquid–liquid phase separation. (16−18) Gal3 is reported to stimulate neutrophil adhesion and migration, spreading of cancer cell lines and, furthermore, promotes focal adhesion turnover and fibronectin fibrillogenesis in tumor cells. (19−22) Gal3 has been linked to various diseases such as cancer, type-1 diabetes, depression, and Alzheimer’s disease. (5,9,23−25) Understanding and regulating the response of Gal3–integrin interactions is a critical biomedical challenge because lectin–integrin signaling is integral to many disease states. (26,27)
Galectins and notably Gal3 have been directly implicated in the regulation of the cell surface homeostasis of glycoproteins. (28) They have been shown to favor the endocytic uptake of these glycoproteins, for example, α5β1 integrin (22,29) via a mechanism that has been termed the glycolipid–lectin or GL–Lect hypothesis. (30) According to this model, monomeric Gal3 in solution oligomerizes upon binding to cell surface glycoproteins such as integrins. Oligomeric Gal3 then acquires the capacity to interact with glycosylated lipids of the glycosphingolipid family in a way such as to drive the formation of tubular endocytic pits from which so-called clathrin-independent endocytic carriers form for the cellular uptake of the cargo glycoproteins. Initially described for Gal3 and the cellular uptake of CD44 and α5β1 integrin, (29) the GL–Lect hypothesis has more recently also been documented for galectin-8 and another specific cargo protein, CD166. (31) Although these findings are in apparent contradiction with earlier ones on galectins inducing lateral lattices that negatively affect endocytosis, (32) we now envision a model in which galectin lattices and the GL–Lect mechanism cooperate to control the homeostasis of glycoproteins at the plasma membrane. (33)
In vitro studies at artificial membrane platforms, such as liposomes and supported lipid bilayers (SLBs), have been successfully applied to interrogate the interaction of membrane lipids as well as membrane-embedded receptors with external biomolecules such as proteins and peptides. (34−36) Although important models, liposomes are somewhat limited in the approaches that can be applied to their analytical interrogation, for example, to study two-dimensional interfaces. Using fluorescence correlation spectroscopy (FCS) to analyze transmembrane proteins in giant unilamellar vesicles (GUVs) is possible because of their large size (10–50 μm). However, challenges with maintaining the focus of the confocal volume can arise due to the movement of the vesicle. (37) Furthermore, as both leaflets of the liposomal bilayer are formed simultaneously, asymmetric leaflet composition is challenging to achieve with precision. (38,39) While compositionally and analytically more versatile, SLBs suffer interference from the interfacial support (substrate effect) due to frictional/pinning on the fluidity and functionality of the bilayer and associated membrane proteins that can limit biomimicry. Tethered or cushioned membranes offer improved biomimicry by introducing an extra layer between the substrate and lower leaflet, through a covalently bound self-assembled monolayer or a physio-absorbed polymer cushion; yet, challenges around friction/lateral movement of proteins still remain. (35,40−43) Nonetheless, the advantages of SLB-based approaches are their greater compositional control and versatility in terms of experimental interrogation compared with liposomes. (44−46) Particularly, when the solid support is conducting, this can be used as an electrode enabling the electrochemical study of lipid–peptide/protein interactions. (47−50)
Alternative approaches have emerged recently, in which membranes supported over buffer-filled periodic pore structures are assembled, which improves membrane fluidity, while maintaining stability. Most importantly, in the case of buffer-filled pore-supported bilayers, they offer the advantage of a relatively deep aqueous reservoir in contact with the proximal leaflet that SLBs lack. (51−55) We recently exploited microcavity array-suspended lipid bilayers (MSLBs) formed at cavity array polydimethylsiloxane (PDMS) and gold electrodes to study receptor-mediated interactions and detection of peripheral proteins such as the B-subunit of cholera toxin, (56) hemagglutinin A1, (57) annexin V, (58) and small-molecule drug permeability, (59−62) across varied lipid membrane compositions, and have demonstrated that they offer the fluidity of liposome/proteoliposomes with the addressability of SLBs. Here, we have applied them to a biophysical study of α5β1 integrin–Gal3 interactions.

Materials and Methods

ARTICLE SECTIONS
Jump To

Equipment and Reagents

Rat livers (Charles River), functionalized wheat germ agglutinin (WGA) agarose resin (Sigma-Aldrich, ref. 61768-5 mL), NHS-activated agarose column functionalized with FN-III9–10 (GE Healthcare, NHS-HiTrap ref. 17071701), pre-casted 4–15% polyacrylamide gels (Bio-Rad), 300–400 mesh carbon-coated copper grids for electron microscopy (EM) (Delta Microscopy, ref. DG400-Cu), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 1 M, pH 7–7.4 (Sigma-Aldrich, ref. H0887), MgCl2, CaCl2, Triton X-100 (Anatrace, ref. T1001-500 mL), n-dodecyl β-d-maltoside (DDM) (Anatrace, ref. D310-25GM), protease inhibitors (Sigma-Aldrich, ref. P8849), Pefabloc (Sigma-Aldrich, ref. 76307), N-acetylglucosamine (GlcNac, Sigma-Aldrich, ref. A8625-5G), sucrose, ethylenediaminetetraacetic acid (EDTA) pH 8, chicken egg phosphatidylcholine (Avanti Polar, ref. 840051C), chicken egg l-α-phosphatidic acid (Avanti Polar, ref. 840101C), rhodamine B 1,2-dihexadécanoyl-sn-glycéro-3-phosphoéthanolamine (Thermo Fisher, ref. L1392), HisPur cobalt resin (Thermo Fisher, ref. 89965), NHS-ATTO488, ATTO655 DOPE, ATTO532 DOPE (ATTO-TEC GmbH), NHS-Alexa647 (Invitrogen), non-reducing sodium dodecyl sulfate (SDS)-loading sample buffer, hamster anti-rat β1 integrin (BioLegend, ref. 102202) primary antibody, HRP-coupled secondary anti-hamster antibody, and ECL. Bio-Beads SM2 adsorbent media (Bio-Rad, ref. 152-3920), Bio-Rad ChemiDoc for protein detection (chemiluminescence).

Purification of α5β1-Integrin from Rat Livers

α5β1 integrin was solubilized and purified as described earlier. (63,64) Briefly, 4 rat livers (about 65 g in total) were cut in small pieces and incubated with lysis buffer [20 mM HEPES, 2 mM MgCl2, 2 mM CaCl2, 2% (v/v) Triton X-100, protease inhibitor cocktail 1/250 dilution, and Pefabloc 1/1000 dilution, pH 7.4], such that the liver tissue weight represents 25% (w/v) of the total final volume of extraction. Tissues were 2 times homogenized using an ultra-Turrax homogenizer and incubated for 60 min at 4 °C on orbital shaker. The extract was centrifuged at 75,000×g for 30 min at 4 °C. The supernatant was then centrifuged again using the same settings. The final lysate supernatant was clarified by filtration through a Whatman paper and loaded on two 25 mL WGA-affinity columns connected in series, at a flow rate of 0.8 mL/min. Columns were washed with washing buffer [20 mM HEPES, 150 mM NaCl, 2 mM MnCl2, 0.2% (v/v) Triton X-100, protease inhibitor cocktail 1/250 dilution, Pefabloc 1/1000 dilution, pH 7.4], at a flow rate of 2 mL/min. Glycosylated proteins were finally eluted with washing buffer supplemented with 300 mM GlcNAc. Protein-enriched fractions were pooled and loaded on a 10 mL FN-III9–10-functionalized column at a flow rate of 0.08 mL/min, to specifically bind α5β1 integrin, using an FPLC purifier system. Unbound material was then removed with washing buffer at a flow rate of 0.2 mL/min. α5β1 integrin was finally eluted with elution buffer [20 mM HEPES, 150 mM NaCl, 10 mM EDTA, 0.2% (v/v) Triton X-100, Pefabloc 1/1000, pH 7.4], at a flow rate of 0.08 mL/min.
For EM and negative staining characterization of purified α5β1 integrin, protein extraction from rat livers was performed using DDM instead of Triton X-100.
Protein purity was determined by SDS-PAGE analysis. Integrin-enriched fractions were then pooled and final concentration determined by the Bradford colorimetric assay. Integrin was snap-frozen and stored at −80 °C.

Negative Staining and Electron Microscopy

α5β1 integrin reconstitution was performed at a lipid/protein ratio (LPR) of 2700 (molmol). 200 μg of eggPC:eggPA (9:1, mol/mol) solubilized in chloroform were mixed and excess of organic solvent evaporated under a nitrogen atmosphere. Lipidic films were dried under vacuum at room temperature for 2 h, and then resuspended in 150 μL of 20 mM HEPES, 150 mM NaCl pH 7.4. Lipids were then solubilized by adding 0.25% (v/v) Triton X-100 and incubated for 10 min at 21 °C under gentle stirring. 20 μg of purified α5β1 integrin was added to the solubilized lipids, the Triton X-100 concentration was adjusted to 0.5% (v/v) in a 200 μL final reconstitution volume and incubated for 10 min at 21 °C under gentle stirring. The detergent was eliminated by three consecutive additions of Bio-Beads SM2 (prepared according to manufacturer’s instruction), 2 times with 10 mg, and 1 time with 20 mg, after 2, 1 ,and 1 h of time intervals, respectively. Lipid and α5β1 integrin final concentrations in small unilamellar vesicles (SUVs) were 1 and 0.1 mg/mL, respectively. This led to complete detergent removal and the formation of unilamellar vesicles. (65) SUVs were transferred to a new tube and stored at 4 °C until use. The size of the integrin reconstituted SUVs was estimated from dynamic light scattering (DLS) using a Malvern, Zetasizer (Figure S1, Supporting Information).

Characterization of α5β1 Integrin Incorporation into SUVs by Floatation in a Sucrose Gradient

For SUV visualization on sucrose gradient, 0.5% (w/w) of rhodamine DHPE was added to the lipid composition of α5β1 integrin/SUVs, prepared, as described above. In a cold room, 100 μL of α5β1 integrin/SUVs were diluted to a final volume of 575 μL in 20 mM HEPES, 150 mM NaCl, 30% (w/v) sucrose, pH 7.4, and placed at the bottom of a 2.5 mL polypropylene ultra-centrifuge tube. Sucrose gradient was then built by gently pouring, from the bottom to the top of the tube, 575 μL of 20, 10, 5, and 0% (w/w) sucrose solutions prepared in 20 mM HEPES and 150 mM NaCl pH 7.4 buffer. The sample was centrifuged at 100,000×g (Beckman TLS55 rotor) for 16 h at 4 °C. Samples were collected from each sucrose density, denatured in non-reducing SDS-sample loading buffer, boiled for 5 min, and loaded on a 4–15% pre-casted polyacrylamide gel. Proteins were then transferred on the nitrocellulose membrane, and the presence of α5β1 integrin in all fractions was immuno-detected by incubation with primary hamster anti-rat β1 integrin antibodies, followed by secondary HRP-coupled anti-hamster antibody.

Characterization of α5β1/SUV Morphology by Cryo-EM

4 μL of α5β1 proteoliposomes were loaded on a glow-discharged lacey carbon 300 mesh grids. Blotting was carried out on the opposite side from the liquid drop and samples were plunge frozen in liquid ethane (EMGP, Leica, Germany). Cryo-EM images were acquired with a Tecnai G2 (Thermo Fisher, USA) Lab6 microscope operated at 200 kV and equipped with a 4k × 4k CMOS camera (F416, TVIPS). Image acquisition was performed under low dose conditions of 10 e–/Å2 at a magnification of 50,000 with a pixel size of 2.14 Å.

Characterization of α5β1 Orientation after Reconstitution in SUVs

5 μL of α5β1/SUVs were diluted with 5 μL of 20 mM HEPES,150 mM NaCl pH 7.4 buffer, and incubated under agitation for 30 min at 20 °C with 10 μL of 200 μg/mL trypsin solution, either complemented with 0.5% (v/v) Triton X-100 (total integrin digestion) or not (surface-exposed integrin digestion). Trypsin was inactivated with 10 μL of non-reducing SDS-sample loading buffer and boiled for 5 min at 95 °C. Samples were loaded on 4–15% pre-casted polyacrylamide gels and proteins were then transferred onto nitrocellulose membranes. The presence of α5β1 integrin was immuno-detected by incubation with primary hamster anti-rat β1 integrin antibody, followed by secondary HRP-coupled anti-hamster antibody.

Functionality of α5β1 Integrin after Reconstitution in SUVs

α5β1 integrin functionality, once incorporated in SUVs, was assessed by its capacity to bind fibronectin. 20 μL of α5β1/SUVs (2 μg of protein and 20 μg of lipids) were incubated for 30 min at 21 °C on a rotating wheel either with 2 mM MgCl2 or MnCl2 in 100 μL of 20 mM HEPES,150 mM NaCl, pH 7.4 buffer. Histidine-tagged FN-III9–10 fibronectin fragment was added at a 10 μM final concentration for 90 min at 21 °C under rotation. Reactions were loaded on 30 μL bed volume of cobalt-coated beads and incubated for one additional hour at 4 °C on a rotating wheel. Unbound materials were then removed, and beads washed with 20 mM HEPES, 150 mM NaCl, pH 7.4 buffer, supplemented or not with 2 mM of MgCl2 or MnCl2. Proteins were eluted from the beads with 30 μL of non-reducing SDS-loading sample buffer and boiled 5 min at 95 °C. Samples were loaded on a 4–15% pre-casted polyacrylamide gel, and proteins were then transferred onto the nitrocellulose membrane. The presence of α5β1 integrin was immuno-detected by incubation with primary hamster anti-rat β1 integrin antibody, followed by secondary HRP-coupled anti-hamster antibody.

Labeling of α5β1 Integrin with ATTO488 Fluorophore

Triton X-100-purified α5β1 integrin [20 mM HEPES, 150 mM NaCl, 0.2% (v/v) Triton X-100] was incubated with a 10 molar excess of NHS-ATTO488 for 2 h at 21 °C under gentle agitation. The reaction was quenched by the addition of 20 mM Tris pH 7.4 for 20 min at 21 °C. Excess of NHS-ATTO488 was then removed using 40 kDa cutoff desalting spin-columns equilibrated with 20 mM HEPES, 150 mM NaCl, 0.2% (v/v) Triton X-100, according to the manufacturer’s instructions. Final protein concentration was assessed by a BCA colorimetric assay.

Labeling of Wild-Type Gal3 and Gal3ΔNter with Alexa647

Wild-Type Gal3 (WTGal3) or Gal3ΔNter at a final concentration of 2 mg/mL were incubated for 2 h at 21 °C under gentle shaking in phosphate-buffered saline (PBS) with 4 molar excess of NHS-Alexa647 dye, in the presence of 10 mM of β-lactose. Reactions were quenched with 20 mM Tris final concentration for 20 min at 21 °C. Unreacted dye was cleared using 7 kDa desalting columns equilibrated with PBS, according to manufacturer’s instructions. Protein concentration and labeling efficiency were determined by measuring OD280 and OD647, respectively.

Fabrication of Microcavity Array Gold and PDMS Substrate

Microcavity SLBs were assembled across periodic pore arrays prepared in PDMS for fluorescence correlation studies, or across gold pore array electrodes for electrochemical studies where the porous arrays were prepared by polystyrene sphere-templating methods as previously described. (56,57,61,66) Briefly, to obtain hexagonally close-packed microcavity array on gold a monolayer of 1 μm polystyrene microspheres was cast using a gravity-assisted method onto pre-cut rectangles of gold-coated silicon wafers (Figure S2, Supporting Information). Gold was electrodeposited onto the interstitial surface between the polystyrene microspheres by applying a reduction potential (−0.6 V, Ag/AgCl) to the gold array in the presence of a cyanide-free gold solution. The electrodeposition was controlled by monitoring the evolution of the current at the gold array until the current reached a minimum value corresponding to the closer distance between the spheres, indicating that the electrodeposition of gold has reached the hemisphere of polystyrene (Figure S2, Supporting Information). After the gold electrodeposition, the arrays were electrochemically cleaned using cyclic voltammetry in 50 mM sulfuric acid for 3 cycles within −0.2 to 1.6 V potential (vs Ag/AgCl) (Figure S2, Supporting Information) and rinsed with deionized water, ethanol, and dried gently under nitrogen flow. The top surface of the gold microcavity arrays was then selectively functionalized with a self-assembled monolayer (SAM) of 6-mercaptohexanol (1 mM) for at least 24 h in ethanol, before removal of the templating spheres, which were subsequently washed out of the array with tetrahydrofuran (THF). (56,62) An electroactive area of ∼5 cm2 was determined for electrodes used in this study for MSLB preparation and subsequent electrochemical experiments (see the Supporting Information).
The PDMS microcavity arrays were prepared by drop-casting 50 μL of ethanol containing 0.1% of 4.61 μm diameter polystyrene spheres (Bangs Laboratories) onto a 1 cm × 1 cm hand cleaved mica sheet, which was glued to a glass coverslip. After ethanol evaporation, PDMS was poured onto the polystyrene microsphere arrays and cured at 90 °C for 1 h. Microcavity arrays were formed after removing the inserted polystyrene microspheres by sonicating the PDMS substrate in THF for 15 min. The substrates were then left to dry overnight. Prior to lipid bilayer formation, the substrates were cleaned using oxygen plasma for 5 min and microcavities were buffer filled before lipid monolayer deposition by sonicating the PDMS substrate in PBS buffer (pH 7.4) for 1 h.

Fabrication of Pore-Suspended Lipid Bilayers

Pore-suspended lipid bilayers were prepared according to a two-step process described previously for a range of bilayer compositions. (55,57−62,66) The proximal leaflet of eggPC monolayers was transferred by the Langmuir–Blodgett (LB) technique. Briefly, approximately 50 μL of eggPC (1 mg/mL in chloroform) were deposited onto the air/water interface of LB trough (NIMA 102D) and the solvent was allowed to evaporate for 15 min. The resulting lipid monolayer at the air/water interface was compressed 4 times to a surface pressure of 33 mN/m at 25 mm/min. Then, the aqueous-filled microcavity arrays were immersed into the LB trough until all of the cavities were submerged completely into the subphase. The micro-cavity array was withdrawn from the trough at a rate of 5 mm/min while the surface pressure of the lipids was maintained at 32 mN/m to ensure an adequate transfer of the eggPC monolayer. To assemble the upper leaflet of the bilayer, liposome (with or without α5β1 integrin) fusion (Figure S2, Supporting Information) was carried out using SUVs prepared as described above, comprised of eggPC:eggPA (90:10). Where integrin was reconstituted into the MSLB, according to the procedure above, a proteoliposome was prepared at a LPR of 2700 and fused to the LB prepared monolayer. Following LB transfer, the fusion processes were carried out within a sealed microfluidic chamber for PDMS arrays. For gold, 150 μL liposome/proteoliposome solution was introduced to the monolayer. The bilayers are not exposed to air/allowed to dry, whether during the liposome wash step or throughout measurement. The resulting PDMS or gold MSLBs were confirmed formed by the fluorescent lifetime imaging (FLIM), FCS, or by electrochemical methods, respectively.

Fluorescence Lifetime Correlation Spectroscopy

Fluorescence lifetime imaging and correlation spectroscopy measurements were performed using a Microtime 200 system (PicoQuant GmBH, Germany) integrated with a FCS module, dual SPD detection unit, time-correlated single photon counting, on an inverted Olympus X1-71 microscope with an Olympus UPlanSApo 60×/1.2 water immersion objective. A single mode optical fiber guided the lasers to the main unit and provided a homogeneous Gaussian profile excitation beam. The lasers were pulsed at 20 MHz, corresponding to an interval of 50 ns. The emitted fluorescence was collected through the microscope objective. A dichroic mirror z532/635rpc blocked the backscattered light, and corresponding filters were used to clean up the signal. A 50 μm pinhole was set to confine the volume of excitation and detection of fluorescence intensity that originated from the fluorescently labeled protein in the axial direction. Fluorescence was detected using a single photon avalanche diode from MPD (PicoQuant). Before the FCLS measurement, backscattered images of the substrate (images were collected using an OD3 density filter) followed by fluorescent lifetime images were acquired to ensure the optimal positioning of the buffer-filled cavities where the bilayers are spanned. Fluorescence lifetime correlation spectroscopy (FLCS) analyzes the time-dependent fluctuations of the fluorescence intensity ∂I(t) recorded over 30 s and analyzed by an autocorrelation function (ACF), G(τ) = (⟨∂I(t)⟩·⟨∂I(t + τ)⟩)/⟨∂I(t)⟩2, where ⟨⟩ denotes the time average, and ⟨∂I(t)⟩ and ⟨∂I(t + τ)⟩ are the fluorescent intensity fluctuations around the mean value at time t and t + τ, respectively. The FLCS autocorrelation data fit to a 2D diffusion model equation defined in eq 1
G(τ)=(1+fτ1−fτe−t/τT)·1⟨N⟩·11+(τ/τD)α
(1)
where, ⟨N⟩ is the average number of diffusing fluorescence particles in the observation volume, fτ and τT are the fraction and the decay time of the triplet state, τD is the transit time, and α is the anomaly coefficient. From the fitting, the τD values were evaluated and accordingly, the diffusion coefficient was obtained from eq 2
D=ω24τD
(2)
where, ω is the observation beam waist diameter, typically obtained from the calibration of a standard dye diffusing in 3D with a known diffusion coefficient value. Point FLCS measurements were then recorded at the center of cavity for a duration of 30 s and an average of 20 cavities were studied for each sample for every FLCS measurements. All FLCS experiments were carried out in triplicate at 20 ± 1 °C.

Electrochemical Impedance Spectroscopy

Electrochemical impedance spectroscopy (EIS) measurement was performed with a CHI760e (CH Instruments, USA). A standard 3-electrode cell was employed for all measurements, comprised of a gold microcavity array covered by the lipid bilayer as a working electrode, an Ag/AgCl (1 M KCl) reference, and a coiled platinum wire counter electrode. The EIS data were recorded across a frequency range of 0.05 to 105 Hz with an AC modulation amplitude of 10 mV at a DC potential bias of 0 V (vs Ag/AgCl). All measurements were carried out in a glass cell (approximate volume of 4 mL of 0.01 M PBS). The EIS of the aqueous-filled microcavity array coated with the lipid bilayer composition alone was measured initially before the addition of galectin to ensure signal stability. The non-Faradaic EIS signal from the integrin-reconstituted MSLBs was evaluated for stability, and it was found that when placed in contact with the buffer, an initial fluctuation of resistance occurred that stabilized within an hour and then remained unchanged over a prolonged 24 h window, which was well beyond our experimental time window (5–6 h) for WTGal3/Gal3ΔNter binding studies. Initially, for integrin-reconstituted MSLBs, a time lag of 90–120 min was allowed to ensure that the membrane had equilibrated in the electrochemical cell (no fluctuation of EIS, i.e., the membrane impedance is unchanged) before proteins were titrated. For each protein aliquot, the designated concentration of proteins was added to the electrochemical cell and an equilibration binding time of 30 min was maintained. This window was confirmed to be sufficient for protein binding to the membrane, as beyond this time, protein binding elicited no further change to a membrane impedance signal. The proteins (WTGal3 and Gal3ΔNter) were initially prepared as a stock solution in buffer and this was aliquoted into the electrochemical cell to achieve the required final concentration and mixed thoroughly. The volume added to the cell never exceeds 200 μL. All measurements were carried out at room temperature (22 ± 1 °C). The impedance of the MSLBs for each protein type, as well as their temporal stability was assessed in triplicate. The measured data were analyzed using Z-View software (Scribner Associates, v3.4e) by fitting the equivalent circuit model (ECM), as shown in Figure 2C. The best fit using the ECM circuit was assessed from both visual inspections of the fit residuals and χ2, typically ∼0.001.

Results and Discussion

ARTICLE SECTIONS
Jump To

Purification, Characterization, and Reconstitution of α5β1 Integrin within Small Unilamellar Vesicles

α5β1 integrin was solubilized in DDM detergent and purified from the rat liver, as described in the Materials and Methods section. We have used EM in the negative staining mode to qualitatively validate the integrity of the DDM-micellar protein particles at the structural level. Purified micellar α5β1 integrin clearly appeared as individual particles on the EM grids that presented the typical shape of integrin heterodimers, including a globular headpiece and thinner leg parts (Figure 1A). Many of these integrin particles were in the bent-closed inactive conformation, 10–15 nm in length and well described for their low affinity for fibronectin (Figure 1A, inset, illustration). This was fully consistent with our elution strategy, where the integrin was initially activated to efficiently bind a FNIII9–10-functionalized column, while the subsequent release was achieved by shifting the protein to the inactive conformation, which led to its elution from the column in the bent-closed conformation.

Figure 1

Figure 1. α5β1 integrin purification and reconstitution into SUVs. (A) Qualitative visualization of α5β1 integrin particles by EM. Negative staining images of α5β1 integrin solubilized in DDM. The inset shows a zoomed image of integrin particles, and an illustration of individual integrins in the bent-closed conformational state. (B) Characterization on sucrose gradients of α5β1 integrin incorporation into vesicles. Gradient fractions F1 to F8 (right cartoon illustration) were collected and submitted to anti-β1 integrin immunoblotting. β1 integrin was expectedly detected at 120 kDa in F1 and F2 fractions. L represents total proteoliposome input. (C) Homogeneity of the proteoliposomes as visualized by cryoEM. Inset shows a magnification view. The proteoliposomes were homogenous in size, with a mean diameter of 150 nm. (D) Analysis of α5β1 integrin orientation within SUVs. Proteoliposomes were subjected or not to trypsin digestion in the presence or absence of Triton X-100. In the absence of detergent (lane 1), the immunoreactive band corresponds to β1 integrin molecules for which the large extracellular domain was oriented into the liposomal lumen and thereby protected from the protease. In the presence of trypsin and detergent (lane 2), no β1 integrin band was detected, since the enzyme now had full access to the whole protein. In the presence of the detergent alone (lane 3), the detected band corresponds to the total amount of β1 integrin. This allowed us to estimate the percentage of correctly oriented α5β1 integrin, which was around 50%. Micellar α5β1 integrin was used as a control. The cartoon illustration summarizes the different conditions. (E) Assessment of the functionality of liposomal α5β1 integrin. To confirm the capacity of α5β1 integrin to become activated. Proteoliposomes were pre-incubated or not (Ctrl) with the indicated activating metal ion salts (MgCl2 or MnCl2) and then submitted to fibronectin FN-III9–10 pull down. As expected, β1 integrin was pulled down in the presence of magnesium (MgCl2) and to a greater extent in the presence of manganese (MnCl2), demonstrating that the SUV-reconstituted integrins were functional.

High Resolution Image
Download MS PowerPoint Slide
We then successfully incorporated the purified micellar-integrin (Triton X-100 micelles) into SUVs at a low protein density with a LPR of 2700 (mol/mol). The rate of integrin incorporation in SUVs was assessed by loading the reconstituted proteoliposomes onto sucrose gradients, followed by immunodetection of the protein in each fraction. Interestingly, integrins were only detected at the middle and the top fractions of the 5% sucrose gradient (Figure 1B). This suggested a homogenous distribution of the protein among the proteoliposome populations and a low density of protein, as expected from the LPR that was used. Moreover, the absence of integrin in the fraction corresponding to the highest concentration of sucrose (here 30%) indicated that no protein aggregation occurred during the reconstitution step.
Cryo-EM analysis was performed to assess both the morphology and size of the α5β1 integrin proteoliposomes. As shown on the micrographs, these liposomes appeared as homogenous unilamellar and spherical vesicles, with a mean diameter of 150 nm (Figure 1C, inset), suitable for subsequent reconstitution onto the MSLBs.
The orientation of α5β1 integrin after reconstitution in SUV was then assessed. It is indeed critical to have a substantial fraction of integrin molecules that are correctly oriented within the proteoliposomes with their cytosolic domain facing into the lumen. The trypsin-accessibility/protection assay was used to address this point. Proteoliposomes were incubated with trypsin in the absence of Triton X-100 for the immunodetection of the luminally orientated integrin fraction [Figure 1D, lane 1, and illustration (top right panel)], or in the presence of Triton X-100 without trypsin [Figure 1D, lane 3, and summarized in the illustration (bottom right panel)] for evaluation of the total amount of incorporated integrin to in fine estimate the proportion of correctly oriented integrin. The efficiency of enzymatic digestion was confirmed by the absence of β1 integrin immunodetection [Figure 1D, lane 2, and illustration (middle right panel)] when SUVs were disrupted by Triton X-100 incubation before trypsin treatment, thus exposing the total pool of integrin to the enzyme. Importantly, integrins were symmetrically oriented in the membrane with 50% of the protein pointing outward from the vesicles.
We then aimed to confirm that our reconstituted α5β1 integrin was functional. For this, we have assessed its capacity to become activated, either using MgCl2 or MnCl2 preincubation for integrin priming, followed by pull-down on fibronectin (FNIII9–10) fragment-coated beads. Immunodetection of β1 integrin in the presence of MgCl2, and to a greater extent with MnCl2 (Figure 1E), confirmed that even in the liposomal environment of SUVs, α5β1 integrin retained its capacity to become activated and to interact efficiently with its natural ligand, fibronectin.

Characterization of Proteoliposomes Using DLS and Fluorescence Life-Time Cross-Correlation Spectroscopy

DLS was used to follow each of the steps of proteoliposome preparation. The initial eggPC:eggPA liposomes resuspended in HEPES buffer formed vesicles of narrow size distribution with a mean diameter of 500 nm (Figure S1A, Supporting Information). Detergent disruption was confirmed from the change in the size distribution of liposomes (Figure S1B, Supporting Information). Following detergent removal, the reformed proteoliposomes containing α5β1 integrin showed a homogeneous diameter of 120 nm (Figure S1C, Supporting Information). Additionally, using fluorescence life-time cross-correlation spectroscopy, the diffusion of labeled α5β1 integrin-ATTO488 and DOPE-ATTO655 in proteoliposomal membranes was evaluated following simultaneous excitation of both fluorophores (Figure S1D, Supporting Information). The ACF showed that α5β1 integrin and DOPE co-diffused, confirming that both were indeed reconstituted into the same proteoliposomes.

Characterization of Integrin-Reconstituted MSLB over Aqueous-Filled Gold Substrate

Gold microcavity-suspended lipid bilayers (MSLB, pore diameter, 1 μm) comprising of eggPC (PC) at the proximal leaflet, eggPC:eggPA (PC:PA) at the distal leaflet, and bilayer-spanning integrin were prepared via LB transfer of a PC monolayer followed by fusion of proteoliposomes, as shown schematically in Figures 2A and S2A (Supporting Information). These MSLBs are hereafter called PC//PC:PA/Int. The proteoliposomes comprised of α5β1 integrin reconstituted into PC:PA (90:10) SUVs (∼120 nm diameter) at a LPR of 2700. Fluorescence lifetime imaging was used to characterize the PC//PC:PA/Int MSLB assembly at the gold microcavity array. To facilitate this, the lower PC leaflet, transferred during the LB deposition was doped with ATTO647-PE (0.03 mol %) and α5β1 integrin was fluorescently labeled with ATTO488.

Figure 2

Figure 2. (A) Schematic illustration of proteoliposome fusion over a lipid monolayer covered gold microcavity array (not to scale) yielding PC//PC:PA/Int MSLBs. Reflectance image (B), FLIM images obtained from the lower PC leaflet doped with 0.03 mol% ATTO647-PE (C), andfrom ATTO488 α5β1 integrin (D). Non-Faradaic Nyquist plot (E) and frequency–normalized complex capacitance plot (F) of the cavity array alone (black), of PC//PC:PA (blue) and of PC//PC:PA/Int MSLBs (red). In panels E and F, zoomed-in areas are shown in insets as indicated by the green boxes. (G) Schematics of MSLB spanned over a microcavity (not to scale) and the associated ECM used to fit EIS data. In the ECM, Rel, and Cstray represent, respectively, solution electrolyte resistance and stray capacitance, RM and QM represent, respectively, membrane resistance and CPE, and Rarray and Qarray are the, respectively, microcavity array resistance and CPE. The corresponding fits to the ECM are shown as solid lines in panels E and F. (H) Relative change in membrane resistance to show the stability of PC//PC:PA membranes without (blue) and with (red) the presence of α5β1 integrin versus time monitored for more than 24 h. The EIS recording at an initial time window of 0–1.5 h shows an increase in membrane resistance, which saturates and remains stable for more than 24 h. EIS measurements were performed in PBS buffer within the frequency ranges between 0.05 and 105 Hz at 0 V DC bias potential vs Ag/AgCl (1 M KCl) with an AC amplitude of 10 mV at 22 ± 1 °C. A three-electrode setup where gold cavity/MSLB, Ag/AgCl (1 M KCl), and Pt wire served as working, reference, and counter electrodes, respectively.

High Resolution Image
Download MS PowerPoint Slide
A representative reflectance image of PC//PC:PA/Int MSLB from a gold microcavity array is shown in Figure 2B. The white circular features show reflected light from the buffer-filled cavities, replicated across the surface. Reflectance imaging indicates that at the gold array, all the cavities are buffer filled. Figure 2C,D shows representative FLIM images obtained from the fluorescently labeled lower leaflet and the integrin, respectively, confirming that a bilayer had formed and that the integrin was reconstituted into the bilayer. The PC//PC:PA:Int bilayer was also characterized by atomic force microscopy (AFM) (Figure S3, Supporting Information). To further confirm bilayer formation from the LB/liposome fusion process, we separately labeled each monolayer of PC//PC:PA membranes that were assembled over gold microcavities. The lower PC leaflet was labeled with ATTO532-PE, and the upper PC:PA leaflet with ATTO647-PE. The corresponding reflectance and FLIM images show, as expected, a continuous fluorescence signal from each leaflet across the entire array surface (Figure S4, Supporting Information).
Exploiting the gold microcavity array as a working electrode, we then characterized the PC//PC:PA/Int MSLB using EIS. Figure 2E shows representative non-Faradaic Nyquist plots obtained from EIS measurements before (black) and after PC//PC:PA/Int (red) spanned over SAM-modified cavity arrays. The corresponding frequency–normalized complex capacitance plots are shown in Figure 2F. The non-Faradaic Nyquist trace shows the sum of real (Z′) and imaginary (−Z″) components of the complex impedance, which reflect any changes to the ion-transfer process across electrode/electrolyte interface. For example, when the Nyquist trace shifts toward the x-axis (Z′), the impedance is decreased, or admittance (ion transport) is increased. Similarly, a shift toward the y-axis (−Z″) implies that the impedance is increased, and admittance decreased. As expected, on PC//PC:PA/Int (red) bilayer formation, the impedance was increased greatly compared to the SAM-functionalized cavity array (black) support (Figure 2E, inset). In addition, the electrode capacitive properties on the assembly of PC//PC:PA/Int can be visualized from the angular frequency–normalized complex capacitance plot (Y″/ω vs Y′/ω), as shown in Figure 2F. The semicircle plot intercept in Y″/ω at ∼40 × 10–6 F for a SAM-modified cavity (black) was significantly reduced to 2.1 × 10–6 F for α5β1 integrin-containing membranes (Figure 2F and inset). As a control, the pristine PC//PC:PA (blue) membrane without α5β1 integrin is also included in panels E and F. When compared to the α5β1 integrin containing membrane, the pristine PC//PC:PA membrane (red) showed a lowering in resistance (blue, Figure 2E) and a modest increase in capacitance (3.2 × 10–6 F). Because capacitance is inversely proportional to the thickness of bilayer (assuming that the bilayer membrane is a parallel plate capacitor) as defined by eq 3; our data suggests that α5β1 integrin containing membranes have a greater thickness than pristine membranes.
capacitance=ϵε0Ad
(3)
where ϵ (=2.1) is the relative dielectric constant, ε0 (=8.854 × 10–12 F/m), d is the thickness of membrane (distance between plates), and A is the area of the plates [electrode onto which dielectric (here MSLB) is spanned]. (44)
Quantitative evaluation of changes to membrane electrical resistance and capacitance properties was achieved by fitting the EIS data to the ECM, as shown in Figure 2G. We previously reported this heuristic approach, applied to the MSLB model. (61,62) Upon fitting the EIS data (solid lines, Figure 2E,F), representative absolute resistance values for SAM-modified cavity arrays without the bilayer, with PC//PC:PA or PC//PC:PA/Int MSLBs were determined to be 0.4 ± 0.15, 7.7 ± 0.5 and 9 ± 0.8 MΩ, respectively. Similarly, the capacitance (Q) values of the constant phase element (CPE) were estimated to be 40 ± 3, 3.4 ± 0.2, and 2.5 ± 0.3 μF·sm–1, respectively. From our fitting, the other components such as electrolyte solution resistance (Rel = ∼40 ± 10 Ω), stray capacitance (Cstray = ∼1 nF) due to the electronic connectors, cavity resistance (Rcavity), and cavity CPE (Qcavity) remained essentially unchanged. The complex capacitance (Q) when CPE is used can be expressed as 1/Q(jω)m, where Q is analogous to the magnitude of capacitance, ω is the angular frequency (rad/s), and m is the homogeneity parameter varying between 0 < m < 1. For an ideal capacitor, m = 1 and for a pure resistor, m = 0. Typically, for MSLBs, m = 0.94 ± 0.02, and for cavity m ≤ 0.5 ± 0.02, which corresponds to the bilayer membrane when CPE approaches an ideal capacitor, and the array CPE becomes a series RC circuit or Warburg impedance. Although, the capacitance (C) can be obtained from the Q value using the expression C(ω) = Qωm–1, it is only valid for a specific ω, limited to the specific ECM and thus, was not used in this study. Furthermore, the membrane resistance and CPE stated above without or with α5β1 integrin containing membranes have not been normalized to the electroactive surface area. This is due to batch-to-batch variations that occur upon microstructuring of surface areas across the ∼1 cm × 1 cm (length × breadth) gold on silicon chips. For example, discontinuities in packing or where PS spheres fail to pack can constitute between 2 and 5% of area. In addition, as the MSLB fabrication step involved a selective SAM modification to the exposed top surface, it was not straightforward to calculate the electroactive area of the electrode using the conventional Randles–Ševčík electrochemical method, as electron-transfer processes would deviate from the reversiblebehavior of a Faradaic electron-transfer redox process. As a result, for lectin binding studies, we reported the relative (Δ) change in membrane resistance and CPE. Nonetheless, the total electroactive area used in our study was 5 ± 0.4 cm2 for a bare cavity array without SAM (Figure S2B,C, Supporting Information). This translated to absolute membrane resistance in the presence and absence of integrin of 45 ± 3 and 35 ± 2 MΩ cm2, respectively. The respective absolute capacitance (CPE, QM) values were thus 0.5 ± 0.07 and 0.68 ± 0.03 μF·sm–1 cm–2, respectively. The respective absolute capacitance (CM) at a fixed frequency (f) of 5 Hz were thus 0.55 ± 0.07 and 0.41 ± 0.03 μF·cm–2, respectively, which agree well with the literature capacitance values of a bilayer membrane. (44,47,67−69)
The non-Faradaic EIS signal after the formation of MSLBs comprised of PC//PC:PA with or without α5β1 integrin was monitored to evaluate membrane stability. When MSLBs were initially placed in contact with PBS buffer, we observed an initial increase in resistance (ΔRM = RMtime=t – RMtime=0) that took 0–1.5 h to equilibrate. Following equilibration, the EIS signal remained stable over a prolonged window of 24 h (Figure 2H). This window of stability was well beyond our experimental time (5–6 h) for the lectin binding studies (vide infra). To be on the safe side, we always allowed membranes to equilibrate for 2 h prior to measurement.

Electrochemical Characterization of Gal3 Binding to α5β1 Integrin-Containing MSLBs

EIS was used as a highly sensitive, label-free method to study the influence of Gal3 on the membrane phase or packing. Following measurement of the impedance of the PC//PC:PA/Int membrane, WTGal3 was incrementally added to the contacting solution. After each WTGal3 addition, an incubation time of ∼30 min was applied before EIS was recorded. The relative changes to membrane resistance and capacitance were extracted by fitting the EIS data to the ECM described above. In each instance, the absolute resistance of the protein-reconstituted membrane was evaluated, to ensure it conformed to the expected RM and QM values of stable lipid bilayers, as described above and previously. (61) As discussed , the relative changes in membrane resistance (ΔR = RM0 – RMlectin) and capacitance (ΔQ = QM0 – QMlectin) were determined, where RM0 and QM0 represent, respectively, the absolute resistance and capacitance of pristine membranes, that is, in the absence of lectin, and RMlectin and QMlectin the respective values when lectin was present in the contacting solution.
Upon binding of WTGal3 to PC//PC:PA/Int MSLBs, the membrane resistance decreased and capacitance increased (Figure 3A,B, black). Although the binding of a Gal3 mutant in which the unstructured N-terminal oligomerization domain was deleted (termed Gal3ΔNter) also reduced the membrane resistance, the magnitude of resistance changes was found to be consistently roughly half of the one that was found or WTGal3. Capacitance values increased with increasing WTGal3 concentration up to 10 nM (Figure 3B, black), whereas a systematic decrease was observed for Gal3ΔNter that saturated at 10 nM (Figure 3B, red).

Figure 3

Figure 3. EIS characterization of integrin-containing membranes upon binding of WTGal3 or Gal3ΔNter. (A,B) Relative changes in (A) resistance, ΔR (filled symbol), and (B) capacitance, ΔQ (open symbols) values obtained upon the addition of different concentrations of WTGal3 (black) or Gal3ΔNter (red) to PC//PC:PA/Int membranes. The solid lines in each panels A and B are shown to guide the eye. Data are means ± SD from triplicate experiments. (C,D) Bar charts showing the change in capacitance values with respect to the pristine PC//PC:PA/Int membrane when 37 nM WTGal3 (C) or 62.5 nM Gal3ΔNter (D) were incubated with α5β1 integrin-containing membranes in the presence (+Lac, 50 mM) or absence of β-lactose. Lactose fully abolished the WTGal3 or Gal3ΔNter effects on ΔQ. In the absence of Gal3, lactose did not affect ΔQ. EIS measurements were performed in PBS buffer, frequency ranges were from 0.05 to 105 Hz at 0 V DC bias potential vs Ag/AgCl (1 M KCl) with an AC amplitude of 10 mV at 22 ± 1 °C. The measurement cell was a three-electrode setup where gold cavity/MSLB, Ag/AgCl (1 M KCl), and Pt wire served as working, reference, and counter electrodes, respectively.

High Resolution Image
Download MS PowerPoint Slide
In the absence of α5β1 integrin, that is, at PC//PC:PA membranes, WTGal3 elicited a systematic decrease in membrane resistance with increasing concentration (Figure S5A, black, Supporting Information) but the response was weaker compared to PC//PC:PA/Int membranes. The decreased membrane resistance was consistent with the increased diffusivity of the membrane reported in FCS studies vide infra and may be due to changes to membrane packing or nanopore formation induced by WTGal3. WTGal3 also caused a very small (∼0.1 μF·sm–1) initial increase to capacitance that stabilized at 10 nM (Figure S5B, black, Supporting Information). In contrast, even at concentrations up to 62.5 nM, Gal3ΔNter did not elicit any measurable change to PC//PC:PA membrane resistance (Figure S5A, red, Supporting Information) or capacitance (Figure S5B, red, Supporting Information).
Overall, the data suggest that WTGal3 interacts with the PC//PC:PA membrane in the absence of integrin and increases membrane ion permeability. This may be mediated through electrostatic interactions between negatively charged egg phosphatidic acid (PA) head groups and the CRD of Gal3, which is positively charged at a physiological pH of 7.4, (15) or via penetration of the partially lipophilic proline-rich N-terminal domain (16) into the lipid bilayer, possibly inducing some surfactant-like nanoporation. (70) That Gal3ΔNter did not affect the electrical membrane properties argues in favor of the latter explanation.
The impact of WTGal3 on membrane electrical properties (Figure S5B, black, Supporting Information) was much greater at integrin-containing membranes (Figure 3B, black), which indicated that the glycoprotein strongly favored the recruitment of the lectin. Because these changes were not observed with Gal3ΔNter (see above), we conclude that they were dependent on the oligomerization capacity of WTGal3. We speculate that the hydrophobic proline-rich N-terminal domain-dependent formation of Gal3 oligomers on α5β1 integrin-perturbed membrane organization, possibly by creating membrane domains and/or pores (see the Conclusions section). WTGal3 has indeed been shown to interact directly with lipid membranes. (70)
Furthermore, at WTGal3 concentrations above 10 nM, membrane resistance stabilized but capacitance gradually decreased suggesting progressive thickening of the membrane (Figure 3B, black). This might have originated from the formation of WTGal3−α5β1 integrin assemblies in clusters that would have grown laterally into lattices as membrane invagination cannot occur in the MSLB system. Of note, the WTGal3 effect on ion permeability persisted under these conditions as resistance remained at a low plateau level (Figure 3A, black) and capacitance─even if decreasing with the continued addition of WTGal3 above 10 nM─globally also remained above the level that was observed in the absence of the integrin (Figure S5B, black, Supporting Information). We speculate that the permeability and thickening effects occurred concomitantly at WTGal3 concentrations above 10 nM.
The disaccharide β-lactose binds into the core pocket of the CRD on galectins. (13) It can, therefore, be used as a competitive inhibitor of interactions that depend on this binding pocket. We found that the effects of WTGal3 and of Gal3ΔNter on the capacitance and resistance of α5β1 integrin-containing membranes were eliminated in the presence of β-lactose (Figures 3C,D and S6, Supporting Information). We confirmed that lactose had no effect on membrane thickness in the absence of Gal3 (Figure 3C,D).
To obtain quantitative empirical insights into the association of WTGal3 and Gal3ΔNter with pristine or α5β1 integrin-containing membranes, the experimental ΔQ data (Figures 3B and S5B, Supporting Information) were fit (Figure S7, Supporting Information) to the empirical Hill–Waud binding model according to eq 4
ΔQ=ΔQsat(C)n(kD)n+(C)n
(4)
where ΔQ is the change in membrane capacitance, ΔQsat is the absorption capacity or change in capacitance at maximum surface loading that relates to the number of available binding sites, kD is the empirical apparent equilibrium dissociation constant, C is the concentration of the galectin (WTGal3 or Gal3ΔNter), and n (dimensionless) is the Hill coefficient, which reflects the steepness of the slope of the binding curve, often related to cooperativity, where it exists, in protein–receptor binding. n < 1 is taken to indicate negative cooperativity, that is, chemically mediated adsorption where binding reduces affinity for further binding events, n = 1 reverts the expression to the Langmuir isotherm, where all binding sites are energetically equal, that is, non-cooperative equilibrium binding, and n > 1 indicates positive cooperativity, where binding promotes affinity for further binding. From the fitting, a kD of 0.2 ± 0.02 nM was obtained when WTGal3 bound to the integrin containing PC//PC:PA membrane versus a kD of 0.76 ± 0.04 nM for Gal3ΔNter binding. The fitting parameter values are reported in Table 1.
Table 1. Evaluation of Dissociation Binding Constant, kD, Cooperativity, n, and Maximum Saturable Absorption Capacity, ΔQsat of WTGal3 and Gal3ΔNter upon Binding to PC//PC:PA or PC//PC:PA/Int Membranesa
lectinmembranekDΔQsatn
WTGal3PC//PC:PA/Int0.2 ± 0.020.35 ± 0.051.7
WTGal3PC//PC:PA0.09 ± 0.020.08 ± 0.11
Gal3ΔNterPC//PC:PA/Int0.76 ± 0.04–0.15 ± 0.010.8
Gal3ΔNterPC//PC:PA---
a

R2 values are 0.95, 0.99, and 0.96 (from top to bottom).

While the comparison between WTGal3 and Gal3ΔNter for the binding to α5β1 integrin was relevant, kD values for binding to membranes with or without α5β1 integrin could not be directly compared as they reflected on different types of interactions. In the first case, the primary binding site of the Gal3 was α5β1 integrin glycans, and its effects on membranes needed to be mediated from there. In contrast, in the absence of α5β1 integrin, the primary binding site was the membrane itself, and the magnitude of EIS signal change was, therefore, expected to be greater. Accordingly, in the presence of integrin, the apparent kD for the WTGal3 is approximately 3 times lower than that of Gal3ΔNter (Table 1). While this finding was qualitatively in agreement with the differential effect of both proteins on the capacitance change (ΔQsat) (Figure 3B), the amplitude difference between WTGal3 and Gal3ΔNter was much higher for ΔQsat than for kD. This indicates much larger changes to membrane packing upon binding of WTGal3. This is notably emphasized by the steep slope of the plot (Figure 3A, open black symbol) and the Hill coefficient (n) in the presence of integrin (Table 1). The n of 1.7 for WTGal3 suggests positive cooperative binding, likely due to Gal3 oligomerization onto the integrin glycoprotein. In contrast, n decreased to 0.8 in the case of the Gal3ΔNter, as expected for this oligomerization deficient mutant.

FCS-Based Characterization of Gal3 Binding to α5β1 Integrin-Reconstituted Membranes

To further support the EIS data, fluorescence lifetime-based imaging and FCS measurements were carried out at MSLBs on optically transparent arrays.
The PC//PC:PA/Int MSLB preparation over PDMS-based cavity arrays was the same as for the gold microcavities. PDMS is used rather than gold for FLCS as a low reflectance medium was required for single molecule fluorescence. The pore apertures were larger for fluorescence measurements (diameter ∼ 2 μm) to facilitate a pore-by-pore study within the confocal volume. (71) The pore array substrates for optical studies were incorporated into microfluidic chambers that permitted introduction of reagents to the MSLB with minimum volume (∼150 μL). (56,57,59,71) The fluidity of the PC//PC:PA MSLBs was evaluated prior to reconstitution of integrin using a fluorescently labeled lipid, ATTO655-DOPE (0.01 mol %), which was doped only at the outer leaflet of the MSLB during liposome preparation, and ATTO532-DOPE (0.01 mol %), which was doped at the inner leaflet. The diffusion coefficients from both leaflets of PC//PC:PA membranes were estimated by fitting the ACF to the 2D diffusion model of eq 1, and the diffusion coefficient at each leaflet were found to be identical as 6.66 ± 0.38 μm2/s with an anomalous factor (α) of 0.94 ± 0.17, indicating Brownian diffusion. The lipid diffusivity at the planar PDMS array (in the absence of underlying aqueous pore cavities) was found to be 2.2 ± 0.3 μm2/s. The identical diffusivity of each leaflet with greater diffusivity than bilayer supported over planar regions of the PDMS substrate confirmed we were measuring membranes that were suspended across aqueous filled pores with no underlying frictional interaction from the solid support. All FLCS measurements were acquired from the center of the pore using the bright reflectance from the aqueous filled well as a guide and Z-scanning to locate the bilayer. Our reported diffusivity data are averages of at least 40–50 measurements across at least three different substrates. The diffusion coefficient for the PC//PC:PA MSLBs was considerably lower than simple dioleoylphosphatidylcholine (DOPC) MSLBs, which were previously reported as approximately 10.5 μm2/s. (56,59) However, our values matched diffusion values in lipid bilayer membranes made of eggPC (72) and thereby the greater viscosity of the natural eggPC:eggPA lipids that were used here. (73)
To study the diffusion of individual molecules, ATTO488-labeled α5β1 integrin and Alexa647-labeled WTGal3 and Gal3ΔNter were used. Successful reconstitution of ATTO488-labeled α5β1 integrin into MSLBs was confirmed by monitoring both FLCS and FLIM at the PDMS platform. Representative FCS autocorrelation data along with FLIM images are shown in Figure 4. The diffusion coefficient of the labeled ATTO488-α5β1 integrin at the MSLB was measured as 1.99 ± 0.56 μm2/s with an α value of 0.86 ± 0.05 in contact with PBS. This value is ∼22× slower when compared to solution diffusivity (46 ± 5 μm2/s) of ATTO488-α5β1 integrin in its micellar form (PBS, 0.2% Triton X-100) at a concentration of 10 nM (Table 2). Compared to the pristine bilayer, the decreased diffusion coefficient of the lipid marker ATTO655-DOPE from 6.66 ± 0.38 to 5.40 ± 0.40 μm2/s in the upper leaflet and to 5.85 ± 0.34 μm2/s in the lower leaflet indicated that α5β1 integrin indeed spanned across the bilayer and influenced membrane viscosity at each leaflet. However, lipid diffusion remained Brownian with an α of 0.98 (Table 2). The diffusion of α5β1 integrin, its molecular brightness, and α value were observed to remain unchanged over a measurement period of 6–8 h, which well exceeded the time window for the lectin binding studies. Interestingly also, the ATTO488-α5β1 integrin diffusion was impacted by the identity of contacting buffer, whereby when HEPES was used, the average diffusion coefficient was measured at 2.76 ± 0.36 μm2/s, instead of 1.99 ± 0.56 μm2/s in PBS. However, α was unchanged. Such buffer effects have been noted previously for other membranes. (58,74)

Figure 4

Figure 4. Representative FLIM and FLCS characterization of α5β1 integrin in MSLBs. (A) Reflectance and (B) FLIM images of ATTO488-labeled α5β1 integrin reconstituted into a PC//PC:PA MSLBs. Brighter white circles in the reflectance image (panel A) resulted from a refractive index mismatch between the buffer and PDMS. (C) FLIM image of ATTO647-DOPE (0.01 mol %) doped in the lower leaflet. Inset depicts a zoomed-in area, with asterisk “*” highlighting the center of cavity’s spatial regimes, where the membrane was suspended over pores and FCS was acquired from. The scale bars in panels A–C are 20 μm. (D) ACFs for ATTO488-α5β1 integrin at different time points (black, red and blue) post-reconstitution into MSLBs, along with 10 nM ATTO488-α5β1 integrin in micellar form diluted in PBS (grey). Solid lines are the fitted data for integrin diffusion across MSLBs and solution using the 2D diffusion model, eq 1, and the pure diffusion model, eq S1 (see the Supporting Information), respectively. The FCS data were collected and averaged from approximately 80–100 points from pores across the substrate. ACF traces showed no changes over 6 h time windows. Both FLCS and FLIM images were taken over PDMS cavity arrays. The substrate was sealed within a microfluidic chamber filled with PBS (pH = 7.4) at 22 ± 1 °C.

High Resolution Image
Download MS PowerPoint Slide
Table 2. Estimated Diffusion Coefficients and Corresponding Anomalous (α) Parameter of ATTO488-α5β1 Integrin in Solution and in MSLBs along with Lipid Diffusion in PC//PC:PA MSLBs in the Presence or Absence of Integrina
diffusing fluorophoresD (μm2/s)α
ATTO488-α5β1 in solution46 ± 51.01 ± 0.03
ATTO488-α5β1 in PC//PC:PA/Int1.99 ± 0.560.86 ± 0.05
ATTO655-DOPE in PC//PC:PA6.66 ± 0.380.94 ± 0.17
ATTO655-DOPE in PC//PC:PA/Int (upper leaflet)5.40 ± 0.400.98 ± 0.10
ATTO655-DOPE in PC//PC:PA/Int (lower leaflet)5.85 ± 0.340.97 ± 0.12
a

PC//PC:PA/Int indicates membranes reconstituted with integrin, and PC//PC:PA membranes without integrin. Data from FLCS studies in PBS at pH 7.4. SDs are from triplicate measurements.

The diffusion coefficient obtained for α5β1 integrin indicated its proper reconstitution into the MSLB, and the values were comparable to previously reported data for reconstituted integrins in GUVs. (75) Our values also compared quite well with reconstituted platelet integrin αIIbβ3 reconstituted into DOPC or into a complex membrane composition at MSLBs, where diffusion coefficients of 3.20 ± 0.59 and 2.80 ± 0.56 μm2/s were reported, respectively, with α coefficients of approximately 1 in these matrices. (55,75) The lower diffusion values obtained here were attributed to the greater viscosity of the eggPC:eggPA mixture and, in particular, to the greater protein loading in the current protocol where in the originating liposomes a LPR of 2700:1 (mol/mol) is expected to be faithfully translated to the MSLB. Moreover, the α was less than one, indicating sub-diffusion of α5β1 integrin under our conditions, which we attribute to the somewhat crowded protein environment compared to our earlier reports where integrin was much less concentrated and the LPR was approximately 8000:1 (mol/mol). (55,75)

WTGal3 and Gal3ΔNter Diffusivity Measurements and Its Impact on α5β1 Integrin and Lipid Diffusivity across MSLBs

Figure 5A depicts representative FLIM images obtained from the integrin-ATTO488 channel of PC//PC:PA/Int membranes (right panel) and from WTGal3-Alexa647 (left panel) at different concentrations. The WTGal3-Alexa467 diffusivity in solution was measured at different concentrations between 3.7 and 37 nM and found consistently to be 83 ± 3 μm2/s in PBS (Figure 5C, gray circle). From the intensity–time traces throughout 20 independent recordings over 10 s, we did not observe any aggregate intensity spikes or any bleaching, suggesting that at the highest concentration of WTGal3 used here, Gal3 remained monomeric in solution (Figure S8, Supporting Information). Using FLIM and FCS, we then studied the association of Alexa647-labeled WTGal3 or of Gal3ΔNter with PC//PC:PA membranes into which α5β1 integrin was reconstituted. Based on the intensity of the FLIM signal, the extent of membrane association of WTGal3 at the α5β1 integrin-containing membrane increased (Figure S9, Supporting Information, for quantification of intensity) in a concentration dependent manner, as depicted in the FLIM images of the Alexa647 channel (Figure 5A, left, top to bottom). The diffusivity of WTGal3 reduced drastically to below 4 μm2/s at membranes when compared to the diffusivity in solution (Table 3). On careful analysis across different cavities, we observed two diffusing components: a slow diffusing component, especially at higher concentrations (1.07 and 0.43 μm2/s for 18.5 and 37 nM WTGal3, respectively; Figure 5C and Table 3) likely indicating clustering, and a mobile fraction with a diffusion coefficient that reached a plateau at highest protein concentration (3.54 ± 0.28 and 3.59 ± 0.11 μm2/s for 18.5 and 37 nM, respectively; Figure 5D and Table 3).

Figure 5

Figure 5. FLIM and FLCS characterization of WTGal3 and Gal3ΔNter upon binding to α5β1 integrin-containing membranes. FLIM images of (A) WTGal3-Alexa647 (left) and (B) Gal3ΔNter-Alexa647 (left) at varying concentrations upon binding to PC//PC:PA/Int membranes. The corresponding ATTO488-α5β1 integrin FLIM images are shown to the right. The arrows in panel A indicate integrin clusters in the presence of Gal3. (C,E) ACFs of WTGal3-Alexa647 (C) and Gal3ΔNter-Alexa647 (E) at varying concentrations upon binding to α5β1 integrin-containing PC//PC:PA membranes. (D,F) ACFs of ATTO488-α5β1 integrin upon incubation with different concentrations of WTGal3-Alexa647 (D) or Gal3ΔNter-Alexa647 (F). (G) ACFs of α5β1 integrin-ATTO488 reconstituted into PC//PC:PA membranes (black circle) in the presence of 50 mM β-lactose (red circles), of 37 nM WTGal3 in the presence of 50 mM β-lactose (blue circles), or after exchanging the contact solution of WTGal3 + Lac with fresh 37 nM WTGal3 (olive circles). In all panels (C–G) open symbols represent the experimental data. Solid lines are the corresponding fits using eq 2 except that a pure diffusion model equation (eq S1, Supporting Information) was used to extract the diffusion coefficient values from Alexa-labeled WTGal3 and Gal3ΔNter in solution. All measurements were carried out in PBS buffer of pH 7.4 and at 22 ± 1 °C.

High Resolution Image
Download MS PowerPoint Slide
Table 3. Averaged Diffusivity Values of α5β1 Integrin-ATTO488 without and with Varying Concentrations of WTGal3-Alexa647 and WTGal3-Alexa647 Diffusion Values from the Same Experiments and the Corresponding Fitsa
protein diffusion type at MSLBD (μm2/s)α
integrin before WTGal31.99 ± 0.560.85 ± 0.26
integrin after 3.7 nM WTGal34.31 ± 0.241.02 ± 0.26
integrin after 18.5 nM WTGal35.01 ± 0.090.93 ± 0.20
integrin after 37 nM WTGal35.26 ± 0.230.92 ± 0.02
3.7 nM WTGal32.51 ± 0.280.96 ± 0.12
18.5 nM WTGal33.54 ± 0.110.96 ± 0.08
 1.07 ± 0.150.84 ± 0.10
37 nM WTGal33.59 ± 0.160.92 ± 0.12
 0.43 ± 0.150.75 ± 0.18
a

The associated anomalous factors are provided. α5β1 integrin was reconstituted into PC:PA (90:10, w/w) membranes at a LPR of 2700 (mol/mol). Values were collected in a single measurement from 80 to 100 cavity pores with each experiment repeated in triplicate at fresh membrane substrates. The SD were extracted from these measurements.

In the absence of α5β1 integrin, WTGal3-Alexa647 adsorbed in a concentration-independent manner at PC//PC:PA membranes (Figure S10, Supporting Information), which was consistent with the earlier EIS data. We also observed the emergence of two distinct populations of WTGal3 diffusivity depending on the concentration of WTGal3: a mobile, lower prevalence population that was observed at low Gal3 concentrations (Figure S10F, filled symbol, Supporting Information), and a dominant population observed at high concentrations that showed very low mobility (Figure S10F, open symbol, Supporting Information). The respective diffusivity values were found to be 6.5 ± 0.15, which matched closely to the diffusivity of the lipid probe (6.6 ± 0.38) and 0.1 ± 0.08 μm2/s, which may have been due to Gal3 self-aggregation or the preferential association of this population with any gel phases within the membrane. Over the described range of concentrations, unlabeled WTGal3 also modestly influenced the diffusivity of the lipid label ATTO655-DOPE, which increased from 6.6 to 6.9 μm2/s (Table S1, Supporting Information). We concluded that membrane packing was somewhat disrupted by WTGal3, which was consistent with the decrease in resistance observed by EIS (Figure S5, Supporting Information). Under identical conditions, much lower association of Gal3ΔNter was observed at the PC//PC:PA membrane (Figure S10E,G, Supporting Information) and no reliable ACF.
Interestingly, when 37 nM of WTGal3-Alexa647 was incubated with PC//PC:PA membranes in the presence of 50 mM β-lactose, we observed very little signal from FLIM imaging, a low ACF signal in FLCS, and a much more modest increase in lipid diffusivity (Figure S11, Supporting Information). Thus, consistent with EIS data, β-lactose seemed to inhibit WTGal3 “non-specific” membrane interaction by preventing its lactose-occupied CRD domain to interact with the membrane. Overall, we speculate that native WTGal3 behaves like an amphiphile: its hydrophobic N-terminus and its positively charged C-terminus may both interact with the lipidic environment, causing the observed decrease in membrane resistance in EIS and altered membrane viscosity. (16) Our observations are highly consistent with the findings of Lukyanov et al. who reported, on the basis of fluorescence studies, that Gal3 binds to phospholipids and can penetrate liposomal and cell membranes. (70) As described, the effect is inhibited by lactose and suggests that the sugar induces structural changes in the galectin beyond the CRD. Indeed it has been reported that self-association between Gal3 molecules (which are not glycosylated) is impeded by lactose. (76,77)
While two mobile WTGal3-Alexa647 populations emerged at the membranes in both the presence and absence of α5β1 integrin, important differences existed. In the absence of integrin, the diffusivity of the mobile population at 6.5 ± 0.15 μm2/s (Table S1, Supporting Information) matched closely that of the ATTO655-DOPE lipid at 6.66 ± 0.38 μm2/s (Table 2). In contrast, in the presence of α5β1, the mobile WTGal3-Alexa647 component exhibited a much lower diffusivity of 2.51± 0.28 and 3.54 ± 0.11 μm2/s at 3.7 and 18.5 nM of the galectin, respectively (Table 3), which followed the same trend as the diffusion coefficients of the integrin. Similarly, for the immobile population of WTGal3-Alexa647, diffusivity increased from 0.1 ± 0.08 μm2/s at the pristine membrane to 0.43 ± 0.15 μm2/s when the integrin was present. The fact that Gal3 diffusivity varied in the presence of α5β1 integrin suggested their co-association in both the slow and fast moving components, where from FCS we observed photobleaching of the integrin (Figure S12A, Supporting Information) when galectin was present, that we attributed to immobilized integrin (see arrow in Figure 5A, right panel). Cross-correlation between integrin and WTGal3-Alexa647 was evident in FCCS studies discussed below for the fast component at MSLBs (Figure S12B, Supporting Information).
WTGal3 was observed to increase the diffusivity of α5β1 integrin in MSLB membranes by at least 2-fold compared to the original diffusion value (Figures 5D and 6, Table 3). This was accompanied by an increase in α to nearly 1 (Table 3), indicating a switch from the sub-diffusion to normal diffusion regime. As described above, the interaction of the WTGal3 with the underlying membrane was observed to decrease its viscosity and this is expected to contribute, at least in part, to this effect. However, both the greater magnitude of the change and alteration in the diffusion regime indicate that other parameters are at play. These may include membrane phase changes or nanopore formation induced by WTGal3 when the integrin is present. However, it is important to note that it is likely that only a subpopulation of integrin remained mobile after galectin addition. α5β1 integrin tied up in immobile lattices would not have contributed to the FCS signal, except as a background bleach which was evident in the time traces. Such immobilization would have depleted the concentration of mobile α5β1 integrin in the bilayer. It has been shown previously that increased concentrations of membrane proteins increase membrane viscosity, thereby slowing the diffusion of lipids and of the proteins themselves. (78−80) This may well explain why we observed an increased diffusion of integrin and an increase in α, as the remaining mobile α5β1 integrin in the bilayer would have been in a less crowded environment after galectin sequestered a sizable fraction of the protein into immobile networks.

Figure 6

Figure 6. Diffusivity of α5β1 integrin and WTGal3. Diffusivity of ATTO488-α5β1 integrin and of WTGal3-Alexa647 at the indicated concentrations of the latter. The blue filled circle shows the diffusion coefficient value for ATTO488-α5β1 integrin at MSLB before WTGal3 addition. Orange circles are the diffusion coefficient values of ATTO488-α5β1 integrin following incremental addition of WTGal3-Alexa647 (3.7, 18.5 and 37 nM). Green squares show the fast component of concentration-dependent diffusion coefficient values for WTGal3-Alexa647 after binding to ATTO488-α5β1 integrin containing PC//PC:PA membranes, and red triangles the ones of the slow diffusing component. Diffusion values in the presence of WTGal3-Alexa647 were obtained following 30 min incubation at the membranes at the indicated concentrations. Dotted oval marks highlight the bimodal diffusivity values of WTGal3-Alexa647.

High Resolution Image
Download MS PowerPoint Slide
Figure 6 traces the trends in α5β1 integrin and galectin diffusivities upon incubation with increasing concentrations of WTGal3. α5β1 integrin and the fast-moving component of WTGal3 followed the same trend toward increased diffusivity when the concentration of WTGal3 was increased. However, the diffusivity values were not identical between integrin and WTGal3. Different explanations may be proposed for this. WTGal3 sub-populations that are not involved in binding to integrin but merely associated with the membrane may have contributed to lowering the mean diffusion values of the fast component of WTGal3 diffusivity. For α5β1 integrin, it needs to be considered that the protein was randomly oriented in the reconstituted membranes (Figure 1), with 50% of the molecules not exposed to WTGal3. Notwithstanding any immobile integrin, the observed diffusivity values for α5β1 integrin were, therefore, likely to be a mean of 2 subpopulations of which only one was in contact with WTGal3. At WTGal3 concentrations of 18.5 nM and above, when the slow diffusivity component of the galectin appeared, α5β1 integrin diffusivity apparently reached a plateau. This behavior could be due to the WTGal3-driven emergence of large α5β1 integrin clusters. These interpretations are in line with a recent study where upon the addition of WTGal3 to HeLa cells, lateral mobility of β1 integrin as well as the size of clusters involving the integrin were increased. (81)

Oligomerization Capacity of Gal3 Is Essential for Efficient α5β1 Integrin–Gal3 Complex Formation

Compared to WTGal3, Gal3ΔNter-Alexa647 associated more weakly with ATTO488-α5β1 integrin containing membranes, as revealed by much weaker signal intensity from the FLIM imaging (Figure 5B, left panel). The solution diffusivity of Gal3ΔNter-Alexa647 was determined as 90 ± 5 μm2/s (Figure 5E, grey circle) and the Gal3ΔNter ACF signal was too weak to reliably measure diffusivity from the membranes (Figure 5E, black, red, and blue symbols). Furthermore, in contrast to WTGal3, the diffusion coefficient of ATTO488-α5β1 integrin was unaffected by incubation with Gal3ΔNter-Alexa647 and remained constant at approximately 1.9 ± 0.2 μm2/s over all Gal3ΔNter concentrations that were explored (Figure 5F). That we saw evidence for weak physisorption of Gal3ΔNter in the EIS measurement but not by FCS can be attributed to the fact that in the EIS experiments, the galectin was retained in the contacting solution during measurements, whereas in the microfluidics device used for FCS, blank buffer was exchanged to reduce any background contribution from unbound fluorophores. If the bilayer interface was not exchanged with fresh buffer, the lectin diffusion stayed the same as in solution (Figure 5C,E, grey; Figure S12B, blue, Supporting Information).
Evidence for association of WTGal3 with the mobile integrin fraction (Figure S12A, Supporting Information) was reflected in modest but significant cross-correlation data (Figure S12B, Supporting Information) obtained from spatial FLCS measurements. The cross-correlation signal was weak, presumably because much of the integrin–galectin was tied up in immobile networks, and the FCCS signal reflected residual integrin–WTGal3 complexes that diffused with the same D as the integrin. It is also important to remember again that 50% of the integrin was in the “upside down” orientation, which prevented it from binding to WTGal3 and becoming immobilized at the galectin network. The weak FCCS signal could, therefore, be attributed to correctly oriented integrin that remained mobile in the membrane and formed a complex with non-networked galectin.
No such cross-correlation was obtained with Gal3ΔNter-Alexa647 on α5β1 integrin-ATTO488 containing membranes (data not shown), which was again consistent with a weak binding of Gal3ΔNter to α5β1 integrin.
Of note, no WTGal3-induced clustering of α5β1 integrin was observed from intensity time traces obtained from multi-channel-scalers when α5β1 integrin was in the micellar form (Figure S12C, Supporting Information). In contrast, the diffusivity of α5β1 integrin was modestly increased in its micellar form upon WTGal3 addition as illustrated from ACFs traces (Figure S12D, Supporting Information), suggesting that Gal3 also oligomerized under these conditions.
In the presence of 50 mM β-lactose (Lac), the increase of ATTO488-α5β1 integrin diffusivity upon WTGal3-Alexa647 addition was no longer observed (Figure 5G). As a control, we showed that the diffusivity of ATTO488-α5β1 integrin itself was not influenced by 50 mM β-lactose incubation and remained stable at 1.9 ± 0.7 μm2/s (Figure 5G). Furthermore, exchange with blank buffer containing freshly added WTGal3-Alexa647 (37 nM) in the absence of β-lactose caused the diffusivity of α5β1 integrin-ATTO488 to increase to 5.5 ± 0.8 μm2/s (α = 0.98) (olive, Figure 5G).
As with EIS experiments, the FCS data established that α5β1 integrin was recognized by WTGal3 at the membrane in a glycan-dependent manner. This efficient α5β1 integrin–Gal3 complex formation and the consecutive measurable membrane electrophysical modulations fully relied on a functional Gal3, where both C-terminal carbohydrate recognition and N-terminal oligomerization domains appeared to be essential features.

Conclusions

ARTICLE SECTIONS
Jump To
We have successfully reconstituted the physiologically purified transmembrane glycoprotein α5β1 integrin into a microcavity-suspended lipid bilayer (MSLB) platform of complex lipid composition and applied electrochemical tools and fluorescence microscopy to study α5β1 integrin–Gal3 complex formation and lateral membrane diffusivity. Use of cavity SLBs with their deep aqueous reservoirs at both membrane interfaces permits native-like lateral fluidity of reconstituted membrane protein and enables versatile multimodal interrogation. This original and sensitive approach allowed us to provide a novel investigative angle to understand the specific functions of the C-terminal glycan binding and the N-terminal oligomerization domains of Gal3, in the presence of a naturally glycosylated cargo protein, α5β1 integrin from the rat liver.
EIS experiments demonstrated that even in the absence of α5β1 integrin, WTGal3 associated with membranes comprised of PC//PC:PA, as deduced from membrane resistance decrease (ΔR) that showed a systematic and saturable response that followed Langmuir behavior with increasing WTGal3 concentrations. Consistent with EIS data, FCS data revealed that the WTGal3 influenced membrane viscosity by modestly increasing the diffusion coefficient of the lipid marker and reducing its α to below 1.
EIS data also showed that even at concentrations below 10 nM, WTGal3 associated with glycosylated α5β1 integrin in a positive cooperative manner (n = 1.7). The simplest interpretation of this data is that WTGal3 oligomerized on individual α5β1 integrin heterodimers. This was then followed by a gradual capacitance decreases at WTGal3 concentrations above 10 nM, which indicated significant thickening of the membrane. This was attributed to an initial WTGal3-driven integrin clustering that further switch to a lateral condensation of α5β1 integrins into lattices, resulting in the observed global thickening of the bilayer. This hypothesis is supported by FLIM imaging, where integrin clusters seemed to reorganize between 3.7 and 18.5 nM of WTGal3 to then increased in size between 18.5 and 37 nM. These changes were not observed with an oligomerization-deficient mutant of Gal3 and Gal3ΔNter and furthermore, they were inhibited in the presence of lactose establishing carbohydrate dependency.
In a natural membrane environment, Gal3 oligomerization on glycoprotein cargoes followed by their locally controlled clustering, would be expected to drive narrow membrane invagination, leading to the formation of tubular endocytic pits, through the GL–Lect mechanism. In contrast, the immobilized population of glycoprotein cargoes would correspond to extended galectin lattices.
FCS measurements confirmed association of the WTGal3 with pristine and integrin-containing membranes and revealed the existence of two Gal3 populations one fast and one slow diffusing that were distinct depending on the presence of α5β1 integrin in the membrane. In the absence of the integrin, the fast-moving component displayed diffusivity values in the range of those observed for the bulk lipid ATTO532-DOPE (6.5 ± 0.15 vs 6.99 ± 0.12 μm2/s respectively), suggesting passive association of the lectin at the pristine membrane. In contrast, diffusivity of the fast component was significantly decreased in α5β1 integrin-containing membranes when compared to the bulk lipid marker (3.54 ± 0.11 vs 5.40 ± 0.40 μm2/s, respectively), which suggested that Gal3 bound to the integrin. Notably, incubation of WTGal3 was observed to increase diffusivity of the integrin at the membrane and also altered the diffusion regime from subdiffusion to Brownian diffusion. However, it was evident from time traces of photobleaching and FLIM imaging that a significant portion of the integrin was immobilized upon WT galectin treatment. The observed increase in diffusion of the mobile α5β1 integrin at the membrane was attributed to this sequestration of a fraction of the total integrin pool into galectin lattices. This would result in a reduced concentration of mobile integrin and thereby to faster protein diffusion by normal Brownian motion in regions of the membrane that now should have been less crowded.
Moreover, the diffusivity rates of both the mobile α5β1 integrin fraction and the fast diffusing sub-population of WTGal3 followed the same trend, though exact matching of integrin and galectin diffusion rates was not observed. That a substantial portion of integrin remained mobile after galectin treatment could be ascribed to the orientation of the integrin within the membrane: the inward oriented pool (roughly 50%) was a priori not exposed to the exogenously added Gal3 and therefore was not expected to be influenced by it, creating an asymmetry in lateral integrin diffusivity. The population of integrin that was oriented inward was only influenced by general membrane parameters. This is evident from cross-correlation FCS data which showed that a sub-population of the mobile integrin co-diffused with galectin. In addition, FCS showed integrin bleaching in the presence of WTGal3, which along with FLIM imaging indicated that a significant sub-population of the integrin indeed became immobilized upon exposure to WTGal3.
In summary, we demonstrate that WTGal3 bound in a carbohydrate-dependent manner to α5β1 integrin in integrin reconstituted into artificial lipid bilayer membranes. EIS and FCS studies indicated that this binding occurred in a cooperative process involving the oligomerization of WTGal3 through its N-terminal domain. Very few biophysical models of integrin–galectin interaction have been reported to date, but with the emerging importance of this interaction across a diverse range of diseases, microcavity-suspended bilayers represent a versatile platform for studying processes such as oligomerization and network formation at the membrane, as they have the compositional versatility and laterally fluidity to facilitate such interactions.

Supporting Information

ARTICLE SECTIONS
Jump To

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpcb.2c05717.

  • Fabrication of gold microcavity array electrodes, DLS, EIS, FLIM, AFM, and FCS data (PDF)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

ARTICLE SECTIONS
Jump To
  • Corresponding Authors
    • Ludger Johannes - Institut Curie, PSL Research University, U1143 INSERM, UMR3666 CNRS, Cellular and Chemical Biology Unit, 75248Paris Cedex 05, France;  Email: [email protected]
    • Tia E. Keyes - School of Chemical Sciences and National Centre for Sensor Research, Dublin City University, DCU Glasnevin Campus, D09 V209Dublin 9, Ireland;  Orcidhttps://orcid.org/0000-0002-4604-5533;  Email: [email protected]
  • Authors
    • Nirod Kumar Sarangi - School of Chemical Sciences and National Centre for Sensor Research, Dublin City University, DCU Glasnevin Campus, D09 V209Dublin 9, Ireland
    • Massiullah Shafaq-Zadah - Institut Curie, PSL Research University, U1143 INSERM, UMR3666 CNRS, Cellular and Chemical Biology Unit, 75248Paris Cedex 05, France;  Orcidhttps://orcid.org/0000-0002-7582-8131
    • Guilherme B. Berselli - School of Chemical Sciences and National Centre for Sensor Research, Dublin City University, DCU Glasnevin Campus, D09 V209Dublin 9, Ireland
    • Jack Robinson - School of Chemical Sciences and National Centre for Sensor Research, Dublin City University, DCU Glasnevin Campus, D09 V209Dublin 9, Ireland
    • Estelle Dransart - Institut Curie, PSL Research University, U1143 INSERM, UMR3666 CNRS, Cellular and Chemical Biology Unit, 75248Paris Cedex 05, France
    • Aurélie Di Cicco - Institut Curie, PSL Research University, UMR 168 CNRS, 75248Paris Cedex 05, France
    • Daniel Lévy - Institut Curie, PSL Research University, UMR 168 CNRS, 75248Paris Cedex 05, France
  • Author Contributions

    N.K.S. and M.S.-Z. contributed equally. G.B.B. and J.R. contributed equally. L.J. and T.E.K. originated the research idea and contributed to experimental design. N.K.S., G.B.B., J.R., M.S.-Z., E.D., A.D.C., and D.L. contributed to experimental design, performed the experiments, and completed the data analyses. L.J. and T.E.K. contributed new reagents or analytic tools. N.K.S., G.B.B., J.R., M.S.-Z., E.D., L.J., and T.E.K. wrote the manuscript. All authors have given approval to the final version of the manuscript.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

ARTICLE SECTIONS
Jump To

L.J., M.S.-Z., E.D., D.L. and A.D.C. gratefully acknowledge the Cell and Tissue Imaging Core Facility (PICT IBiSA), Institut Curie, member of the French National Research Infrastructure France-BioImaging (ANR10-INBS-04). T.E.K., J.R., G.B.B., and N.K.S. gratefully acknowledge Science Foundation Ireland under [14/IA/2488] and [12/RC/2276_P2], T.E.K. and J.R. gratefully acknowledge The Irish Research Council for Postgraduate Studentship funding, and L.J. gratefully acknowledges Mizutani Foundation for Glycosciences (reference no. 200014), Agence National de la Recherche ANR (ANR-19-CE13-0001-01 and ANR-20-CE15-0009-01), and Fondation pour la Recherche Médicale (EQU202103012926). L.J., M.S.-Z., E.D., D.L., and A.D.C. gratefully acknowledge the Cell and Tissue Imaging Core Facility (PICT IBiSA), Institut Curie, member of the French National Research Infrastructure France-BioImaging (ANR10-INBS-04).

Abbreviations

ARTICLE SECTIONS
Jump To
PC

egg phosphatidylcholine

PA

egg phosphatidic acid

DOPC

dioleoylphosphatidylcholine

LUV

large unilamellar vesicle

EIS

electrochemical impedance spectroscopy

FLIM

fluorescence lifetime imaging

FCS

fluorescence correlation spectroscopy

FLCS

fluorescence lifetime correlation spectroscopy

FLCCS

fluorescence cross-correlation spectroscopy

ACF

auto-correlation function

Int

α5β1 integrin

WTGal3

wild-type galectin-3

Gal3ΔNter

N-terminal truncated galectin-3

References

ARTICLE SECTIONS
Jump To

This article references 81 other publications.

  1. 1
    Mouw, J. K.; Ou, G.; Weaver, V. M. Extracellular Matrix Assembly: A Multiscale Deconstruction. Nat. Rev. Mol. Cell Biol. 2014, 15, 771– 785,  DOI: 10.1038/nrm3902
    Google Scholar
    1
    Extracellular matrix assembly: a multiscale deconstruction
    Mouw, Janna K.; Ou, Guanqing; Weaver, Valerie M.
    Nature Reviews Molecular Cell Biology (2014), 15 (12), 771-785CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
    The biochem. and biophys. properties of the extracellular matrix (ECM) dictate tissue-specific cell behavior. The mols. that are assocd. with the ECM of each tissue, including collagens, proteoglycans, laminins and fibronectin, and the manner in which they are assembled det. the structure and the organization of the resultant ECM. The product is a specific ECM signature that is comprised of unique compositional and topog. features that both reflect and facilitate the functional requirements of the tissue.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvFKmur7L&md5=5418b0ab81bde5077f540e5c8cd652b7
  2. 2
    Lee, J.-O.; Bankston, L. A.; Robert C Liddington, M. A. A. Two Conformations of the Integrin A-Domain (I-Domain): A Pathway for Activation?. Structure 1995, 3, 1333– 1340,  DOI: 10.1016/S0969-2126(01)00271-4
    Google Scholar
    2
    Two conformations of the integrin A-domain (I-domain): a pathway for activation?
    Lee, Jie-Oh; Bankston, Laurie Anne; Arnaout, M. Amin; Liddington, Robert C.
    Structure (London) (1995), 3 (12), 1333-40CODEN: STRUE6; ISSN:0969-2126. (Current Biology)
    Integrins are plasma membrane proteins that mediate adhesion to other cells and to components of the extracellular matrix. Most integrins are constitutively inactive in resting cells, but are rapidly and reversibly activated in response to agonists, leading to highly regulated cell adhesion. This activation is assocd. with conformational changes in their extracellular portions, but the nature of the structural changes that lead to a change in adhesiveness is not understood. The interactions of several integrins with their extracellular ligands are mediated by an A-type domain (generally called the I-domain in integrins). Binding of the I-domain to protein ligands is dependent on divalent cations. The authors have described previously the structure of the I-domain from complement receptor 3 with bound Mg2+, in which the glutamate side chain from a second I-domain completes the octahedral coordination sphere of the metal, acting as a ligand mimetic. The authors now describe a new crystal form of the I-domain with bound Mn2+, in which water completes the metal coordination sphere and there is no equiv. of the glutamate ligand. Comparison of the two crystal forms reveals a change in metal coordination which is linked to a large (10 Å) shift of the C-terminal helix and the burial of two phenylalanine residues into the hydrophobic core of the Mn2+ form. These structural changes, analogous to those seen in the signal-transducing G-proteins, alter the electrophilicity of the metal, reducing its ability to bind ligand-assocd. acidic residues, and dramatically alter the surface of the protein implicated in binding ligand. The authors' observations provide the first at. resoln. view of conformational changes in an integrin domain, and suggest how these changes are linked to a change in integrin adhesiveness. The authors propose that the Mg2+ form represents the conformation of the domain in the active state and the Mn2+ form the conformation in the inactive state of the integrin.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XitFGgtA%253D%253D&md5=b8be7fae31062245f970a70aed2792b3
  3. 3
    Margadant, C.; van den Bout, I.; van Boxtel, A. L.; Thijssen, V. L.; Sonnenberg, A. Epigenetic Regulation of Galectin-3 Expression by Β1 Integrins Promotes Cell Adhesion and Migration. J. Biol. Chem. 2012, 287, 44684– 44693,  DOI: 10.1074/jbc.M112.426445
    Google Scholar
    3
    Epigenetic regulation of galectin-3 Expression by β1 integrins promotes cell adhesion and migration
    Margadant, Coert; van den Bout, Iman; van Boxtel, Antonius L.; Thijssen, Victor L.; Sonnenberg, Arnoud
    Journal of Biological Chemistry (2012), 287 (53), 44684-44693CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Introduction of the integrin β1- but not the β3-subunit in GE11 cells induces an epithelial-mesenchymal-transition (EMT)-like phenomenon that is characterized by the loss of cell-cell contacts, cell scattering, increased cell migration and RhoA activity, and fibronectin fibrillogenesis. Because galactose-binding lectins (galectins) have been implicated in these phenomena, we investigated whether galectins are involved in the β1-induced phenotype. We examd. 9 galectins and, intriguingly, found that the expression of galectin-3 (Gal-3) is specifically induced by β1 but not by β3. Using β1-β3 chimeric integrins, we show that the induction of Gal-3 expression requires the hypervariable region in the extracellular domain of β1, but not its cytoplasmic tail. Furthermore, Gal-3 expression does not depend on RhoA signaling, serum factors, or any of the major signal transduction pathways involving protein kinase C (PKC), p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase-1/-2 (ERK-1/2), phosphatidylinositol-3-OH kinase (PI3-K), or Src kinases. Instead, Gal-3 expression is controlled in an epigenetic manner. Whereas DNA methylation of the Lgals3 promoter maintains Gal-3 silencing in GE11 cells, expression of β1 causes its demethylation, leading to transcriptional activation of the Lgals3 gene. In turn, Gal-3 expression enhances β1 integrin-mediated cell adhesion to fibronectin (FN) and laminin (LN), as well as cell migration. Gal-3 also promotes β1-mediated cell adhesion to LN and Collagen-1 (Col)-1 in cells that endogenously express Gal-3 and β1 integrins. In conclusion, we identify a functional feedback-loop between β1 integrins and Gal-3 that involves the epigenetic induction of Gal-3 expression during integrin-induced EMT and cell scattering.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjvVaq&md5=b65c0929261f0fa5d175b1bc868b7dd2
  4. 4
    Seguin, L.; Desgrosellier, J. S.; Weis, S. M.; Cheresh, D. A. Integrins and Cancer: Regulators of Cancer Stemness, Metastasis, and Drug Resistance. Trends Cell Biol. 2015, 25, 234– 240,  DOI: 10.1016/j.tcb.2014.12.006
    Google Scholar
    4
    Integrins and cancer: regulators of cancer stemness, metastasis, and drug resistance
    Seguin, Laetitia; Desgrosellier, Jay S.; Weis, Sara M.; Cheresh, David A.
    Trends in Cell Biology (2015), 25 (4), 234-240CODEN: TCBIEK; ISSN:0962-8924. (Elsevier Ltd.)
    Interactions between cancer cells and their surroundings can trigger essential signaling cues that det. cell fate and influence the evolution of the malignant phenotype. As the primary receptors involved in cell-matrix adhesion, integrins present on the surface of tumor and stromal cells have a profound impact on the ability to survive in specific locations, but in some cases, these receptors can also function in the absence of ligand binding to promote stemness and survival in the presence of environmental and therapeutic stresses. Understanding how integrin expression and function is regulated in this context will enable the development of new therapeutic approaches to sensitize tumors to therapy and suppress their metastatic phenotype.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXlslCqu7o%253D&md5=04150b9842cbe69bec0c8bee7f0a6050
  5. 5
    Aoudjit, F.; Vuori, K. Integrin Signaling in Cancer Cell Survival and Chemoresistance. Chemother. Res. Pract. 2012, 2012, 1– 16,  DOI: 10.1155/2012/283181
    Google Scholar
    There is no corresponding record for this reference.
  6. 6
    Bänfer, S.; Schneider, D.; Dewes, J.; Strauss, M. T.; Freibert, S.-A.; Heimerl, T.; Maier, U. G.; Elsässer, H.-P.; Jungmann, R.; Jacob, R. Molecular Mechanism to Recruit Galectin-3 into Multivesicular Bodies for Polarized Exosomal Secretion. Proc. Natl. Acad. Sci. U.S.A. 2018, 115, E4396– E4405,  DOI: 10.1073/pnas.1718921115
    Google Scholar
    There is no corresponding record for this reference.
  7. 7
    Seelenmeyer, C.; Wegehingel, S.; Tews, I.; Künzler, M.; Aebi, M.; Nickel, W. Cell Surface Counter Receptors Are Essential Components of the Unconventional Export Machinery of Galectin-1. J. Cell Biol. 2005, 171, 373– 381,  DOI: 10.1083/jcb.200506026
    Google Scholar
    7
    Cell surface counter receptors are essential components of the unconventional export machinery of galectin-1
    Seelenmeyer, Claudia; Wegehingel, Sabine; Tews, Ivo; Kuenzler, Markus; Aebi, Markus; Nickel, Walter
    Journal of Cell Biology (2005), 171 (2), 373-381CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)
    Galectin-1 is a component of the extracellular matrix as well as a ligand of cell surface counter receptors such as β-galactoside-contg. glycolipids, however, the mol. mechanism of galectin-1 secretion has remained elusive. Based on a nonbiased screen for galectin-1 export mutants we have identified 26 single amino acid changes that cause a defect of both export and binding to counter receptors. When wild-type galectin-1 was analyzed in CHO clone 13 cells, a mutant cell line incapable of expressing functional galectin-1 counter receptors, secretion was blocked. Intriguingly, we also find that a distant relative of galectin-1, the fungal lectin CGL-2, is a substrate for nonclassical export from Chinese hamster ovary (CHO) cells. Alike mammalian galectin-1, a CGL-2 mutant defective in β-galactoside binding, does not get exported from CHO cells. We conclude that the β-galactoside binding site represents the primary targeting motif of galectins defining a galectin export machinery that makes use of β-galactoside-contg. surface mols. as export receptors for intracellular galectin-1.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtFGktbnJ&md5=fc4e3a7f16a321952fb3d9a907c949cc
  8. 8
    Dumic, J.; Dabelic, S.; Flögel, M. Galectin-3: An Open-Ended Story. Biochim. Biophys. Acta, Gen. Subj. 2006, 1760, 616– 635,  DOI: 10.1016/j.bbagen.2005.12.020
    Google Scholar
    8
    Galectin-3: An open-ended story
    Dumic, Jerka; Dabelic, Sanja; Floegel, Mirna
    Biochimica et Biophysica Acta, General Subjects (2006), 1760 (4), 616-635CODEN: BBGSB3; ISSN:0304-4165. (Elsevier B.V.)
    A review. Galectins, an ancient lectin family, are characterized by specific binding of β-galactosides through the evolutionary conserved sequence elements of a carbohydrate-recognition domain (CRD). A structurally unique member of the family is galectin-3 (I); in addn. to the CRD it contains a proline- and glycine-rich N-terminal domain (ND) through which it is able to form oligomers. I is widely spread among different types of cells and tissues, found intracellularly in the nucleus and cytoplasm or secreted via a non-classical pathway outside of the cell, thus being found on the cell surface or in the extracellular space. Through specific interactions with a variety of intracellular and extracellular proteins, I affects numerous biol. processes and seems to be involved in different physiol. and pathophysiol. conditions, such as development, immune reactions, and neoplastic transformation and metastasis. Here, the authors attempt to summarize existing information on structural, biochem., and intriguing functional properties of I.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xjsl2mtrw%253D&md5=54287fc34bc267fd22ed4eb69ee9607a
  9. 9
    Liu, F.-T.; Rabinovich, G. A. Galectins as Modulators of Tumour Progression. Nat. Rev. Cancer 2005, 5, 29– 41,  DOI: 10.1038/nrc1527
    Google Scholar
    9
    Galectins as modulators of tumor progression
    Liu, Fu-Tong; Rabinovich, Gabriel A.
    Nature Reviews Cancer (2005), 5 (1), 29-41CODEN: NRCAC4; ISSN:1474-175X. (Nature Publishing Group)
    A review. Galectins are a family of animal lectins with diverse biol. activities. They function both extracellularly, by interacting with cell-surface and extracellular matrix glycoproteins and glycolipids, and intracellularly, by interacting with cytoplasmic and nuclear proteins to modulate signalling pathways. Current research indicates that galectins have important roles in cancer; they contribute to neoplastic transformation, tumor cell survival, angiogenesis and tumor metastasis. They can modulate the immune and inflammatory responses and might have a key role helping tumors to escape immune surveillance. How do the different members of the Galectin family contribute to these diverse aspects of tumor biol.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXlvVem&md5=4b98b28336810c194a336bd940fc02c0
  10. 10
    Houzelstein, D.; Gonçalves, I. R.; Fadden, A. J.; Sidhu, S. S.; Cooper, D. N. W.; Drickamer, K.; Leffler, H.; Poirier, F. Phylogenetic Analysis of the Vertebrate Galectin Family. Mol. Biol. Evol. 2004, 21, 1177– 1187,  DOI: 10.1093/molbev/msh082
    Google Scholar
    10
    Phylogenetic analysis of the vertebrate galectin family
    Houzelstein, Denis; Goncalves, Isabelle R.; Fadden, Andrew J.; Sidhu, Sukhvinder S.; Cooper, Douglas N. W.; Drickamer, Kurt; Leffler, Hakon; Poirier, Francoise
    Molecular Biology and Evolution (2004), 21 (7), 1177-1187CODEN: MBEVEO; ISSN:0737-4038. (Oxford University Press)
    Galectins form a family of structurally related carbohydrate binding proteins (lectins) that have been identified in a large variety of metazoan phyla. They are involved in many biol. processes such as morphogenesis, control of cell death, immunol. response, and cancer. To elucidate the evolutionary history of galectins and galectin-like proteins in chordates, we have exploited three independent lines of evidence: (i) location of galectin encoding genes (LGALS) in the human genome; (ii) exon-intron organization of galectin encoding genes; and (iii) sequence comparison of carbohydrate recognition domains (CRDs) of chordate galectins. Our results suggest that a duplication of a mono-CRD galectin gene gave rise to an original bi-CRD galectin gene, before or early in chordate evolution. The N-terminal and C-terminal CRDs of this original galectin subsequently diverged into two different subtypes, defined by exon-intron structure (F4-CRD and F3-CRD). We show that all vertebrate mono-CRD galectins known to date belong to either the F3- or F4- subtype. A sequence of duplication and divergence events of the different galectins in chordates is proposed.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXltlKlsro%253D&md5=25c5d56ff8d4ede969b2f6d95f3ca416
  11. 11
    Popa, S. J.; Stewart, S. E.; Moreau, K. Unconventional Secretion of Annexins and Galectins. Semin. Cell Dev. Biol. 2018, 83, 42– 50,  DOI: 10.1016/j.semcdb.2018.02.022
    Google Scholar
    11
    Unconventional secretion of annexins and galectins
    Popa, Stephanie J.; Stewart, Sarah E.; Moreau, Kevin
    Seminars in Cell & Developmental Biology (2018), 83 (), 42-50CODEN: SCDBFX; ISSN:1084-9521. (Elsevier Ltd.)
    A review. Eukaryotic cells have a highly evolved system of protein secretion, and dysfunction in this pathway is assocd. with many diseases including cancer, infection, metabolic disease and neurol. disorders. Most proteins are secreted using the conventional endoplasmic reticulum (ER)/Golgi network and as such, this pathway is well-characterised. However, several cytosolic proteins have now been documented as secreted by unconventional transport pathways. This review focuses on two of these proteins families: annexins and galectins. The extracellular functions of these proteins are well documented, as are assocns. of their perturbed secretion with several diseases. However, the mechanisms and regulation of their secretion remain poorly characterised, and are discussed in this review.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXkslWkt7w%253D&md5=ed0616b196d586d567e5a248b57c628d
  12. 12
    Hayashi, Y.; Jia, W.; Kidoya, H.; Muramatsu, F.; Tsukada, Y.; Takakura, N. Galectin-3 Inhibits Cancer Metastasis by Negatively Regulating Integrin Β3 Expression. Am. J. Pathol. 2019, 189, 900– 910,  DOI: 10.1016/j.ajpath.2018.12.005
    Google Scholar
    12
    Galectin-3 Inhibits Cancer Metastasis by Negatively Regulating Integrin β3 Expression
    Hayashi, Yumiko; Jia, Weizhen; Kidoya, Hiroyasu; Muramatsu, Fumitaka; Tsukada, Yohei; Takakura, Nobuyuki
    American Journal of Pathology (2019), 189 (4), 900-910CODEN: AJPAA4; ISSN:0002-9440. (Elsevier B.V.)
    Galectin-3 (Gal-3; gene LGALS3) is a member of the β-galactose-binding lectin family. Previous studies showed that Gal-3 is expressed in several tissues across species and functions as a regulator of cell proliferation, apoptosis, adhesion, and migration, thus affecting many aspects of events, such as angiogenesis and tumorigenesis. Although several reports have suggested that the level of Gal-3 expression correlates pos. with tumor progression, herein we show that highly metastatic mouse melanoma B16/BL6 cells express less Gal-3 than B16 cells with a lower metastatic potential. It was found that overexpression of Gal-3 in melanoma cells in fact suppresses metastasis. In contrast, knocking out Gal-3 expression in cancer cells promoted cell aggregation mediated through interactions with platelets and fibrinogen in vitro and increased the no. of metastatic foci in vivo. Thus, reduced Gal-3 expression results in the up-regulation of β3 integrin expression, and this contributes to metastatic potential. These findings indicate that changes of Gal-3 expression in cancer cells during tumor progression influence the characteristics of metastatic cells.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXivFSmtr0%253D&md5=70f33ca776716d7b1e330e52b2acf259
  13. 13
    Johannes, L.; Jacob, R.; Leffler, H. Galectins at a Glance. J. Cell Sci. 2018, 131, jcs208884,  DOI: 10.1242/jcs.208884
    Google Scholar
    There is no corresponding record for this reference.
  14. 14
    Ruvolo, P. P. Galectin 3 as a Guardian of the Tumor Microenvironment. Biochim. Biophys. Acta, Mol. Cell Res. 2016, 1863, 427– 437,  DOI: 10.1016/j.bbamcr.2015.08.008
    Google Scholar
    14
    Galectin 3 as a guardian of the tumor microenvironment
    Ruvolo, Peter P.
    Biochimica et Biophysica Acta, Molecular Cell Research (2016), 1863 (3), 427-437CODEN: BBAMCO; ISSN:0167-4889. (Elsevier B.V.)
    Galectin 3 is a member of a family of β-galactoside binding proteins and has emerged as an important regulator of diverse functions crit. in cancer biol. including apoptosis, metastasis, immune surveillance, mol. trafficking, mRNA splicing, gene expression, and inflammation. Galectin 3's ability to support cancer cell survival by intra-cellular and extra-cellular mechanisms suggests this mol. is an important component of the tumor microenvironment that potentially could be targeted for therapy. Data is emerging that Galectin 3 is elevated in many cancers including solid tumors and the cancers of the blood. Galectin 3 also appears to be a key mol. produced by tumor microenvironment support cells including mesenchymal stromal cells (MSC) to suppress immune surveillance by killing T cells and interfering with NK cell function and by supporting metastasis. Levels of Galectin 3 increase in the MSC of aging mice and perhaps this contributes to the development of cancer in the elderly. Galectin 3 modulates surface protein expression of a diverse set of glycoproteins including CD44 by regulating endocytosis of these proteins. In addn., Galectin 3 binding to receptor kinases such as CD45 and the T cell receptor is crit. in the regulation of their function. In this review I will examine the various mechanisms how Galectin 3 supports chemoresistance and metastasis in solid tumors and in leukemia and lymphoma. I will also discuss possible therapeutic strategies to target this Galectin for cancer therapy. This article is part of a Special Issue entitled: Tumor Microenvironment Regulation of Cancer Cell Survival, Metastasis, Inflammation, and Immune Surveillance.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtleksrnM&md5=f37b5229716ede0729e577b3d23b8c2e
  15. 15
    Barondes, S. H.; Cooper, D. N.; Gitt, M. A.; Leffler, H. Galectins. Structure and Function of a Large Family of Animal Lectins. J. Biol. Chem. 1994, 269, 20807– 20810,  DOI: 10.1016/S0021-9258(17)31891-4
    Google Scholar
    15
    Galectins. Structure and function of a large family of animal lectins
    Barondes, Samuel H.; Cooper, Douglas N. W.; Gitt, Michael A.; Leffler, Hakon
    Journal of Biological Chemistry (1994), 269 (33), 20807-10CODEN: JBCHA3; ISSN:0021-9258.
    A review with 80 refs. Lectins are proteins that bind to specific carbohydrate structures and can thus recognize particular glycoconjugates among the vast array expressed in animal tissues. Most animal lectins can be classified into four distinct families (1): C-type lectins (including the selectins); P-type lectins; pentraxins; and galectins, formerly known as S-type or S-Lac lectins. The purpose of this short review is to provide a framework for integrating the rapid increase in knowledge of the diversity, structure, and function of the galectins. While the emphasis here is on mammalian galectins, important advances are also being made in studies of galectins in other species, including nematode and sponge. The review deals specifically with the structural classification and properties, carbohydrate binding specificity, non-classical secretion, biol. functions, ligands, and major known sites of expression of mammalian galectins.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXlsFWgurc%253D&md5=b92e79e0e3bf4d112c3a5876f3c6fc69
  16. 16
    Zhao, Z.; Xu, X.; Cheng, H.; Miller, M. C.; He, Z.; Gu, H.; Zhang, Z.; Raz, A.; Mayo, K. H.; Tai, G.; Zhou, Y. Galectin-3 N-Terminal Tail Prolines Modulate Cell Activity and Glycan-Mediated Oligomerization/Phase Separation. Proc. Natl. Acad. Sci. U.S.A. 2021, 118, e2021074118  DOI: 10.1073/pnas.2021074118
    Google Scholar
    16
    Galectin-3 N-terminal tail prolines modulate cell activity and glycan-mediated oligomerization/phase separation
    Zhao, Zihan; Xu, Xuejiao; Cheng, Hairong; Miller, Michelle C.; He, Zhen; Gu, Hongming; Zhang, Zhongyu; Raz, Avraham; Mayo, Kevin H.; Tai, Guihua; Zhou, Yifa
    Proceedings of the National Academy of Sciences of the United States of America (2021), 118 (19), e2021074118CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Galectin-3 (Gal-3) has a long, aperiodic, and dynamic proline-rich N-terminal tail (NT). The functional role of the NT with its numerous prolines has remained enigmatic since its discovery. To provide some resoln. to this puzzle, we individually mutated all 14 NT prolines over the first 68 residues and assessed their effects on various Gal-3-mediated functions. Our findings show that mutation of any single proline (esp. P37A, P55A, P60A, P64A/H, and P67A) dramatically and differentially inhibits Gal-3-mediated cellular activities (i.e., cell migration, activation, endocytosis, and hemagglutination). For mechanistic insight, we investigated the role of prolines in mediating Gal-3 oligomerization, a fundamental process required for these cell activities. We showed that Gal-3 oligomerization triggered by binding to glycoproteins is a dynamic process analogous to liq.-liq. phase sepn. (LLPS). The compn. of these heterooligomers is dependent on the concn. of Gal-3 as well as on the concn. and type of glycoprotein. LLPS-like Gal-3 oligomerization/condensation was also obsd. on the plasma membrane and disrupted endomembranes. Mol.- and cell-based assays indicate that glycan binding-triggered Gal-3 LLPS (or LLPS-like) is driven mainly by dynamic intermol. interactions between the Gal-3 NT and the carbohydrate recognition domain (CRD) F-face, although NT-NT interactions appear to contribute to a lesser extent. Mutation of each proline within the NT differentially controls NT-CRD interactions, consequently affecting glycan binding, LLPS, and cellular activities. Our results unveil the role of proline polymorphisms (e.g., at P64) assocd. with many diseases and suggest that the function of glycosylated cell surface receptors is dynamically regulated by Gal-3.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhtVyhtb7L&md5=bd2024509914b2505ac4807de67368d0
  17. 17
    Chiu, Y.-P.; Sun, Y.-C.; Qiu, D.-C.; Lin, Y.-H.; Chen, Y.-Q.; Kuo, J.-C.; Huang, J. Liquid-Liquid Phase Separation and Extracellular Multivalent Interactions in the Tale of Galectin-3. Nat. Commun. 2020, 11, 1229,  DOI: 10.1038/s41467-020-15007-3
    Google Scholar
    17
    Liquid-liquid phase separation and extracellular multivalent interactions in the tale of galectin-3
    Chiu, Yi-Ping; Sun, Yung-Chen; Qiu, De-Chen; Lin, Yu-Hao; Chen, Yin-Quan; Kuo, Jean-Cheng; Huang, Jie-rong
    Nature Communications (2020), 11 (1), 1229CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Abstr.: Liq.-liq. phase sepn. (LLPS) explains many intracellular activities, but its role in extracellular functions has not been studied to the same extent. Here we report how LLPS mediates the extracellular function of galectin-3, the only monomeric member of the galectin family. The mechanism through which galectin-3 agglutinates (acting as a "bridge" to aggregate glycosylated mols.) is largely unknown. Our data show that its N-terminal domain (NTD) undergoes LLPS driven by interactions between its arom. residues (two tryptophans and 10 tyrosines). Our lipopolysaccharide (LPS) micelle model shows that the NTDs form multiple weak interactions to other galectin-3 and then aggregate LPS micelles. Aggregation is reversed when interactions between the LPS and the carbohydrate recognition domains are blocked by lactose. The proposed mechanism explains many of galectin-3's functions and suggests that the arom. residues in the NTD are interesting drug design targets.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXlt1Sgt7o%253D&md5=14b0b011324679d48f71f852ed6e9da0
  18. 18
    Lin, Y.-H.; Qiu, D.-C.; Chang, W.-H.; Yeh, Y.-Q.; Jeng, U.-S.; Liu, F.-T.; Huang, J. The Intrinsically Disordered N-Terminal Domain of Galectin-3 Dynamically Mediates Multisite Self-Association of the Protein through Fuzzy Interactions. J. Biol. Chem. 2017, 292, 17845– 17856,  DOI: 10.1074/jbc.M117.802793
    Google Scholar
    18
    The intrinsically disordered N-terminal domain of galectin-3 dynamically mediates multisite self-association of the protein through fuzzy interactions
    Lin, Yu-Hao; Qiu, De-Chen; Chang, Wen-Han; Yeh, Yi-Qi; Jeng, U-Ser; Liu, Fu-Tong; Huang, Jie-rong
    Journal of Biological Chemistry (2017), 292 (43), 17845-17856CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Galectins are a family of lectins that bind β-galactosides through their conserved carbohydrate recognition domain (CRD) and can induce aggregation with glycoproteins or glycolipids on the cell surface and thereby regulate cell activation, migration, adhesion, and signaling. Galectin-3 has an intrinsically disordered N-terminal domain and a canonical CRD. Unlike the other 14 known galectins in mammalian cells, which have dimeric or tandem-repeated CRDs enabling multivalency for various functions, galectin-3 is monomeric, and its functional multivalency therefore is somewhat of a mystery. Here, we used NMR spectroscopy, mutagenesis, small-angle X-ray scattering, and computational modeling to study the self-assocn.-related multivalency of galectin-3 at the residue-specific level. We show that the disordered N-terminal domain (residues ∼20-100) interacts with itself and with a part of the CRD not involved in carbohydrate recognition (β-strands 7-9; residues ∼200-220), forming a fuzzy complex via inter- and intramol. interactions, mainly through hydrophobicity. These fuzzy interactions are characteristic of intrinsically disordered proteins to achieve liq.-liq. phase sepn., and we demonstrated that galectin-3 can also undergo liq.-liq. phase sepn. We propose that galectin-3 may achieve multivalency through this multisite self-assocn. mechanism facilitated by fuzzy interactions.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslehs7zO&md5=1bf3159cabfa56c346e8a632e457bc80
  19. 19
    Friedrichs, J.; Manninen, A.; Muller, D. J.; Helenius, J. Galectin-3 Regulates Integrin α2β1-Mediated Adhesion to Collagen-I and -IV. J. Biol. Chem. 2008, 283, 32264– 32272,  DOI: 10.1074/jbc.M803634200
    Google Scholar
    19
    Galectin-3 Regulates Integrin α2β1-mediated Adhesion to Collagen-I and -IV
    Friedrichs, Jens; Manninen, Aki; Muller, Daniel J.; Helenius, Jonne
    Journal of Biological Chemistry (2008), 283 (47), 32264-32272CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Galectins are a taxonomically widespread family of galactose-binding proteins of which galectin-3 is known to modulate cell adhesion. Using single cell force spectroscopy, the contribution of galectin-3 to the adhesion of Madin-Darby canine kidney (MDCK) cells to different extracellular matrix proteins was investigated. When adhering to collagen-I or -IV, some cells rapidly entered an enhanced adhesion state, marked by a significant increase in the force required for cell detachment. Galectin-3-depleted cells had an increased probability of entering the enhanced adhesion state. Adhesion enhancement was specific to integrin α2β1, as it was not obsd. when cells adhered to extracellular matrix substrates by other integrins. The adhesion phenotype of galectin-3-depleted cells was mimicked in a galactoside-deficient MDCK cell line and could be complemented by the addn. of recombinant galectin-3. We propose that galectin-3 influences integrin α2β1-mediated adhesion complex formation by altering receptor clustering.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtlOjtL%252FN&md5=aec191a18da391835c81d5319945373d
  20. 20
    Rao, S. P.; Wang, Z.; Zuberi, R. I.; Sikora, L.; Bahaie, N. S.; Zuraw, B. L.; Liu, F.-T.; Sriramarao, P. Galectin-3 Functions as an Adhesion Molecule to Support Eosinophil Rolling and Adhesion under Conditions of Flow. J. Immunol. 2007, 179, 7800– 7807,  DOI: 10.4049/jimmunol.179.11.7800
    Google Scholar
    20
    Galectin-3 functions as an adhesion molecule to support eosinophil rolling and adhesion under conditions of flow
    Rao, Savita P.; Wang, Zhuangzhi; Zuberi, Riaz I.; Sikora, Lyudmila; Bahaie, Nooshin S.; Zuraw, Bruce L.; Liu, Fu-Tong; Sriramarao, P.
    Journal of Immunology (2007), 179 (11), 7800-7807CODEN: JOIMA3; ISSN:0022-1767. (American Association of Immunologists)
    Allergic inflammation involves the mobilization and trafficking of eosinophils to sites of inflammation. Galectin-3 (Gal-3) has been shown to play a crit. role in eosinophil recruitment and airway allergic inflammation in vivo. The role played by Gal-3 in human eosinophil trafficking was investigated. Eosinophils from allergic donors expressed elevated levels of Gal-3 and demonstrated increased rolling and firm adhesion on immobilized VCAM-1 and, more surprisingly, on Gal-3 under conditions of flow. Inhibition studies with specific mAbs as well as lactose demonstrated that: (1) eosinophil-expressed Gal-3 mediates rolling and adhesion on VCAM-1; (2) α4 integrin mediates eosinophil rolling on immobilized Gal-3; and (3) eosinophil-expressed Gal-3 interacts with immobilized Gal-3 through the carbohydrate recognition domain of Gal-3 during eosinophil trafficking. These findings were further confirmed using inflamed endothelial cells. Interestingly, Gal-3 was found to bind to α4 integrin by ELISA, and the two mols. exhibited colocalized expression on the cell surface of eosinophils from allergic donors. Thus, Gal-3 functions as a cell surface adhesion mol. to support eosinophil rolling and adhesion under conditions of flow.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtlajt7jL&md5=fcd4ea11b0a703e2974b3bb11c248d62
  21. 21
    Goetz, J. G.; Joshi, B.; Lajoie, P.; Strugnell, S. S.; Scudamore, T.; Kojic, L. D.; Nabi, I. R. Concerted Regulation of Focal Adhesion Dynamics by Galectin-3 and Tyrosine-Phosphorylated Caveolin-1. J. Cell Biol. 2008, 180, 1261– 1275,  DOI: 10.1083/jcb.200709019
    Google Scholar
    21
    Concerted regulation of focal adhesion dynamics by galectin-3 and tyrosine-phosphorylated caveolin-1
    Goetz, Jacky G.; Joshi, Bharat; Lajoie, Patrick; Strugnell, Scott S.; Scudamore, Trevor; Kojic, Liliana D.; Nabi, Ivan R.
    Journal of Cell Biology (2008), 180 (6), 1261-1275CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)
    Both tyrosine-phosphorylated caveolin-1 (pY14Cav1) and GlcNAc-transferase V (Mga5) are linked with focal adhesions (FAs); however, their function in this context is unknown. Here, we show that galectin-3 binding to Mgat5-modified N-glycans functions together with pY14Cav1 to stabilize focal adhesion kinase (FAK) within FAs, and thereby promotes FA disassembly and turnover. Expression of the Mgat5/galectin lattice alone induces FAs and cell spreading. However, FAK stabilization in FAs also requires expression of pY14Cav1. In cells lacking the Mgat5/galectin lattice, pY14Cav1 is not sufficient to promote FAK stabilization, FA disassembly, and turnover. In human MDA-435 cancer cells, Cav1 expression, but not mutant Y14FCav1, stabilizes FAK exchange and stimulates de novo FA formation in protrusive cellular regions. Thus, transmembrane crosstalk between the galectin lattice and pY14Cav1 promotes FA turnover by stabilizing FAK within FAs defining previously unknown, interdependent roles for galectin-3 and pY14Cav1 in tumor cell migration.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXktFGlsrk%253D&md5=433a50aca5f9da9f811119144c0302b1
  22. 22
    Furtak, V.; Hatcher, F.; Ochieng, J. Galectin-3 Mediates the Endocytosis of β-1 Integrins by Breast Carcinoma Cells. Biochem. Biophys. Res. Commun. 2001, 289, 845– 850,  DOI: 10.1006/bbrc.2001.6064
    Google Scholar
    22
    Galectin-3 Mediates the Endocytosis of β-1 Integrins by Breast Carcinoma Cells
    Furtak, Vyacheslav; Hatcher, Frank; Ochieng, Josiah
    Biochemical and Biophysical Research Communications (2001), 289 (4), 845-850CODEN: BBRCA9; ISSN:0006-291X. (Academic Press)
    Galectin-3, a β-galactoside binding lectin, has been demonstrated to play a key role(s) in cell to extracellular matrix interaction. The precise mechanism by which it modulates cellular adhesion is presently unclear and warrants further studies. We hereby report that galectin-3 mediates the endocytosis of β-1 integrins in a lactose-dependent manner. Interestingly we obsd. that galectin-3 was also rapidly internalized by the cells via the same pathway and the internalization was completely blocked by lactose. The endocytosis process was temp. dependent and was inhibited by filipin but not chlorpromazine. The endocytosis of galectin-3 and β-1 integrins by the cells was accompanied by rapid cell spreading due to cytoskeletal reorganization. The data suggest a novel mechanism by which galectin-3 and β-1 integrins are internalized into breast carcinoma cells via a caveolae-like pathway of endocytosis. (c) 2001 Academic Press.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXovVWiur0%253D&md5=b1a3949778f27c95bc7545fc41d49714
  23. 23
    King, D. R.; Salako, D. C.; Arthur-Bentil, S. K.; Rubin, A. E.; Italiya, J. B.; Tan, J. S.; Macris, D. G.; Neely, H. K.; Palka, J. M.; Grodin, J. L.; Davis-Bordovsky, K.; Faubion, M.; North, C. S.; Brown, E. S. Relationship between Novel Inflammatory Biomarker Galectin-3 and Depression Symptom Severity in a Large Community-Based Sample. J. Affective Disord. 2021, 281, 384– 389,  DOI: 10.1016/j.jad.2020.12.050
    Google Scholar
    23
    Relationship between novel inflammatory biomarker galectin-3 and depression symptom severity in a large community-based sample
    King, Darlene R.; Salako, Damilola C.; Arthur-Bentil, Samia Kate; Rubin, Arielle E.; Italiya, Jay B.; Tan, Jenny S.; Macris, Dimitri G.; Neely, Hunter K.; Palka, Jayme M.; Grodin, Justin L.; Davis-Bordovsky, Kaylee; Faubion, Matthew; North, Carol S.; Brown, E. Sherwood
    Journal of Affective Disorders (2021), 281 (), 384-389CODEN: JADID7; ISSN:0165-0327. (Elsevier Inc.)
    Major depressive disorder is assocd. with pro-inflammatory markers, such as cytokines TNF-alpha, IL-6, IL-1ss, and C-reactive protein. Galectin-3 is a novel emerging biomarker with pro-inflammatory properties. It is a saccharide binding protein distributed throughout many tissues with varying functions and is a predictor of poor outcomes in patients with heart failure and stroke. However, its role as a predictor in depressive symptom severity remains undefined. Data from the community-based Dallas Heart Study (n = 2554) were examd. using a multiple linear regression anal. to evaluate the relationship between galectin-3 and depressive symptom severity as assessed with Quick Inventory of Depressive Symptomatol. Self-Report (QIDS-SR) scores. Addnl. covariates included age, sex, race/ethnicity, body mass index (BMI), years of education, serum creatinine, history of diabetes, and smoking history. Galectin-3 levels statistically significantly predicted QIDS-SR depressive symptom severity (β = 0.055, p = .015). Female sex, smoking status, and BMI were found to be statistically significant pos. predictors of depression severity, while age, years of education, non-Hispanic White race, and Hispanic ethnicity were neg. predictors of depressive symptom severity. In this large sample, higher galectin-3 levels were assocd. with higher levels of depressive symptoms. The findings suggest that galectin-3 may be a new and useful inflammatory biomarker assocd. with depression.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXit1ais7s%253D&md5=0e4919f831829c56a1ab6abd69666dbf
  24. 24
    Melin, E. O.; Dereke, J.; Thunander, M.; Hillman, M. Depression in Type 1 Diabetes Was Associated with High Levels of Circulating Galectin-3. Endocr. Connect. 2018, 7, 819– 828,  DOI: 10.1530/EC-18-0108
    Google Scholar
    24
    Depression in type 1 diabetes was associated with high levels of circulating galectin-3
    Melin, Eva Olga; Dereke, Jonatan; Thunander, Maria; Hillman, Magnus
    Endocrine Connections (2018), 7 (6), 819-828CODEN: ECNOCX; ISSN:2049-3614. (BioScientifica Ltd.)
    Objective: Neuroinflammatory responses are implicated in depression. The aim was to explore whether depression in patients with type 1 diabetes (T1D) was assocd. with high circulating galectin-3, controlling for metabolic variables, s-creatinine, life style factors, medication and cardiovascular complications. Design: Cross-sectional. Methods: Participants were T1D patients (n = 283, 56% men, age 18-59 years, diabetes duration ≥1 yr). Depression was assessed by Hospital Anxiety and Depression Scale-depression subscale. Blood samples, anthropometrics and blood pressure were collected, and supplemented with data from medical records and the Swedish National Diabetes Registry. Galectin-3 ≥2.562μg/l, corresponding to the 85th percentile, was defined as high galectin-3. Results: Median (quartile1, quartile3) galectin-3 (μg/l) was 1.3 (0.8, 2.9) for the 30 depressed patients, and 0.9 (0.5, 1.6) for the 253 non-depressed, P = 0.009. Depression was assocd. with high galectin-3 in all the 283 patients (adjusted odds ratio (AOR) 3.5), in the 161 men (AOR 3.4), and in the 122 women (AOR 3.9). HbA1c, s-lipids, s-creatinine, blood pressure, obesity, smoking, phys. inactivity, cardiovascular complications and drugs (antihypertensive, lipid lowering, oral antidiabetic drugs and antidepressants) were not assocd. with high galectin-3. Conclusions: This is the first study to show an assocn. between depression and galectin-3. Depression was the only explored parameter assocd. with high circulating galectin-3 levels in 283 T1D patients. High galectin-3 levels might contribute to the increased risk for Alzheimer's disease, cardiovascular and all-cause mortality obsd. in persons with depression. Potentially, in the future, treatment targeting galactin-3 might improve the prognosis for patients with high galectin-3 levels.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXlsVKitbY%253D&md5=e6fd58d490469beded1f486969d0be66
  25. 25
    Tao, C.-C.; Cheng, K.-M.; Ma, Y.-L.; Hsu, W.-L.; Chen, Y.-C.; Fuh, J.-L.; Lee, W.-J.; Chao, C.-C.; Lee, E. H. Y. Galectin-3 Promotes Aβ Oligomerization and Aβ Toxicity in a Mouse Model of Alzheimer’s Disease. Cell Death Differ. 2020, 27, 192– 209,  DOI: 10.1038/s41418-019-0348-z
    Google Scholar
    25
    Galectin-3 promotes Aβ oligomerization and Aβ toxicity in a mouse model of Alzheimer's disease
    Tao, Chih-Chieh; Cheng, Kuang-Min; Ma, Yun-Li; Hsu, Wei-Lun; Chen, Yan-Chu; Fuh, Jong-Ling; Lee, Wei-Ju; Chao, Chih-Chang; Lee, Eminy H. Y.
    Cell Death & Differentiation (2020), 27 (1), 192-209CODEN: CDDIEK; ISSN:1350-9047. (Nature Research)
    Amyloid-β (Aβ) oligomers largely initiate the cascade underlying the pathol. of Alzheimers disease (AD). Galectin-3 (Gal-3), which is a member of the galectin protein family, promotes inflammatory responses and enhances the homotypic aggregation of cancer cells. Here, we examd. the role and action mechanism of Gal-3 in Aβ oligomerization and Aβ toxicities. Wild-type (WT) and Gal-3-knockout (KO) mice, APP/PS1;WT mice, APP/PS1;Gal-3+/- mice and brain tissues from normal subjects and AD patients were used. We found that Aβ oligomerization is reduced in Gal-3 KO mice injected with Aβ, whereas overexpression of Gal-3 enhances Aβ oligomerization in the hippocampi of Aβ-injected mice. Gal-3 expression shows an age-dependent increase that parallels endogenous Aβ oligomerization in APP/PS1 mice. Moreover, Aβ oligomerization, Iba1 expression, GFAP expression and amyloid plaque accumulation are reduced in APP/PS1;Gal-3+/- mice compared with APP/PS1;WT mice. APP/PS1;Gal-3+/- mice also show better acquisition and retention performance compared to APP/PS1;WT mice. In studying the mechanism underlying Gal-3-promoted Aβ oligomerization, we found that Gal-3 primarily co-localizes with Iba1, and that microglia-secreted Gal-3 directly interacts with Aβ. Gal-3 also interacts with triggering receptor expressed on myeloid cells-2, which then mediates the ability of Gal-3 to activate microglia for further Gal-3 expression. Immunohistochem. analyses show that the distribution of Gal-3 overlaps with that of endogenous Aβ in APP/PS1 mice and partially overlaps with that of amyloid plaque. Moreover, the expression of the Aβ-degrading enzyme, neprilysin, is increased in Gal-3 KO mice and this is assocd. with enhanced integrin-mediated signaling. Consistently, Gal-3 expression is also increased in the frontal lobe of AD patients, in parallel with Aβ oligomerization. Because Gal-3 expression is dramatically increased as early as 3 mo of age in APP/PS1 mice and anti-Aβ oligomerization is believed to protect against Aβ toxicity, Gal-3 could be considered a novel therapeutic target in efforts to combat AD.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtFSmtr7I&md5=3acae686f7adcb28e1eb2a6131ce9604
  26. 26
    Radosavljevic, G.; Volarevic, V.; Jovanovic, I.; Milovanovic, M.; Pejnovic, N.; Arsenijevic, N.; Hsu, D. K.; Lukic, M. L. The Roles of Galectin-3 in Autoimmunity and Tumor Progression. Immunol. Res. 2012, 52, 100– 110,  DOI: 10.1007/s12026-012-8286-6
    Google Scholar
    26
    The roles of Galectin-3 in autoimmunity and tumor progression
    Radosavljevic, Gordana; Volarevic, Vladislav; Jovanovic, Ivan; Milovanovic, Marija; Pejnovic, Nada; Arsenijevic, Nebojsa; Hsu, Daniel K.; Lukic, Miodrag L.
    Immunologic Research (2012), 52 (1-2), 100-110CODEN: IMRSEB; ISSN:0257-277X. (Humana Press Inc.)
    A review. Galectin-3, a unique chimera-type member of the β-galactoside-binding sol. lectin family, is widely expressed in numerous cells. Here, we discuss the role of Galectin-3 in T-cell-mediated inflammatory (auto) immunity and tumor rejection by using Galectin-3-deficient mice and four disease models of human pathol.: exptl. autoimmune encephalomyelitis (EAE), Con-A-induced hepatitis, multiple low-dose streptozotocin-induced diabetes (MLD-STZ diabetes) and metastatic melanoma. We present evidence which suggest that Galectin-3 plays an important pro-inflammatory role in Con-A-induced hepatitis by promoting the activation of T lymphocytes, NKT cells and DCs, cytokine secretion, prevention of M2 macrophage polarization and apoptosis of mononuclear cells, and it leads to severe liver injury. In addn., expts. in Galectin-3-"knock-out" mice indicate that Galectin-3 is also involved in immune-mediated β-cell damage and is required for diabetogenesis in MLD-STZ model by promoting the expression of IFN-gamma, TNF-alpha, IL-17 and iNOS in immune and accessory effector cells. Next, our data demonstrated that Galectin-3 plays an important disease-exacerbating role in EAE through its multifunctional roles in preventing cell apoptosis and increasing IL-17 and IFN-gamma synthesis, but decreasing IL-10 prodn. Finally, based on our findings, we postulated that expression of Galectin-3 in the host may also facilitate melanoma metastasis by affecting tumor cell adhesion and modulating anti-melanoma immune response, in particular innate antitumor immunity. Taken together, we discuss the evidence of pro-inflammatory and antitumor activities of Galectin-3 and suggest that Galectin-3 may be an important therapeutic target.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XlvVSisb4%253D&md5=dfc6dc92c6e02d5e0777458f930e82ca
  27. 27
    Sonnino, S.; Mauri, L.; Chigorno, V.; Prinetti, A. Gangliosides as Components of Lipid Membrane Domains. Glycobiology 2007, 17, 1R– 13R,  DOI: 10.1093/glycob/cwl052
    Google Scholar
    27
    Gangliosides as components of lipid membrane domains
    Sonnino, Sandro; Mauri, Laura; Chigorno, Vanna; Prinetti, Alessandro
    Glycobiology (2006), 17 (1), 1R-13RCODEN: GLYCE3; ISSN:0959-6658. (Oxford University Press)
    A review. Cell membrane components are organized as specialized domains involved in membrane-assocd. events such as cell signaling, cell adhesion, and protein sorting. These membrane domains are enriched in sphingolipids and cholesterol but display a low protein content. Theor. considerations and exptl. data suggest that some properties of gangliosides play an important role in the formation and stabilization of specific cell lipid membrane domains. Gangliosides are glycolipids with strong amphiphilic character and are particularly abundant in the plasma membranes, where they are inserted into the external leaflet with the hydrophobic ceramide moiety and with the oligosaccharide chain protruding into the extracellular medium. The geometry of the monomer inserted into the membrane, largely detd. by the very large surface area occupied by the oligosaccharide chain, the ability of the ceramide amide linkage to form a network of hydrogen bonds at the water-lipid interface of cell membranes, the Δ4 double bond of sphingosine proximal to the water-lipid interface, the capability of the oligosaccharide chain to interact with water, and the absence of double bonds into the double-tailed hydrophobic moiety are the ganglioside features that will be discussed in this review, to show how gangliosides are responsible for the formation of cell lipid membrane domains characterized by a strong pos. curvature.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtlChtbrP&md5=a084b4668f7526e126a39c184eb3e31a
  28. 28
    Johannes, L.; Billet, A. Glycosylation and Raft Endocytosis in Cancer. Cancer Metastasis Rev. 2020, 39, 375– 396,  DOI: 10.1007/s10555-020-09880-z
    Google Scholar
    28
    Glycosylation and raft endocytosis in cancer
    Johannes, Ludger; Billet, Anne
    Cancer and Metastasis Reviews (2020), 39 (2), 375-396CODEN: CMRED4; ISSN:0167-7659. (Springer)
    A review. Changes in glycosylation on proteins or lipids are one of the hallmarks of tumorigenesis. In many cases, it is still not understood how glycan information is translated into biol. function. In this review, we discuss at the example of specific cancer-related glycoproteins how their endocytic uptake into eukaryotic cells is tuned by carbohydrate modifications. For this, we not only focus on overall uptake rates, but also illustrate how different uptake processes-dependent or not on the conventional clathrin machinery-are used under given glycosylation conditions. Furthermore, we discuss the role of certain sugar-binding proteins, termed galectins, to tune glycoprotein uptake by inducing their crosslinking into lattices, or by co-clustering them with glycolipids into raft-type membrane nanodomains from which the so-called clathrin-independent carriers (CLICs) are formed for glycoprotein internalization into cells. The latter process has been termed glycolipid-lectin (GL-Lect) hypothesis, which operates in a complementary manner to the clathrin pathway and galectin lattices.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXptVers74%253D&md5=3d12f6406c663ba4a7e8f87f9f950514
  29. 29
    Lakshminarayan, R.; Wunder, C.; Becken, U.; Howes, M. T.; Benzing, C.; Arumugam, S.; Sales, S.; Ariotti, N.; Chambon, V.; Lamaze, C.; Loew, D.; Shevchenko, A.; Gaus, K.; Parton, R. G.; Johannes, L. Galectin-3 Drives Glycosphingolipid-Dependent Biogenesis of Clathrin-Independent Carriers. Nat. Cell Biol. 2014, 16, 592– 603,  DOI: 10.1038/ncb2970
    Google Scholar
    There is no corresponding record for this reference.
  30. 30
    Johannes, L.; Wunder, C.; Shafaq-Zadah, M. Glycolipids and Lectins in Endocytic Uptake Processes. J. Mol. Biol. 2016, 428, 4792– 4818,  DOI: 10.1016/j.jmb.2016.10.027
    Google Scholar
    30
    Glycolipids and lectins in endocytic uptake processes
    Johannes, Ludger; Wunder, Christian; Shafaq-Zadah, Massiullah
    Journal of Molecular Biology (2016), 428 (24_Part_A), 4792-4818CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    A review. A host of endocytic processes has been described at the plasma membrane of eukaryotic cells. Their categorization has most commonly referenced cytosolic machinery, of which the clathrin coat has occupied a preponderant position. In what concerns the intramembrane constituents, the focus of interest has been on phosphatidylinositol lipids and their capacity to orchestrate endocytic events on the cytosolic leaflet of the membrane. The contribution of extracellular determinants to the construction of endocytic pits has received much less attention, despite the fact that (glyco)sphingolipids are exoplasmic leaflet fabric of membrane domains, termed rafts, whose contributions to predominantly clathrin-independent internalization processes are well-recognized. Furthermore, sugar-binding proteins, termed lectins, and sugar modifications on extracellular domains of proteins have also been linked to the uptake of endocytic cargos at the plasma membrane. Here, the authors summarize these contributions by extracellular determinants to the endocytic process. The authors propose a mol. hypothesis, termed the GL-Lect hypothesis, on how glycolipids and lectins drive the formation of compositional nano-environments from which the endocytic uptake of glycosylated cargo proteins is operated via clathrin-independent carriers. Finally, the authors position this hypothesis within the global context of endocytic pathway proposals that have emerged in the recent years.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhslKksbvL&md5=9f011e970fe2a12f2ade88b6f90b9533
  31. 31
    Renard, H.-F.; Tyckaert, F.; Lo Giudice, C.; Hirsch, T.; Valades-Cruz, C. A.; Lemaigre, C.; Shafaq-Zadah, M.; Wunder, C.; Wattiez, R.; Johannes, L.; van der Bruggen, P.; Alsteens, D.; Morsomme, P. Endophilin-A3 and Galectin-8 Control the Clathrin-Independent Endocytosis of CD166. Nat. Commun. 2020, 11, 1457,  DOI: 10.1038/s41467-020-15303-y
    Google Scholar
    31
    Endophilin-A3 and Galectin-8 control the clathrin-independent endocytosis of CD166
    Renard, Henri-Francois; Tyckaert, Francois; Lo Giudice, Cristina; Hirsch, Thibault; Valades-Cruz, Cesar Augusto; Lemaigre, Camille; Shafaq-Zadah, Massiullah; Wunder, Christian; Wattiez, Ruddy; Johannes, Ludger; van der Bruggen, Pierre; Alsteens, David; Morsomme, Pierre
    Nature Communications (2020), 11 (1), 1457CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Abstr.: While several clathrin-independent endocytic processes have been described so far, their biol. relevance often remains elusive, esp. in pathophysiol. contexts such as cancer. In this study, we find that the tumor marker CD166/ALCAM (Activated Leukocyte Cell Adhesion Mol.) is a clathrin-independent cargo. We show that endophilin-A3-but neither A1 nor A2 isoforms-functionally assocs. with CD166-contg. early endocytic carriers and phys. interacts with the cargo. Our data further demonstrates that the three endophilin-A isoforms control the uptake of distinct subsets of cargoes. In addn., we provide strong evidence that the construction of endocytic sites from which CD166 is taken up in an endophilin-A3-dependent manner is driven by extracellular galectin-8. Taken together, our data reveal the existence of a previously uncharacterized clathrin-independent endocytic modality, that modulates the abundance of CD166 at the cell surface, and regulates adhesive and migratory properties of cancer cells.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXlvFCrsLw%253D&md5=4b99c3cf616ceb5d9d0b292158a9e602
  32. 32
    Nabi, I. R.; Shankar, J.; Dennis, J. W. The Galectin Lattice at a Glance. J. Cell Sci. 2015, 128, 2213– 2219,  DOI: 10.1242/jcs.151159
    Google Scholar
    32
    The galectin lattice at a glance
    Nabi, Ivan R.; Shankar, Jay; Dennis, James W.
    Journal of Cell Science (2015), 128 (13), 2213-2219CODEN: JNCSAI; ISSN:0021-9533. (Company of Biologists Ltd.)
    Galectins are a family of widely expressed β-galactoside-binding lectins in metazoans. The 15 mammalian galectins have either one or two conserved carbohydrate recognition domains (CRDs), with galectin-3 being able to pentamerize; they form complexes that crosslink glycosylated ligands to form a dynamic lattice. The galectin lattice regulates the diffusion, compartmentalization and endocytosis of plasma membrane glycoproteins and glycolipids. The galectin lattice also regulates the selection, activation and arrest of T cells, receptor kinase signaling and the functionality of membrane receptors, including the glucagon receptor, glucose and amino acid transporters, cadherins and integrins. The affinity of transmembrane glycoproteins to the galectin lattice is proportional to the no. and branching of their N-glycans; with branching being mediated by Golgi N-acetylglucosaminyltransferase-branching enzymes and the supply of UDP-GlcNAc through metabolite flux through the hexosamine biosynthesis pathway. The relative affinities of glycoproteins for the galectin lattice depend on the activities of the Golgi enzymes that generate the epitopes of their ligands and, thus, provide a means to analyze biol. function of lectins and of the 'glycome' more broadly.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtlSktLfL&md5=7e049fa9dbe95319e6fbdb37691a3f6b
  33. 33
    Mathew, M. P.; Donaldson, J. G. Distinct Cargo-Specific Response Landscapes Underpin the Complex and Nuanced Role of Galectin–Glycan Interactions in Clathrin-Independent Endocytosis. J. Biol. Chem. 2018, 293, 7222– 7237,  DOI: 10.1074/jbc.RA118.001802
    Google Scholar
    33
    Distinct cargo-specific response landscapes underpin the complex and nuanced role of galectin-glycan interactions in clathrin-independent endocytosis
    Mathew, Mohit P.; Donaldson, Julie G.
    Journal of Biological Chemistry (2018), 293 (19), 7222-7237CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Clathrin-independent endocytosis (CIE) is a form of endocytosis that lacks a defined cytoplasmic machinery. Here, we asked whether glycan interactions, acting from the outside, could be a part of that endocytic machinery. We show that the perturbation of global cellular patterns of protein glycosylation by modulation of metabolic flux affects CIE. Interestingly, these changes in glycosylation had cargo-specific effects. For example, in HeLa cells, GlcNAc treatment, which increases glycan branching, increased major histocompatibility complex class I (MHCI) internalization but inhibited CIE of the glycoprotein CD59 mol. (CD59). The effects of knocking down the expression of galectin 3, a carbohydrate-binding protein and an important player in galectin-glycan interactions, were also cargo-specific and stimulated CD59 uptake. By contrast, inhibition of all galectin-glycan interactions by lactose inhibited CIE of both MHCI and CD59. None of these treatments affected clathrin-mediated endocytosis, implying that glycosylation changes specifically affect CIE. We also found that the galectin lattice tailors membrane fluidity and cell spreading. Furthermore, changes in membrane dynamics mediated by the galectin lattice affected macropinocytosis, an altered form of CIE, in HT1080 cells. Our results suggest that glycans play an important and nuanced role in CIE, with each cargo being affected uniquely by alterations in galectin and glycan profiles and their interactions. We conclude that galectin-driven effects exist on a continuum from stimulatory to inhibitory, with distinct CIE cargo proteins having unique response landscapes and with different cell types starting at different positions on these conceptual landscapes.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXptlaqsrs%253D&md5=26118498d162fda9bfd30009601a11ba
  34. 34
    Wagner, M. L.; Tamm, L. K. Tethered Polymer-Supported Planar Lipid Bilayers for Reconstitution of Integral Membrane Proteins: Silane-Polyethyleneglycol-Lipid as a Cushion and Covalent Linker. Biophys. J. 2000, 79, 1400– 1414,  DOI: 10.1016/S0006-3495(00)76392-2
    Google Scholar
    34
    Tethered polymer-supported planar lipid bilayers for reconstitution of integral membrane proteins: silane-polyethyleneglycol-lipid as a cushion and covalent linker
    Wagner, Michael L.; Tamm, Lukas K.
    Biophysical Journal (2000), 79 (3), 1400-1414CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
    There is increasing interest in supported membranes as models of biol. membranes and as a physiol. matrix for studying the structure and function of membrane proteins and receptors. A common problem of protein-lipid bilayers that are directly supported on a hydrophilic substrate is nonphysiol. interactions of integral membrane proteins with the solid support to the extent that they will not diffuse in the plane of the membrane. To alleviate some of these problems we have developed a new tethered polymer-supported planar lipid bilayer system, which permitted us to reconstitute integral membrane proteins in a laterally mobile form. We have supported lipid bilayers on a newly designed polyethyleneglycol cushion, which provided a soft support and, for increased stability, covalent linkage of the membranes to the supporting quartz or glass substrates. The formation and morphol. of the bilayers were followed by total internal reflection and epifluorescence microscopy, and the lateral diffusion of the lipids and proteins in the bilayer was monitored by fluorescence recovery after photobleaching. Uniform bilayers with high lateral lipid diffusion coeffs. (0.8-1.2×10-8 cm2/s) were obsd. when the polymer concn. was kept slightly below the mushroom-to-brush transition. Cytochrome b5 and annexin V were used as first test proteins in this system. When reconstituted in supported bilayers that were directly supported on quartz, both proteins were largely immobile with mobile fractions < 25%. However, two populations of laterally mobile proteins were obsd. in the polymer-supported bilayers. Approx. 25% of cytochrome b5 diffused with a diffusion coeff. of ≥ 1×10-8 cm2/s, and 50-60% diffused with a diffusion coeff. of ∼2×10-10 cm2/s. Similarly, one-third of annexin V diffused with a diffusion coeff. of ∼ 3×10-9 cm2/s, and two-thirds diffused with a diffusion coeff. of ∼4×10-10 cm2/s. A model for the interaction of these proteins with the underlying polymer is discussed.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXmsVars70%253D&md5=fe3f7affd4f65df6cb8605e7008fea81
  35. 35
    Hussain, N. F.; Siegel, A. P.; Ge, Y.; Jordan, R.; Naumann, C. A. Bilayer Asymmetry Influences Integrin Sequestering in Raft-Mimicking Lipid Mixtures. Biophys. J. 2013, 104, 2212– 2221,  DOI: 10.1016/j.bpj.2013.04.020
    Google Scholar
    35
    Bilayer Asymmetry Influences Integrin Sequestering in Raft-Mimicking Lipid Mixtures
    Hussain, Noor F.; Siegel, Amanda P.; Ge, Yifan; Jordan, Rainer; Naumann, Christoph A.
    Biophysical Journal (2013), 104 (10), 2212-2221CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    There is growing recognition that lipid heterogeneities in cellular membranes play an important role in the distribution and functionality of membrane proteins. However, the detection and characterization of such heterogeneities at the cellular level remains challenging. Here we report on the poorly understood relationship between lipid bilayer asymmetry and membrane protein sequestering in raft-mimicking model membrane mixts. using a powerful exptl. platform comprised of confocal spectroscopy XY-scan and photon-counting histogram analyses. This exptl. approach is utilized to probe the domain-specific sequestering and oligomerization state of αvβ3 and α5β1 integrins in bilayers, which contain coexisting liq.-disordered/liq.-ordered (ld/lo) phase regions exclusively in the top leaflet of the bilayer (bottom leaflet contains ld phase). Comparison with previously reported integrin sequestering data in bilayer-spanning lo-ld phase sepns. demonstrates that bilayer asymmetry has a profound influence on αvβ3 and α5β1 sequestering behavior. For example, both integrins sequester preferentially to the lo phase in asym. bilayers, but to the ld phase in their sym. counterparts. Furthermore, our data show that bilayer asymmetry significantly influences the role of native ligands in integrin sequestering.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXotlynsr8%253D&md5=cd19583e9a04ee9188b55d8eac0a5356
  36. 36
    Peetla, C.; Stine, A.; Labhasetwar, V. Biophysical Interactions with Model Lipid Membranes: Applications in Drug Discovery and Drug Delivery. Mol. Pharm. 2009, 6, 1264– 1276,  DOI: 10.1021/mp9000662
    Google Scholar
    36
    Biophysical Interactions with Model Lipid Membranes: Applications in Drug Discovery and Drug Delivery
    Peetla, Chiranjeevi; Stine, Andrew; Labhasetwar, Vinod
    Molecular Pharmaceutics (2009), 6 (5), 1264-1276CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)
    A review. The transport of drugs or drug delivery systems across the cell membrane is a complex biol. process, often difficult to understand because of its dynamic nature. In this regard, model lipid membranes, which mimic many aspects of cell-membrane lipids, have been very useful in helping investigators to discern the roles of lipids in cellular interactions. One can use drug-lipid interactions to predict pharmacokinetic properties of drugs, such as their transport, biodistribution, accumulation, and hence efficacy. These interactions can also be used to study the mechanisms of transport, based on the structure and hydrophilicity/hydrophobicity of drug mols. In recent years, model lipid membranes have also been explored to understand their mechanisms of interactions with peptides, polymers, and nanocarriers. These interaction studies can be used to design and develop efficient drug delivery systems. Changes in the lipid compn. of cells and tissue in certain disease conditions may alter biophys. interactions, which could be explored to develop target-specific drugs and drug delivery systems. In this review, we discuss different model membranes, drug-lipid interactions and their significance, studies of model membrane interactions with nanocarriers, and how biophys. interaction studies with lipid model membranes could play an important role in drug discovery and drug delivery.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXmtlKju7o%253D&md5=18b63d6d84e976e48c4780911ab5bf20
  37. 37
    Ramadurai, S.; Holt, A.; Krasnikov, V.; van den Bogaart, G.; Killian, J. A.; Poolman, B. Lateral Diffusion of Membrane Proteins. J. Am. Chem. Soc. 2009, 131, 12650– 12656,  DOI: 10.1021/ja902853g
    Google Scholar
    37
    Lateral Diffusion of Membrane Proteins
    Ramadurai, Sivaramakrishnan; Holt, Andrea; Krasnikov, Victor; van den Bogaart, Geert; Killian, J. Antoinette; Poolman, Bert
    Journal of the American Chemical Society (2009), 131 (35), 12650-12656CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    We measured the lateral mobility of integral membrane proteins reconstituted in giant unilamellar vesicles (GUVs), using fluorescence correlation spectroscopy. Receptor, channel, and transporter proteins with 1-36 transmembrane segments (lateral radii ranging from 0.5 to 4 nm) and a α-helical peptide (radius of 0.5 nm) were fluorescently labeled and incorporated into GUVs. At low protein-to-lipid ratios (i.e., 10-100 proteins per μm2 of membrane surface), the diffusion coeff. D displayed a weak dependence on the hydrodynamic radius (R) of the proteins [D scaled with ln(1/R)], consistent with the Saffman-Delbruck model. At higher protein-to lipid ratios (up to 3000 μm-2), the lateral diffusion coeff. of the mols. decreased linearly with increasing the protein concn. in the membrane. The implications of our findings for protein mobility in biol. membranes (protein crowding of ∼25,000 μm-2) and use of diffusion measurements for protein geometry (size, oligomerization) detns. are discussed.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXpslaiurs%253D&md5=0d1993b5cb282585e36c07594e0d6583
  38. 38
    Li, B.; London, E. Preparation and Drug Entrapment Properties of Asymmetric Liposomes Containing Cationic and Anionic Lipids. Langmuir 2020, 36, 12521– 12531,  DOI: 10.1021/acs.langmuir.0c01968
    Google Scholar
    38
    Preparation and Drug Entrapment Properties of Asymmetric Liposomes Containing Cationic and Anionic Lipids
    Li, Bingchen; London, Erwin
    Langmuir (2020), 36 (42), 12521-12531CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    We have developed cyclodextrin-catalyzed lipid exchange methods to prep. large unilamellar vesicles (LUVs) with asym. charge distributions, i.e., with different net charges on the lipids in the inner and outer leaflets. LUVs contained a mixt. of a zwitterionic lipid (phosphatidylcholine), cholesterol, and various cationic lipids (O-Et phosphatidylcholine or dioleoyl-3-trimethylammonium propane) or anionic lipids (phosphatidylglycerol, phosphatidylserine, or phosphatidic acid). Sym. and asym. LUVs with a wide variety of lipid combinations were prepd. The asym. LUVs contained cationic or anionic outer leaflets and inner leaflets that had either the opposite charge or were uncharged. The behavior of sym. LUVs prepd. with zwitterionic, anionic, or cationic leaflets was compared to those of asym. LUVs. Lipid exchange was confirmed by quant. thin-layer chromatog., and lipid asymmetry by a novel assay measuring binding of a cationic fluorescent probe to the LUV outer leaflet. For both sym. and asym. LUVs, the level of entrapment of the cationic drug doxorubicin was controlled by the charge on the inner leaflet, with the greatest entrapment and slowest leakage in vesicles with an anionic inner leaflet. This shows that it is possible to choose inner leaflet lipids to maximize liposomal loading of charged drugs independently of the identity of outer-leaflet lipids. This implies that it should also be possible to independently vary outer-leaflet lipids to, for example, impart favorable bioavailability and biodistribution properties to lipid vesicles.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitVyrsrzJ&md5=0b91852927fcc0419cfbbe093351275d
  39. 39
    Cheng, H.-T.; Megha; London, E. Preparation and Properties of Asymmetric Vesicles That Mimic Cell Membranes. J. Biol. Chem. 2009, 284, 6079– 6092,  DOI: 10.1074/jbc.M806077200
    Google Scholar
    39
    Preparation and Properties of Asymmetric Vesicles That Mimic Cell Membranes: effect upon lipid raft formation and transmembrane helix orientation
    Cheng, Hui-Ting; Megha; London, Erwin
    Journal of Biological Chemistry (2009), 284 (10), 6079-6092CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    A methyl-β-cyclodextrin-induced lipid exchange technique was devised to prep. small unilamellar vesicles with stable asym. lipid compns. Asym. vesicles that mimic biol. membranes were prepd. with sphingomyelin (SM) or SM mixed with 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) as the predominant lipids in the outer leaflet and dioleoylphosphatidylcholine (DOPC), POPC, 1-palmitoyl-2-oleoyl-phosphatidyl-L-serine (POPS), or POPS mixed with 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) in the inner leaflet. Fluorescence-based assays were developed to confirm lipid asymmetry. Cholesterol was introduced into these vesicles using a second methyl-β-cyclodextrin exchange step. In asym. vesicles composed of SM outside, DOPC inside (SMo/DOPCi) or SM outside, 2:1 mol:mol POPE:POPS inside (SMo/2:1 POPE:POPSi) the outer leaflet SM formed an ordered state with a thermal stability similar to that in pure SM vesicles and significantly greater than that in sym. vesicles with the same overall lipid compn. Analogous behavior was obsd. in vesicles contg. cholesterol. This shows that an asym. lipid distribution like that in eukaryotic plasma membranes can be conducive to ordered domain (raft) formation. Furthermore asym. vesicles contg. ∼25 mol % cholesterol formed ordered domains more thermally stable than those in asym. vesicles lacking cholesterol, showing that the crucial ability of cholesterol to stabilize ordered domain formation is likely to contribute to ordered domain formation in cell membranes. Addnl. studies demonstrated that hydrophobic helix orientation is affected by lipid asymmetry with asymmetry favoring formation of the transmembrane configuration. The ability to form asym. vesicles represents an important improvement in model membrane studies and should find many applications in the future.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXisVSksbw%253D&md5=462c2fdcec4f096814084bfd8bc05b92
  40. 40
    Zhang, L.; Granick, S. Lipid Diffusion Compared in Outer and Inner Leaflets of Planar Supported Bilayers. J. Chem. Phys. 2005, 123, 211104,  DOI: 10.1063/1.2138699
    Google Scholar
    40
    Lipid diffusion compared in outer and inner leaflets of planar supported bilayers
    Zhang, Liangfang; Granick, Steve
    Journal of Chemical Physics (2005), 123 (21), 211104/1-211104/4CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
    The translational diffusion coeff. (D) of lipids located in the outer and inner leaflets of planar supported DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine) bilayers in the fluid phase was measured using fluorescence correlation spectroscopy of dye-labeled lipids at the low concn. of 0.001% and using iodide quenching of dyes in the outer leaflet to distinguish diffusion in the inner leaflet from that in the outer leaflet. To confirm the generality of these findings, the bilayers were prepd. not only by vesicle fusion but also by Langmuir-Blodgett deposition. We conclude that regardless of whether the bilayers were supported on quartz or on a polymer cushion, D in the inner and outer leaflets was the same within an exptl. uncertainty of ±10% but with a small systematic tendency to be slower (by <5%) within the inner leaflet.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlCgtbvE&md5=27dbf9c7d3ead51a6e600d70c256043e
  41. 41
    Murray, D. H.; Tamm, L. K.; Kiessling, V. Supported Double Membranes. J. Struct. Biol. 2009, 168, 183– 189,  DOI: 10.1016/j.jsb.2009.02.008
    Google Scholar
    41
    Supported double membranes
    Murray, David H.; Tamm, Lukas K.; Kiessling, Volker
    Journal of Structural Biology (2009), 168 (1), 183-189CODEN: JSBIEM; ISSN:1047-8477. (Elsevier B.V.)
    A review. Planar model membranes, like supported lipid bilayers and surface-tethered vesicles, have been proven to be useful tools for the investigation of complex biol. functions in a significantly less complex membrane environment. In this study, we introduce a supported double membrane system that should be useful for studies that target biol. processes in the proximity of two lipid bilayers such as the periplasm of bacteria and mitochondria or the small cleft between pre- and postsynaptic neuronal membranes. Large unilamellar vesicles (LUV) were tethered to a preformed supported bilayer by a biotin-streptavidin tether. We show from single particle tracking (SPT) expts. that these vesicle are mobile above the plane of the supported membrane. At higher concns., the tethered vesicles fuse to form a second continuous bilayer on top of the supported bilayer. The distance between the two bilayers was detd. by fluorescence interference contrast (FLIC) microscopy to be between 16 and 24 nm. The lateral diffusion of labeled lipids in the second bilayer was very similar to that in supported membranes. SPT expts. with reconstituted syntaxin-1A show that the mobility of transmembrane proteins was not improved when compared with solid supported membranes.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtVKkur7M&md5=4dbc0bcb5ed5670d365ae515a32b794a
  42. 42
    McGillivray, D. J.; Valincius, G.; Vanderah, D. J.; Febo-Ayala, W.; Woodward, J. T.; Heinrich, F.; Kasianowicz, J. J.; Lösche, M. Molecular-Scale Structural and Functional Characterization of Sparsely Tethered Bilayer Lipid Membranes. Biointerphases 2007, 2, 21– 33,  DOI: 10.1116/1.2709308
    Google Scholar
    42
    Molecular-scale structural and functional characterization of sparsely tethered bilayer lipid membranes
    McGillivray, Duncan J.; Valincius, Gintaras; Vanderah, David J.; Febo-Ayala, Wilma; Woodward, John T.; Heinrich, Frank; Kasianowicz, John J.; Losche, Mathias
    Biointerphases (2007), 2 (1), 21-33CODEN: BJIOBN; ISSN:1559-4106. (AVS-Science and Technology of Materials, Interfaces and Processing)
    Surface-tethered biomimetic bilayer membranes (tethered bilayer lipid membranes (tBLMs)) were formed on gold surfaces from phospholipids and a synthetic 1-thiahexa(ethylene oxide) lipid, WC14. They were characterized using electrochem. impedance spectroscopy, neutron reflection (NR), and Fourier-transform IR reflection-absorption spectroscopy (FT-IRRAS) to obtain functional and structural information. The authors found that elec. insulating membranes (conductance and capacitance as low as 1 μS cm-2 and 0.6 μF cm-2, resp.) with high surface coverage (>95% completion of the outer leaflet) can be formed from a range of lipids in a simple two-step process that consists of the formation of a self-assembled monolayer (SAM) and bilayer completion by "rapid solvent exchange.". NR provided a molecularly resolved characterization of the interface architecture and, in particular, the constitution of the space between the tBLM and the solid support. In tBLMs based on SAMs of pure WC14, the hexa(ethylene oxide) tether region had low hydration even though FT-IRRAS showed that this region is structurally disordered. However, on mixed SAMs made from the coadsorption of WC14 with a short-chain "backfiller," β-mercaptoethanol, the submembrane spaces between the tBLM and the substrates contained up to 60% exchangeable solvent by vol., as judged from NR and contrast variation of the solvent. Complete and stable "sparsely tethered" BLMs (stBLMs) can be readily prepd. from SAMs chemisorbed from solns. with low WC14 proportions. Phospholipids with unsatd. or satd., straight or branched chains all formed qual. similar stBLMs.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXksFGrtLg%253D&md5=b9d8a3517735a5276fe489dc40eecdc6
  43. 43
    Sarangi, N. K.; Patnaik, A. L-Tryptophan-Induced Electron Transport across Supported Lipid Bilayers: An Alkyl-Chain Tilt-Angle, and Bilayer-Symmetry Dependence. ChemPhysChem 2012, 13, 4258– 4270,  DOI: 10.1002/cphc.201200655
    Google Scholar
    43
    L-Tryptophan-Induced Electron Transport across Supported Lipid Bilayers: an Alkyl-Chain Tilt-Angle, and Bilayer-Symmetry Dependence
    Sarangi, Nirod Kumar; Patnaik, Archita
    ChemPhysChem (2012), 13 (18), 4258-4270CODEN: CPCHFT; ISSN:1439-4235. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Mol. orientation-dependent electron transport across supported 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid bilayers (SLBs) on semiconducting indium tin oxide (ITO) is reported with an aim towards potential nanobiotechnol. applications. A bifunctional strategy is adopted to form sym. and asym. bilayers of DPPC that interact with L-tryptophan, and are analyzed by surface manometry and at. force microscopy. Polarization-dependent real-time Fourier transform IR reflection absorption spectroscopy (FT-IRRAS) anal. of these SLBs reveals electrostatic, hydrogen-bonding, and cation-π interactions between the polar head groups of the lipid and the indole side chains. Consequently, a mol. tilt arises from the effective interface dipole, facilitating electron transport across the ITO-anchored SLBs in the presence of an internal Fe(CN)64-/3- redox probe. The incorporation of tryptophan enhances the voltammetric features of the SLBs. The estd. electron-transfer rate consts. for sym. and asym. bilayers (ks=2.0 × 10-2 and 2.8 × 10-2 s-1) across the two-dimensional (2D) ordered DPPC/tryptophan SLBs are higher compared to pure DPPC SLBs (ks=3.2 × 10-3 and 3.9 × 10-3 s-1). In addn., they are mol. tilt-dependent, as it is the case with the std. apparent rate consts., estd. from electrochem. impedance spectroscopy and bipotentiostatic expts. with a Pt ultramicroelectrode. Lower magnitudes of ks imply that electrochem. reactions across the ITO-SLB electrodes are kinetically limited and consequently governed by electron tunneling across the SLBs. Std. theor. rate consts. accrued upon electron tunneling comply with the potential-independent electron-tunneling coeff. β=0.15 Å-1. Insulator-semiconductor transitions moving from a liq.-expanded to a condensed 2D-phase state of the SLBs are noted, adding a new dimension to their transport behavior. These results highlight the role of tryptophan in expediting electron transfer across lipid bilayer membranes in a cellular environment and can provide potential clues towards patterned lipid nanocomposites and devices.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsFKns7vL&md5=b73f88353fdd53982b4abd099db1ac48
  44. 44
    Gufler, P. C.; Pum, D.; Sleytr, U. B.; Schuster, B. Highly Robust Lipid Membranes on Crystalline S-Layer Supports Investigated by Electrochemical Impedance Spectroscopy. Biochim. Biophys. Acta, Biomembr. 2004, 1661, 154– 165,  DOI: 10.1016/j.bbamem.2003.12.009
    Google Scholar
    44
    Highly robust lipid membranes on crystalline S-layer supports investigated by electrochemical impedance spectroscopy
    Gufler, Petra C.; Pum, Dietmar; Sleytr, Uwe B.; Schuster, Bernhard
    Biochimica et Biophysica Acta, Biomembranes (2004), 1661 (2), 154-165CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
    In the present work, S-layer supported lipid membranes formed by a modified Langmuir-Blodgett technique were investigated by electrochem. impedance spectroscopy (EIS). Basically two intermediate hydrophilic supports for phospholipid- (DPhyPC) and bipolar tetraetherlipid- (MPL from Thermoplasma acidophilum) membranes have been applied: first, the S-layer protein SbpA isolated from Bacillus sphaericus CCM 2177 recrystd. onto a gold electrode; and second, as a ref. support, an S-layer ultrafiltration membrane (SUM), which consists of a microfiltration membrane (MFM) with deposited S-layer carrying cell wall fragments. The electrochem. properties and the stability of DPhyPC and MPL membranes were found to depend on the support employed. The specific capacitances were 0.53 and 0.69 μF/cm2 for DPhyPC bilayers and 0.75 and 0.77 μF/cm2 for MPL monolayers resting on SbpA and SUM, resp. Membrane resistances of up to 80 MΩ cm2 were obsd. for DPhyPC bilayers on SbpA. In addn., membranes supported by SbpA exhibited a remarkable long-term robustness of up to 2 days. The membrane functionality could be demonstrated by reconstitution of membrane-active peptides such as valinomycin and alamethicin. The present results recommend S-layer-supported lipid membranes as promising structures for membrane protein-based biosensor technol.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhvFagsL4%253D&md5=8aae1cbbfd1b0702c465e8e514a5263c
  45. 45
    Hillman, A. R.; Ryder, K. S.; Madrid, E.; Burley, A. W.; Wiltshire, R. J.; Merotra, J.; Grau, M.; Horswell, S. L.; Glidle, A.; Dalgliesh, R. M.; Hughes, A.; Cubitt, R.; Wildes, A. Structure and Dynamics of Phospholipid Bilayer Films under Electrochemical Control. Faraday Discuss. 2010, 145, 357– 379,  DOI: 10.1039/b911246b
    Google Scholar
    45
    Structure and dynamics of phospholipid bilayer films under electrochemical control
    Hillman, A. Robert; Ryder, Karl S.; Madrid, Elena; Burley, Andrew W.; Wiltshire, Richard J.; Merotra, James; Grau, Michaela; Horswell, Sarah L.; Glidle, Andrew; Dalgliesh, Robert M.; Hughes, Arwel; Cubitt, Robert; Wildes, Andrew
    Faraday Discussions (2010), 145 (), 357-379CODEN: FDISE6; ISSN:1359-6640. (Royal Society of Chemistry)
    Vesicle fusion was used to deposit mixed dimyristoyl phosphatidylethanolamine-dimyristoyl phosphatidylserine (DMPE-DMPS) phospholipid bilayers on Au electrodes. Bilayer structure and compn., when exposed to aq. NaF and subject to an applied electrochem. potential, were studied using electrochem., spectroscopic and neutron reflectivity (NR) techniques. Interfacial capacitance data indicate the formation of compact films. Chronocolometric data show that surface charge is significantly altered by the presence of lipid in the potential range -0.75 < E/V (Ag|AgCl) < 0.35. NR measurements were made on lipid films in which the hydrocarbon tails were either fully hydrogenous (h-DMPE-h-DMPS) or perdeuterated (d-DMPE-d-DMPS), in each case serially exposed to D2O and H2O electrolytes and subject to different applied potentials. Guided by simulations of candidate interfacial structures, these yield the spatial distributions of lipid and solvent within the layers. Adjacent to the electrode, a compact inner leaflet is formed, with potential-dependent solvent vol. fraction in the range 0.09 < ΦS < 0.19; there was no evidence of an intervening water layer. The outer leaflet contains rather more solvent, 0.52 < ΦS < 0.55. NR-derived film thickness and PM-IRRAS intensity data show that the lipid mols. are tilted from the surface normal by ca. 26°. Bilayer solvation and charge data show a strong correlation for the inner leaflet and very little for the outer leaflet.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXntlKntw%253D%253D&md5=ecb50d07b01dd7705ab5c515f8c28a3d
  46. 46
    Steinem, C.; Janshoff, A.; Ulrich, W. P.; Sieber, M.; Galla, H. J. Impedance Analysis of Supported Lipid Bilayer Membranes: A Scrutiny of Different Preparation Techniques. Biochim. Biophys. Acta, Biomembr. 1996, 1279, 169– 180,  DOI: 10.1016/0005-2736(95)00274-X
    Google Scholar
    46
    Impedance analysis of supported lipid bilayer membranes: a scrutiny of different preparation techniques
    Steinem, Claudia; Janshoff, Andreas; Ulrich, Wolf-Peter; Sieber, Manfred; Galla, Hans-Joachim
    Biochimica et Biophysica Acta, Biomembranes (1996), 1279 (2), 169-80CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
    One topic of this study is the comparison of different prepn. techniques to build up solid supported lipid bilayers onto gold substrates. The deposited lipid bilayers were investigated by a.c. impedance spectroscopy. Three different strategies were applied: (1) The gold surface was initially covered with a chemisorbed monolayer of octadecanethiol or 1,2-dimyristoyl-sn-glycero-3-phosphothioethanol (DMPTE). The second monolayer consisting of phospholipids was then deposited onto this hydrophobic surface by (i) the Langmuir-Schaefer-technique, (ii) from lipid soln. in n-decane/isobutanol, (iii) by the lipid/detergent diln. technique or (i.v.) by fusion of vesicles. (2) Charged mols. carrying thiol-anchors for attachment to the gold surface by chemisorption were used. Neg. charged surfaces of 3-mercaptopropionic acid were excellent substrates that allow the attachment of planar lipid bilayers by applying pos. charged dimethyldioctadecylammoniumbromide (DODAB) vesicles or neg. charged 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol vesicles in the presence of chelating Ca2+-ions. If pos. charged first monolayers of mercaptoethylammoniumhydrochloride were used the authors were able to attach mixed 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol/1,2-dimyristoyl-sn -glycero-3-phosphoethanolamine vesicles to form planar lipid bilayers via electrostatic interaction. (3) Direct deposition of lipid bilayers is possible from vesicles contg. 1,2-dimyristoyl-sn-glycero-3-phosphothioethanol (DMPTE). A crit. amt. of more than 50 mol% of DMPTE was necessary to form a solid supported lipid bilayer. Bilayers obtained with these different prepn. techniques were scrutinized with respect to their capacitances, kinetics of formation and their long-term stabilities by impedance spectroscopy. The second feature of this paper is the application of the supported bilayers to study ion transport through channel-forming peptides. The authors used a DODAB-bilayer for the reconstitution of gramicidin D channels. By CD measurements the authors verified that the peptide is in its channel conformation. The ion transport of Cs+-ions through the channels was recorded by impedance anal.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XhsFOjtbc%253D&md5=f62efc4a6b801c312e9ebef4e6951cbc
  47. 47
    McGillivray, D. J.; Valincius, G.; Heinrich, F.; Robertson, J. W. F.; Vanderah, D. J.; Febo-Ayala, W.; Ignatjev, I.; Lösche, M.; Kasianowicz, J. J. Structure of Functional Staphylococcus Aureus α-Hemolysin Channels in Tethered Bilayer Lipid Membranes. Biophys. J. 2009, 96, 1547– 1553,  DOI: 10.1016/j.bpj.2008.11.020
    Google Scholar
    47
    Structure of functional Staphylococcus aureus α-hemolysin channels in tethered bilayer lipid membranes
    McGillivray, Duncan J.; Valincius, Gintaras; Heinrich, Frank; Robertson, Joseph W. F.; Vanderah, David J.; Febo-Ayala, Wilma; Ignatjev, Ilja; Losche, Mathias; Kasianowicz, John J.
    Biophysical Journal (2009), 96 (4), 1547-1553CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
    We demonstrate a method for simultaneous structure and function detn. of integral membrane proteins. Elec. impedance spectroscopy (EIS) shows that Staphylococcus aureus α-hemolysin (α-HL) channels in membranes tethered to gold have the same properties as those formed in free-standing bilayer lipid membranes. Neutron reflectometry (NR) provides high-resoln. structural information on the interaction between the channel and the disordered membrane, validating predictions based on the channel's x-ray crystal structure. The robust nature of the membrane enabled the precise localization of the protein within 1.1 Å. The channel's extramembranous cap domain affects the lipid headgroup region and the alkyl chains in the outer membrane leaflet and significantly dehydrates the headgroups. The results suggest that this technique could be used to elucidate mol. details of the assocn. of other proteins with membranes and may provide structural information on domain organization and stimuli-responsive reorganization for transmembrane proteins in membrane mimics.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXnvVersb0%253D&md5=bd1a5b223aef364b888e49b0d300268b
  48. 48
    Wiegand, G.; Arribas-Layton, N.; Hillebrandt, H.; Sackmann, E.; Wagner, P. Electrical Properties of Supported Lipid Bilayer Membranes. J. Phys. Chem. B 2002, 106, 4245– 4254,  DOI: 10.1021/jp014337e
    Google Scholar
    48
    Electrical properties of supported lipid bilayer membranes
    Wiegand, Gerald; Arribas-Layton, Noah; Hillebrandt, Heiko; Sackmann, Erich; Wagner, Peter
    Journal of Physical Chemistry B (2002), 106 (16), 4245-4254CODEN: JPCBFK; ISSN:1089-5647. (American Chemical Society)
    This paper describes a study of the elec. properties of supported lipid bilayer membranes on semiconductor and gold surfaces. The study is aimed to foster the understanding of supported membrane systems and to allow the rational design of biosensor assays for ion channel anal. Impedance spectroscopy was applied for the elec. characterization of the supported membrane systems. A novel equiv. circuit model is introduced for the data evaluation, which accounts for the deviation of the impedance response of supported membranes from that of an ideal RC element. As a result of the improved accordance of model and data, the resistance and the capacity of supported membranes can be detd. more accurately and independently from each other. Exptl. results describe the phenomenol. of the elec. properties of supported bilayers regarding variations in prepn., compn., and environmental conditions. We discuss the findings in terms of membrane-substrate interactions and models of membrane permeability. The important role of the electrostatics between the lipid bilayer and the solid substrate for the formation of an elec. dense supported membrane is identified. Bilayer permeability models explain the correlation between the structure of the lipid bilayer and its insulating properties. These models are also in accordance with the obsd. dependence of the elec. resistance of the lipid bilayer on the temp. and the ion concn. of the electrolyte.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XitlCis78%253D&md5=266b61310169b8fe0daf2a60ce9ecc4f
  49. 49
    Su, Z.; Leitch, J. J.; Lipkowski, J. Electrode-Supported Biomimetic Membranes: An Electrochemical and Surface Science Approach for Characterizing Biological Cell Membranes. Curr. Opin. Electrochem. 2018, 12, 60– 72,  DOI: 10.1016/J.COELEC.2018.05.020
    Google Scholar
    49
    Electrode-supported biomimetic membranes: An electrochemical and surface science approach for characterizing biological cell membranes
    Su, ZhangFei; Leitch, J. Jay; Lipkowski, Jacek
    Current Opinion in Electrochemistry (2018), 12 (), 60-72CODEN: COEUCY; ISSN:2451-9111. (Elsevier B.V.)
    A review. Planar solid-supported lipid bilayers have been developed as simplified biol. membranes to model the phys. properties of cell membrane processes. Lipid bilayer membranes supported at conductive metal substrates provide a unique opportunity to investigate the effect of the static elec. field on the membrane structure and function. The insights gained from this research can be used to develop novel biosensors and biomedical devices. This review summarizes the recent developments in metal-supported biomimetic membrane systems. It provides an overview of the various models, such as metal-supported monolayers and bilayers, hybrid bilayers, tethered bilayers, and floating bilayers, used to study membrane processes at electrode surfaces, such as metal-supported monolayers and bilayers, hybrid, tethered, and floating bilayers. The paper discusses the recent advancements in these biomimetic models and describes the fundamental knowledge about membrane processes that has been extd. from these different platforms. The potential for the design and improvement of biomedical devices using metal-supported bilayers is also discussed. Metal-supported bilayers allow for the application of a plethora of spectroscopic and surface imaging techniques to obtain information about the voltage-dependent properties of biomols. at the mol. level. The underlying methodol. of these anal. techniques and the structural, chem. and kinetic information extd. are reviewed.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXisF2lu73P&md5=7ecfb661116ec1bafeb8fa37d5ae7ad9
  50. 50
    Abbasi, F.; Leitch, J. J.; Su, Z.; Szymanski, G.; Lipkowski, J. Direct Visualization of Alamethicin Ion Pores Formed in a Floating Phospholipid Membrane Supported on a Gold Electrode Surface. Electrochim. Acta 2018, 267, 195– 205,  DOI: 10.1016/J.ELECTACTA.2018.02.057
    Google Scholar
    50
    Direct visualization of alamethicin ion pores formed in a floating phospholipid membrane supported on a gold electrode surface
    Abbasi, Fatemeh; Leitch, J. Jay; Su, ZhangFei; Szymanski, Grzegorz; Lipkowski, Jacek
    Electrochimica Acta (2018), 267 (), 195-205CODEN: ELCAAV; ISSN:0013-4686. (Elsevier Ltd.)
    Unilamellar DMPC/DMPG vesicles in the absence and presence of alamethicin were fused onto the surface of a Au electrode modified with a 1-thio-β-D-glucose self-assembled monolayer. The resulting floating bilayer lipid membranes (fBLMs) were studied using at. force microscopy (AFM) and electrochem. impedance spectroscopy (EIS). A corrugated film structure was obsd. for the pure DMPC/DMPG fBLMs due to surface stress between the tightly packed lipids. These corrugations are removed by the addn. of alamethicin suggesting the lipid-peptide interactions alleviate the overall surface stress creating a more uniform bilayer. Both DMPC/DMPG films in the absence and presence of alamethicin had thickness of 5.5 ± 0.9 nm demonstrating that alamethicin has a minimal effect on the overall bilayer thickness. However, a significant decrease in membrane resistivity was obsd. when alamethicin was inserted into the fBLM indicating that the peptides are forming ion conducting pores. A direct visualization of the alamethicin pores was obtained by mol. resoln. AFM images revealing that the pores are not randomly dispersed throughout the bilayer, but instead form hexagonal aggregates. The diam. of an individual pore within the aggregates is 2.3 ± 0.3 nm, which is consistent with the size of a hexameric pore predicted by mol. dynamics simulations. Addnl., the image revealed a broad size distribution of alamethicin aggregates, which explains the origin of multiple cond. states obsd. for the incorporation of alamethicin into free standing bilayer lipid membranes.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjtVantb0%253D&md5=1ee9c727bbdb290d91b783e110330793
  51. 51
    Drexler, J.; Steinem, C. Pore-Suspending Lipid Bilayers on Porous Alumina Investigated by Electrical Impedance Spectroscopy. J. Phys. Chem. B 2003, 107, 11245– 11254,  DOI: 10.1021/jp030762r
    Google Scholar
    51
    Pore-Suspending Lipid Bilayers on Porous Alumina Investigated by Electrical Impedance Spectroscopy
    Drexler, Janine; Steinem, Claudia
    Journal of Physical Chemistry B (2003), 107 (40), 11245-11254CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)
    Nonordered and ordered porous alumina substrates with pore diams. of 20 and 50 nm, resp., were utilized to immobilize lipid membranes spanning the pores of the porous material. The substrates were characterized by means of interferometry and elec. impedance spectroscopy. For impedance data redn., an equiv. circuit representing the elec. behavior of porous alumina was developed on the basis of the parallel layer model. It turned out that the elec. parameters of the as prepd. alumina substrates prevent a sensitive monitoring of the formation of immobilized lipid membranes. Thus, we established a technique to modify the substrates with respect to their elec. properties, leading to a significantly increased capacitance of porous alumina, which allowed for a sensitive detection of pore-spanning lipid bilayers by impedance spectroscopy. Two different membrane prepn. techniques based on vesicle spreading were investigated. First, neg. charged giant liposomes were spread onto the porous alumina surface under an applied dc voltage of +100 mV. Second, large unilamellar vesicles contg. lipids bearing a thiol anchor were used to chemisorb on gold functionalized porous alumina substrates and subsequently rupture to form planar pore-spanning membranes. For both techniques, impedance spectra were obtained, which indicate the formation of lipid bilayers on top of the porous alumina substrates.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXnt1Oktbw%253D&md5=81bcd70aed28801d79a1d049adecfd23
  52. 52
    Orth, A.; Johannes, L.; Römer, W.; Steinem, C. Creating and Modulating Microdomains in Pore-Spanning Membranes. ChemPhysChem 2012, 13, 108– 114,  DOI: 10.1002/cphc.201100644
    Google Scholar
    52
    Creating and Modulating Microdomains in Pore-Spanning Membranes
    Orth, Alexander; Johannes, Ludger; Roemer, Winfried; Steinem, Claudia
    ChemPhysChem (2012), 13 (1), 108-114CODEN: CPCHFT; ISSN:1439-4235. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The architecture of the plasma membrane is not only detd. by the lipid and protein compn., but is also influenced by its attachment to the underlying cytoskeleton. Herein, the authors show that microscopic phase sepn. of "raft-like" lipid mixts. in pore-spanning bilayers is strongly detd. by the underlying highly ordered porous substrate. In detail, lipid membranes composed of DOPC/sphingomyelin/cholesterol/Gb3 were prepd. on ordered pore arrays in silicon with pore diams. of 0.8, 1.2 and 2 μm, resp., by spreading and fusion of giant unilamellar vesicles. The upper part of the silicon substrate was first coated with gold and then functionalized with a thiol-bearing cholesterol deriv. rendering the surface hydrophobic, which is prerequisite for membrane formation. Confocal laser scanning fluorescence microscopy was used to investigate the phase behavior of the obtained pore-spanning membranes. Coexisting liq.-ordered- (lo) and liq.-disordered (ld) domains were visualized for DOPC/sphingomyelin/cholesterol/Gb3 (40:35:20:5) membranes. The size of the lo-phase domains was strongly affected by the underlying pore size of the silicon substrate and could be controlled by temp., and the cholesterol content in the membrane, which was modulated by the addn. of methyl-β-cyclodextrin. Binding of Shiga toxin B-pentamers to the Gb3-doped membranes increased the lo-phase considerably and even induced lo-phase domains in non-phase sepd. bilayers composed of DOPC/sphingomyelin/cholesterol/Gb3 (65:10:20:5).
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsFGjsbfM&md5=15e5dde1008ee608b32aaf28dff91999
  53. 53
    Ronen, R.; Kaufman, Y.; Freger, V. Formation of Pore-Spanning Lipid Membrane and Cross-Membrane Water and Ion Transport. J. Membr. Sci. 2017, 523, 247– 254,  DOI: 10.1016/j.memsci.2016.09.059
    Google Scholar
    53
    Formation of pore-spanning lipid membrane and cross-membrane water and ion transport
    Ronen, Rona; Kaufman, Yair; Freger, Viatcheslav
    Journal of Membrane Science (2017), 523 (), 247-254CODEN: JMESDO; ISSN:0376-7388. (Elsevier B.V.)
    The authors report on (1) the formation mechanism of an array of pore-spanning phospholipid membranes via 'vesicle fusion', and (2) a microfluidic device that is used to assess the stability of the pore-spanning lipid membrane under flow and osmotic gradient. It is shown that the formation of pore-spanning lipid membranes via 'vesicles fusion' proceeds in three steps: first, small vesicles merge into giant ones of about the size of the substrate pore size. The giant vesicles then settle at the pore mouths and flatten. Last, the flattened giant vesicles rupture and form a lipid membrane that closes the pore. Exposing the membrane to combined transmembrane osmotic and tangential shear flows in a microfluidic device, which simulates common osmotic process conditions, shows that, in addn. to remaining open pores, a fraction of pore-spanning membranes ruptures. Possible ways to avoid such rupture and minimize fraction of open pores are discussed.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhslShs7jN&md5=0776caf39dae9fbac8d7662f544aa49d
  54. 54
    Jose, B.; Mallon, C. T.; Forster, R. J.; Blackledge, C.; Keyes, T. E. Lipid Bilayer Assembly at a Gold Nanocavity Array. Chem. Commun. 2011, 47, 12530,  DOI: 10.1039/c1cc15709d
    Google Scholar
    54
    Lipid bilayer assembly at a gold nanocavity array
    Jose, Bincy; Mallon, Colm T.; Forster, Robert J.; Blackledge, Chuck; Keyes, Tia E.
    Chemical Communications (Cambridge, United Kingdom) (2011), 47 (46), 12530-12532CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    The assembly of lipid bilayer membranes, using ultrasonic disruption of liposomes of L-α-dimyristoylphosphatidylcholine, across 820 nm diam. spherical cap gold cavity arrays is demonstrated.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsVGrs7jE&md5=73a9425ab66f901dc4eda4d1eded7c9f
  55. 55
    Basit, H.; Gaul, V.; Maher, S.; Forster, R. J.; Keyes, T. E. Aqueous-Filled Polymer Microcavity Arrays: Versatile & Stable Lipid Bilayer Platforms Offering High Lateral Mobility to Incorporated Membrane Proteins. Analyst 2015, 140, 3012– 3018,  DOI: 10.1039/C4AN02317J
    Google Scholar
    55
    Aqueous-filled polymer microcavity arrays: versatile & stable lipid bilayer platforms offering high lateral mobility to incorporated membrane proteins
    Basit, Hajra; Gaul, Vinnie; Maher, Sean; Forster, Robert J.; Keyes, Tia E.
    Analyst (Cambridge, United Kingdom) (2015), 140 (9), 3012-3018CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
    A key prerequisite in an ideal supported lipid bilayer based cell membrane model is that the mobility of both the lipid matrix and its components are unhindered by the underlying support. This is not trivial and with the exception of liposomes, many of even the most advanced approaches, although accomplishing lipid mobility, fail to achieve complete mobility of incorporated membrane proteins. This is addressed in a novel platform comprising lipid bilayers assembled over buffer-filled arrays of spherical cap microcavities formed from microsphere template polydimethylsiloxane. Prior to bilayer assembly the PDMS is rendered hydrophilic by plasma treatment and the lipid bilayer prepd. using Langmuir Blodgett assembly followed by liposome/proteoliposome fusion. Fluorescence Lifetime Correlation Spectroscopy confirmed the pore suspended lipid bilayer exhibits diffusion coeffs. comparable to free-standing vesicles in soln. The bilayer modified arrays are highly reproducible and stable over days. As the bilayers are suspended over deep aq. reservoirs, reconstituted membrane proteins experience an aq. interface at both membrane interfaces and attain full lateral mobility. Their utility as membrane protein platforms was exemplified in two case studies with proteins of different dimensions in their extracellular and cytoplasmic domains reconstituted into DOPC lipid bilayers; Glycophorin A, and Integrin αIIbβ3. In both cases, the proteins exhibited 100% mobility with high lateral diffusion coeffs.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXkt1OrsLs%253D&md5=4555de38bfa1f1605591077d10438f4d
  56. 56
    Berselli, G. B.; Sarangi, N. K.; Ramadurai, S.; Murphy, P. V.; Keyes, T. E. Microcavity-Supported Lipid Membranes: Versatile Platforms for Building Asymmetric Lipid Bilayers and for Protein Recognition. ACS Appl. Bio Mater. 2019, 2, 3404– 3417,  DOI: 10.1021/acsabm.9b00378
    Google Scholar
    56
    Microcavity-Supported Lipid Membranes: Versatile Platforms for Building Asymmetric Lipid Bilayers and for Protein Recognition
    Berselli, Guilherme B.; Sarangi, Nirod Kumar; Ramadurai, Sivaramakrishnan; Murphy, Paul V.; Keyes, Tia E.
    ACS Applied Bio Materials (2019), 2 (8), 3404-3417CODEN: AABMCB; ISSN:2576-6422. (American Chemical Society)
    Microcavity-supported lipid bilayers (MSLBs) are contact-free membranes suspended across aq.-filled pores that maintain the lipid bilayer in a highly fluidic state, free from frictional interactions with substrate. Such platforms offer the prospect of liposome-like fluidity with the compositional versatility and addressability of supported lipid bilayers and thus offer a significant opportunity to model membrane asymmetry, protein-membrane interactions, and aggregation at the membrane interface. Herein we evaluate their performance by studying the effect of transmembrane lipid asymmetry on lipid diffusivity, membrane viscosity, and cholera toxin-ganglioside recognition across six sym. and asym. membranes including binary compns. contg. both fluid and gel phases, and ternary phase-sepd. membrane compns. Fluorescence lifetime correlation spectroscopy was used to det. the lateral mobility of the lipid and the protein, and electrochem. impedance spectroscopy enabled the detection of the protein-membrane assembly over the nanomolar range. Transmembrane leaflet asymmetry was obsd. to have a profound impact on membrane electrochem. resistance, where the resistance of a ternary sym. phase-sepd. bilayer was found to be at least 2.6 times higher than the asym. bilayer with analogous compn. in the distal leaflet but where the lower leaflet comprised only 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Similarly, the diffusion coeff. for MSLBs was obsd. to be 2.5 times faster for asym. MSLBs where the lower leaflet is DOPC alone. Our results demonstrate that the interplay of lipid packing across both membrane leaflets and the concn. of GM1 both affect the extent of cholera toxin aggregation and the consequent diffusion of the cholera-GM1 aggregates. Given that true biomembranes are both fluidic and asym., MSLBs offer the opportunity to build greater biomimicry into biophys. models, and the approach described demonstrates the value of MSLBs in studying aggregation and the membrane-assocd. multivalent interactions prevalent in many carbohydrate-mediated processes.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXht1Oqtr7L&md5=45629dfffab81e7cca5609d09704fa92
  57. 57
    Berselli, G. B.; Sarangi, N. K.; Gimenez, A. V.; Murphy, P. V.; Keyes, T. E. Microcavity Array Supported Lipid Bilayer Models of Ganglioside─Influenza Hemagglutinin1binding. Chem. Commun. 2020, 56, 11251– 11254,  DOI: 10.1039/d0cc04276e
    Google Scholar
    57
    Microcavity array supported lipid bilayer models of ganglioside - influenza hemagglutinin1 binding
    Berselli, Guilherme B.; Sarangi, Nirod Kumar; Gimenez, Aurelien V.; Murphy, Paul V.; Keyes, Tia E.
    Chemical Communications (Cambridge, United Kingdom) (2020), 56 (76), 11251-11254CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
    The binding of influenza receptor (HA1) to membranes contg. different glycosphingolipid receptors was studied at Microcavity Supported Lipid Bilayers (MSLBs). HA1 preferentially binds to GD1a but the diffusion coeff. of the assocd. complex at lipid bilayer is approx. double that of the complexes formed by HA1 GM1 or GM3.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFGgs7%252FM&md5=21de2dc3ca350829450d6d5466e5cc37
  58. 58
    Robinson, J.; Berselli, G. B.; Ryadnov, M. G.; Keyes, T. E. Annexin v Drives Stabilization of Damaged Asymmetric Phospholipid Bilayers. Langmuir 2020, 36, 5454– 5465,  DOI: 10.1021/acs.langmuir.0c00035
    Google Scholar
    58
    Annexin V Drives Stabilization of Damaged Asymmetric Phospholipid Bilayers
    Robinson, Jack; Berselli, Guilherme B.; Ryadnov, Maxim G.; Keyes, Tia E.
    Langmuir (2020), 36 (19), 5454-5465CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    Annexins are sol. membrane-binding proteins that assoc. in a calcium dependent manner with anionic phospholipids. They play roles in membrane organization, signaling and vesicle transport and in several disease states including thrombosis and inflammation. Annexin V is believed to be involved in membrane repair. Mediated through binding to phosphatidylserine exposed at damaged plasma membrane, the protein forms cryst. networks that seal or stabilize small membrane tears. Herein, we model this biochem. mechanism to simulate membrane healing at microcavity array supported, transversally asym., lipid bilayers (MSLBs) comprising 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS). Varying annexin V concn., lipid compn., and DOPS presence at each leaflet, fluorescence imaging and correlation spectroscopy confirmed that when DOPS was present at the external, annexin V, contacting leaflet, the protein assembled rapidly at the membrane interface to form a layer. From electrochem. impedance studies, the annexin layer decreased membrane capacitance while reducing resistance. With DOPS incorporated only at the lower (proximal) leaflet, no appreciable annexin assembly was obsd. over the first 21 h. This suggests that membrane asymmetry is preserved over this window and transversal diffusion of DOPS is slow. Intense laser light applied to the membrane, in which DOPS is initially isolated at the lower leaflet, was found to simulate membrane damage, stimulating the rapid assembly of annexin V at the membrane interface confirmed by fluorescence imaging, correlation spectroscopy, and electrochem. impedance measurements. The damage induced by light increased impedance and decreased membrane resistance. The resulting bilayer annexin V patched bilayer showed better temporal stability toward impedance changes when compared with that of the parent membrane. In summary, this simple model of annexin V assembly in a fluidic lipid membrane provides new insights into the assembly of annexins as well as an empirical basis for building patch-repair mechanisms into interfacial bilayer membrane assemblies.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXnslehtLw%253D&md5=b48dade43ea0a2d3dd6aad1f18ae0113
  59. 59
    Ramadurai, S.; Sarangi, N. K.; Maher, S.; MacConnell, N.; Bond, A. M.; McDaid, D.; Flynn, D.; Keyes, T. E. Microcavity-Supported Lipid Bilayers; Evaluation of Drug–Lipid Membrane Interactions by Electrochemical Impedance and Fluorescence Correlation Spectroscopy. Langmuir 2019, 35, 8095– 8109,  DOI: 10.1021/acs.langmuir.9b01028
    Google Scholar
    59
    Microcavity-Supported Lipid Bilayers; Evaluation of Drug-Lipid Membrane Interactions by Electrochemical Impedance and Fluorescence Correlation Spectroscopy
    Ramadurai, Sivaramakrishnan; Sarangi, Nirod Kumar; Maher, Sean; MacConnell, Nicola; Bond, Alan M.; McDaid, Dennis; Flynn, Damien; Keyes, Tia E.
    Langmuir (2019), 35 (24), 8095-8109CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    Many drugs have intracellular or membrane-assocd. targets, thus understanding their interaction with the cell membrane is of value in drug development. Cell-free tools used to predict membrane interactions should replicate the mol. organization of the membrane. Microcavity array-supported lipid bilayer (MSLB) platforms are versatile biophys. models of the cell membrane that combine liposome-like membrane fluidity with stability and addressability. We used an MSLB herein to interrogate drug-membrane interactions across seven drugs from different classes, including nonsteroidal anti-inflammatories: ibuprofen (Ibu) and diclofenac (Dic); antibiotics: rifampicin (Rif), levofloxacin (Levo), and pefloxacin (Pef); and bisphosphonates: alendronate (Ale) and clodronate (Clo). Fluorescence lifetime correlation spectroscopy (FLCS) and electrochem. impedance spectroscopy (EIS) were used to evaluate the impact of drug on 1,2-dioleyl-sn-glycerophosphocholine and binary bilayers over physiol. relevant drug concns. Although FLCS data revealed Ibu, Levo, Pef, Ale, and Clo had no impact on lipid lateral mobility, EIS, which is more sensitive to membrane structural change, indicated modest but significant decreases to membrane resistivity consistent with adsorption but weak penetration of drugs at the membrane. Ale and Clo, evaluated at pH 5.25, did not impact the impedance of the membrane except at concns. exceeding 4 mM. Conversely, Dic and Rif dramatically altered bilayer fluidity, suggesting their translocation through the bilayer, and EIS data showed that resistivity of the membrane decreased substantially with increasing drug concn. Capacitance changes to the bilayer in most cases were insignificant. Using a Langmuir-Freundlich model to fit the EIS data, we propose Rsat as an empirical value that reflects permeation. Overall, the data indicate that Ibu, Levo, and Pef adsorb at the interface of the lipid membrane but Dic and Rif interact strongly, permeating the membrane core modifying the water/ion permeability of the bilayer structure. These observations are discussed in the context of previously reported data on drug permeability and log P.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtVektrjE&md5=94d657b20e39245d3b1f59c6442110ea
  60. 60
    Ramadurai, S.; Kohut, A.; Sarangi, N. K.; Zholobko, O.; Baulin, V. A.; Voronov, A.; Keyes, T. E. Macromolecular Inversion-Driven Polymer Insertion into Model Lipid Bilayer Membranes. J. Colloid Interface Sci. 2019, 542, 483– 494,  DOI: 10.1016/j.jcis.2019.01.093
    Google Scholar
    60
    Macromolecular inversion-driven polymer insertion into model lipid bilayer membranes
    Ramadurai, Sivaramakrishnan; Kohut, Ananiy; Sarangi, Nirod Kumar; Zholobko, Oksana; Baulin, Vladimir A.; Voronov, Andriy; Keyes, Tia E.
    Journal of Colloid and Interface Science (2019), 542 (), 483-494CODEN: JCISA5; ISSN:0021-9797. (Elsevier B.V.)
    Macromols. of amphiphilic invertible polymers (AIPs) are capable of self-assembly into micellar assemblies of various morphologies in solvents of different polarities. The micellar assemblies in aq. media are capable of encapsulating poorly aq. sol. cargo and can undergo inverse conformational change and cargo release in contact with non-polar media, including potentially, cell membranes. Thus, invertible micellar assemblies have significant potential in drug delivery and related domains. However, to date there have been few investigations into their interactions with lipid membranes. Herein, we investigate the interactions of three recently developed AIPs of varying hydrophobicity/hydrophilicity balance with a highly fluidic microcavity supported 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer. We combined electrochem. impedance spectroscopy (EIS) with fluorescence correlation spectroscopy (FCS) to understand how the AIP micellar assemblies impacted bilayer permeability and fluidity resp., across polymer concns. above and below their crit. micelle concns. (cmcs). At concn. as above their cmcs, all of the AIPs explored increased permeability and decreased the fluidity of the lipid membrane. The extent of impact depended on the hydrophobicity of the AIP. PEG600-PTHF650, the most hydrophobic of the polymers, synthesized from PEG (mol. wt. 600 g/mol) and PTHF (mol. wt. 650 g/mol) exerted the greatest influence on the bilayer's phys. properties and fluorescence imaging and correlation data indicate that PEG600-PTHF650 micelles loaded with BODIPY probes adsorb and invert at the lipid membrane with release of cargo into the bilayer. This study should help inform future advancement of AIPs for membrane mol. delivery.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXjtVKju7s%253D&md5=3a16b5df1721ab3f2b617ffacf0f953c
  61. 61
    Sarangi, N. K.; Prabhakaran, A.; Keyes, T. E. Interaction of Miltefosine with Microcavity Supported Lipid Membrane: Biophysical Insights from Electrochemical Impedance Spectroscopy. Electroanalysis 2020, 32, 2936– 2945,  DOI: 10.1002/elan.202060424
    Google Scholar
    61
    Interaction of Miltefosine with Microcavity Supported Lipid Membrane: Biophysical Insights from Electrochemical Impedance Spectroscopy
    Sarangi, Nirod Kumar; Prabhakaran, Amrutha; Keyes, Tia E.
    Electroanalysis (2020), 32 (12), 2936-2945CODEN: ELANEU; ISSN:1040-0397. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Miltefosine, an alkylphosphocholine analog, is the only drug taken orally for the treatment of leishmaniasis - a parasitic disease caused by sandflies. Although it is believed that Miltefosine exerts its activity by acting at the lipid membrane, detailed understanding of the interaction of this drug with eukaryotic membranes is still lacking. Herein, we exploit microcavity pore suspended lipid bilayers (MSLBs) as a biomimetic platform in combination with a highly sensitive label-free electrochem. impedance spectroscopy (EIS) technique to gain biophys. insight into the interaction of Miltefosine with host cell membrane as a function of lipid membranes compn. Four membrane compns. with increasing complexity were evaluated; DOPC, DOPC : Chol (75 : 25), domain forming DOPC : SM : Chol (40 : 40 : 20) and mammalian plasma membrane (MPM) mimetic DOPC:DOPE:Chol:SM:DOPS (32 : 25 : 20 : 15 : 8) and used to study the interaction of Miltefosine in a concn.-dependent manner using EIS. The membrane resistance changes in response to Miltefosine were modelled by an empirical Langmuir isotherm binding model to provide ests. of binding satn. and equil. assocn. const. Miltefosine was found to have greatest impact on electrochem. properties of the simpler membrane systems; DOPC and DOPC : Chol, where these membranes were found to be more susceptible to membrane thinning, attributed to strong permeation/penetration of the drug while, compns. that included both Chol and SM, expected to contain large liq.-ordered domains exhibited weaker changes to membrane resistance but strongest drug assocn. In contrast, at the MPM membrane, Miltefosine exerts weakest assocn., which is tentatively attributed to electrostatic effects from the anionic DOPS but some membrane thinning is obsd. reflected in change in resistance and capacitance values attributed to some weak permeation.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitV2lsr3N&md5=6c0fdd5a9b7f4f97f1c1b1d982045d61
  62. 62
    Sarangi, N. K.; Stalcup, A.; Keyes, T. E. The Impact of Membrane Composition and Co-Drug Synergistic Effects on Vancomycin Association with Model Membranes from Electrochemical Impedance Spectroscopy. ChemElectroChem 2020, 7, 4535– 4542,  DOI: 10.1002/celc.202000818
    Google Scholar
    62
    The Impact of Membrane Composition and Co-Drug Synergistic Effects on Vancomycin Association with Model Membranes from Electrochemical Impedance Spectroscopy
    Sarangi, Nirod Kumar; Stalcup, Apryll; Keyes, Tia E.
    ChemElectroChem (2020), 7 (22), 4535-4542CODEN: CHEMRA; ISSN:2196-0216. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The interaction of the antibacterial drug, vancomycin with microcavity suspended lipid bilayers (MSLB) was investigated using non-Faradaic electrochem. impedance spectroscopy (EIS). Five MSLB compns. were prepd. with increasing complexity at gold substrates: DOPC, DOPC:Chol, DOPC:SM:Chol, mammalian plasma membrane mimetic (MPM), and E. coli polar lipid ext. The latter two, are intended to mimic eukaryotic and bacterial inner membrane compns. resp. The extent of vancomycin assocn. and the impact drug assocn. has on the electrochem. properties of the membrane depended on biomembrane compn. Trends were similar across all membranes but the E. coli membrane compn. Vancomycin increased membrane resistance and decreased capacitance in a saturable, concn. dependent manner, but for E. coli membrane resistance change was negligible and capacitance increased. Membrane resistance data was fit to the Langmuir-Freundlich model to give quant. insight into the relative extent of assocn. of drug with membrane as a function of membrane compn. Overall, electrochem. data indicates that vancomycin assocs. at the interface of lipid membranes rather than penetrates the layer and this is promoted by the presence of anionic phospholipid. Interestingly, co-incubation of the E. coli ext. bilayer with both rifampicin and vancomycin, known to act synergistically, significantly promotes vancomycin assocn. to E. coli membrane.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFyhtr3E&md5=b3bfb0afbd578f49a209a207e0d979be
  63. 63
    Johansson, S.; Forsberg, E.; Lundgren, B. Comparison of Fibronectin Receptors from Rat Hepatocytes and Fibroblasts. J. Biol. Chem. 1987, 262, 7819– 7824,  DOI: 10.1016/S0021-9258(18)47641-7
    Google Scholar
    63
    Comparison of fibronectin receptors from rat hepatocytes and fibroblasts
    Johansson, Staffan; Forsberg, Erik; Lundgren, Bjoern
    Journal of Biological Chemistry (1987), 262 (16), 7819-24CODEN: JBCHA3; ISSN:0021-9258.
    A cell-surface fibronectin receptor was isolated from primary rat hepatocytes by affinity chromatog. on Sepharose conjugated with the cell-binding domain (105 kilodalton, kDa) of fibronectin. The receptor remained bound to the affinity column in the presence of 1M NaCl but was eluted by 1.5 mM of glycyl-arginyl-glycyl-aspartyl-seryl-cysteine peptide or by lowering the pH to 4. The eluted material migrated under nonreducing conditions in SDS-PAGE as 2 bands: the α- and β-components had apparent mol. wts. of 155,000 and 115,000, resp. After redn. the 155-kDa component gave rise to 2 peptides of Mr 145,000 and 20,000, whereas the 115-kDa component shifted migration to a Mr of 130,000. Antibodies specifically recognizing the 155- and 115-kDa proteins from hepatocytes inhibited the attachment of these cells to fibronectin-coated dishes, whereas attachment to dishes coated with collagen or laminin was unaffected. A fibronectin receptor isolated from rat fibroblasts showed closely similar, but not identical, migration in SDS-PAGE as the hepatocyte receptor. Furthermore, only the β-subunit of the fibroblast receptor reacted with the antibodies. The results suggest that distinct α-subunits of the fibronectin receptors may be the basis for the different fibronectin-binding properties of these cells.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2sXktlGgtrs%253D&md5=afe6b29fa22b2b7c22c43b86789c71ba
  64. 64
    Dransart, E.; Di Cicco, A.; El Marjou, A.; Lévy, D.; Johansson, S.; Johannes, L.; Shafaq-Zadah, M. Solubilization and Purification of α5β1 Integrin from Rat Liver for Reconstitution into Nanodiscs. In Heterologous Expression of Membrane Proteins; Methods in Molecular Biology, Vol 2507; Springer US: New York, NY, 2022. DOI: 10.1007/978-1-0716-2368-8_1
    Google Scholar
    There is no corresponding record for this reference.
  65. 65
    Lévy, D.; Bluzat, A.; Seigneuret, M.; Rigaud, J.-L. A Systematic Study of Liposome and Proteoliposome Reconstitution Involving Bio-Bead-Mediated Triton X-100 Removal. Biochim. Biophys. Acta, Biomembr. 1990, 1025, 179– 190,  DOI: 10.1016/0005-2736(90)90096-7
    Google Scholar
    64
    A systematic study of liposome and proteoliposome reconstitution involving Bio-Bead-mediated Triton X-100 removal
    Levy, Daniel; Bluzat, Aline; Seigneuret, Michel; Rigaud, Jean Louis
    Biochimica et Biophysica Acta, Biomembranes (1990), 1025 (2), 179-90CODEN: BBBMBS; ISSN:0005-2736.
    Equil. and kinetic aspects of Triton X 100 adsorption onto hydrophobic Bio-Beads SM2 were investigated in detail using the batch procedure originally described by P. W. Holloway (1973). The results demonstrated the importance of the initial detergent concn., the amt. of beads, the com. source of the detergent, the temp. and the presence of phospholipids in detg. the rates of Triton X 100 adsorption onto Bio-Beads. One of the main findings was that Bio-Beads allowed the almost complete removal of Triton X 100, whatever the initial exptl. conditions. It was shown that monomeric as well as micellar detergent could be adsorbed and that a key factor in detg. the rate of detergent removal was the availability of the free bead surface. Rates of detergent removal were found to be linearly related to the amt. of beads even for bead concns. above those sufficient to remove all the detergent initially present. Adsorptive capacity of phospholipids onto Bio-Beads SM2 was also analyzed and found to be much smaller (2 mg lipid/g of wet beads) than that of Triton X 100 (185 mg TX 100 per g of wet beads). A more general aspect of this work was that the use of Bio-Beads SM2 provided a convenient way for varying and controlling the time course of Triton X 100 removal. The method was further extended to the formation of liposomes from phospholipid-Triton X 100 micelles and the size of the liposomes was found to be critically dependent upon the rate of detergent removal. A general procedure was described to prep. homogeneous populations of vesicles. Freeze-fracture electron microscopy and permeability studies indicated that the liposomes thus obtained were unilamellar, relatively large and impermeable. Noteworthy, this new procedure was shown to be well suited for the reconstitution of different membrane transport proteins such as bacteriorhodopsin, Ca2+-ATPase, and H+-ATPase.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXkvFCgs7k%253D&md5=a4761d4d5578f937a349f54ef9e0cf07
  66. 66
    Maher, S.; Basit, H.; Forster, R. J.; Keyes, T. E. Micron Dimensioned Cavity Array Supported Lipid Bilayers for the Electrochemical Investigation of Ionophore Activity. Bioelectrochemistry 2016, 112, 16– 23,  DOI: 10.1016/j.bioelechem.2016.07.002
    Google Scholar
    65
    Micron dimensioned cavity array supported lipid bilayers for the electrochemical investigation of ionophore activity
    Maher, Sean; Basit, Hajra; Forster, Robert J.; Keyes, Tia E.
    Bioelectrochemistry (2016), 112 (), 16-23CODEN: BIOEFK; ISSN:1567-5394. (Elsevier B.V.)
    Microcavity supported lipid bilayers, MSLBs, were applied to an electrochem. investigation of ionophore mediated ion transport. The arrays comprise of 1 cm2 gold electrode imprinted with ordered array of uniform spherical-cap pores of 2.8μm diam. prepd. by gold electrodeposition through polystyrene templating spheres. The pores were pre-filled with aq. buffer prior to Langmuir-Blodgett assembly of a 1,2-dioleoyl-sn-glycero-3-phosphocholine bilayer. Fluorescence lifetime correlation spectroscopy enabled by micron dimensions of the pores permitted study of lipid diffusion across single apertures, yielding a diffusion coeff. of 12.58 ± 1.28μm2 s-1 and anomalous exponent of 1.03 ± 0.02, consistent with Brownian motion. From FLCS, the MSLBs were stable over 3 days and electrochem. impedance spectroscopy of the membrane with and without ionic gradient over exptl. windows of 6 h showed excellent stability. Two ionophores were studied at the MSLBs; Valinomycin, a K+ uniporter and Nigericin, a K+/H+ antiporter. Ionophore reconstituted into the DOPC bilayer resulted in a decrease and increase in membrane resistance and capacitance resp. Significant increases in Valinomycin and Nigericin activity were obsd., reflected in large decreases in membrane resistance when K+ was present in the contacting buffer and in the presence of H+ ionic gradient across the membrane resp.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtFKmurzJ&md5=a6aa5c0452ecdd654f25b424606bb403
  67. 67
    Khan, M. S.; Dosoky, N. S.; Williams, J. D. Engineering Lipid Bilayer Membranes for Protein Studies. Int. J. Mol. Sci. 2013, 14, 21561– 21597,  DOI: 10.3390/ijms141121561
    Google Scholar
    66
    Engineering lipid bilayer membranes for protein studies
    Khan, Muhammad Shuja; Dosoky, Noura Sayed; Williams, John Dalton
    International Journal of Molecular Sciences (2013), 14 (11), 21561-21597, 37 pp.CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
    A review. Lipid membranes regulate the flow of nutrients and communication signaling between cells and protect the sub-cellular structures. Recent attempts to fabricate artificial systems using nanostructures that mimic the physiol. properties of natural lipid bilayer membranes (LBM) fused with transmembrane proteins have helped demonstrate the importance of temp., pH, ionic strength, adsorption behavior, conformational reorientation and surface d. in cellular membranes which all affect the incorporation of proteins on solid surfaces. Much of this work is performed on artificial templates made of polymer sponges or porous materials based on alumina, mica and porous silicon (PSi) surfaces. For example, porous silicon materials have high biocompatibility, biodegradability, and photoluminescence, which allow them to be used both as a support structure for lipid bilayers or a template to measure the electrochem. functionality of living cells grown over the surface as in vivo. The variety of these media, coupled with the complex physiol. conditions present in living systems, warrant a summary and prospectus detailing which artificial systems provide the most promise for different biol. conditions. This study summarizes the use of electrochem. impedance spectroscopy (EIS) data on artificial biol. membranes that are closely matched with previously published biol. systems using both black lipid membrane and patch clamp techniques.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXltlOntLY%253D&md5=b1eb59c9c2e41daed3608181f8343c50
  68. 68
    Khan, M. S.; Dosoky, N. S.; Berdiev, B. K.; Williams, J. D. Electrochemical Impedance Spectroscopy for Black Lipid Membranes Fused with Channel Protein Supported on Solid-State Nanopore. Eur. Biophys. J. 2016, 45, 843– 852,  DOI: 10.1007/s00249-016-1156-8
    Google Scholar
    67
    Electrochemical impedance spectroscopy for black lipid membranes fused with channel protein supported on solid-state nanopore
    Khan, Muhammad S.; Dosoky, Noura S.; Berdiev, Bakhrom K.; Williams, John D.
    European Biophysics Journal (2016), 45 (8), 843-852CODEN: EBJOE8; ISSN:0175-7571. (Springer)
    Black lipid membranes (BLMs) have been used for detecting single-channel activities of pore-forming peptides and ion channels. However, the short lifetimes and poor mech. stability of suspended bilayers limit their applications in high throughput electrophysiol. expts. In this work, we present a synthetic solid-state nanopore functionalized with BLM fused with channel protein. A nanopore with diam. of ∼180 nm was electrochem. fabricated in a thin silicon membrane. Folding and painting techniques were demonstrated for prodn. of stable suspended BLMs followed by incorporation of transmembrane protein, ENaC. Membrane formation was confirmed by employing electrochem. impedance spectroscopy (EIS) in the frequency regime of 10-2-105 Hz. Results show that electrochem. fabricated solid state nanopore support resulted in excellent membrane stability, with >1 GΩ of up to 72 and 41 h for painting and folding techniques, resp. After fusion of ENaC channel protein, the BLM exhibits the stability of ∼5 h. We anticipate that such a solid-state nanopore with diam. in the range of 150-200 nm and thickness <1 μm could be a potential platform to enhance the throughput of ion-channel characterization using BLMs.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtlWjtr7N&md5=43c3472f72c4958b00bfc3523366b694
  69. 69
    Daniels, J. S.; Pourmand, N. Label-Free Impedance Biosensors: Opportunities and Challenges. Electroanalysis 2007, 19, 1239– 1257,  DOI: 10.1002/elan.200603855
    Google Scholar
    68
    Label-free impedance biosensors: opportunities and challenges
    Daniels, Jonathan S.; Pourmand, Nader
    Electroanalysis (2007), 19 (12), 1239-1257CODEN: ELANEU; ISSN:1040-0397. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. Impedance biosensors are a class of elec. biosensors that show promise for point-of-care and other applications due to low cost, ease of miniaturization, and label-free operation. Unlabeled DNA and protein targets can be detected by monitoring changes in surface impedance when a target mol. binds to an immobilized probe. The affinity capture step leads to challenges shared by all label-free affinity biosensors; these challenges are discussed along with others unique to impedance readout. Various possible mechanisms for impedance change upon target binding are discussed. The authors critically summarize accomplishments of past label-free impedance biosensors and identify areas for future research.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXntlCgtLg%253D&md5=ff30599e2516efceaf1d69a839aa5fe8
  70. 70
    Lukyanov, P.; Furtak, V.; Ochieng, J. Galectin-3 Interacts with Membrane Lipids and Penetrates the Lipid Bilayer. Biochem. Biophys. Res. Commun. 2005, 338, 1031– 1036,  DOI: 10.1016/j.bbrc.2005.10.033
    Google Scholar
    69
    Galectin-3 interacts with membrane lipids and penetrates the lipid bilayer
    Lukyanov, Pavel; Furtak, Vyacheslav; Ochieng, Josiah
    Biochemical and Biophysical Research Communications (2005), 338 (2), 1031-1036CODEN: BBRCA9; ISSN:0006-291X. (Elsevier)
    The precise mechanism by which galectin-3 and other cytosolic proteins that lack signal peptides are secreted is yet to be elucidated. In the present analyses, we detd. that galectin-3, a β-galactoside binding protein, can interact directly with membrane lipids in solid phase binding assays. More interestingly, we detd. by spectrophotometric methods that it can spontaneously penetrate the lipid bilayer of liposomes in either direction. These findings suggest that galectin-3 on its own has the capacity to traverse the lipid bilayer. Whereas the situation is rather simplified in liposomes, the interaction of galectin-3 with the plasma membrane may involve cholesterol-rich membrane domains where galectin-3 can be concd. and form multimers or interact covalently with other proteins.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXht1WjurrO&md5=61474ea225fb5a6798a4c57d015b043c
  71. 71
    Sarangi, N. K.; Prabhakaran, A.; Keyes, T. E. Multimodal Investigation into the Interaction of Quinacrine with Microcavity-Supported Lipid Bilayers. Langmuir 2022, 38, 6411– 6424,  DOI: 10.1021/acs.langmuir.2c00524
    Google Scholar
    70
    Multimodal Investigation into the Interaction of Quinacrine with Microcavity-Supported Lipid Bilayers
    Sarangi, Nirod Kumar; Prabhakaran, Amrutha; Keyes, Tia E.
    Langmuir (2022), 38 (20), 6411-6424CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    Quinacrine is a versatile drug that is widely recognized for its antimalarial action through its inhibition of the phospholipase enzyme. It also has antianthelmintic and antiprotozoan activities and is a strong DNA binder that may be used to combat multidrug resistance in cancer. Despite extensive cell-based studies, a detailed understanding of quinacrine's influence on the cell membrane, including permeability, binding, and rearrangement at the mol. level, is lacking. Herein, we apply microcavity-suspended lipid bilayers (MSLBs) as in vitro models of the cell membrane comprising DOPC, DOPC:Chol(3:1), and DOPC:SM:Chol(2:2:1) to investigate the influence of cholesterol and intrinsic phase heterogeneity induced by mixed-lipid compn. on the membrane interactions of quinacrine. Using electrochem. impedance spectroscopy (EIS) and surface-enhanced Raman spectroscopy (SERS) as label-free surface-sensitive techniques, we have studied quinacrine interaction and permeability across the different MSLBs. Our EIS data reveal that the drug is permeable through ternary DOPC:SM:Chol and DOPC-only bilayer compns. In contrast, the binary cholesterol/DOPC membrane arrested permeation, yet the drug binds or intercalates at this membrane as reflected by an increase in membrane impedance. SERS supported the EIS data, which was utilized to gain structural insights into the drug-membrane interaction. Our SERS data also provides a simple but powerful label-free assessment of drug permeation because a significant SERS enhancement of the drug's Raman signature was obsd. only if the drug accessed the plasmonic interior of the pore cavity passing through the membrane. Fluorescent lifetime correlation spectroscopy (FLCS) provides further biophys. insight, revealing that quinacrine binding increases the lipid diffusivity of DOPC and the ternary membrane while remarkably decreasing the lipid diffusivity of the DOPC:Chol membrane. Overall, because of its adaptability to multimodal approaches, the MSLB platform provides rich and detailed insights into drug-membrane interactions, making it a powerful tool for in vitro drug screening.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xht1KrurjJ&md5=d2e7575d74217350051f227b00705c3f
  72. 72
    Chung, M.; Lowe, R. D.; Chan, Y.-H. M.; Ganesan, P. V.; Boxer, S. G. DNA-Tethered Membranes Formed by Giant Vesicle Rupture. J. Struct. Biol. 2009, 168, 190– 199,  DOI: 10.1016/j.jsb.2009.06.015
    Google Scholar
    71
    DNA-tethered membranes formed by giant vesicle rupture
    Chung, Minsub; Lowe, Randall D.; Chan, Yee-Hung M.; Ganesan, Prasad V.; Boxer, Steven G.
    Journal of Structural Biology (2009), 168 (1), 190-199CODEN: JSBIEM; ISSN:1047-8477. (Elsevier B.V.)
    We have developed a strategy for prepg. tethered lipid bilayer membrane patches on solid surfaces by DNA hybridization. In this way, the tethered membrane patch is held at a controllable distance from the surface by varying the length of the DNA used. Two basic strategies are described. In the first, single-stranded DNA strands are immobilized by click chem. to a silica surface, whose remaining surface is passivated to prevent direct assembly of a solid supported bilayer. Then giant unilamellar vesicles (GUVs) displaying the antisense strand, using a DNA-lipid conjugate developed in an earlier work. Lipid-anchored DNA mediates vesicle fusion as obsd. by lipid and content mixing are allowed to tether, spread and rupture to form tethered bilayer patches. In the second, a supported lipid bilayer displaying DNA using the DNA-lipid conjugate is first assembled on the surface. Then GUVs displaying the antisense strand are allowed to tether, spread and rupture to form tethered bilayer patches. The essential difference between these methods is that the tethering hybrid DNA is immobile in the first, while it is mobile in the second. Both strategies are successful; however, with mobile DNA hybrids as tethers, the patches are unstable, while in the first strategy stable patches can be formed. In the case of mobile tethers, if different length DNA hybrids are present, lateral segregation by length occurs and can be visualized by fluorescence interference contrast microscopy making this an interesting model for interactions that occur in cell junctions. In both cases, lipid mobility is high and there is a negligible immobile fraction. Thus, these architectures offer a flexible platform for the assembly of lipid bilayers at a well-defined distance from a solid support.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtVKkur7O&md5=c3667ad44f1bbe7e08d63aec16da9882
  73. 73
    Weng, K. C.; Kanter, J. L.; Robinson, W. H.; Frank, C. W. Fluid Supported Lipid Bilayers Containing Monosialoganglioside GM1: A QCM-D and FRAP Study. Colloids Surf., B 2006, 50, 76– 84,  DOI: 10.1016/j.colsurfb.2006.03.010
    Google Scholar
    72
    Fluid supported lipid bilayers containing monosialoganglioside GM1: A QCM-D and FRAP study
    Weng, Kevin C.; Kanter, Jennifer L.; Robinson, William H.; Frank, Curtis W.
    Colloids and Surfaces, B: Biointerfaces (2006), 50 (1), 76-84CODEN: CSBBEQ; ISSN:0927-7765. (Elsevier B.V.)
    In an effort to use model fluid membranes for immunol. studies, the authors compared the formation of planar phospholipid bilayers supported on silicon dioxide surfaces with and without incorporation of glycolipids as the antigen for in situ antibody binding. Dynamic light scattering measurements did not differentiate the hydrodynamic vols. of extruded small unilamellar vesicles (E-SUVs) contg. physiol. relevant concns. (0.5-5 mol%) of monosialoganglioside GM1 from exclusive egg yolk L-α-phosphatidylcholine (egg PC) E-SUVs. However, quantifiable differences in deposition mass and dissipative energy loss emerged in the transformation of 5 mol% GM1/95 mol% egg PC E-SUVs to planar supported lipid bilayers (PSLBs) by vesicle fusion on thermally evapd. SiO2, as monitored by the quartz crystal microbalance with dissipation (QCM-D) technique. Compared to the 100 mol% egg PC bilayers on the same surface, E-SUVs contg. 5 mol% GM1 reached a ∼12% higher mass and a lower dissipative energy loss during bilayer transformation. PSLBs with 5 mol% GM1 are ∼18% heavier than 100 mol% egg PC and ∼11% smaller in projected area per lipid, indicating an increased rigidity and a tighter packing. Subsequent binding of polyclonal IgG anti-GM1 to the PSLBs was performed in situ and showed specificity. The anti-GM1 to GM1 ratios at equil. were roughly proportional to the concns. of anti-GM1 administered in the soln. Fluorescence recovery after photobleaching was utilized to verify the retained, albeit reduced lateral fluidity of the supported membranes. Five moles percentage of GM1 membranes (GM1 to PC ratio ∼1:19) decorated with 1 mol% N-(Texas Red sulfonyl)-1,2-dihexadecanoyl-sn-glycerol-3-phosphoethanolamine (Texas Red DHPE) exhibited an approx. 16% lower diffusion coeff. of 1.32±0.06 μM2/s, compared to 1.58±0.04 μM2/s for egg PC membranes without GM1 (p < 0.01). The changes in vesicle properties and membrane lateral fluidity are attributed to the interactions of GM1 with itself and GM1 with other membrane lipids. This system allows for mols. of interest such as GM1 to exist on a more biol. relevant surface than those used in conventional methods such as ELISA. The authors' anal. of rabbit serum antibodies binding to GM1 demonstrates this platform can be used to test for the presence of anti-lipid antibodies in serum.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XltlKisro%253D&md5=20a14c6720e4797318b7391a6528c9cf
  74. 74
    Johnson, M. A.; Seifert, S.; Petrache, H. I.; Kimble-Hill, A. C. Phase Coexistence in Single-Lipid Membranes Induced by Buffering Agents. Langmuir 2014, 30, 9880– 9885,  DOI: 10.1021/la5018938
    Google Scholar
    73
    Phase coexistence in single-lipid membranes induced by buffering agents
    Johnson, Merrell A.; Seifert, Soenke; Petrache, Horia I.; Kimble-Hill, Ann C.
    Langmuir (2014), 30 (33), 9880-9885CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    Recent literature has shown that buffers affect the interaction between lipid bilayers through a mechanism that involves van der Waals forces, electrostatics, hydration forces, and membrane bending rigidity. Here, the authors show an addnl. peculiar effect of buffers on mixed-chain POPC lipid bilayers, namely phase coexistence similar to what was previously reported for alkali chlorides. The data presented suggested that one phase appeared to dehydrate below the value in pure water, while the other phase swelled as the concn. of buffer was increased. However, since the 2 phases must be in osmotic equil. with one another, this behavior challenges theor. models of lipid interactions.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtlaisLnN&md5=270c8a26f3919cc62e9db4a492051ab4
  75. 75
    Gaul, V.; Lopez, S. G.; Lentz, B. R.; Moran, N.; Forster, R. J.; Keyes, T. E. The Lateral Diffusion and Fibrinogen Induced Clustering of Platelet Integrin ΑiIbβ3 Reconstituted into Physiologically Mimetic GUVs. Integr. Biol. 2015, 7, 402– 411,  DOI: 10.1039/c5ib00003c
    Google Scholar
    74
    The lateral diffusion and fibrinogen induced clustering of platelet integrin αIIbβ3 reconstituted into physiologically mimetic GUVs
    Gaul, Vinnie; Lopez, Sergio G.; Lentz, Barry R.; Moran, Niamh; Forster, Robert J.; Keyes, Tia E.
    Integrative Biology (2015), 7 (4), 402-411CODEN: IBNIFL; ISSN:1757-9694. (Royal Society of Chemistry)
    Platelet integrin αIIbβ3 is a key mediator of platelet activation and thrombosis. Upon activation αIIbβ3 undergoes significant conformational rearrangement, inducing complex bidirectional signalling and protein recruitment leading to platelet activation. Reconstituted lipid models of the integrin can enhance our understanding of the structural and mechanistic details of αIIbβ3 behavior away from the complexity of the platelet machinery. Here, a novel method of αIIbβ3 insertion into Giant Unilamellar Vesicles (GUVs) is described that allows for effective integrin reconstitution unrestricted by lipid compn. αIIbβ3 was inserted into two GUV lipid compns. that seek to better mimic the platelet membrane. First, "nature's own", comprising 32% DOPC, 25% DOPE, 20% CH, 15% SM and 8% DOPS, intended to mimic the platelet cell membrane. Fluorescence Lifetime Correlation Spectroscopy (FLCS) reveals that exposure of the integrin to the activators Mn2+ or DTT does not influence the diffusion coeff. of αIIbβ3. Similarly, exposure to αIIbβ3's primary ligand fibrinogen (Fg) alone does not affect αIIbβ3's diffusion coeff. However, addn. of Fg with either activator reduces the integrin diffusion coeff. from 2.52 ± 0.29 to μm2 s-1 to 1.56 ± 0.26 (Mn2+) or 1.49 ± 0.41 μm2 s-1 (DTT) which is consistent with aggregation of activated αIIbβ3 induced by fibrinogen binding. The Multichannel Scaler (MCS) trace shows that the integrin-Fg complex diffuses through the confocal vol. in clusters. Using the Saffman-Delbr.ovrddot.uck model as a first approxn., the diffusion coeff. of the complex suggests at least a 20-fold increase in the radius of membrane bound protein, consistent with integrin clustering. Second, αIIbβ3 was also reconstituted into a "raft forming" GUV with well defined liq. disordered (Ld) and liq. ordered (Lo) phases. Using confocal microscopy and lipid partitioning dyes, αIIbβ3 showed an affinity for the DOPC rich Ld phase of the raft forming GUVs, and was effectively excluded from the cholesterol and sphingomyelin rich Lo phase. Activation and Fg binding of the integrin did not alter the distribution of αIIbβ3 between the lipid phases. This observation suggests partitioning of the activated fibrinogen bound αIIbβ3 into cholesterol rich domains is not responsible for the integrin clustering obsd.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXivFKitLg%253D&md5=989f815fb1f24c2aabbaeb83db55a787
  76. 76
    Yang, R.-Y.; Hill, P. N.; Hsu, D. K.; Liu, F.-T. Role of the Carboxyl-Terminal Lectin Domain in Self-Association of Galectin-3. Biochemistry 1998, 37, 4086– 4092,  DOI: 10.1021/bi971409c
    Google Scholar
    75
    Role of the carboxyl-terminal lectin domain in self-association of galectin 3
    Yang, Ri-Yao; Hill, Paul N.; Hsu, Daniel K.; Liu, Fu-Tong
    Biochemistry (1998), 37 (12), 4086-4092CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    Galectin 3 is a member of a large family of β-galactoside-binding animal lectins and is composed of a C-terminal lectin domain connected to an N-terminal nonlectin part. Previous exptl. results suggested that when bound to multivalent glycoconjugates, galectin 3 self-assocs. through intermol. interactions involving the N-terminal domain. Here, the authors obtained evidence suggesting that the protein self-assocs. in the absence of its saccharide ligands, in a manner that is dependent on the C-terminal domain. This mode of self-assocn. was inhibitable by the lectin's saccharide ligands. Specifically, recombinant human galectin 3 was found to bind to galectin 3C (the C-terminal domain fragment) conjugated to Sepharose 4B and the binding was inhibitable by lactose. In addn., biotinylated galectin 3 bound to galectin 3 immobilized on plastic surfaces and the binding could also be inhibited by various saccharide ligands of the lectin. A mutant with a Trp → Leu replacement in the C-terminal domain, which exhibited diminished carbohydrate-binding activity, did not bind to galectin 3C-Sepharose 4B. Furthermore, galectin 3C formed covalent homodimers when it was treated with a chem. crosslinker and the dimer formation was completely inhibited by lactose. Therefore, galectin 3 can self-assoc. through intermol. interactions involving both the N- and the C-terminal domains and the relative contribution of each depends on whether the lectin is bound to its saccharide ligands.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXhsVOksLY%253D&md5=e70c4a4d3c309ec89ab1288029a6b76c
  77. 77
    Lepur, A.; Salomonsson, E.; Nilsson, U. J.; Leffler, H. Ligand Induced Galectin-3 Protein Self-Association. J. Biol. Chem. 2012, 287, 21751– 21756,  DOI: 10.1074/jbc.C112.358002
    Google Scholar
    76
    Ligand induced galectin-3 protein self-association
    Lepur, Adriana; Salomonsson, Emma; Nilsson, Ulf J.; Leffler, Hakon
    Journal of Biological Chemistry (2012), 287 (26), 21751-21756CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Many functions of galectin-3 entail binding of its carbohydrate recognition site to glycans of a glycoprotein, resulting in crosslinking thought to be mediated by its N-terminal noncarbohydrate-binding domain. Here, the authors studied the interaction of galectin-3 with the model glycoprotein, asialofetuin (ASF), using a fluorescence anisotropy assay to measure the concn. of free galectin carbohydrate recognition sites in soln. Surprisingly, in the presence of ASF, this remained low even at high galectin-3 concns., showing that many more galectin-3 mols. were engaged than expected due to the ∼9 known glycan-based binding sites per ASF mol. This suggested that after ASF-induced nucleation, galectin-3 assocd. with itself by the carbohydrate recognition site binding to another galectin-3 mol., possibly forming oligomers. The authors named this type-C self-assocn. to distinguish it from the previously proposed models (type-N) where galectin-3 mols. bind to each other through the N-terminal domain, and all carbohydrate recognition sites are available for binding glycans. Both types of self-assocn. could result in ppts., as measured here by turbidimetry and dynamic light scattering. Type-C self-assocn. and pptn. occurred even with a galectin-3 mutant (R186S) that bound poorly to ASF but required a much higher concn. (∼50 μM) as compared with wild type (∼1 μM). ASF also induced weaker type-C self-assocn. of galectin-3 lacking its N-terminal domains, but as expected, no pptn. Neither a monovalent nor a divalent N-acetyl-D-lactosamine-contg. glycan induced type-C self-assocn., even if the latter gave ppts. with high concns. of galectin-3 (> ∼50 μM) in agreement with published results and perhaps due to type-N self-assocn.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XptVWrtro%253D&md5=3bba1bbe7b159bdea7ae0f1dee647ef5
  78. 78
    Vaz, W. L. C.; Goodsaid-Zalduondo, F.; Jacobson, K. Lateral Diffusion of Lipids and Proteins in Bilayer Membranes. FEBS Lett. 1984, 174, 199– 207,  DOI: 10.1016/0014-5793(84)81157-6
    Google Scholar
    77
    Lateral diffusion of lipids and proteins in bilayer membranes
    Vaz, Winchil L. C.; Goodsaid-Zalduondo, Federico; Jacobson, Ken
    FEBS Letters (1984), 174 (2), 199-207CODEN: FEBLAL; ISSN:0014-5793.
    A review with 37 refs.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXls1GlsbY%253D&md5=ef8b7dc46e215bae9a0d3ea2a1b53e05
  79. 79
    Saxton, M. J. Lateral Diffusion in an Archipelago. The Effect of Mobile Obstacles. Biophys. J. 1987, 52, 989– 997,  DOI: 10.1016/S0006-3495(87)83291-5
    Google Scholar
    78
    Lateral diffusion in an archipelago. The effect of mobile obstacles
    Saxton, Michael J.
    Biophysical Journal (1987), 52 (6), 989-97CODEN: BIOJAU; ISSN:0006-3495.
    Lateral diffusion of mobile proteins and lipids (tracers) in a membrane is hindered by the presence of proteins (obstacles) in the membrane. If the obstacles are immobile, their effect may be described by percolation theory, which states that the long-range diffusion const. of the tracers goes to zero when the area fraction of obstacles is greater than the percolation threshold. If the obstacles are themselves mobile, the diffusion const. of the tracers depends on the area fraction of obstacles and the relative jump rate of tracers and obstacles. Monte Carlo calcns. are presented of diffusion consts. on square and triangular lattices as a function of the concn. of obstacles and the relative jump rate. The diffusion const. for particles of various sizes is also obtained. Calcd. values of the concn.-dependent diffusion const. are compared with obsd. values for gramicidin and bacteriorhodopsin. The effect of the proteins as inert obstacles is significant, but other factors, such as protein-protein interactions and perturbation of lipid viscosity by proteins, are of comparable importance. Potential applications include the diffusion of proteins at high concns. (such as rhodopsin in rod outer segments), the modulation of diffusion by release of membrane proteins from cytoskeletal attachment, and the diffusion of mobile redox carriers in mitochondria, chloroplasts, and endoplasmic reticulum.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXptVWgsg%253D%253D&md5=8737507970a84533ffa5b30975d5caeb
  80. 80
    Pearce, K. H.; Hof, M.; Lentz, B. R.; Thompson, N. L. Comparison of the Membrane Binding Kinetics of Bovine Prothrombin and Its Fragment 1. J. Biol. Chem. 1993, 268, 22984– 22991,  DOI: 10.1016/S0021-9258(19)49415-5
    Google Scholar
    79
    Comparison of the membrane binding kinetics of bovine prothrombin and its fragment 1
    Pearce, Kenneth H.; Hof, Martin; Lentz, Barry R.; Thompson, Nancy L.
    Journal of Biological Chemistry (1993), 268 (31), 22984-91CODEN: JBCHA3; ISSN:0021-9258.
    Total internal reflection fluorescence microscopy was used to compare the membrane-binding characteristics of fluorescein-labeled bovine prothrombin and fluorescein-labeled bovine prothrombin fragment 1. The Ca2+-dependent assocn. of these proteins with quartz-supported planar membranes composed of mixts. of phosphatidylserine (2-10 mol %) and phosphatidylcholine was examd. Equil. binding measurements showed that the apparent equil. dissocn. consts. increased with decreasing molar fractions of phosphatidylserine and that the dissocn. consts. were somewhat lower for intact prothrombin. Kinetic measurements, using fluorescence photobleaching recovery, showed that the measured dissocn. rates were approx. equiv. for prothrombin and fragment 1 and did not change with the protein soln. concn. or the molar fraction of phosphatidylserine. The kinetic data also implied that the surface-binding mechanism for both proteins is more complex than a simple reversible reaction between monovalent proteins and monovalent surface sites. Measured equil. and kinetic consts. are reported and compared for prothrombin and fragment 1 on planar membranes.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXlslyhs7Y%253D&md5=ded44c3c88afdc71e4abbb8ba93dfda2
  81. 81
    Yang, E. H.; Rode, J.; Howlader, M. A.; Eckermann, M.; Santos, J. T.; Hernandez Armada, D.; Zheng, R.; Zou, C.; Cairo, C. W. Galectin-3 Alters the Lateral Mobility and Clustering of Β1-Integrin Receptors. PLoS One 2017, 12, e0184378  DOI: 10.1371/journal.pone.0184378
    Google Scholar
    80
    Galectin-3 alters the lateral mobility and clustering of β1-integrin receptors
    Yang, Esther H.; Rode, Julia; Howlader, Md. Amran; Eckermann, Marina; Santos, Jobette T.; Armada, Daniel Hernandez; Zheng, Ruixiang; Zou, Chunxia; Cairo, Christopher W.
    PLoS One (2017), 12 (10), e0184378/1-e0184378/17CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Glycoprotein receptors are influenced by myriad intermol. interactions at the cell surface. Specific glycan structures may interact with endogenous lectins that enforce or disrupt receptor-receptor interactions. Glycoproteins bound by multivalent lectins may form extended oligomers or lattices, altering the lateral mobility of the receptor and influencing its function through endocytosis or changes in activation. In this study, we have examd. the interaction of Galectin-3 (Gal-3), a human lectin, with adhesion receptors. We measured the effect of recombinant Gal-3 added exogenously on the lateral mobility of the α5β1 integrin on HeLa cells. Using single-particle tracking (SPT) we detected increased lateral mobility of the integrin in the presence of Gal-3, while its truncated C-terminal domain (Gal-3C) showed only minor redns. in lateral mobility. Treatment of cells with Gal-3 increased β1-integrin mediated migration with no apparent changes in viability. In contrast, Gal-3C decreased both cell migration and viability. Fluorescence microscopy allowed us to confirm that exogenous Gal-3 resulted in reorganization of the integrin into larger clusters. We used a proteomics anal. to confirm that cells expressed endogenous Gal-3, and found that addn. of competitive oligosaccharide ligands for the lectin altered the lateral mobility of the integrin. Together, our results are consistent with a Gal-3-integrin lattice model of binding and confirm that the lateral mobility of integrins is natively regulated, in part, by galectins.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXpvVOktbc%253D&md5=5399528cf1dc4c395ae87febc6548e51

Cited By

ARTICLE SECTIONS
Jump To

This article has not yet been cited by other publications.

Download PDF

  • Abstract

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 1

    Figure 1. α5β1 integrin purification and reconstitution into SUVs. (A) Qualitative visualization of α5β1 integrin particles by EM. Negative staining images of α5β1 integrin solubilized in DDM. The inset shows a zoomed image of integrin particles, and an illustration of individual integrins in the bent-closed conformational state. (B) Characterization on sucrose gradients of α5β1 integrin incorporation into vesicles. Gradient fractions F1 to F8 (right cartoon illustration) were collected and submitted to anti-β1 integrin immunoblotting. β1 integrin was expectedly detected at 120 kDa in F1 and F2 fractions. L represents total proteoliposome input. (C) Homogeneity of the proteoliposomes as visualized by cryoEM. Inset shows a magnification view. The proteoliposomes were homogenous in size, with a mean diameter of 150 nm. (D) Analysis of α5β1 integrin orientation within SUVs. Proteoliposomes were subjected or not to trypsin digestion in the presence or absence of Triton X-100. In the absence of detergent (lane 1), the immunoreactive band corresponds to β1 integrin molecules for which the large extracellular domain was oriented into the liposomal lumen and thereby protected from the protease. In the presence of trypsin and detergent (lane 2), no β1 integrin band was detected, since the enzyme now had full access to the whole protein. In the presence of the detergent alone (lane 3), the detected band corresponds to the total amount of β1 integrin. This allowed us to estimate the percentage of correctly oriented α5β1 integrin, which was around 50%. Micellar α5β1 integrin was used as a control. The cartoon illustration summarizes the different conditions. (E) Assessment of the functionality of liposomal α5β1 integrin. To confirm the capacity of α5β1 integrin to become activated. Proteoliposomes were pre-incubated or not (Ctrl) with the indicated activating metal ion salts (MgCl2 or MnCl2) and then submitted to fibronectin FN-III9–10 pull down. As expected, β1 integrin was pulled down in the presence of magnesium (MgCl2) and to a greater extent in the presence of manganese (MnCl2), demonstrating that the SUV-reconstituted integrins were functional.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 2

    Figure 2. (A) Schematic illustration of proteoliposome fusion over a lipid monolayer covered gold microcavity array (not to scale) yielding PC//PC:PA/Int MSLBs. Reflectance image (B), FLIM images obtained from the lower PC leaflet doped with 0.03 mol% ATTO647-PE (C), andfrom ATTO488 α5β1 integrin (D). Non-Faradaic Nyquist plot (E) and frequency–normalized complex capacitance plot (F) of the cavity array alone (black), of PC//PC:PA (blue) and of PC//PC:PA/Int MSLBs (red). In panels E and F, zoomed-in areas are shown in insets as indicated by the green boxes. (G) Schematics of MSLB spanned over a microcavity (not to scale) and the associated ECM used to fit EIS data. In the ECM, Rel, and Cstray represent, respectively, solution electrolyte resistance and stray capacitance, RM and QM represent, respectively, membrane resistance and CPE, and Rarray and Qarray are the, respectively, microcavity array resistance and CPE. The corresponding fits to the ECM are shown as solid lines in panels E and F. (H) Relative change in membrane resistance to show the stability of PC//PC:PA membranes without (blue) and with (red) the presence of α5β1 integrin versus time monitored for more than 24 h. The EIS recording at an initial time window of 0–1.5 h shows an increase in membrane resistance, which saturates and remains stable for more than 24 h. EIS measurements were performed in PBS buffer within the frequency ranges between 0.05 and 105 Hz at 0 V DC bias potential vs Ag/AgCl (1 M KCl) with an AC amplitude of 10 mV at 22 ± 1 °C. A three-electrode setup where gold cavity/MSLB, Ag/AgCl (1 M KCl), and Pt wire served as working, reference, and counter electrodes, respectively.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 3

    Figure 3. EIS characterization of integrin-containing membranes upon binding of WTGal3 or Gal3ΔNter. (A,B) Relative changes in (A) resistance, ΔR (filled symbol), and (B) capacitance, ΔQ (open symbols) values obtained upon the addition of different concentrations of WTGal3 (black) or Gal3ΔNter (red) to PC//PC:PA/Int membranes. The solid lines in each panels A and B are shown to guide the eye. Data are means ± SD from triplicate experiments. (C,D) Bar charts showing the change in capacitance values with respect to the pristine PC//PC:PA/Int membrane when 37 nM WTGal3 (C) or 62.5 nM Gal3ΔNter (D) were incubated with α5β1 integrin-containing membranes in the presence (+Lac, 50 mM) or absence of β-lactose. Lactose fully abolished the WTGal3 or Gal3ΔNter effects on ΔQ. In the absence of Gal3, lactose did not affect ΔQ. EIS measurements were performed in PBS buffer, frequency ranges were from 0.05 to 105 Hz at 0 V DC bias potential vs Ag/AgCl (1 M KCl) with an AC amplitude of 10 mV at 22 ± 1 °C. The measurement cell was a three-electrode setup where gold cavity/MSLB, Ag/AgCl (1 M KCl), and Pt wire served as working, reference, and counter electrodes, respectively.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 4

    Figure 4. Representative FLIM and FLCS characterization of α5β1 integrin in MSLBs. (A) Reflectance and (B) FLIM images of ATTO488-labeled α5β1 integrin reconstituted into a PC//PC:PA MSLBs. Brighter white circles in the reflectance image (panel A) resulted from a refractive index mismatch between the buffer and PDMS. (C) FLIM image of ATTO647-DOPE (0.01 mol %) doped in the lower leaflet. Inset depicts a zoomed-in area, with asterisk “*” highlighting the center of cavity’s spatial regimes, where the membrane was suspended over pores and FCS was acquired from. The scale bars in panels A–C are 20 μm. (D) ACFs for ATTO488-α5β1 integrin at different time points (black, red and blue) post-reconstitution into MSLBs, along with 10 nM ATTO488-α5β1 integrin in micellar form diluted in PBS (grey). Solid lines are the fitted data for integrin diffusion across MSLBs and solution using the 2D diffusion model, eq 1, and the pure diffusion model, eq S1 (see the Supporting Information), respectively. The FCS data were collected and averaged from approximately 80–100 points from pores across the substrate. ACF traces showed no changes over 6 h time windows. Both FLCS and FLIM images were taken over PDMS cavity arrays. The substrate was sealed within a microfluidic chamber filled with PBS (pH = 7.4) at 22 ± 1 °C.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 5

    Figure 5. FLIM and FLCS characterization of WTGal3 and Gal3ΔNter upon binding to α5β1 integrin-containing membranes. FLIM images of (A) WTGal3-Alexa647 (left) and (B) Gal3ΔNter-Alexa647 (left) at varying concentrations upon binding to PC//PC:PA/Int membranes. The corresponding ATTO488-α5β1 integrin FLIM images are shown to the right. The arrows in panel A indicate integrin clusters in the presence of Gal3. (C,E) ACFs of WTGal3-Alexa647 (C) and Gal3ΔNter-Alexa647 (E) at varying concentrations upon binding to α5β1 integrin-containing PC//PC:PA membranes. (D,F) ACFs of ATTO488-α5β1 integrin upon incubation with different concentrations of WTGal3-Alexa647 (D) or Gal3ΔNter-Alexa647 (F). (G) ACFs of α5β1 integrin-ATTO488 reconstituted into PC//PC:PA membranes (black circle) in the presence of 50 mM β-lactose (red circles), of 37 nM WTGal3 in the presence of 50 mM β-lactose (blue circles), or after exchanging the contact solution of WTGal3 + Lac with fresh 37 nM WTGal3 (olive circles). In all panels (C–G) open symbols represent the experimental data. Solid lines are the corresponding fits using eq 2 except that a pure diffusion model equation (eq S1, Supporting Information) was used to extract the diffusion coefficient values from Alexa-labeled WTGal3 and Gal3ΔNter in solution. All measurements were carried out in PBS buffer of pH 7.4 and at 22 ± 1 °C.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 6

    Figure 6. Diffusivity of α5β1 integrin and WTGal3. Diffusivity of ATTO488-α5β1 integrin and of WTGal3-Alexa647 at the indicated concentrations of the latter. The blue filled circle shows the diffusion coefficient value for ATTO488-α5β1 integrin at MSLB before WTGal3 addition. Orange circles are the diffusion coefficient values of ATTO488-α5β1 integrin following incremental addition of WTGal3-Alexa647 (3.7, 18.5 and 37 nM). Green squares show the fast component of concentration-dependent diffusion coefficient values for WTGal3-Alexa647 after binding to ATTO488-α5β1 integrin containing PC//PC:PA membranes, and red triangles the ones of the slow diffusing component. Diffusion values in the presence of WTGal3-Alexa647 were obtained following 30 min incubation at the membranes at the indicated concentrations. Dotted oval marks highlight the bimodal diffusivity values of WTGal3-Alexa647.

    High Resolution Image
    Download MS PowerPoint Slide

  • References

    ARTICLE SECTIONS
    Jump To

    This article references 81 other publications.

    1. 1
      Mouw, J. K.; Ou, G.; Weaver, V. M. Extracellular Matrix Assembly: A Multiscale Deconstruction. Nat. Rev. Mol. Cell Biol. 2014, 15, 771– 785,  DOI: 10.1038/nrm3902
      Google Scholar
      1
      Extracellular matrix assembly: a multiscale deconstruction
      Mouw, Janna K.; Ou, Guanqing; Weaver, Valerie M.
      Nature Reviews Molecular Cell Biology (2014), 15 (12), 771-785CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
      The biochem. and biophys. properties of the extracellular matrix (ECM) dictate tissue-specific cell behavior. The mols. that are assocd. with the ECM of each tissue, including collagens, proteoglycans, laminins and fibronectin, and the manner in which they are assembled det. the structure and the organization of the resultant ECM. The product is a specific ECM signature that is comprised of unique compositional and topog. features that both reflect and facilitate the functional requirements of the tissue.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvFKmur7L&md5=5418b0ab81bde5077f540e5c8cd652b7
    2. 2
      Lee, J.-O.; Bankston, L. A.; Robert C Liddington, M. A. A. Two Conformations of the Integrin A-Domain (I-Domain): A Pathway for Activation?. Structure 1995, 3, 1333– 1340,  DOI: 10.1016/S0969-2126(01)00271-4
      Google Scholar
      2
      Two conformations of the integrin A-domain (I-domain): a pathway for activation?
      Lee, Jie-Oh; Bankston, Laurie Anne; Arnaout, M. Amin; Liddington, Robert C.
      Structure (London) (1995), 3 (12), 1333-40CODEN: STRUE6; ISSN:0969-2126. (Current Biology)
      Integrins are plasma membrane proteins that mediate adhesion to other cells and to components of the extracellular matrix. Most integrins are constitutively inactive in resting cells, but are rapidly and reversibly activated in response to agonists, leading to highly regulated cell adhesion. This activation is assocd. with conformational changes in their extracellular portions, but the nature of the structural changes that lead to a change in adhesiveness is not understood. The interactions of several integrins with their extracellular ligands are mediated by an A-type domain (generally called the I-domain in integrins). Binding of the I-domain to protein ligands is dependent on divalent cations. The authors have described previously the structure of the I-domain from complement receptor 3 with bound Mg2+, in which the glutamate side chain from a second I-domain completes the octahedral coordination sphere of the metal, acting as a ligand mimetic. The authors now describe a new crystal form of the I-domain with bound Mn2+, in which water completes the metal coordination sphere and there is no equiv. of the glutamate ligand. Comparison of the two crystal forms reveals a change in metal coordination which is linked to a large (10 Å) shift of the C-terminal helix and the burial of two phenylalanine residues into the hydrophobic core of the Mn2+ form. These structural changes, analogous to those seen in the signal-transducing G-proteins, alter the electrophilicity of the metal, reducing its ability to bind ligand-assocd. acidic residues, and dramatically alter the surface of the protein implicated in binding ligand. The authors' observations provide the first at. resoln. view of conformational changes in an integrin domain, and suggest how these changes are linked to a change in integrin adhesiveness. The authors propose that the Mg2+ form represents the conformation of the domain in the active state and the Mn2+ form the conformation in the inactive state of the integrin.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XitFGgtA%253D%253D&md5=b8be7fae31062245f970a70aed2792b3
    3. 3
      Margadant, C.; van den Bout, I.; van Boxtel, A. L.; Thijssen, V. L.; Sonnenberg, A. Epigenetic Regulation of Galectin-3 Expression by Β1 Integrins Promotes Cell Adhesion and Migration. J. Biol. Chem. 2012, 287, 44684– 44693,  DOI: 10.1074/jbc.M112.426445
      Google Scholar
      3
      Epigenetic regulation of galectin-3 Expression by β1 integrins promotes cell adhesion and migration
      Margadant, Coert; van den Bout, Iman; van Boxtel, Antonius L.; Thijssen, Victor L.; Sonnenberg, Arnoud
      Journal of Biological Chemistry (2012), 287 (53), 44684-44693CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Introduction of the integrin β1- but not the β3-subunit in GE11 cells induces an epithelial-mesenchymal-transition (EMT)-like phenomenon that is characterized by the loss of cell-cell contacts, cell scattering, increased cell migration and RhoA activity, and fibronectin fibrillogenesis. Because galactose-binding lectins (galectins) have been implicated in these phenomena, we investigated whether galectins are involved in the β1-induced phenotype. We examd. 9 galectins and, intriguingly, found that the expression of galectin-3 (Gal-3) is specifically induced by β1 but not by β3. Using β1-β3 chimeric integrins, we show that the induction of Gal-3 expression requires the hypervariable region in the extracellular domain of β1, but not its cytoplasmic tail. Furthermore, Gal-3 expression does not depend on RhoA signaling, serum factors, or any of the major signal transduction pathways involving protein kinase C (PKC), p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase-1/-2 (ERK-1/2), phosphatidylinositol-3-OH kinase (PI3-K), or Src kinases. Instead, Gal-3 expression is controlled in an epigenetic manner. Whereas DNA methylation of the Lgals3 promoter maintains Gal-3 silencing in GE11 cells, expression of β1 causes its demethylation, leading to transcriptional activation of the Lgals3 gene. In turn, Gal-3 expression enhances β1 integrin-mediated cell adhesion to fibronectin (FN) and laminin (LN), as well as cell migration. Gal-3 also promotes β1-mediated cell adhesion to LN and Collagen-1 (Col)-1 in cells that endogenously express Gal-3 and β1 integrins. In conclusion, we identify a functional feedback-loop between β1 integrins and Gal-3 that involves the epigenetic induction of Gal-3 expression during integrin-induced EMT and cell scattering.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXjvVaq&md5=b65c0929261f0fa5d175b1bc868b7dd2
    4. 4
      Seguin, L.; Desgrosellier, J. S.; Weis, S. M.; Cheresh, D. A. Integrins and Cancer: Regulators of Cancer Stemness, Metastasis, and Drug Resistance. Trends Cell Biol. 2015, 25, 234– 240,  DOI: 10.1016/j.tcb.2014.12.006
      Google Scholar
      4
      Integrins and cancer: regulators of cancer stemness, metastasis, and drug resistance
      Seguin, Laetitia; Desgrosellier, Jay S.; Weis, Sara M.; Cheresh, David A.
      Trends in Cell Biology (2015), 25 (4), 234-240CODEN: TCBIEK; ISSN:0962-8924. (Elsevier Ltd.)
      Interactions between cancer cells and their surroundings can trigger essential signaling cues that det. cell fate and influence the evolution of the malignant phenotype. As the primary receptors involved in cell-matrix adhesion, integrins present on the surface of tumor and stromal cells have a profound impact on the ability to survive in specific locations, but in some cases, these receptors can also function in the absence of ligand binding to promote stemness and survival in the presence of environmental and therapeutic stresses. Understanding how integrin expression and function is regulated in this context will enable the development of new therapeutic approaches to sensitize tumors to therapy and suppress their metastatic phenotype.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXlslCqu7o%253D&md5=04150b9842cbe69bec0c8bee7f0a6050
    5. 5
      Aoudjit, F.; Vuori, K. Integrin Signaling in Cancer Cell Survival and Chemoresistance. Chemother. Res. Pract. 2012, 2012, 1– 16,  DOI: 10.1155/2012/283181
      Google Scholar
      There is no corresponding record for this reference.
    6. 6
      Bänfer, S.; Schneider, D.; Dewes, J.; Strauss, M. T.; Freibert, S.-A.; Heimerl, T.; Maier, U. G.; Elsässer, H.-P.; Jungmann, R.; Jacob, R. Molecular Mechanism to Recruit Galectin-3 into Multivesicular Bodies for Polarized Exosomal Secretion. Proc. Natl. Acad. Sci. U.S.A. 2018, 115, E4396– E4405,  DOI: 10.1073/pnas.1718921115
      Google Scholar
      There is no corresponding record for this reference.
    7. 7
      Seelenmeyer, C.; Wegehingel, S.; Tews, I.; Künzler, M.; Aebi, M.; Nickel, W. Cell Surface Counter Receptors Are Essential Components of the Unconventional Export Machinery of Galectin-1. J. Cell Biol. 2005, 171, 373– 381,  DOI: 10.1083/jcb.200506026
      Google Scholar
      7
      Cell surface counter receptors are essential components of the unconventional export machinery of galectin-1
      Seelenmeyer, Claudia; Wegehingel, Sabine; Tews, Ivo; Kuenzler, Markus; Aebi, Markus; Nickel, Walter
      Journal of Cell Biology (2005), 171 (2), 373-381CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)
      Galectin-1 is a component of the extracellular matrix as well as a ligand of cell surface counter receptors such as β-galactoside-contg. glycolipids, however, the mol. mechanism of galectin-1 secretion has remained elusive. Based on a nonbiased screen for galectin-1 export mutants we have identified 26 single amino acid changes that cause a defect of both export and binding to counter receptors. When wild-type galectin-1 was analyzed in CHO clone 13 cells, a mutant cell line incapable of expressing functional galectin-1 counter receptors, secretion was blocked. Intriguingly, we also find that a distant relative of galectin-1, the fungal lectin CGL-2, is a substrate for nonclassical export from Chinese hamster ovary (CHO) cells. Alike mammalian galectin-1, a CGL-2 mutant defective in β-galactoside binding, does not get exported from CHO cells. We conclude that the β-galactoside binding site represents the primary targeting motif of galectins defining a galectin export machinery that makes use of β-galactoside-contg. surface mols. as export receptors for intracellular galectin-1.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtFGktbnJ&md5=fc4e3a7f16a321952fb3d9a907c949cc
    8. 8
      Dumic, J.; Dabelic, S.; Flögel, M. Galectin-3: An Open-Ended Story. Biochim. Biophys. Acta, Gen. Subj. 2006, 1760, 616– 635,  DOI: 10.1016/j.bbagen.2005.12.020
      Google Scholar
      8
      Galectin-3: An open-ended story
      Dumic, Jerka; Dabelic, Sanja; Floegel, Mirna
      Biochimica et Biophysica Acta, General Subjects (2006), 1760 (4), 616-635CODEN: BBGSB3; ISSN:0304-4165. (Elsevier B.V.)
      A review. Galectins, an ancient lectin family, are characterized by specific binding of β-galactosides through the evolutionary conserved sequence elements of a carbohydrate-recognition domain (CRD). A structurally unique member of the family is galectin-3 (I); in addn. to the CRD it contains a proline- and glycine-rich N-terminal domain (ND) through which it is able to form oligomers. I is widely spread among different types of cells and tissues, found intracellularly in the nucleus and cytoplasm or secreted via a non-classical pathway outside of the cell, thus being found on the cell surface or in the extracellular space. Through specific interactions with a variety of intracellular and extracellular proteins, I affects numerous biol. processes and seems to be involved in different physiol. and pathophysiol. conditions, such as development, immune reactions, and neoplastic transformation and metastasis. Here, the authors attempt to summarize existing information on structural, biochem., and intriguing functional properties of I.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xjsl2mtrw%253D&md5=54287fc34bc267fd22ed4eb69ee9607a
    9. 9
      Liu, F.-T.; Rabinovich, G. A. Galectins as Modulators of Tumour Progression. Nat. Rev. Cancer 2005, 5, 29– 41,  DOI: 10.1038/nrc1527
      Google Scholar
      9
      Galectins as modulators of tumor progression
      Liu, Fu-Tong; Rabinovich, Gabriel A.
      Nature Reviews Cancer (2005), 5 (1), 29-41CODEN: NRCAC4; ISSN:1474-175X. (Nature Publishing Group)
      A review. Galectins are a family of animal lectins with diverse biol. activities. They function both extracellularly, by interacting with cell-surface and extracellular matrix glycoproteins and glycolipids, and intracellularly, by interacting with cytoplasmic and nuclear proteins to modulate signalling pathways. Current research indicates that galectins have important roles in cancer; they contribute to neoplastic transformation, tumor cell survival, angiogenesis and tumor metastasis. They can modulate the immune and inflammatory responses and might have a key role helping tumors to escape immune surveillance. How do the different members of the Galectin family contribute to these diverse aspects of tumor biol.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXlvVem&md5=4b98b28336810c194a336bd940fc02c0
    10. 10
      Houzelstein, D.; Gonçalves, I. R.; Fadden, A. J.; Sidhu, S. S.; Cooper, D. N. W.; Drickamer, K.; Leffler, H.; Poirier, F. Phylogenetic Analysis of the Vertebrate Galectin Family. Mol. Biol. Evol. 2004, 21, 1177– 1187,  DOI: 10.1093/molbev/msh082
      Google Scholar
      10
      Phylogenetic analysis of the vertebrate galectin family
      Houzelstein, Denis; Goncalves, Isabelle R.; Fadden, Andrew J.; Sidhu, Sukhvinder S.; Cooper, Douglas N. W.; Drickamer, Kurt; Leffler, Hakon; Poirier, Francoise
      Molecular Biology and Evolution (2004), 21 (7), 1177-1187CODEN: MBEVEO; ISSN:0737-4038. (Oxford University Press)
      Galectins form a family of structurally related carbohydrate binding proteins (lectins) that have been identified in a large variety of metazoan phyla. They are involved in many biol. processes such as morphogenesis, control of cell death, immunol. response, and cancer. To elucidate the evolutionary history of galectins and galectin-like proteins in chordates, we have exploited three independent lines of evidence: (i) location of galectin encoding genes (LGALS) in the human genome; (ii) exon-intron organization of galectin encoding genes; and (iii) sequence comparison of carbohydrate recognition domains (CRDs) of chordate galectins. Our results suggest that a duplication of a mono-CRD galectin gene gave rise to an original bi-CRD galectin gene, before or early in chordate evolution. The N-terminal and C-terminal CRDs of this original galectin subsequently diverged into two different subtypes, defined by exon-intron structure (F4-CRD and F3-CRD). We show that all vertebrate mono-CRD galectins known to date belong to either the F3- or F4- subtype. A sequence of duplication and divergence events of the different galectins in chordates is proposed.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXltlKlsro%253D&md5=25c5d56ff8d4ede969b2f6d95f3ca416
    11. 11
      Popa, S. J.; Stewart, S. E.; Moreau, K. Unconventional Secretion of Annexins and Galectins. Semin. Cell Dev. Biol. 2018, 83, 42– 50,  DOI: 10.1016/j.semcdb.2018.02.022
      Google Scholar
      11
      Unconventional secretion of annexins and galectins
      Popa, Stephanie J.; Stewart, Sarah E.; Moreau, Kevin
      Seminars in Cell & Developmental Biology (2018), 83 (), 42-50CODEN: SCDBFX; ISSN:1084-9521. (Elsevier Ltd.)
      A review. Eukaryotic cells have a highly evolved system of protein secretion, and dysfunction in this pathway is assocd. with many diseases including cancer, infection, metabolic disease and neurol. disorders. Most proteins are secreted using the conventional endoplasmic reticulum (ER)/Golgi network and as such, this pathway is well-characterised. However, several cytosolic proteins have now been documented as secreted by unconventional transport pathways. This review focuses on two of these proteins families: annexins and galectins. The extracellular functions of these proteins are well documented, as are assocns. of their perturbed secretion with several diseases. However, the mechanisms and regulation of their secretion remain poorly characterised, and are discussed in this review.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXkslWkt7w%253D&md5=ed0616b196d586d567e5a248b57c628d
    12. 12
      Hayashi, Y.; Jia, W.; Kidoya, H.; Muramatsu, F.; Tsukada, Y.; Takakura, N. Galectin-3 Inhibits Cancer Metastasis by Negatively Regulating Integrin Β3 Expression. Am. J. Pathol. 2019, 189, 900– 910,  DOI: 10.1016/j.ajpath.2018.12.005
      Google Scholar
      12
      Galectin-3 Inhibits Cancer Metastasis by Negatively Regulating Integrin β3 Expression
      Hayashi, Yumiko; Jia, Weizhen; Kidoya, Hiroyasu; Muramatsu, Fumitaka; Tsukada, Yohei; Takakura, Nobuyuki
      American Journal of Pathology (2019), 189 (4), 900-910CODEN: AJPAA4; ISSN:0002-9440. (Elsevier B.V.)
      Galectin-3 (Gal-3; gene LGALS3) is a member of the β-galactose-binding lectin family. Previous studies showed that Gal-3 is expressed in several tissues across species and functions as a regulator of cell proliferation, apoptosis, adhesion, and migration, thus affecting many aspects of events, such as angiogenesis and tumorigenesis. Although several reports have suggested that the level of Gal-3 expression correlates pos. with tumor progression, herein we show that highly metastatic mouse melanoma B16/BL6 cells express less Gal-3 than B16 cells with a lower metastatic potential. It was found that overexpression of Gal-3 in melanoma cells in fact suppresses metastasis. In contrast, knocking out Gal-3 expression in cancer cells promoted cell aggregation mediated through interactions with platelets and fibrinogen in vitro and increased the no. of metastatic foci in vivo. Thus, reduced Gal-3 expression results in the up-regulation of β3 integrin expression, and this contributes to metastatic potential. These findings indicate that changes of Gal-3 expression in cancer cells during tumor progression influence the characteristics of metastatic cells.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXivFSmtr0%253D&md5=70f33ca776716d7b1e330e52b2acf259
    13. 13
      Johannes, L.; Jacob, R.; Leffler, H. Galectins at a Glance. J. Cell Sci. 2018, 131, jcs208884,  DOI: 10.1242/jcs.208884
      Google Scholar
      There is no corresponding record for this reference.
    14. 14
      Ruvolo, P. P. Galectin 3 as a Guardian of the Tumor Microenvironment. Biochim. Biophys. Acta, Mol. Cell Res. 2016, 1863, 427– 437,  DOI: 10.1016/j.bbamcr.2015.08.008
      Google Scholar
      14
      Galectin 3 as a guardian of the tumor microenvironment
      Ruvolo, Peter P.
      Biochimica et Biophysica Acta, Molecular Cell Research (2016), 1863 (3), 427-437CODEN: BBAMCO; ISSN:0167-4889. (Elsevier B.V.)
      Galectin 3 is a member of a family of β-galactoside binding proteins and has emerged as an important regulator of diverse functions crit. in cancer biol. including apoptosis, metastasis, immune surveillance, mol. trafficking, mRNA splicing, gene expression, and inflammation. Galectin 3's ability to support cancer cell survival by intra-cellular and extra-cellular mechanisms suggests this mol. is an important component of the tumor microenvironment that potentially could be targeted for therapy. Data is emerging that Galectin 3 is elevated in many cancers including solid tumors and the cancers of the blood. Galectin 3 also appears to be a key mol. produced by tumor microenvironment support cells including mesenchymal stromal cells (MSC) to suppress immune surveillance by killing T cells and interfering with NK cell function and by supporting metastasis. Levels of Galectin 3 increase in the MSC of aging mice and perhaps this contributes to the development of cancer in the elderly. Galectin 3 modulates surface protein expression of a diverse set of glycoproteins including CD44 by regulating endocytosis of these proteins. In addn., Galectin 3 binding to receptor kinases such as CD45 and the T cell receptor is crit. in the regulation of their function. In this review I will examine the various mechanisms how Galectin 3 supports chemoresistance and metastasis in solid tumors and in leukemia and lymphoma. I will also discuss possible therapeutic strategies to target this Galectin for cancer therapy. This article is part of a Special Issue entitled: Tumor Microenvironment Regulation of Cancer Cell Survival, Metastasis, Inflammation, and Immune Surveillance.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtleksrnM&md5=f37b5229716ede0729e577b3d23b8c2e
    15. 15
      Barondes, S. H.; Cooper, D. N.; Gitt, M. A.; Leffler, H. Galectins. Structure and Function of a Large Family of Animal Lectins. J. Biol. Chem. 1994, 269, 20807– 20810,  DOI: 10.1016/S0021-9258(17)31891-4
      Google Scholar
      15
      Galectins. Structure and function of a large family of animal lectins
      Barondes, Samuel H.; Cooper, Douglas N. W.; Gitt, Michael A.; Leffler, Hakon
      Journal of Biological Chemistry (1994), 269 (33), 20807-10CODEN: JBCHA3; ISSN:0021-9258.
      A review with 80 refs. Lectins are proteins that bind to specific carbohydrate structures and can thus recognize particular glycoconjugates among the vast array expressed in animal tissues. Most animal lectins can be classified into four distinct families (1): C-type lectins (including the selectins); P-type lectins; pentraxins; and galectins, formerly known as S-type or S-Lac lectins. The purpose of this short review is to provide a framework for integrating the rapid increase in knowledge of the diversity, structure, and function of the galectins. While the emphasis here is on mammalian galectins, important advances are also being made in studies of galectins in other species, including nematode and sponge. The review deals specifically with the structural classification and properties, carbohydrate binding specificity, non-classical secretion, biol. functions, ligands, and major known sites of expression of mammalian galectins.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXlsFWgurc%253D&md5=b92e79e0e3bf4d112c3a5876f3c6fc69
    16. 16
      Zhao, Z.; Xu, X.; Cheng, H.; Miller, M. C.; He, Z.; Gu, H.; Zhang, Z.; Raz, A.; Mayo, K. H.; Tai, G.; Zhou, Y. Galectin-3 N-Terminal Tail Prolines Modulate Cell Activity and Glycan-Mediated Oligomerization/Phase Separation. Proc. Natl. Acad. Sci. U.S.A. 2021, 118, e2021074118  DOI: 10.1073/pnas.2021074118
      Google Scholar
      16
      Galectin-3 N-terminal tail prolines modulate cell activity and glycan-mediated oligomerization/phase separation
      Zhao, Zihan; Xu, Xuejiao; Cheng, Hairong; Miller, Michelle C.; He, Zhen; Gu, Hongming; Zhang, Zhongyu; Raz, Avraham; Mayo, Kevin H.; Tai, Guihua; Zhou, Yifa
      Proceedings of the National Academy of Sciences of the United States of America (2021), 118 (19), e2021074118CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Galectin-3 (Gal-3) has a long, aperiodic, and dynamic proline-rich N-terminal tail (NT). The functional role of the NT with its numerous prolines has remained enigmatic since its discovery. To provide some resoln. to this puzzle, we individually mutated all 14 NT prolines over the first 68 residues and assessed their effects on various Gal-3-mediated functions. Our findings show that mutation of any single proline (esp. P37A, P55A, P60A, P64A/H, and P67A) dramatically and differentially inhibits Gal-3-mediated cellular activities (i.e., cell migration, activation, endocytosis, and hemagglutination). For mechanistic insight, we investigated the role of prolines in mediating Gal-3 oligomerization, a fundamental process required for these cell activities. We showed that Gal-3 oligomerization triggered by binding to glycoproteins is a dynamic process analogous to liq.-liq. phase sepn. (LLPS). The compn. of these heterooligomers is dependent on the concn. of Gal-3 as well as on the concn. and type of glycoprotein. LLPS-like Gal-3 oligomerization/condensation was also obsd. on the plasma membrane and disrupted endomembranes. Mol.- and cell-based assays indicate that glycan binding-triggered Gal-3 LLPS (or LLPS-like) is driven mainly by dynamic intermol. interactions between the Gal-3 NT and the carbohydrate recognition domain (CRD) F-face, although NT-NT interactions appear to contribute to a lesser extent. Mutation of each proline within the NT differentially controls NT-CRD interactions, consequently affecting glycan binding, LLPS, and cellular activities. Our results unveil the role of proline polymorphisms (e.g., at P64) assocd. with many diseases and suggest that the function of glycosylated cell surface receptors is dynamically regulated by Gal-3.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhtVyhtb7L&md5=bd2024509914b2505ac4807de67368d0
    17. 17
      Chiu, Y.-P.; Sun, Y.-C.; Qiu, D.-C.; Lin, Y.-H.; Chen, Y.-Q.; Kuo, J.-C.; Huang, J. Liquid-Liquid Phase Separation and Extracellular Multivalent Interactions in the Tale of Galectin-3. Nat. Commun. 2020, 11, 1229,  DOI: 10.1038/s41467-020-15007-3
      Google Scholar
      17
      Liquid-liquid phase separation and extracellular multivalent interactions in the tale of galectin-3
      Chiu, Yi-Ping; Sun, Yung-Chen; Qiu, De-Chen; Lin, Yu-Hao; Chen, Yin-Quan; Kuo, Jean-Cheng; Huang, Jie-rong
      Nature Communications (2020), 11 (1), 1229CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Abstr.: Liq.-liq. phase sepn. (LLPS) explains many intracellular activities, but its role in extracellular functions has not been studied to the same extent. Here we report how LLPS mediates the extracellular function of galectin-3, the only monomeric member of the galectin family. The mechanism through which galectin-3 agglutinates (acting as a "bridge" to aggregate glycosylated mols.) is largely unknown. Our data show that its N-terminal domain (NTD) undergoes LLPS driven by interactions between its arom. residues (two tryptophans and 10 tyrosines). Our lipopolysaccharide (LPS) micelle model shows that the NTDs form multiple weak interactions to other galectin-3 and then aggregate LPS micelles. Aggregation is reversed when interactions between the LPS and the carbohydrate recognition domains are blocked by lactose. The proposed mechanism explains many of galectin-3's functions and suggests that the arom. residues in the NTD are interesting drug design targets.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXlt1Sgt7o%253D&md5=14b0b011324679d48f71f852ed6e9da0
    18. 18
      Lin, Y.-H.; Qiu, D.-C.; Chang, W.-H.; Yeh, Y.-Q.; Jeng, U.-S.; Liu, F.-T.; Huang, J. The Intrinsically Disordered N-Terminal Domain of Galectin-3 Dynamically Mediates Multisite Self-Association of the Protein through Fuzzy Interactions. J. Biol. Chem. 2017, 292, 17845– 17856,  DOI: 10.1074/jbc.M117.802793
      Google Scholar
      18
      The intrinsically disordered N-terminal domain of galectin-3 dynamically mediates multisite self-association of the protein through fuzzy interactions
      Lin, Yu-Hao; Qiu, De-Chen; Chang, Wen-Han; Yeh, Yi-Qi; Jeng, U-Ser; Liu, Fu-Tong; Huang, Jie-rong
      Journal of Biological Chemistry (2017), 292 (43), 17845-17856CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Galectins are a family of lectins that bind β-galactosides through their conserved carbohydrate recognition domain (CRD) and can induce aggregation with glycoproteins or glycolipids on the cell surface and thereby regulate cell activation, migration, adhesion, and signaling. Galectin-3 has an intrinsically disordered N-terminal domain and a canonical CRD. Unlike the other 14 known galectins in mammalian cells, which have dimeric or tandem-repeated CRDs enabling multivalency for various functions, galectin-3 is monomeric, and its functional multivalency therefore is somewhat of a mystery. Here, we used NMR spectroscopy, mutagenesis, small-angle X-ray scattering, and computational modeling to study the self-assocn.-related multivalency of galectin-3 at the residue-specific level. We show that the disordered N-terminal domain (residues ∼20-100) interacts with itself and with a part of the CRD not involved in carbohydrate recognition (β-strands 7-9; residues ∼200-220), forming a fuzzy complex via inter- and intramol. interactions, mainly through hydrophobicity. These fuzzy interactions are characteristic of intrinsically disordered proteins to achieve liq.-liq. phase sepn., and we demonstrated that galectin-3 can also undergo liq.-liq. phase sepn. We propose that galectin-3 may achieve multivalency through this multisite self-assocn. mechanism facilitated by fuzzy interactions.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslehs7zO&md5=1bf3159cabfa56c346e8a632e457bc80
    19. 19
      Friedrichs, J.; Manninen, A.; Muller, D. J.; Helenius, J. Galectin-3 Regulates Integrin α2β1-Mediated Adhesion to Collagen-I and -IV. J. Biol. Chem. 2008, 283, 32264– 32272,  DOI: 10.1074/jbc.M803634200
      Google Scholar
      19
      Galectin-3 Regulates Integrin α2β1-mediated Adhesion to Collagen-I and -IV
      Friedrichs, Jens; Manninen, Aki; Muller, Daniel J.; Helenius, Jonne
      Journal of Biological Chemistry (2008), 283 (47), 32264-32272CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Galectins are a taxonomically widespread family of galactose-binding proteins of which galectin-3 is known to modulate cell adhesion. Using single cell force spectroscopy, the contribution of galectin-3 to the adhesion of Madin-Darby canine kidney (MDCK) cells to different extracellular matrix proteins was investigated. When adhering to collagen-I or -IV, some cells rapidly entered an enhanced adhesion state, marked by a significant increase in the force required for cell detachment. Galectin-3-depleted cells had an increased probability of entering the enhanced adhesion state. Adhesion enhancement was specific to integrin α2β1, as it was not obsd. when cells adhered to extracellular matrix substrates by other integrins. The adhesion phenotype of galectin-3-depleted cells was mimicked in a galactoside-deficient MDCK cell line and could be complemented by the addn. of recombinant galectin-3. We propose that galectin-3 influences integrin α2β1-mediated adhesion complex formation by altering receptor clustering.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhtlOjtL%252FN&md5=aec191a18da391835c81d5319945373d
    20. 20
      Rao, S. P.; Wang, Z.; Zuberi, R. I.; Sikora, L.; Bahaie, N. S.; Zuraw, B. L.; Liu, F.-T.; Sriramarao, P. Galectin-3 Functions as an Adhesion Molecule to Support Eosinophil Rolling and Adhesion under Conditions of Flow. J. Immunol. 2007, 179, 7800– 7807,  DOI: 10.4049/jimmunol.179.11.7800
      Google Scholar
      20
      Galectin-3 functions as an adhesion molecule to support eosinophil rolling and adhesion under conditions of flow
      Rao, Savita P.; Wang, Zhuangzhi; Zuberi, Riaz I.; Sikora, Lyudmila; Bahaie, Nooshin S.; Zuraw, Bruce L.; Liu, Fu-Tong; Sriramarao, P.
      Journal of Immunology (2007), 179 (11), 7800-7807CODEN: JOIMA3; ISSN:0022-1767. (American Association of Immunologists)
      Allergic inflammation involves the mobilization and trafficking of eosinophils to sites of inflammation. Galectin-3 (Gal-3) has been shown to play a crit. role in eosinophil recruitment and airway allergic inflammation in vivo. The role played by Gal-3 in human eosinophil trafficking was investigated. Eosinophils from allergic donors expressed elevated levels of Gal-3 and demonstrated increased rolling and firm adhesion on immobilized VCAM-1 and, more surprisingly, on Gal-3 under conditions of flow. Inhibition studies with specific mAbs as well as lactose demonstrated that: (1) eosinophil-expressed Gal-3 mediates rolling and adhesion on VCAM-1; (2) α4 integrin mediates eosinophil rolling on immobilized Gal-3; and (3) eosinophil-expressed Gal-3 interacts with immobilized Gal-3 through the carbohydrate recognition domain of Gal-3 during eosinophil trafficking. These findings were further confirmed using inflamed endothelial cells. Interestingly, Gal-3 was found to bind to α4 integrin by ELISA, and the two mols. exhibited colocalized expression on the cell surface of eosinophils from allergic donors. Thus, Gal-3 functions as a cell surface adhesion mol. to support eosinophil rolling and adhesion under conditions of flow.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtlajt7jL&md5=fcd4ea11b0a703e2974b3bb11c248d62
    21. 21
      Goetz, J. G.; Joshi, B.; Lajoie, P.; Strugnell, S. S.; Scudamore, T.; Kojic, L. D.; Nabi, I. R. Concerted Regulation of Focal Adhesion Dynamics by Galectin-3 and Tyrosine-Phosphorylated Caveolin-1. J. Cell Biol. 2008, 180, 1261– 1275,  DOI: 10.1083/jcb.200709019
      Google Scholar
      21
      Concerted regulation of focal adhesion dynamics by galectin-3 and tyrosine-phosphorylated caveolin-1
      Goetz, Jacky G.; Joshi, Bharat; Lajoie, Patrick; Strugnell, Scott S.; Scudamore, Trevor; Kojic, Liliana D.; Nabi, Ivan R.
      Journal of Cell Biology (2008), 180 (6), 1261-1275CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)
      Both tyrosine-phosphorylated caveolin-1 (pY14Cav1) and GlcNAc-transferase V (Mga5) are linked with focal adhesions (FAs); however, their function in this context is unknown. Here, we show that galectin-3 binding to Mgat5-modified N-glycans functions together with pY14Cav1 to stabilize focal adhesion kinase (FAK) within FAs, and thereby promotes FA disassembly and turnover. Expression of the Mgat5/galectin lattice alone induces FAs and cell spreading. However, FAK stabilization in FAs also requires expression of pY14Cav1. In cells lacking the Mgat5/galectin lattice, pY14Cav1 is not sufficient to promote FAK stabilization, FA disassembly, and turnover. In human MDA-435 cancer cells, Cav1 expression, but not mutant Y14FCav1, stabilizes FAK exchange and stimulates de novo FA formation in protrusive cellular regions. Thus, transmembrane crosstalk between the galectin lattice and pY14Cav1 promotes FA turnover by stabilizing FAK within FAs defining previously unknown, interdependent roles for galectin-3 and pY14Cav1 in tumor cell migration.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXktFGlsrk%253D&md5=433a50aca5f9da9f811119144c0302b1
    22. 22
      Furtak, V.; Hatcher, F.; Ochieng, J. Galectin-3 Mediates the Endocytosis of β-1 Integrins by Breast Carcinoma Cells. Biochem. Biophys. Res. Commun. 2001, 289, 845– 850,  DOI: 10.1006/bbrc.2001.6064
      Google Scholar
      22
      Galectin-3 Mediates the Endocytosis of β-1 Integrins by Breast Carcinoma Cells
      Furtak, Vyacheslav; Hatcher, Frank; Ochieng, Josiah
      Biochemical and Biophysical Research Communications (2001), 289 (4), 845-850CODEN: BBRCA9; ISSN:0006-291X. (Academic Press)
      Galectin-3, a β-galactoside binding lectin, has been demonstrated to play a key role(s) in cell to extracellular matrix interaction. The precise mechanism by which it modulates cellular adhesion is presently unclear and warrants further studies. We hereby report that galectin-3 mediates the endocytosis of β-1 integrins in a lactose-dependent manner. Interestingly we obsd. that galectin-3 was also rapidly internalized by the cells via the same pathway and the internalization was completely blocked by lactose. The endocytosis process was temp. dependent and was inhibited by filipin but not chlorpromazine. The endocytosis of galectin-3 and β-1 integrins by the cells was accompanied by rapid cell spreading due to cytoskeletal reorganization. The data suggest a novel mechanism by which galectin-3 and β-1 integrins are internalized into breast carcinoma cells via a caveolae-like pathway of endocytosis. (c) 2001 Academic Press.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXovVWiur0%253D&md5=b1a3949778f27c95bc7545fc41d49714
    23. 23
      King, D. R.; Salako, D. C.; Arthur-Bentil, S. K.; Rubin, A. E.; Italiya, J. B.; Tan, J. S.; Macris, D. G.; Neely, H. K.; Palka, J. M.; Grodin, J. L.; Davis-Bordovsky, K.; Faubion, M.; North, C. S.; Brown, E. S. Relationship between Novel Inflammatory Biomarker Galectin-3 and Depression Symptom Severity in a Large Community-Based Sample. J. Affective Disord. 2021, 281, 384– 389,  DOI: 10.1016/j.jad.2020.12.050
      Google Scholar
      23
      Relationship between novel inflammatory biomarker galectin-3 and depression symptom severity in a large community-based sample
      King, Darlene R.; Salako, Damilola C.; Arthur-Bentil, Samia Kate; Rubin, Arielle E.; Italiya, Jay B.; Tan, Jenny S.; Macris, Dimitri G.; Neely, Hunter K.; Palka, Jayme M.; Grodin, Justin L.; Davis-Bordovsky, Kaylee; Faubion, Matthew; North, Carol S.; Brown, E. Sherwood
      Journal of Affective Disorders (2021), 281 (), 384-389CODEN: JADID7; ISSN:0165-0327. (Elsevier Inc.)
      Major depressive disorder is assocd. with pro-inflammatory markers, such as cytokines TNF-alpha, IL-6, IL-1ss, and C-reactive protein. Galectin-3 is a novel emerging biomarker with pro-inflammatory properties. It is a saccharide binding protein distributed throughout many tissues with varying functions and is a predictor of poor outcomes in patients with heart failure and stroke. However, its role as a predictor in depressive symptom severity remains undefined. Data from the community-based Dallas Heart Study (n = 2554) were examd. using a multiple linear regression anal. to evaluate the relationship between galectin-3 and depressive symptom severity as assessed with Quick Inventory of Depressive Symptomatol. Self-Report (QIDS-SR) scores. Addnl. covariates included age, sex, race/ethnicity, body mass index (BMI), years of education, serum creatinine, history of diabetes, and smoking history. Galectin-3 levels statistically significantly predicted QIDS-SR depressive symptom severity (β = 0.055, p = .015). Female sex, smoking status, and BMI were found to be statistically significant pos. predictors of depression severity, while age, years of education, non-Hispanic White race, and Hispanic ethnicity were neg. predictors of depressive symptom severity. In this large sample, higher galectin-3 levels were assocd. with higher levels of depressive symptoms. The findings suggest that galectin-3 may be a new and useful inflammatory biomarker assocd. with depression.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXit1ais7s%253D&md5=0e4919f831829c56a1ab6abd69666dbf
    24. 24
      Melin, E. O.; Dereke, J.; Thunander, M.; Hillman, M. Depression in Type 1 Diabetes Was Associated with High Levels of Circulating Galectin-3. Endocr. Connect. 2018, 7, 819– 828,  DOI: 10.1530/EC-18-0108
      Google Scholar
      24
      Depression in type 1 diabetes was associated with high levels of circulating galectin-3
      Melin, Eva Olga; Dereke, Jonatan; Thunander, Maria; Hillman, Magnus
      Endocrine Connections (2018), 7 (6), 819-828CODEN: ECNOCX; ISSN:2049-3614. (BioScientifica Ltd.)
      Objective: Neuroinflammatory responses are implicated in depression. The aim was to explore whether depression in patients with type 1 diabetes (T1D) was assocd. with high circulating galectin-3, controlling for metabolic variables, s-creatinine, life style factors, medication and cardiovascular complications. Design: Cross-sectional. Methods: Participants were T1D patients (n = 283, 56% men, age 18-59 years, diabetes duration ≥1 yr). Depression was assessed by Hospital Anxiety and Depression Scale-depression subscale. Blood samples, anthropometrics and blood pressure were collected, and supplemented with data from medical records and the Swedish National Diabetes Registry. Galectin-3 ≥2.562μg/l, corresponding to the 85th percentile, was defined as high galectin-3. Results: Median (quartile1, quartile3) galectin-3 (μg/l) was 1.3 (0.8, 2.9) for the 30 depressed patients, and 0.9 (0.5, 1.6) for the 253 non-depressed, P = 0.009. Depression was assocd. with high galectin-3 in all the 283 patients (adjusted odds ratio (AOR) 3.5), in the 161 men (AOR 3.4), and in the 122 women (AOR 3.9). HbA1c, s-lipids, s-creatinine, blood pressure, obesity, smoking, phys. inactivity, cardiovascular complications and drugs (antihypertensive, lipid lowering, oral antidiabetic drugs and antidepressants) were not assocd. with high galectin-3. Conclusions: This is the first study to show an assocn. between depression and galectin-3. Depression was the only explored parameter assocd. with high circulating galectin-3 levels in 283 T1D patients. High galectin-3 levels might contribute to the increased risk for Alzheimer's disease, cardiovascular and all-cause mortality obsd. in persons with depression. Potentially, in the future, treatment targeting galactin-3 might improve the prognosis for patients with high galectin-3 levels.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXlsVKitbY%253D&md5=e6fd58d490469beded1f486969d0be66
    25. 25
      Tao, C.-C.; Cheng, K.-M.; Ma, Y.-L.; Hsu, W.-L.; Chen, Y.-C.; Fuh, J.-L.; Lee, W.-J.; Chao, C.-C.; Lee, E. H. Y. Galectin-3 Promotes Aβ Oligomerization and Aβ Toxicity in a Mouse Model of Alzheimer’s Disease. Cell Death Differ. 2020, 27, 192– 209,  DOI: 10.1038/s41418-019-0348-z
      Google Scholar
      25
      Galectin-3 promotes Aβ oligomerization and Aβ toxicity in a mouse model of Alzheimer's disease
      Tao, Chih-Chieh; Cheng, Kuang-Min; Ma, Yun-Li; Hsu, Wei-Lun; Chen, Yan-Chu; Fuh, Jong-Ling; Lee, Wei-Ju; Chao, Chih-Chang; Lee, Eminy H. Y.
      Cell Death & Differentiation (2020), 27 (1), 192-209CODEN: CDDIEK; ISSN:1350-9047. (Nature Research)
      Amyloid-β (Aβ) oligomers largely initiate the cascade underlying the pathol. of Alzheimers disease (AD). Galectin-3 (Gal-3), which is a member of the galectin protein family, promotes inflammatory responses and enhances the homotypic aggregation of cancer cells. Here, we examd. the role and action mechanism of Gal-3 in Aβ oligomerization and Aβ toxicities. Wild-type (WT) and Gal-3-knockout (KO) mice, APP/PS1;WT mice, APP/PS1;Gal-3+/- mice and brain tissues from normal subjects and AD patients were used. We found that Aβ oligomerization is reduced in Gal-3 KO mice injected with Aβ, whereas overexpression of Gal-3 enhances Aβ oligomerization in the hippocampi of Aβ-injected mice. Gal-3 expression shows an age-dependent increase that parallels endogenous Aβ oligomerization in APP/PS1 mice. Moreover, Aβ oligomerization, Iba1 expression, GFAP expression and amyloid plaque accumulation are reduced in APP/PS1;Gal-3+/- mice compared with APP/PS1;WT mice. APP/PS1;Gal-3+/- mice also show better acquisition and retention performance compared to APP/PS1;WT mice. In studying the mechanism underlying Gal-3-promoted Aβ oligomerization, we found that Gal-3 primarily co-localizes with Iba1, and that microglia-secreted Gal-3 directly interacts with Aβ. Gal-3 also interacts with triggering receptor expressed on myeloid cells-2, which then mediates the ability of Gal-3 to activate microglia for further Gal-3 expression. Immunohistochem. analyses show that the distribution of Gal-3 overlaps with that of endogenous Aβ in APP/PS1 mice and partially overlaps with that of amyloid plaque. Moreover, the expression of the Aβ-degrading enzyme, neprilysin, is increased in Gal-3 KO mice and this is assocd. with enhanced integrin-mediated signaling. Consistently, Gal-3 expression is also increased in the frontal lobe of AD patients, in parallel with Aβ oligomerization. Because Gal-3 expression is dramatically increased as early as 3 mo of age in APP/PS1 mice and anti-Aβ oligomerization is believed to protect against Aβ toxicity, Gal-3 could be considered a novel therapeutic target in efforts to combat AD.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtFSmtr7I&md5=3acae686f7adcb28e1eb2a6131ce9604
    26. 26
      Radosavljevic, G.; Volarevic, V.; Jovanovic, I.; Milovanovic, M.; Pejnovic, N.; Arsenijevic, N.; Hsu, D. K.; Lukic, M. L. The Roles of Galectin-3 in Autoimmunity and Tumor Progression. Immunol. Res. 2012, 52, 100– 110,  DOI: 10.1007/s12026-012-8286-6
      Google Scholar
      26
      The roles of Galectin-3 in autoimmunity and tumor progression
      Radosavljevic, Gordana; Volarevic, Vladislav; Jovanovic, Ivan; Milovanovic, Marija; Pejnovic, Nada; Arsenijevic, Nebojsa; Hsu, Daniel K.; Lukic, Miodrag L.
      Immunologic Research (2012), 52 (1-2), 100-110CODEN: IMRSEB; ISSN:0257-277X. (Humana Press Inc.)
      A review. Galectin-3, a unique chimera-type member of the β-galactoside-binding sol. lectin family, is widely expressed in numerous cells. Here, we discuss the role of Galectin-3 in T-cell-mediated inflammatory (auto) immunity and tumor rejection by using Galectin-3-deficient mice and four disease models of human pathol.: exptl. autoimmune encephalomyelitis (EAE), Con-A-induced hepatitis, multiple low-dose streptozotocin-induced diabetes (MLD-STZ diabetes) and metastatic melanoma. We present evidence which suggest that Galectin-3 plays an important pro-inflammatory role in Con-A-induced hepatitis by promoting the activation of T lymphocytes, NKT cells and DCs, cytokine secretion, prevention of M2 macrophage polarization and apoptosis of mononuclear cells, and it leads to severe liver injury. In addn., expts. in Galectin-3-"knock-out" mice indicate that Galectin-3 is also involved in immune-mediated β-cell damage and is required for diabetogenesis in MLD-STZ model by promoting the expression of IFN-gamma, TNF-alpha, IL-17 and iNOS in immune and accessory effector cells. Next, our data demonstrated that Galectin-3 plays an important disease-exacerbating role in EAE through its multifunctional roles in preventing cell apoptosis and increasing IL-17 and IFN-gamma synthesis, but decreasing IL-10 prodn. Finally, based on our findings, we postulated that expression of Galectin-3 in the host may also facilitate melanoma metastasis by affecting tumor cell adhesion and modulating anti-melanoma immune response, in particular innate antitumor immunity. Taken together, we discuss the evidence of pro-inflammatory and antitumor activities of Galectin-3 and suggest that Galectin-3 may be an important therapeutic target.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XlvVSisb4%253D&md5=dfc6dc92c6e02d5e0777458f930e82ca
    27. 27
      Sonnino, S.; Mauri, L.; Chigorno, V.; Prinetti, A. Gangliosides as Components of Lipid Membrane Domains. Glycobiology 2007, 17, 1R– 13R,  DOI: 10.1093/glycob/cwl052
      Google Scholar
      27
      Gangliosides as components of lipid membrane domains
      Sonnino, Sandro; Mauri, Laura; Chigorno, Vanna; Prinetti, Alessandro
      Glycobiology (2006), 17 (1), 1R-13RCODEN: GLYCE3; ISSN:0959-6658. (Oxford University Press)
      A review. Cell membrane components are organized as specialized domains involved in membrane-assocd. events such as cell signaling, cell adhesion, and protein sorting. These membrane domains are enriched in sphingolipids and cholesterol but display a low protein content. Theor. considerations and exptl. data suggest that some properties of gangliosides play an important role in the formation and stabilization of specific cell lipid membrane domains. Gangliosides are glycolipids with strong amphiphilic character and are particularly abundant in the plasma membranes, where they are inserted into the external leaflet with the hydrophobic ceramide moiety and with the oligosaccharide chain protruding into the extracellular medium. The geometry of the monomer inserted into the membrane, largely detd. by the very large surface area occupied by the oligosaccharide chain, the ability of the ceramide amide linkage to form a network of hydrogen bonds at the water-lipid interface of cell membranes, the Δ4 double bond of sphingosine proximal to the water-lipid interface, the capability of the oligosaccharide chain to interact with water, and the absence of double bonds into the double-tailed hydrophobic moiety are the ganglioside features that will be discussed in this review, to show how gangliosides are responsible for the formation of cell lipid membrane domains characterized by a strong pos. curvature.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtlChtbrP&md5=a084b4668f7526e126a39c184eb3e31a
    28. 28
      Johannes, L.; Billet, A. Glycosylation and Raft Endocytosis in Cancer. Cancer Metastasis Rev. 2020, 39, 375– 396,  DOI: 10.1007/s10555-020-09880-z
      Google Scholar
      28
      Glycosylation and raft endocytosis in cancer
      Johannes, Ludger; Billet, Anne
      Cancer and Metastasis Reviews (2020), 39 (2), 375-396CODEN: CMRED4; ISSN:0167-7659. (Springer)
      A review. Changes in glycosylation on proteins or lipids are one of the hallmarks of tumorigenesis. In many cases, it is still not understood how glycan information is translated into biol. function. In this review, we discuss at the example of specific cancer-related glycoproteins how their endocytic uptake into eukaryotic cells is tuned by carbohydrate modifications. For this, we not only focus on overall uptake rates, but also illustrate how different uptake processes-dependent or not on the conventional clathrin machinery-are used under given glycosylation conditions. Furthermore, we discuss the role of certain sugar-binding proteins, termed galectins, to tune glycoprotein uptake by inducing their crosslinking into lattices, or by co-clustering them with glycolipids into raft-type membrane nanodomains from which the so-called clathrin-independent carriers (CLICs) are formed for glycoprotein internalization into cells. The latter process has been termed glycolipid-lectin (GL-Lect) hypothesis, which operates in a complementary manner to the clathrin pathway and galectin lattices.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXptVers74%253D&md5=3d12f6406c663ba4a7e8f87f9f950514
    29. 29
      Lakshminarayan, R.; Wunder, C.; Becken, U.; Howes, M. T.; Benzing, C.; Arumugam, S.; Sales, S.; Ariotti, N.; Chambon, V.; Lamaze, C.; Loew, D.; Shevchenko, A.; Gaus, K.; Parton, R. G.; Johannes, L. Galectin-3 Drives Glycosphingolipid-Dependent Biogenesis of Clathrin-Independent Carriers. Nat. Cell Biol. 2014, 16, 592– 603,  DOI: 10.1038/ncb2970
      Google Scholar
      There is no corresponding record for this reference.
    30. 30
      Johannes, L.; Wunder, C.; Shafaq-Zadah, M. Glycolipids and Lectins in Endocytic Uptake Processes. J. Mol. Biol. 2016, 428, 4792– 4818,  DOI: 10.1016/j.jmb.2016.10.027
      Google Scholar
      30
      Glycolipids and lectins in endocytic uptake processes
      Johannes, Ludger; Wunder, Christian; Shafaq-Zadah, Massiullah
      Journal of Molecular Biology (2016), 428 (24_Part_A), 4792-4818CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      A review. A host of endocytic processes has been described at the plasma membrane of eukaryotic cells. Their categorization has most commonly referenced cytosolic machinery, of which the clathrin coat has occupied a preponderant position. In what concerns the intramembrane constituents, the focus of interest has been on phosphatidylinositol lipids and their capacity to orchestrate endocytic events on the cytosolic leaflet of the membrane. The contribution of extracellular determinants to the construction of endocytic pits has received much less attention, despite the fact that (glyco)sphingolipids are exoplasmic leaflet fabric of membrane domains, termed rafts, whose contributions to predominantly clathrin-independent internalization processes are well-recognized. Furthermore, sugar-binding proteins, termed lectins, and sugar modifications on extracellular domains of proteins have also been linked to the uptake of endocytic cargos at the plasma membrane. Here, the authors summarize these contributions by extracellular determinants to the endocytic process. The authors propose a mol. hypothesis, termed the GL-Lect hypothesis, on how glycolipids and lectins drive the formation of compositional nano-environments from which the endocytic uptake of glycosylated cargo proteins is operated via clathrin-independent carriers. Finally, the authors position this hypothesis within the global context of endocytic pathway proposals that have emerged in the recent years.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhslKksbvL&md5=9f011e970fe2a12f2ade88b6f90b9533
    31. 31
      Renard, H.-F.; Tyckaert, F.; Lo Giudice, C.; Hirsch, T.; Valades-Cruz, C. A.; Lemaigre, C.; Shafaq-Zadah, M.; Wunder, C.; Wattiez, R.; Johannes, L.; van der Bruggen, P.; Alsteens, D.; Morsomme, P. Endophilin-A3 and Galectin-8 Control the Clathrin-Independent Endocytosis of CD166. Nat. Commun. 2020, 11, 1457,  DOI: 10.1038/s41467-020-15303-y
      Google Scholar
      31
      Endophilin-A3 and Galectin-8 control the clathrin-independent endocytosis of CD166
      Renard, Henri-Francois; Tyckaert, Francois; Lo Giudice, Cristina; Hirsch, Thibault; Valades-Cruz, Cesar Augusto; Lemaigre, Camille; Shafaq-Zadah, Massiullah; Wunder, Christian; Wattiez, Ruddy; Johannes, Ludger; van der Bruggen, Pierre; Alsteens, David; Morsomme, Pierre
      Nature Communications (2020), 11 (1), 1457CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Abstr.: While several clathrin-independent endocytic processes have been described so far, their biol. relevance often remains elusive, esp. in pathophysiol. contexts such as cancer. In this study, we find that the tumor marker CD166/ALCAM (Activated Leukocyte Cell Adhesion Mol.) is a clathrin-independent cargo. We show that endophilin-A3-but neither A1 nor A2 isoforms-functionally assocs. with CD166-contg. early endocytic carriers and phys. interacts with the cargo. Our data further demonstrates that the three endophilin-A isoforms control the uptake of distinct subsets of cargoes. In addn., we provide strong evidence that the construction of endocytic sites from which CD166 is taken up in an endophilin-A3-dependent manner is driven by extracellular galectin-8. Taken together, our data reveal the existence of a previously uncharacterized clathrin-independent endocytic modality, that modulates the abundance of CD166 at the cell surface, and regulates adhesive and migratory properties of cancer cells.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXlvFCrsLw%253D&md5=4b99c3cf616ceb5d9d0b292158a9e602
    32. 32
      Nabi, I. R.; Shankar, J.; Dennis, J. W. The Galectin Lattice at a Glance. J. Cell Sci. 2015, 128, 2213– 2219,  DOI: 10.1242/jcs.151159
      Google Scholar
      32
      The galectin lattice at a glance
      Nabi, Ivan R.; Shankar, Jay; Dennis, James W.
      Journal of Cell Science (2015), 128 (13), 2213-2219CODEN: JNCSAI; ISSN:0021-9533. (Company of Biologists Ltd.)
      Galectins are a family of widely expressed β-galactoside-binding lectins in metazoans. The 15 mammalian galectins have either one or two conserved carbohydrate recognition domains (CRDs), with galectin-3 being able to pentamerize; they form complexes that crosslink glycosylated ligands to form a dynamic lattice. The galectin lattice regulates the diffusion, compartmentalization and endocytosis of plasma membrane glycoproteins and glycolipids. The galectin lattice also regulates the selection, activation and arrest of T cells, receptor kinase signaling and the functionality of membrane receptors, including the glucagon receptor, glucose and amino acid transporters, cadherins and integrins. The affinity of transmembrane glycoproteins to the galectin lattice is proportional to the no. and branching of their N-glycans; with branching being mediated by Golgi N-acetylglucosaminyltransferase-branching enzymes and the supply of UDP-GlcNAc through metabolite flux through the hexosamine biosynthesis pathway. The relative affinities of glycoproteins for the galectin lattice depend on the activities of the Golgi enzymes that generate the epitopes of their ligands and, thus, provide a means to analyze biol. function of lectins and of the 'glycome' more broadly.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtlSktLfL&md5=7e049fa9dbe95319e6fbdb37691a3f6b
    33. 33
      Mathew, M. P.; Donaldson, J. G. Distinct Cargo-Specific Response Landscapes Underpin the Complex and Nuanced Role of Galectin–Glycan Interactions in Clathrin-Independent Endocytosis. J. Biol. Chem. 2018, 293, 7222– 7237,  DOI: 10.1074/jbc.RA118.001802
      Google Scholar
      33
      Distinct cargo-specific response landscapes underpin the complex and nuanced role of galectin-glycan interactions in clathrin-independent endocytosis
      Mathew, Mohit P.; Donaldson, Julie G.
      Journal of Biological Chemistry (2018), 293 (19), 7222-7237CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Clathrin-independent endocytosis (CIE) is a form of endocytosis that lacks a defined cytoplasmic machinery. Here, we asked whether glycan interactions, acting from the outside, could be a part of that endocytic machinery. We show that the perturbation of global cellular patterns of protein glycosylation by modulation of metabolic flux affects CIE. Interestingly, these changes in glycosylation had cargo-specific effects. For example, in HeLa cells, GlcNAc treatment, which increases glycan branching, increased major histocompatibility complex class I (MHCI) internalization but inhibited CIE of the glycoprotein CD59 mol. (CD59). The effects of knocking down the expression of galectin 3, a carbohydrate-binding protein and an important player in galectin-glycan interactions, were also cargo-specific and stimulated CD59 uptake. By contrast, inhibition of all galectin-glycan interactions by lactose inhibited CIE of both MHCI and CD59. None of these treatments affected clathrin-mediated endocytosis, implying that glycosylation changes specifically affect CIE. We also found that the galectin lattice tailors membrane fluidity and cell spreading. Furthermore, changes in membrane dynamics mediated by the galectin lattice affected macropinocytosis, an altered form of CIE, in HT1080 cells. Our results suggest that glycans play an important and nuanced role in CIE, with each cargo being affected uniquely by alterations in galectin and glycan profiles and their interactions. We conclude that galectin-driven effects exist on a continuum from stimulatory to inhibitory, with distinct CIE cargo proteins having unique response landscapes and with different cell types starting at different positions on these conceptual landscapes.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXptlaqsrs%253D&md5=26118498d162fda9bfd30009601a11ba
    34. 34
      Wagner, M. L.; Tamm, L. K. Tethered Polymer-Supported Planar Lipid Bilayers for Reconstitution of Integral Membrane Proteins: Silane-Polyethyleneglycol-Lipid as a Cushion and Covalent Linker. Biophys. J. 2000, 79, 1400– 1414,  DOI: 10.1016/S0006-3495(00)76392-2
      Google Scholar
      34
      Tethered polymer-supported planar lipid bilayers for reconstitution of integral membrane proteins: silane-polyethyleneglycol-lipid as a cushion and covalent linker
      Wagner, Michael L.; Tamm, Lukas K.
      Biophysical Journal (2000), 79 (3), 1400-1414CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
      There is increasing interest in supported membranes as models of biol. membranes and as a physiol. matrix for studying the structure and function of membrane proteins and receptors. A common problem of protein-lipid bilayers that are directly supported on a hydrophilic substrate is nonphysiol. interactions of integral membrane proteins with the solid support to the extent that they will not diffuse in the plane of the membrane. To alleviate some of these problems we have developed a new tethered polymer-supported planar lipid bilayer system, which permitted us to reconstitute integral membrane proteins in a laterally mobile form. We have supported lipid bilayers on a newly designed polyethyleneglycol cushion, which provided a soft support and, for increased stability, covalent linkage of the membranes to the supporting quartz or glass substrates. The formation and morphol. of the bilayers were followed by total internal reflection and epifluorescence microscopy, and the lateral diffusion of the lipids and proteins in the bilayer was monitored by fluorescence recovery after photobleaching. Uniform bilayers with high lateral lipid diffusion coeffs. (0.8-1.2×10-8 cm2/s) were obsd. when the polymer concn. was kept slightly below the mushroom-to-brush transition. Cytochrome b5 and annexin V were used as first test proteins in this system. When reconstituted in supported bilayers that were directly supported on quartz, both proteins were largely immobile with mobile fractions < 25%. However, two populations of laterally mobile proteins were obsd. in the polymer-supported bilayers. Approx. 25% of cytochrome b5 diffused with a diffusion coeff. of ≥ 1×10-8 cm2/s, and 50-60% diffused with a diffusion coeff. of ∼2×10-10 cm2/s. Similarly, one-third of annexin V diffused with a diffusion coeff. of ∼ 3×10-9 cm2/s, and two-thirds diffused with a diffusion coeff. of ∼4×10-10 cm2/s. A model for the interaction of these proteins with the underlying polymer is discussed.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXmsVars70%253D&md5=fe3f7affd4f65df6cb8605e7008fea81
    35. 35
      Hussain, N. F.; Siegel, A. P.; Ge, Y.; Jordan, R.; Naumann, C. A. Bilayer Asymmetry Influences Integrin Sequestering in Raft-Mimicking Lipid Mixtures. Biophys. J. 2013, 104, 2212– 2221,  DOI: 10.1016/j.bpj.2013.04.020
      Google Scholar
      35
      Bilayer Asymmetry Influences Integrin Sequestering in Raft-Mimicking Lipid Mixtures
      Hussain, Noor F.; Siegel, Amanda P.; Ge, Yifan; Jordan, Rainer; Naumann, Christoph A.
      Biophysical Journal (2013), 104 (10), 2212-2221CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      There is growing recognition that lipid heterogeneities in cellular membranes play an important role in the distribution and functionality of membrane proteins. However, the detection and characterization of such heterogeneities at the cellular level remains challenging. Here we report on the poorly understood relationship between lipid bilayer asymmetry and membrane protein sequestering in raft-mimicking model membrane mixts. using a powerful exptl. platform comprised of confocal spectroscopy XY-scan and photon-counting histogram analyses. This exptl. approach is utilized to probe the domain-specific sequestering and oligomerization state of αvβ3 and α5β1 integrins in bilayers, which contain coexisting liq.-disordered/liq.-ordered (ld/lo) phase regions exclusively in the top leaflet of the bilayer (bottom leaflet contains ld phase). Comparison with previously reported integrin sequestering data in bilayer-spanning lo-ld phase sepns. demonstrates that bilayer asymmetry has a profound influence on αvβ3 and α5β1 sequestering behavior. For example, both integrins sequester preferentially to the lo phase in asym. bilayers, but to the ld phase in their sym. counterparts. Furthermore, our data show that bilayer asymmetry significantly influences the role of native ligands in integrin sequestering.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXotlynsr8%253D&md5=cd19583e9a04ee9188b55d8eac0a5356
    36. 36
      Peetla, C.; Stine, A.; Labhasetwar, V. Biophysical Interactions with Model Lipid Membranes: Applications in Drug Discovery and Drug Delivery. Mol. Pharm. 2009, 6, 1264– 1276,  DOI: 10.1021/mp9000662
      Google Scholar
      36
      Biophysical Interactions with Model Lipid Membranes: Applications in Drug Discovery and Drug Delivery
      Peetla, Chiranjeevi; Stine, Andrew; Labhasetwar, Vinod
      Molecular Pharmaceutics (2009), 6 (5), 1264-1276CODEN: MPOHBP; ISSN:1543-8384. (American Chemical Society)
      A review. The transport of drugs or drug delivery systems across the cell membrane is a complex biol. process, often difficult to understand because of its dynamic nature. In this regard, model lipid membranes, which mimic many aspects of cell-membrane lipids, have been very useful in helping investigators to discern the roles of lipids in cellular interactions. One can use drug-lipid interactions to predict pharmacokinetic properties of drugs, such as their transport, biodistribution, accumulation, and hence efficacy. These interactions can also be used to study the mechanisms of transport, based on the structure and hydrophilicity/hydrophobicity of drug mols. In recent years, model lipid membranes have also been explored to understand their mechanisms of interactions with peptides, polymers, and nanocarriers. These interaction studies can be used to design and develop efficient drug delivery systems. Changes in the lipid compn. of cells and tissue in certain disease conditions may alter biophys. interactions, which could be explored to develop target-specific drugs and drug delivery systems. In this review, we discuss different model membranes, drug-lipid interactions and their significance, studies of model membrane interactions with nanocarriers, and how biophys. interaction studies with lipid model membranes could play an important role in drug discovery and drug delivery.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXmtlKju7o%253D&md5=18b63d6d84e976e48c4780911ab5bf20
    37. 37
      Ramadurai, S.; Holt, A.; Krasnikov, V.; van den Bogaart, G.; Killian, J. A.; Poolman, B. Lateral Diffusion of Membrane Proteins. J. Am. Chem. Soc. 2009, 131, 12650– 12656,  DOI: 10.1021/ja902853g
      Google Scholar
      37
      Lateral Diffusion of Membrane Proteins
      Ramadurai, Sivaramakrishnan; Holt, Andrea; Krasnikov, Victor; van den Bogaart, Geert; Killian, J. Antoinette; Poolman, Bert
      Journal of the American Chemical Society (2009), 131 (35), 12650-12656CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      We measured the lateral mobility of integral membrane proteins reconstituted in giant unilamellar vesicles (GUVs), using fluorescence correlation spectroscopy. Receptor, channel, and transporter proteins with 1-36 transmembrane segments (lateral radii ranging from 0.5 to 4 nm) and a α-helical peptide (radius of 0.5 nm) were fluorescently labeled and incorporated into GUVs. At low protein-to-lipid ratios (i.e., 10-100 proteins per μm2 of membrane surface), the diffusion coeff. D displayed a weak dependence on the hydrodynamic radius (R) of the proteins [D scaled with ln(1/R)], consistent with the Saffman-Delbruck model. At higher protein-to lipid ratios (up to 3000 μm-2), the lateral diffusion coeff. of the mols. decreased linearly with increasing the protein concn. in the membrane. The implications of our findings for protein mobility in biol. membranes (protein crowding of ∼25,000 μm-2) and use of diffusion measurements for protein geometry (size, oligomerization) detns. are discussed.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXpslaiurs%253D&md5=0d1993b5cb282585e36c07594e0d6583
    38. 38
      Li, B.; London, E. Preparation and Drug Entrapment Properties of Asymmetric Liposomes Containing Cationic and Anionic Lipids. Langmuir 2020, 36, 12521– 12531,  DOI: 10.1021/acs.langmuir.0c01968
      Google Scholar
      38
      Preparation and Drug Entrapment Properties of Asymmetric Liposomes Containing Cationic and Anionic Lipids
      Li, Bingchen; London, Erwin
      Langmuir (2020), 36 (42), 12521-12531CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      We have developed cyclodextrin-catalyzed lipid exchange methods to prep. large unilamellar vesicles (LUVs) with asym. charge distributions, i.e., with different net charges on the lipids in the inner and outer leaflets. LUVs contained a mixt. of a zwitterionic lipid (phosphatidylcholine), cholesterol, and various cationic lipids (O-Et phosphatidylcholine or dioleoyl-3-trimethylammonium propane) or anionic lipids (phosphatidylglycerol, phosphatidylserine, or phosphatidic acid). Sym. and asym. LUVs with a wide variety of lipid combinations were prepd. The asym. LUVs contained cationic or anionic outer leaflets and inner leaflets that had either the opposite charge or were uncharged. The behavior of sym. LUVs prepd. with zwitterionic, anionic, or cationic leaflets was compared to those of asym. LUVs. Lipid exchange was confirmed by quant. thin-layer chromatog., and lipid asymmetry by a novel assay measuring binding of a cationic fluorescent probe to the LUV outer leaflet. For both sym. and asym. LUVs, the level of entrapment of the cationic drug doxorubicin was controlled by the charge on the inner leaflet, with the greatest entrapment and slowest leakage in vesicles with an anionic inner leaflet. This shows that it is possible to choose inner leaflet lipids to maximize liposomal loading of charged drugs independently of the identity of outer-leaflet lipids. This implies that it should also be possible to independently vary outer-leaflet lipids to, for example, impart favorable bioavailability and biodistribution properties to lipid vesicles.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitVyrsrzJ&md5=0b91852927fcc0419cfbbe093351275d
    39. 39
      Cheng, H.-T.; Megha; London, E. Preparation and Properties of Asymmetric Vesicles That Mimic Cell Membranes. J. Biol. Chem. 2009, 284, 6079– 6092,  DOI: 10.1074/jbc.M806077200
      Google Scholar
      39
      Preparation and Properties of Asymmetric Vesicles That Mimic Cell Membranes: effect upon lipid raft formation and transmembrane helix orientation
      Cheng, Hui-Ting; Megha; London, Erwin
      Journal of Biological Chemistry (2009), 284 (10), 6079-6092CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      A methyl-β-cyclodextrin-induced lipid exchange technique was devised to prep. small unilamellar vesicles with stable asym. lipid compns. Asym. vesicles that mimic biol. membranes were prepd. with sphingomyelin (SM) or SM mixed with 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) as the predominant lipids in the outer leaflet and dioleoylphosphatidylcholine (DOPC), POPC, 1-palmitoyl-2-oleoyl-phosphatidyl-L-serine (POPS), or POPS mixed with 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) in the inner leaflet. Fluorescence-based assays were developed to confirm lipid asymmetry. Cholesterol was introduced into these vesicles using a second methyl-β-cyclodextrin exchange step. In asym. vesicles composed of SM outside, DOPC inside (SMo/DOPCi) or SM outside, 2:1 mol:mol POPE:POPS inside (SMo/2:1 POPE:POPSi) the outer leaflet SM formed an ordered state with a thermal stability similar to that in pure SM vesicles and significantly greater than that in sym. vesicles with the same overall lipid compn. Analogous behavior was obsd. in vesicles contg. cholesterol. This shows that an asym. lipid distribution like that in eukaryotic plasma membranes can be conducive to ordered domain (raft) formation. Furthermore asym. vesicles contg. ∼25 mol % cholesterol formed ordered domains more thermally stable than those in asym. vesicles lacking cholesterol, showing that the crucial ability of cholesterol to stabilize ordered domain formation is likely to contribute to ordered domain formation in cell membranes. Addnl. studies demonstrated that hydrophobic helix orientation is affected by lipid asymmetry with asymmetry favoring formation of the transmembrane configuration. The ability to form asym. vesicles represents an important improvement in model membrane studies and should find many applications in the future.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXisVSksbw%253D&md5=462c2fdcec4f096814084bfd8bc05b92
    40. 40
      Zhang, L.; Granick, S. Lipid Diffusion Compared in Outer and Inner Leaflets of Planar Supported Bilayers. J. Chem. Phys. 2005, 123, 211104,  DOI: 10.1063/1.2138699
      Google Scholar
      40
      Lipid diffusion compared in outer and inner leaflets of planar supported bilayers
      Zhang, Liangfang; Granick, Steve
      Journal of Chemical Physics (2005), 123 (21), 211104/1-211104/4CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
      The translational diffusion coeff. (D) of lipids located in the outer and inner leaflets of planar supported DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine) bilayers in the fluid phase was measured using fluorescence correlation spectroscopy of dye-labeled lipids at the low concn. of 0.001% and using iodide quenching of dyes in the outer leaflet to distinguish diffusion in the inner leaflet from that in the outer leaflet. To confirm the generality of these findings, the bilayers were prepd. not only by vesicle fusion but also by Langmuir-Blodgett deposition. We conclude that regardless of whether the bilayers were supported on quartz or on a polymer cushion, D in the inner and outer leaflets was the same within an exptl. uncertainty of ±10% but with a small systematic tendency to be slower (by <5%) within the inner leaflet.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtlCgtbvE&md5=27dbf9c7d3ead51a6e600d70c256043e
    41. 41
      Murray, D. H.; Tamm, L. K.; Kiessling, V. Supported Double Membranes. J. Struct. Biol. 2009, 168, 183– 189,  DOI: 10.1016/j.jsb.2009.02.008
      Google Scholar
      41
      Supported double membranes
      Murray, David H.; Tamm, Lukas K.; Kiessling, Volker
      Journal of Structural Biology (2009), 168 (1), 183-189CODEN: JSBIEM; ISSN:1047-8477. (Elsevier B.V.)
      A review. Planar model membranes, like supported lipid bilayers and surface-tethered vesicles, have been proven to be useful tools for the investigation of complex biol. functions in a significantly less complex membrane environment. In this study, we introduce a supported double membrane system that should be useful for studies that target biol. processes in the proximity of two lipid bilayers such as the periplasm of bacteria and mitochondria or the small cleft between pre- and postsynaptic neuronal membranes. Large unilamellar vesicles (LUV) were tethered to a preformed supported bilayer by a biotin-streptavidin tether. We show from single particle tracking (SPT) expts. that these vesicle are mobile above the plane of the supported membrane. At higher concns., the tethered vesicles fuse to form a second continuous bilayer on top of the supported bilayer. The distance between the two bilayers was detd. by fluorescence interference contrast (FLIC) microscopy to be between 16 and 24 nm. The lateral diffusion of labeled lipids in the second bilayer was very similar to that in supported membranes. SPT expts. with reconstituted syntaxin-1A show that the mobility of transmembrane proteins was not improved when compared with solid supported membranes.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtVKkur7M&md5=4dbc0bcb5ed5670d365ae515a32b794a
    42. 42
      McGillivray, D. J.; Valincius, G.; Vanderah, D. J.; Febo-Ayala, W.; Woodward, J. T.; Heinrich, F.; Kasianowicz, J. J.; Lösche, M. Molecular-Scale Structural and Functional Characterization of Sparsely Tethered Bilayer Lipid Membranes. Biointerphases 2007, 2, 21– 33,  DOI: 10.1116/1.2709308
      Google Scholar
      42
      Molecular-scale structural and functional characterization of sparsely tethered bilayer lipid membranes
      McGillivray, Duncan J.; Valincius, Gintaras; Vanderah, David J.; Febo-Ayala, Wilma; Woodward, John T.; Heinrich, Frank; Kasianowicz, John J.; Losche, Mathias
      Biointerphases (2007), 2 (1), 21-33CODEN: BJIOBN; ISSN:1559-4106. (AVS-Science and Technology of Materials, Interfaces and Processing)
      Surface-tethered biomimetic bilayer membranes (tethered bilayer lipid membranes (tBLMs)) were formed on gold surfaces from phospholipids and a synthetic 1-thiahexa(ethylene oxide) lipid, WC14. They were characterized using electrochem. impedance spectroscopy, neutron reflection (NR), and Fourier-transform IR reflection-absorption spectroscopy (FT-IRRAS) to obtain functional and structural information. The authors found that elec. insulating membranes (conductance and capacitance as low as 1 μS cm-2 and 0.6 μF cm-2, resp.) with high surface coverage (>95% completion of the outer leaflet) can be formed from a range of lipids in a simple two-step process that consists of the formation of a self-assembled monolayer (SAM) and bilayer completion by "rapid solvent exchange.". NR provided a molecularly resolved characterization of the interface architecture and, in particular, the constitution of the space between the tBLM and the solid support. In tBLMs based on SAMs of pure WC14, the hexa(ethylene oxide) tether region had low hydration even though FT-IRRAS showed that this region is structurally disordered. However, on mixed SAMs made from the coadsorption of WC14 with a short-chain "backfiller," β-mercaptoethanol, the submembrane spaces between the tBLM and the substrates contained up to 60% exchangeable solvent by vol., as judged from NR and contrast variation of the solvent. Complete and stable "sparsely tethered" BLMs (stBLMs) can be readily prepd. from SAMs chemisorbed from solns. with low WC14 proportions. Phospholipids with unsatd. or satd., straight or branched chains all formed qual. similar stBLMs.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXksFGrtLg%253D&md5=b9d8a3517735a5276fe489dc40eecdc6
    43. 43
      Sarangi, N. K.; Patnaik, A. L-Tryptophan-Induced Electron Transport across Supported Lipid Bilayers: An Alkyl-Chain Tilt-Angle, and Bilayer-Symmetry Dependence. ChemPhysChem 2012, 13, 4258– 4270,  DOI: 10.1002/cphc.201200655
      Google Scholar
      43
      L-Tryptophan-Induced Electron Transport across Supported Lipid Bilayers: an Alkyl-Chain Tilt-Angle, and Bilayer-Symmetry Dependence
      Sarangi, Nirod Kumar; Patnaik, Archita
      ChemPhysChem (2012), 13 (18), 4258-4270CODEN: CPCHFT; ISSN:1439-4235. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Mol. orientation-dependent electron transport across supported 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid bilayers (SLBs) on semiconducting indium tin oxide (ITO) is reported with an aim towards potential nanobiotechnol. applications. A bifunctional strategy is adopted to form sym. and asym. bilayers of DPPC that interact with L-tryptophan, and are analyzed by surface manometry and at. force microscopy. Polarization-dependent real-time Fourier transform IR reflection absorption spectroscopy (FT-IRRAS) anal. of these SLBs reveals electrostatic, hydrogen-bonding, and cation-π interactions between the polar head groups of the lipid and the indole side chains. Consequently, a mol. tilt arises from the effective interface dipole, facilitating electron transport across the ITO-anchored SLBs in the presence of an internal Fe(CN)64-/3- redox probe. The incorporation of tryptophan enhances the voltammetric features of the SLBs. The estd. electron-transfer rate consts. for sym. and asym. bilayers (ks=2.0 × 10-2 and 2.8 × 10-2 s-1) across the two-dimensional (2D) ordered DPPC/tryptophan SLBs are higher compared to pure DPPC SLBs (ks=3.2 × 10-3 and 3.9 × 10-3 s-1). In addn., they are mol. tilt-dependent, as it is the case with the std. apparent rate consts., estd. from electrochem. impedance spectroscopy and bipotentiostatic expts. with a Pt ultramicroelectrode. Lower magnitudes of ks imply that electrochem. reactions across the ITO-SLB electrodes are kinetically limited and consequently governed by electron tunneling across the SLBs. Std. theor. rate consts. accrued upon electron tunneling comply with the potential-independent electron-tunneling coeff. β=0.15 Å-1. Insulator-semiconductor transitions moving from a liq.-expanded to a condensed 2D-phase state of the SLBs are noted, adding a new dimension to their transport behavior. These results highlight the role of tryptophan in expediting electron transfer across lipid bilayer membranes in a cellular environment and can provide potential clues towards patterned lipid nanocomposites and devices.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsFKns7vL&md5=b73f88353fdd53982b4abd099db1ac48
    44. 44
      Gufler, P. C.; Pum, D.; Sleytr, U. B.; Schuster, B. Highly Robust Lipid Membranes on Crystalline S-Layer Supports Investigated by Electrochemical Impedance Spectroscopy. Biochim. Biophys. Acta, Biomembr. 2004, 1661, 154– 165,  DOI: 10.1016/j.bbamem.2003.12.009
      Google Scholar
      44
      Highly robust lipid membranes on crystalline S-layer supports investigated by electrochemical impedance spectroscopy
      Gufler, Petra C.; Pum, Dietmar; Sleytr, Uwe B.; Schuster, Bernhard
      Biochimica et Biophysica Acta, Biomembranes (2004), 1661 (2), 154-165CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
      In the present work, S-layer supported lipid membranes formed by a modified Langmuir-Blodgett technique were investigated by electrochem. impedance spectroscopy (EIS). Basically two intermediate hydrophilic supports for phospholipid- (DPhyPC) and bipolar tetraetherlipid- (MPL from Thermoplasma acidophilum) membranes have been applied: first, the S-layer protein SbpA isolated from Bacillus sphaericus CCM 2177 recrystd. onto a gold electrode; and second, as a ref. support, an S-layer ultrafiltration membrane (SUM), which consists of a microfiltration membrane (MFM) with deposited S-layer carrying cell wall fragments. The electrochem. properties and the stability of DPhyPC and MPL membranes were found to depend on the support employed. The specific capacitances were 0.53 and 0.69 μF/cm2 for DPhyPC bilayers and 0.75 and 0.77 μF/cm2 for MPL monolayers resting on SbpA and SUM, resp. Membrane resistances of up to 80 MΩ cm2 were obsd. for DPhyPC bilayers on SbpA. In addn., membranes supported by SbpA exhibited a remarkable long-term robustness of up to 2 days. The membrane functionality could be demonstrated by reconstitution of membrane-active peptides such as valinomycin and alamethicin. The present results recommend S-layer-supported lipid membranes as promising structures for membrane protein-based biosensor technol.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhvFagsL4%253D&md5=8aae1cbbfd1b0702c465e8e514a5263c
    45. 45
      Hillman, A. R.; Ryder, K. S.; Madrid, E.; Burley, A. W.; Wiltshire, R. J.; Merotra, J.; Grau, M.; Horswell, S. L.; Glidle, A.; Dalgliesh, R. M.; Hughes, A.; Cubitt, R.; Wildes, A. Structure and Dynamics of Phospholipid Bilayer Films under Electrochemical Control. Faraday Discuss. 2010, 145, 357– 379,  DOI: 10.1039/b911246b
      Google Scholar
      45
      Structure and dynamics of phospholipid bilayer films under electrochemical control
      Hillman, A. Robert; Ryder, Karl S.; Madrid, Elena; Burley, Andrew W.; Wiltshire, Richard J.; Merotra, James; Grau, Michaela; Horswell, Sarah L.; Glidle, Andrew; Dalgliesh, Robert M.; Hughes, Arwel; Cubitt, Robert; Wildes, Andrew
      Faraday Discussions (2010), 145 (), 357-379CODEN: FDISE6; ISSN:1359-6640. (Royal Society of Chemistry)
      Vesicle fusion was used to deposit mixed dimyristoyl phosphatidylethanolamine-dimyristoyl phosphatidylserine (DMPE-DMPS) phospholipid bilayers on Au electrodes. Bilayer structure and compn., when exposed to aq. NaF and subject to an applied electrochem. potential, were studied using electrochem., spectroscopic and neutron reflectivity (NR) techniques. Interfacial capacitance data indicate the formation of compact films. Chronocolometric data show that surface charge is significantly altered by the presence of lipid in the potential range -0.75 < E/V (Ag|AgCl) < 0.35. NR measurements were made on lipid films in which the hydrocarbon tails were either fully hydrogenous (h-DMPE-h-DMPS) or perdeuterated (d-DMPE-d-DMPS), in each case serially exposed to D2O and H2O electrolytes and subject to different applied potentials. Guided by simulations of candidate interfacial structures, these yield the spatial distributions of lipid and solvent within the layers. Adjacent to the electrode, a compact inner leaflet is formed, with potential-dependent solvent vol. fraction in the range 0.09 < ΦS < 0.19; there was no evidence of an intervening water layer. The outer leaflet contains rather more solvent, 0.52 < ΦS < 0.55. NR-derived film thickness and PM-IRRAS intensity data show that the lipid mols. are tilted from the surface normal by ca. 26°. Bilayer solvation and charge data show a strong correlation for the inner leaflet and very little for the outer leaflet.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXntlKntw%253D%253D&md5=ecb50d07b01dd7705ab5c515f8c28a3d
    46. 46
      Steinem, C.; Janshoff, A.; Ulrich, W. P.; Sieber, M.; Galla, H. J. Impedance Analysis of Supported Lipid Bilayer Membranes: A Scrutiny of Different Preparation Techniques. Biochim. Biophys. Acta, Biomembr. 1996, 1279, 169– 180,  DOI: 10.1016/0005-2736(95)00274-X
      Google Scholar
      46
      Impedance analysis of supported lipid bilayer membranes: a scrutiny of different preparation techniques
      Steinem, Claudia; Janshoff, Andreas; Ulrich, Wolf-Peter; Sieber, Manfred; Galla, Hans-Joachim
      Biochimica et Biophysica Acta, Biomembranes (1996), 1279 (2), 169-80CODEN: BBBMBS; ISSN:0005-2736. (Elsevier B.V.)
      One topic of this study is the comparison of different prepn. techniques to build up solid supported lipid bilayers onto gold substrates. The deposited lipid bilayers were investigated by a.c. impedance spectroscopy. Three different strategies were applied: (1) The gold surface was initially covered with a chemisorbed monolayer of octadecanethiol or 1,2-dimyristoyl-sn-glycero-3-phosphothioethanol (DMPTE). The second monolayer consisting of phospholipids was then deposited onto this hydrophobic surface by (i) the Langmuir-Schaefer-technique, (ii) from lipid soln. in n-decane/isobutanol, (iii) by the lipid/detergent diln. technique or (i.v.) by fusion of vesicles. (2) Charged mols. carrying thiol-anchors for attachment to the gold surface by chemisorption were used. Neg. charged surfaces of 3-mercaptopropionic acid were excellent substrates that allow the attachment of planar lipid bilayers by applying pos. charged dimethyldioctadecylammoniumbromide (DODAB) vesicles or neg. charged 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol vesicles in the presence of chelating Ca2+-ions. If pos. charged first monolayers of mercaptoethylammoniumhydrochloride were used the authors were able to attach mixed 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol/1,2-dimyristoyl-sn -glycero-3-phosphoethanolamine vesicles to form planar lipid bilayers via electrostatic interaction. (3) Direct deposition of lipid bilayers is possible from vesicles contg. 1,2-dimyristoyl-sn-glycero-3-phosphothioethanol (DMPTE). A crit. amt. of more than 50 mol% of DMPTE was necessary to form a solid supported lipid bilayer. Bilayers obtained with these different prepn. techniques were scrutinized with respect to their capacitances, kinetics of formation and their long-term stabilities by impedance spectroscopy. The second feature of this paper is the application of the supported bilayers to study ion transport through channel-forming peptides. The authors used a DODAB-bilayer for the reconstitution of gramicidin D channels. By CD measurements the authors verified that the peptide is in its channel conformation. The ion transport of Cs+-ions through the channels was recorded by impedance anal.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XhsFOjtbc%253D&md5=f62efc4a6b801c312e9ebef4e6951cbc
    47. 47
      McGillivray, D. J.; Valincius, G.; Heinrich, F.; Robertson, J. W. F.; Vanderah, D. J.; Febo-Ayala, W.; Ignatjev, I.; Lösche, M.; Kasianowicz, J. J. Structure of Functional Staphylococcus Aureus α-Hemolysin Channels in Tethered Bilayer Lipid Membranes. Biophys. J. 2009, 96, 1547– 1553,  DOI: 10.1016/j.bpj.2008.11.020
      Google Scholar
      47
      Structure of functional Staphylococcus aureus α-hemolysin channels in tethered bilayer lipid membranes
      McGillivray, Duncan J.; Valincius, Gintaras; Heinrich, Frank; Robertson, Joseph W. F.; Vanderah, David J.; Febo-Ayala, Wilma; Ignatjev, Ilja; Losche, Mathias; Kasianowicz, John J.
      Biophysical Journal (2009), 96 (4), 1547-1553CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)
      We demonstrate a method for simultaneous structure and function detn. of integral membrane proteins. Elec. impedance spectroscopy (EIS) shows that Staphylococcus aureus α-hemolysin (α-HL) channels in membranes tethered to gold have the same properties as those formed in free-standing bilayer lipid membranes. Neutron reflectometry (NR) provides high-resoln. structural information on the interaction between the channel and the disordered membrane, validating predictions based on the channel's x-ray crystal structure. The robust nature of the membrane enabled the precise localization of the protein within 1.1 Å. The channel's extramembranous cap domain affects the lipid headgroup region and the alkyl chains in the outer membrane leaflet and significantly dehydrates the headgroups. The results suggest that this technique could be used to elucidate mol. details of the assocn. of other proteins with membranes and may provide structural information on domain organization and stimuli-responsive reorganization for transmembrane proteins in membrane mimics.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXnvVersb0%253D&md5=bd1a5b223aef364b888e49b0d300268b
    48. 48
      Wiegand, G.; Arribas-Layton, N.; Hillebrandt, H.; Sackmann, E.; Wagner, P. Electrical Properties of Supported Lipid Bilayer Membranes. J. Phys. Chem. B 2002, 106, 4245– 4254,  DOI: 10.1021/jp014337e
      Google Scholar
      48
      Electrical properties of supported lipid bilayer membranes
      Wiegand, Gerald; Arribas-Layton, Noah; Hillebrandt, Heiko; Sackmann, Erich; Wagner, Peter
      Journal of Physical Chemistry B (2002), 106 (16), 4245-4254CODEN: JPCBFK; ISSN:1089-5647. (American Chemical Society)
      This paper describes a study of the elec. properties of supported lipid bilayer membranes on semiconductor and gold surfaces. The study is aimed to foster the understanding of supported membrane systems and to allow the rational design of biosensor assays for ion channel anal. Impedance spectroscopy was applied for the elec. characterization of the supported membrane systems. A novel equiv. circuit model is introduced for the data evaluation, which accounts for the deviation of the impedance response of supported membranes from that of an ideal RC element. As a result of the improved accordance of model and data, the resistance and the capacity of supported membranes can be detd. more accurately and independently from each other. Exptl. results describe the phenomenol. of the elec. properties of supported bilayers regarding variations in prepn., compn., and environmental conditions. We discuss the findings in terms of membrane-substrate interactions and models of membrane permeability. The important role of the electrostatics between the lipid bilayer and the solid substrate for the formation of an elec. dense supported membrane is identified. Bilayer permeability models explain the correlation between the structure of the lipid bilayer and its insulating properties. These models are also in accordance with the obsd. dependence of the elec. resistance of the lipid bilayer on the temp. and the ion concn. of the electrolyte.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD38XitlCis78%253D&md5=266b61310169b8fe0daf2a60ce9ecc4f
    49. 49
      Su, Z.; Leitch, J. J.; Lipkowski, J. Electrode-Supported Biomimetic Membranes: An Electrochemical and Surface Science Approach for Characterizing Biological Cell Membranes. Curr. Opin. Electrochem. 2018, 12, 60– 72,  DOI: 10.1016/J.COELEC.2018.05.020
      Google Scholar
      49
      Electrode-supported biomimetic membranes: An electrochemical and surface science approach for characterizing biological cell membranes
      Su, ZhangFei; Leitch, J. Jay; Lipkowski, Jacek
      Current Opinion in Electrochemistry (2018), 12 (), 60-72CODEN: COEUCY; ISSN:2451-9111. (Elsevier B.V.)
      A review. Planar solid-supported lipid bilayers have been developed as simplified biol. membranes to model the phys. properties of cell membrane processes. Lipid bilayer membranes supported at conductive metal substrates provide a unique opportunity to investigate the effect of the static elec. field on the membrane structure and function. The insights gained from this research can be used to develop novel biosensors and biomedical devices. This review summarizes the recent developments in metal-supported biomimetic membrane systems. It provides an overview of the various models, such as metal-supported monolayers and bilayers, hybrid bilayers, tethered bilayers, and floating bilayers, used to study membrane processes at electrode surfaces, such as metal-supported monolayers and bilayers, hybrid, tethered, and floating bilayers. The paper discusses the recent advancements in these biomimetic models and describes the fundamental knowledge about membrane processes that has been extd. from these different platforms. The potential for the design and improvement of biomedical devices using metal-supported bilayers is also discussed. Metal-supported bilayers allow for the application of a plethora of spectroscopic and surface imaging techniques to obtain information about the voltage-dependent properties of biomols. at the mol. level. The underlying methodol. of these anal. techniques and the structural, chem. and kinetic information extd. are reviewed.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXisF2lu73P&md5=7ecfb661116ec1bafeb8fa37d5ae7ad9
    50. 50
      Abbasi, F.; Leitch, J. J.; Su, Z.; Szymanski, G.; Lipkowski, J. Direct Visualization of Alamethicin Ion Pores Formed in a Floating Phospholipid Membrane Supported on a Gold Electrode Surface. Electrochim. Acta 2018, 267, 195– 205,  DOI: 10.1016/J.ELECTACTA.2018.02.057
      Google Scholar
      50
      Direct visualization of alamethicin ion pores formed in a floating phospholipid membrane supported on a gold electrode surface
      Abbasi, Fatemeh; Leitch, J. Jay; Su, ZhangFei; Szymanski, Grzegorz; Lipkowski, Jacek
      Electrochimica Acta (2018), 267 (), 195-205CODEN: ELCAAV; ISSN:0013-4686. (Elsevier Ltd.)
      Unilamellar DMPC/DMPG vesicles in the absence and presence of alamethicin were fused onto the surface of a Au electrode modified with a 1-thio-β-D-glucose self-assembled monolayer. The resulting floating bilayer lipid membranes (fBLMs) were studied using at. force microscopy (AFM) and electrochem. impedance spectroscopy (EIS). A corrugated film structure was obsd. for the pure DMPC/DMPG fBLMs due to surface stress between the tightly packed lipids. These corrugations are removed by the addn. of alamethicin suggesting the lipid-peptide interactions alleviate the overall surface stress creating a more uniform bilayer. Both DMPC/DMPG films in the absence and presence of alamethicin had thickness of 5.5 ± 0.9 nm demonstrating that alamethicin has a minimal effect on the overall bilayer thickness. However, a significant decrease in membrane resistivity was obsd. when alamethicin was inserted into the fBLM indicating that the peptides are forming ion conducting pores. A direct visualization of the alamethicin pores was obtained by mol. resoln. AFM images revealing that the pores are not randomly dispersed throughout the bilayer, but instead form hexagonal aggregates. The diam. of an individual pore within the aggregates is 2.3 ± 0.3 nm, which is consistent with the size of a hexameric pore predicted by mol. dynamics simulations. Addnl., the image revealed a broad size distribution of alamethicin aggregates, which explains the origin of multiple cond. states obsd. for the incorporation of alamethicin into free standing bilayer lipid membranes.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXjtVantb0%253D&md5=1ee9c727bbdb290d91b783e110330793
    51. 51
      Drexler, J.; Steinem, C. Pore-Suspending Lipid Bilayers on Porous Alumina Investigated by Electrical Impedance Spectroscopy. J. Phys. Chem. B 2003, 107, 11245– 11254,  DOI: 10.1021/jp030762r
      Google Scholar
      51
      Pore-Suspending Lipid Bilayers on Porous Alumina Investigated by Electrical Impedance Spectroscopy
      Drexler, Janine; Steinem, Claudia
      Journal of Physical Chemistry B (2003), 107 (40), 11245-11254CODEN: JPCBFK; ISSN:1520-6106. (American Chemical Society)
      Nonordered and ordered porous alumina substrates with pore diams. of 20 and 50 nm, resp., were utilized to immobilize lipid membranes spanning the pores of the porous material. The substrates were characterized by means of interferometry and elec. impedance spectroscopy. For impedance data redn., an equiv. circuit representing the elec. behavior of porous alumina was developed on the basis of the parallel layer model. It turned out that the elec. parameters of the as prepd. alumina substrates prevent a sensitive monitoring of the formation of immobilized lipid membranes. Thus, we established a technique to modify the substrates with respect to their elec. properties, leading to a significantly increased capacitance of porous alumina, which allowed for a sensitive detection of pore-spanning lipid bilayers by impedance spectroscopy. Two different membrane prepn. techniques based on vesicle spreading were investigated. First, neg. charged giant liposomes were spread onto the porous alumina surface under an applied dc voltage of +100 mV. Second, large unilamellar vesicles contg. lipids bearing a thiol anchor were used to chemisorb on gold functionalized porous alumina substrates and subsequently rupture to form planar pore-spanning membranes. For both techniques, impedance spectra were obtained, which indicate the formation of lipid bilayers on top of the porous alumina substrates.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXnt1Oktbw%253D&md5=81bcd70aed28801d79a1d049adecfd23
    52. 52
      Orth, A.; Johannes, L.; Römer, W.; Steinem, C. Creating and Modulating Microdomains in Pore-Spanning Membranes. ChemPhysChem 2012, 13, 108– 114,  DOI: 10.1002/cphc.201100644
      Google Scholar
      52
      Creating and Modulating Microdomains in Pore-Spanning Membranes
      Orth, Alexander; Johannes, Ludger; Roemer, Winfried; Steinem, Claudia
      ChemPhysChem (2012), 13 (1), 108-114CODEN: CPCHFT; ISSN:1439-4235. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The architecture of the plasma membrane is not only detd. by the lipid and protein compn., but is also influenced by its attachment to the underlying cytoskeleton. Herein, the authors show that microscopic phase sepn. of "raft-like" lipid mixts. in pore-spanning bilayers is strongly detd. by the underlying highly ordered porous substrate. In detail, lipid membranes composed of DOPC/sphingomyelin/cholesterol/Gb3 were prepd. on ordered pore arrays in silicon with pore diams. of 0.8, 1.2 and 2 μm, resp., by spreading and fusion of giant unilamellar vesicles. The upper part of the silicon substrate was first coated with gold and then functionalized with a thiol-bearing cholesterol deriv. rendering the surface hydrophobic, which is prerequisite for membrane formation. Confocal laser scanning fluorescence microscopy was used to investigate the phase behavior of the obtained pore-spanning membranes. Coexisting liq.-ordered- (lo) and liq.-disordered (ld) domains were visualized for DOPC/sphingomyelin/cholesterol/Gb3 (40:35:20:5) membranes. The size of the lo-phase domains was strongly affected by the underlying pore size of the silicon substrate and could be controlled by temp., and the cholesterol content in the membrane, which was modulated by the addn. of methyl-β-cyclodextrin. Binding of Shiga toxin B-pentamers to the Gb3-doped membranes increased the lo-phase considerably and even induced lo-phase domains in non-phase sepd. bilayers composed of DOPC/sphingomyelin/cholesterol/Gb3 (65:10:20:5).
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsFGjsbfM&md5=15e5dde1008ee608b32aaf28dff91999
    53. 53
      Ronen, R.; Kaufman, Y.; Freger, V. Formation of Pore-Spanning Lipid Membrane and Cross-Membrane Water and Ion Transport. J. Membr. Sci. 2017, 523, 247– 254,  DOI: 10.1016/j.memsci.2016.09.059
      Google Scholar
      53
      Formation of pore-spanning lipid membrane and cross-membrane water and ion transport
      Ronen, Rona; Kaufman, Yair; Freger, Viatcheslav
      Journal of Membrane Science (2017), 523 (), 247-254CODEN: JMESDO; ISSN:0376-7388. (Elsevier B.V.)
      The authors report on (1) the formation mechanism of an array of pore-spanning phospholipid membranes via 'vesicle fusion', and (2) a microfluidic device that is used to assess the stability of the pore-spanning lipid membrane under flow and osmotic gradient. It is shown that the formation of pore-spanning lipid membranes via 'vesicles fusion' proceeds in three steps: first, small vesicles merge into giant ones of about the size of the substrate pore size. The giant vesicles then settle at the pore mouths and flatten. Last, the flattened giant vesicles rupture and form a lipid membrane that closes the pore. Exposing the membrane to combined transmembrane osmotic and tangential shear flows in a microfluidic device, which simulates common osmotic process conditions, shows that, in addn. to remaining open pores, a fraction of pore-spanning membranes ruptures. Possible ways to avoid such rupture and minimize fraction of open pores are discussed.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhslShs7jN&md5=0776caf39dae9fbac8d7662f544aa49d
    54. 54
      Jose, B.; Mallon, C. T.; Forster, R. J.; Blackledge, C.; Keyes, T. E. Lipid Bilayer Assembly at a Gold Nanocavity Array. Chem. Commun. 2011, 47, 12530,  DOI: 10.1039/c1cc15709d
      Google Scholar
      54
      Lipid bilayer assembly at a gold nanocavity array
      Jose, Bincy; Mallon, Colm T.; Forster, Robert J.; Blackledge, Chuck; Keyes, Tia E.
      Chemical Communications (Cambridge, United Kingdom) (2011), 47 (46), 12530-12532CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      The assembly of lipid bilayer membranes, using ultrasonic disruption of liposomes of L-α-dimyristoylphosphatidylcholine, across 820 nm diam. spherical cap gold cavity arrays is demonstrated.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsVGrs7jE&md5=73a9425ab66f901dc4eda4d1eded7c9f
    55. 55
      Basit, H.; Gaul, V.; Maher, S.; Forster, R. J.; Keyes, T. E. Aqueous-Filled Polymer Microcavity Arrays: Versatile & Stable Lipid Bilayer Platforms Offering High Lateral Mobility to Incorporated Membrane Proteins. Analyst 2015, 140, 3012– 3018,  DOI: 10.1039/C4AN02317J
      Google Scholar
      55
      Aqueous-filled polymer microcavity arrays: versatile & stable lipid bilayer platforms offering high lateral mobility to incorporated membrane proteins
      Basit, Hajra; Gaul, Vinnie; Maher, Sean; Forster, Robert J.; Keyes, Tia E.
      Analyst (Cambridge, United Kingdom) (2015), 140 (9), 3012-3018CODEN: ANALAO; ISSN:0003-2654. (Royal Society of Chemistry)
      A key prerequisite in an ideal supported lipid bilayer based cell membrane model is that the mobility of both the lipid matrix and its components are unhindered by the underlying support. This is not trivial and with the exception of liposomes, many of even the most advanced approaches, although accomplishing lipid mobility, fail to achieve complete mobility of incorporated membrane proteins. This is addressed in a novel platform comprising lipid bilayers assembled over buffer-filled arrays of spherical cap microcavities formed from microsphere template polydimethylsiloxane. Prior to bilayer assembly the PDMS is rendered hydrophilic by plasma treatment and the lipid bilayer prepd. using Langmuir Blodgett assembly followed by liposome/proteoliposome fusion. Fluorescence Lifetime Correlation Spectroscopy confirmed the pore suspended lipid bilayer exhibits diffusion coeffs. comparable to free-standing vesicles in soln. The bilayer modified arrays are highly reproducible and stable over days. As the bilayers are suspended over deep aq. reservoirs, reconstituted membrane proteins experience an aq. interface at both membrane interfaces and attain full lateral mobility. Their utility as membrane protein platforms was exemplified in two case studies with proteins of different dimensions in their extracellular and cytoplasmic domains reconstituted into DOPC lipid bilayers; Glycophorin A, and Integrin αIIbβ3. In both cases, the proteins exhibited 100% mobility with high lateral diffusion coeffs.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXkt1OrsLs%253D&md5=4555de38bfa1f1605591077d10438f4d
    56. 56
      Berselli, G. B.; Sarangi, N. K.; Ramadurai, S.; Murphy, P. V.; Keyes, T. E. Microcavity-Supported Lipid Membranes: Versatile Platforms for Building Asymmetric Lipid Bilayers and for Protein Recognition. ACS Appl. Bio Mater. 2019, 2, 3404– 3417,  DOI: 10.1021/acsabm.9b00378
      Google Scholar
      56
      Microcavity-Supported Lipid Membranes: Versatile Platforms for Building Asymmetric Lipid Bilayers and for Protein Recognition
      Berselli, Guilherme B.; Sarangi, Nirod Kumar; Ramadurai, Sivaramakrishnan; Murphy, Paul V.; Keyes, Tia E.
      ACS Applied Bio Materials (2019), 2 (8), 3404-3417CODEN: AABMCB; ISSN:2576-6422. (American Chemical Society)
      Microcavity-supported lipid bilayers (MSLBs) are contact-free membranes suspended across aq.-filled pores that maintain the lipid bilayer in a highly fluidic state, free from frictional interactions with substrate. Such platforms offer the prospect of liposome-like fluidity with the compositional versatility and addressability of supported lipid bilayers and thus offer a significant opportunity to model membrane asymmetry, protein-membrane interactions, and aggregation at the membrane interface. Herein we evaluate their performance by studying the effect of transmembrane lipid asymmetry on lipid diffusivity, membrane viscosity, and cholera toxin-ganglioside recognition across six sym. and asym. membranes including binary compns. contg. both fluid and gel phases, and ternary phase-sepd. membrane compns. Fluorescence lifetime correlation spectroscopy was used to det. the lateral mobility of the lipid and the protein, and electrochem. impedance spectroscopy enabled the detection of the protein-membrane assembly over the nanomolar range. Transmembrane leaflet asymmetry was obsd. to have a profound impact on membrane electrochem. resistance, where the resistance of a ternary sym. phase-sepd. bilayer was found to be at least 2.6 times higher than the asym. bilayer with analogous compn. in the distal leaflet but where the lower leaflet comprised only 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Similarly, the diffusion coeff. for MSLBs was obsd. to be 2.5 times faster for asym. MSLBs where the lower leaflet is DOPC alone. Our results demonstrate that the interplay of lipid packing across both membrane leaflets and the concn. of GM1 both affect the extent of cholera toxin aggregation and the consequent diffusion of the cholera-GM1 aggregates. Given that true biomembranes are both fluidic and asym., MSLBs offer the opportunity to build greater biomimicry into biophys. models, and the approach described demonstrates the value of MSLBs in studying aggregation and the membrane-assocd. multivalent interactions prevalent in many carbohydrate-mediated processes.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXht1Oqtr7L&md5=45629dfffab81e7cca5609d09704fa92
    57. 57
      Berselli, G. B.; Sarangi, N. K.; Gimenez, A. V.; Murphy, P. V.; Keyes, T. E. Microcavity Array Supported Lipid Bilayer Models of Ganglioside─Influenza Hemagglutinin1binding. Chem. Commun. 2020, 56, 11251– 11254,  DOI: 10.1039/d0cc04276e
      Google Scholar
      57
      Microcavity array supported lipid bilayer models of ganglioside - influenza hemagglutinin1 binding
      Berselli, Guilherme B.; Sarangi, Nirod Kumar; Gimenez, Aurelien V.; Murphy, Paul V.; Keyes, Tia E.
      Chemical Communications (Cambridge, United Kingdom) (2020), 56 (76), 11251-11254CODEN: CHCOFS; ISSN:1359-7345. (Royal Society of Chemistry)
      The binding of influenza receptor (HA1) to membranes contg. different glycosphingolipid receptors was studied at Microcavity Supported Lipid Bilayers (MSLBs). HA1 preferentially binds to GD1a but the diffusion coeff. of the assocd. complex at lipid bilayer is approx. double that of the complexes formed by HA1 GM1 or GM3.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFGgs7%252FM&md5=21de2dc3ca350829450d6d5466e5cc37
    58. 58
      Robinson, J.; Berselli, G. B.; Ryadnov, M. G.; Keyes, T. E. Annexin v Drives Stabilization of Damaged Asymmetric Phospholipid Bilayers. Langmuir 2020, 36, 5454– 5465,  DOI: 10.1021/acs.langmuir.0c00035
      Google Scholar
      58
      Annexin V Drives Stabilization of Damaged Asymmetric Phospholipid Bilayers
      Robinson, Jack; Berselli, Guilherme B.; Ryadnov, Maxim G.; Keyes, Tia E.
      Langmuir (2020), 36 (19), 5454-5465CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      Annexins are sol. membrane-binding proteins that assoc. in a calcium dependent manner with anionic phospholipids. They play roles in membrane organization, signaling and vesicle transport and in several disease states including thrombosis and inflammation. Annexin V is believed to be involved in membrane repair. Mediated through binding to phosphatidylserine exposed at damaged plasma membrane, the protein forms cryst. networks that seal or stabilize small membrane tears. Herein, we model this biochem. mechanism to simulate membrane healing at microcavity array supported, transversally asym., lipid bilayers (MSLBs) comprising 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS). Varying annexin V concn., lipid compn., and DOPS presence at each leaflet, fluorescence imaging and correlation spectroscopy confirmed that when DOPS was present at the external, annexin V, contacting leaflet, the protein assembled rapidly at the membrane interface to form a layer. From electrochem. impedance studies, the annexin layer decreased membrane capacitance while reducing resistance. With DOPS incorporated only at the lower (proximal) leaflet, no appreciable annexin assembly was obsd. over the first 21 h. This suggests that membrane asymmetry is preserved over this window and transversal diffusion of DOPS is slow. Intense laser light applied to the membrane, in which DOPS is initially isolated at the lower leaflet, was found to simulate membrane damage, stimulating the rapid assembly of annexin V at the membrane interface confirmed by fluorescence imaging, correlation spectroscopy, and electrochem. impedance measurements. The damage induced by light increased impedance and decreased membrane resistance. The resulting bilayer annexin V patched bilayer showed better temporal stability toward impedance changes when compared with that of the parent membrane. In summary, this simple model of annexin V assembly in a fluidic lipid membrane provides new insights into the assembly of annexins as well as an empirical basis for building patch-repair mechanisms into interfacial bilayer membrane assemblies.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXnslehtLw%253D&md5=b48dade43ea0a2d3dd6aad1f18ae0113
    59. 59
      Ramadurai, S.; Sarangi, N. K.; Maher, S.; MacConnell, N.; Bond, A. M.; McDaid, D.; Flynn, D.; Keyes, T. E. Microcavity-Supported Lipid Bilayers; Evaluation of Drug–Lipid Membrane Interactions by Electrochemical Impedance and Fluorescence Correlation Spectroscopy. Langmuir 2019, 35, 8095– 8109,  DOI: 10.1021/acs.langmuir.9b01028
      Google Scholar
      59
      Microcavity-Supported Lipid Bilayers; Evaluation of Drug-Lipid Membrane Interactions by Electrochemical Impedance and Fluorescence Correlation Spectroscopy
      Ramadurai, Sivaramakrishnan; Sarangi, Nirod Kumar; Maher, Sean; MacConnell, Nicola; Bond, Alan M.; McDaid, Dennis; Flynn, Damien; Keyes, Tia E.
      Langmuir (2019), 35 (24), 8095-8109CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      Many drugs have intracellular or membrane-assocd. targets, thus understanding their interaction with the cell membrane is of value in drug development. Cell-free tools used to predict membrane interactions should replicate the mol. organization of the membrane. Microcavity array-supported lipid bilayer (MSLB) platforms are versatile biophys. models of the cell membrane that combine liposome-like membrane fluidity with stability and addressability. We used an MSLB herein to interrogate drug-membrane interactions across seven drugs from different classes, including nonsteroidal anti-inflammatories: ibuprofen (Ibu) and diclofenac (Dic); antibiotics: rifampicin (Rif), levofloxacin (Levo), and pefloxacin (Pef); and bisphosphonates: alendronate (Ale) and clodronate (Clo). Fluorescence lifetime correlation spectroscopy (FLCS) and electrochem. impedance spectroscopy (EIS) were used to evaluate the impact of drug on 1,2-dioleyl-sn-glycerophosphocholine and binary bilayers over physiol. relevant drug concns. Although FLCS data revealed Ibu, Levo, Pef, Ale, and Clo had no impact on lipid lateral mobility, EIS, which is more sensitive to membrane structural change, indicated modest but significant decreases to membrane resistivity consistent with adsorption but weak penetration of drugs at the membrane. Ale and Clo, evaluated at pH 5.25, did not impact the impedance of the membrane except at concns. exceeding 4 mM. Conversely, Dic and Rif dramatically altered bilayer fluidity, suggesting their translocation through the bilayer, and EIS data showed that resistivity of the membrane decreased substantially with increasing drug concn. Capacitance changes to the bilayer in most cases were insignificant. Using a Langmuir-Freundlich model to fit the EIS data, we propose Rsat as an empirical value that reflects permeation. Overall, the data indicate that Ibu, Levo, and Pef adsorb at the interface of the lipid membrane but Dic and Rif interact strongly, permeating the membrane core modifying the water/ion permeability of the bilayer structure. These observations are discussed in the context of previously reported data on drug permeability and log P.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXhtVektrjE&md5=94d657b20e39245d3b1f59c6442110ea
    60. 60
      Ramadurai, S.; Kohut, A.; Sarangi, N. K.; Zholobko, O.; Baulin, V. A.; Voronov, A.; Keyes, T. E. Macromolecular Inversion-Driven Polymer Insertion into Model Lipid Bilayer Membranes. J. Colloid Interface Sci. 2019, 542, 483– 494,  DOI: 10.1016/j.jcis.2019.01.093
      Google Scholar
      60
      Macromolecular inversion-driven polymer insertion into model lipid bilayer membranes
      Ramadurai, Sivaramakrishnan; Kohut, Ananiy; Sarangi, Nirod Kumar; Zholobko, Oksana; Baulin, Vladimir A.; Voronov, Andriy; Keyes, Tia E.
      Journal of Colloid and Interface Science (2019), 542 (), 483-494CODEN: JCISA5; ISSN:0021-9797. (Elsevier B.V.)
      Macromols. of amphiphilic invertible polymers (AIPs) are capable of self-assembly into micellar assemblies of various morphologies in solvents of different polarities. The micellar assemblies in aq. media are capable of encapsulating poorly aq. sol. cargo and can undergo inverse conformational change and cargo release in contact with non-polar media, including potentially, cell membranes. Thus, invertible micellar assemblies have significant potential in drug delivery and related domains. However, to date there have been few investigations into their interactions with lipid membranes. Herein, we investigate the interactions of three recently developed AIPs of varying hydrophobicity/hydrophilicity balance with a highly fluidic microcavity supported 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer. We combined electrochem. impedance spectroscopy (EIS) with fluorescence correlation spectroscopy (FCS) to understand how the AIP micellar assemblies impacted bilayer permeability and fluidity resp., across polymer concns. above and below their crit. micelle concns. (cmcs). At concn. as above their cmcs, all of the AIPs explored increased permeability and decreased the fluidity of the lipid membrane. The extent of impact depended on the hydrophobicity of the AIP. PEG600-PTHF650, the most hydrophobic of the polymers, synthesized from PEG (mol. wt. 600 g/mol) and PTHF (mol. wt. 650 g/mol) exerted the greatest influence on the bilayer's phys. properties and fluorescence imaging and correlation data indicate that PEG600-PTHF650 micelles loaded with BODIPY probes adsorb and invert at the lipid membrane with release of cargo into the bilayer. This study should help inform future advancement of AIPs for membrane mol. delivery.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXjtVKju7s%253D&md5=3a16b5df1721ab3f2b617ffacf0f953c
    61. 61
      Sarangi, N. K.; Prabhakaran, A.; Keyes, T. E. Interaction of Miltefosine with Microcavity Supported Lipid Membrane: Biophysical Insights from Electrochemical Impedance Spectroscopy. Electroanalysis 2020, 32, 2936– 2945,  DOI: 10.1002/elan.202060424
      Google Scholar
      61
      Interaction of Miltefosine with Microcavity Supported Lipid Membrane: Biophysical Insights from Electrochemical Impedance Spectroscopy
      Sarangi, Nirod Kumar; Prabhakaran, Amrutha; Keyes, Tia E.
      Electroanalysis (2020), 32 (12), 2936-2945CODEN: ELANEU; ISSN:1040-0397. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Miltefosine, an alkylphosphocholine analog, is the only drug taken orally for the treatment of leishmaniasis - a parasitic disease caused by sandflies. Although it is believed that Miltefosine exerts its activity by acting at the lipid membrane, detailed understanding of the interaction of this drug with eukaryotic membranes is still lacking. Herein, we exploit microcavity pore suspended lipid bilayers (MSLBs) as a biomimetic platform in combination with a highly sensitive label-free electrochem. impedance spectroscopy (EIS) technique to gain biophys. insight into the interaction of Miltefosine with host cell membrane as a function of lipid membranes compn. Four membrane compns. with increasing complexity were evaluated; DOPC, DOPC : Chol (75 : 25), domain forming DOPC : SM : Chol (40 : 40 : 20) and mammalian plasma membrane (MPM) mimetic DOPC:DOPE:Chol:SM:DOPS (32 : 25 : 20 : 15 : 8) and used to study the interaction of Miltefosine in a concn.-dependent manner using EIS. The membrane resistance changes in response to Miltefosine were modelled by an empirical Langmuir isotherm binding model to provide ests. of binding satn. and equil. assocn. const. Miltefosine was found to have greatest impact on electrochem. properties of the simpler membrane systems; DOPC and DOPC : Chol, where these membranes were found to be more susceptible to membrane thinning, attributed to strong permeation/penetration of the drug while, compns. that included both Chol and SM, expected to contain large liq.-ordered domains exhibited weaker changes to membrane resistance but strongest drug assocn. In contrast, at the MPM membrane, Miltefosine exerts weakest assocn., which is tentatively attributed to electrostatic effects from the anionic DOPS but some membrane thinning is obsd. reflected in change in resistance and capacitance values attributed to some weak permeation.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitV2lsr3N&md5=6c0fdd5a9b7f4f97f1c1b1d982045d61
    62. 62
      Sarangi, N. K.; Stalcup, A.; Keyes, T. E. The Impact of Membrane Composition and Co-Drug Synergistic Effects on Vancomycin Association with Model Membranes from Electrochemical Impedance Spectroscopy. ChemElectroChem 2020, 7, 4535– 4542,  DOI: 10.1002/celc.202000818
      Google Scholar
      62
      The Impact of Membrane Composition and Co-Drug Synergistic Effects on Vancomycin Association with Model Membranes from Electrochemical Impedance Spectroscopy
      Sarangi, Nirod Kumar; Stalcup, Apryll; Keyes, Tia E.
      ChemElectroChem (2020), 7 (22), 4535-4542CODEN: CHEMRA; ISSN:2196-0216. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The interaction of the antibacterial drug, vancomycin with microcavity suspended lipid bilayers (MSLB) was investigated using non-Faradaic electrochem. impedance spectroscopy (EIS). Five MSLB compns. were prepd. with increasing complexity at gold substrates: DOPC, DOPC:Chol, DOPC:SM:Chol, mammalian plasma membrane mimetic (MPM), and E. coli polar lipid ext. The latter two, are intended to mimic eukaryotic and bacterial inner membrane compns. resp. The extent of vancomycin assocn. and the impact drug assocn. has on the electrochem. properties of the membrane depended on biomembrane compn. Trends were similar across all membranes but the E. coli membrane compn. Vancomycin increased membrane resistance and decreased capacitance in a saturable, concn. dependent manner, but for E. coli membrane resistance change was negligible and capacitance increased. Membrane resistance data was fit to the Langmuir-Freundlich model to give quant. insight into the relative extent of assocn. of drug with membrane as a function of membrane compn. Overall, electrochem. data indicates that vancomycin assocs. at the interface of lipid membranes rather than penetrates the layer and this is promoted by the presence of anionic phospholipid. Interestingly, co-incubation of the E. coli ext. bilayer with both rifampicin and vancomycin, known to act synergistically, significantly promotes vancomycin assocn. to E. coli membrane.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFyhtr3E&md5=b3bfb0afbd578f49a209a207e0d979be
    63. 63
      Johansson, S.; Forsberg, E.; Lundgren, B. Comparison of Fibronectin Receptors from Rat Hepatocytes and Fibroblasts. J. Biol. Chem. 1987, 262, 7819– 7824,  DOI: 10.1016/S0021-9258(18)47641-7
      Google Scholar
      63
      Comparison of fibronectin receptors from rat hepatocytes and fibroblasts
      Johansson, Staffan; Forsberg, Erik; Lundgren, Bjoern
      Journal of Biological Chemistry (1987), 262 (16), 7819-24CODEN: JBCHA3; ISSN:0021-9258.
      A cell-surface fibronectin receptor was isolated from primary rat hepatocytes by affinity chromatog. on Sepharose conjugated with the cell-binding domain (105 kilodalton, kDa) of fibronectin. The receptor remained bound to the affinity column in the presence of 1M NaCl but was eluted by 1.5 mM of glycyl-arginyl-glycyl-aspartyl-seryl-cysteine peptide or by lowering the pH to 4. The eluted material migrated under nonreducing conditions in SDS-PAGE as 2 bands: the α- and β-components had apparent mol. wts. of 155,000 and 115,000, resp. After redn. the 155-kDa component gave rise to 2 peptides of Mr 145,000 and 20,000, whereas the 115-kDa component shifted migration to a Mr of 130,000. Antibodies specifically recognizing the 155- and 115-kDa proteins from hepatocytes inhibited the attachment of these cells to fibronectin-coated dishes, whereas attachment to dishes coated with collagen or laminin was unaffected. A fibronectin receptor isolated from rat fibroblasts showed closely similar, but not identical, migration in SDS-PAGE as the hepatocyte receptor. Furthermore, only the β-subunit of the fibroblast receptor reacted with the antibodies. The results suggest that distinct α-subunits of the fibronectin receptors may be the basis for the different fibronectin-binding properties of these cells.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2sXktlGgtrs%253D&md5=afe6b29fa22b2b7c22c43b86789c71ba
    64. 64
      Dransart, E.; Di Cicco, A.; El Marjou, A.; Lévy, D.; Johansson, S.; Johannes, L.; Shafaq-Zadah, M. Solubilization and Purification of α5β1 Integrin from Rat Liver for Reconstitution into Nanodiscs. In Heterologous Expression of Membrane Proteins; Methods in Molecular Biology, Vol 2507; Springer US: New York, NY, 2022. DOI: 10.1007/978-1-0716-2368-8_1
      Google Scholar
      There is no corresponding record for this reference.
    65. 65
      Lévy, D.; Bluzat, A.; Seigneuret, M.; Rigaud, J.-L. A Systematic Study of Liposome and Proteoliposome Reconstitution Involving Bio-Bead-Mediated Triton X-100 Removal. Biochim. Biophys. Acta, Biomembr. 1990, 1025, 179– 190,  DOI: 10.1016/0005-2736(90)90096-7
      Google Scholar
      64
      A systematic study of liposome and proteoliposome reconstitution involving Bio-Bead-mediated Triton X-100 removal
      Levy, Daniel; Bluzat, Aline; Seigneuret, Michel; Rigaud, Jean Louis
      Biochimica et Biophysica Acta, Biomembranes (1990), 1025 (2), 179-90CODEN: BBBMBS; ISSN:0005-2736.
      Equil. and kinetic aspects of Triton X 100 adsorption onto hydrophobic Bio-Beads SM2 were investigated in detail using the batch procedure originally described by P. W. Holloway (1973). The results demonstrated the importance of the initial detergent concn., the amt. of beads, the com. source of the detergent, the temp. and the presence of phospholipids in detg. the rates of Triton X 100 adsorption onto Bio-Beads. One of the main findings was that Bio-Beads allowed the almost complete removal of Triton X 100, whatever the initial exptl. conditions. It was shown that monomeric as well as micellar detergent could be adsorbed and that a key factor in detg. the rate of detergent removal was the availability of the free bead surface. Rates of detergent removal were found to be linearly related to the amt. of beads even for bead concns. above those sufficient to remove all the detergent initially present. Adsorptive capacity of phospholipids onto Bio-Beads SM2 was also analyzed and found to be much smaller (2 mg lipid/g of wet beads) than that of Triton X 100 (185 mg TX 100 per g of wet beads). A more general aspect of this work was that the use of Bio-Beads SM2 provided a convenient way for varying and controlling the time course of Triton X 100 removal. The method was further extended to the formation of liposomes from phospholipid-Triton X 100 micelles and the size of the liposomes was found to be critically dependent upon the rate of detergent removal. A general procedure was described to prep. homogeneous populations of vesicles. Freeze-fracture electron microscopy and permeability studies indicated that the liposomes thus obtained were unilamellar, relatively large and impermeable. Noteworthy, this new procedure was shown to be well suited for the reconstitution of different membrane transport proteins such as bacteriorhodopsin, Ca2+-ATPase, and H+-ATPase.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXkvFCgs7k%253D&md5=a4761d4d5578f937a349f54ef9e0cf07
    66. 66
      Maher, S.; Basit, H.; Forster, R. J.; Keyes, T. E. Micron Dimensioned Cavity Array Supported Lipid Bilayers for the Electrochemical Investigation of Ionophore Activity. Bioelectrochemistry 2016, 112, 16– 23,  DOI: 10.1016/j.bioelechem.2016.07.002
      Google Scholar
      65
      Micron dimensioned cavity array supported lipid bilayers for the electrochemical investigation of ionophore activity
      Maher, Sean; Basit, Hajra; Forster, Robert J.; Keyes, Tia E.
      Bioelectrochemistry (2016), 112 (), 16-23CODEN: BIOEFK; ISSN:1567-5394. (Elsevier B.V.)
      Microcavity supported lipid bilayers, MSLBs, were applied to an electrochem. investigation of ionophore mediated ion transport. The arrays comprise of 1 cm2 gold electrode imprinted with ordered array of uniform spherical-cap pores of 2.8μm diam. prepd. by gold electrodeposition through polystyrene templating spheres. The pores were pre-filled with aq. buffer prior to Langmuir-Blodgett assembly of a 1,2-dioleoyl-sn-glycero-3-phosphocholine bilayer. Fluorescence lifetime correlation spectroscopy enabled by micron dimensions of the pores permitted study of lipid diffusion across single apertures, yielding a diffusion coeff. of 12.58 ± 1.28μm2 s-1 and anomalous exponent of 1.03 ± 0.02, consistent with Brownian motion. From FLCS, the MSLBs were stable over 3 days and electrochem. impedance spectroscopy of the membrane with and without ionic gradient over exptl. windows of 6 h showed excellent stability. Two ionophores were studied at the MSLBs; Valinomycin, a K+ uniporter and Nigericin, a K+/H+ antiporter. Ionophore reconstituted into the DOPC bilayer resulted in a decrease and increase in membrane resistance and capacitance resp. Significant increases in Valinomycin and Nigericin activity were obsd., reflected in large decreases in membrane resistance when K+ was present in the contacting buffer and in the presence of H+ ionic gradient across the membrane resp.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtFKmurzJ&md5=a6aa5c0452ecdd654f25b424606bb403
    67. 67
      Khan, M. S.; Dosoky, N. S.; Williams, J. D. Engineering Lipid Bilayer Membranes for Protein Studies. Int. J. Mol. Sci. 2013, 14, 21561– 21597,  DOI: 10.3390/ijms141121561
      Google Scholar
      66
      Engineering lipid bilayer membranes for protein studies
      Khan, Muhammad Shuja; Dosoky, Noura Sayed; Williams, John Dalton
      International Journal of Molecular Sciences (2013), 14 (11), 21561-21597, 37 pp.CODEN: IJMCFK; ISSN:1422-0067. (MDPI AG)
      A review. Lipid membranes regulate the flow of nutrients and communication signaling between cells and protect the sub-cellular structures. Recent attempts to fabricate artificial systems using nanostructures that mimic the physiol. properties of natural lipid bilayer membranes (LBM) fused with transmembrane proteins have helped demonstrate the importance of temp., pH, ionic strength, adsorption behavior, conformational reorientation and surface d. in cellular membranes which all affect the incorporation of proteins on solid surfaces. Much of this work is performed on artificial templates made of polymer sponges or porous materials based on alumina, mica and porous silicon (PSi) surfaces. For example, porous silicon materials have high biocompatibility, biodegradability, and photoluminescence, which allow them to be used both as a support structure for lipid bilayers or a template to measure the electrochem. functionality of living cells grown over the surface as in vivo. The variety of these media, coupled with the complex physiol. conditions present in living systems, warrant a summary and prospectus detailing which artificial systems provide the most promise for different biol. conditions. This study summarizes the use of electrochem. impedance spectroscopy (EIS) data on artificial biol. membranes that are closely matched with previously published biol. systems using both black lipid membrane and patch clamp techniques.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXltlOntLY%253D&md5=b1eb59c9c2e41daed3608181f8343c50
    68. 68
      Khan, M. S.; Dosoky, N. S.; Berdiev, B. K.; Williams, J. D. Electrochemical Impedance Spectroscopy for Black Lipid Membranes Fused with Channel Protein Supported on Solid-State Nanopore. Eur. Biophys. J. 2016, 45, 843– 852,  DOI: 10.1007/s00249-016-1156-8
      Google Scholar
      67
      Electrochemical impedance spectroscopy for black lipid membranes fused with channel protein supported on solid-state nanopore
      Khan, Muhammad S.; Dosoky, Noura S.; Berdiev, Bakhrom K.; Williams, John D.
      European Biophysics Journal (2016), 45 (8), 843-852CODEN: EBJOE8; ISSN:0175-7571. (Springer)
      Black lipid membranes (BLMs) have been used for detecting single-channel activities of pore-forming peptides and ion channels. However, the short lifetimes and poor mech. stability of suspended bilayers limit their applications in high throughput electrophysiol. expts. In this work, we present a synthetic solid-state nanopore functionalized with BLM fused with channel protein. A nanopore with diam. of ∼180 nm was electrochem. fabricated in a thin silicon membrane. Folding and painting techniques were demonstrated for prodn. of stable suspended BLMs followed by incorporation of transmembrane protein, ENaC. Membrane formation was confirmed by employing electrochem. impedance spectroscopy (EIS) in the frequency regime of 10-2-105 Hz. Results show that electrochem. fabricated solid state nanopore support resulted in excellent membrane stability, with >1 GΩ of up to 72 and 41 h for painting and folding techniques, resp. After fusion of ENaC channel protein, the BLM exhibits the stability of ∼5 h. We anticipate that such a solid-state nanopore with diam. in the range of 150-200 nm and thickness <1 μm could be a potential platform to enhance the throughput of ion-channel characterization using BLMs.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtlWjtr7N&md5=43c3472f72c4958b00bfc3523366b694
    69. 69
      Daniels, J. S.; Pourmand, N. Label-Free Impedance Biosensors: Opportunities and Challenges. Electroanalysis 2007, 19, 1239– 1257,  DOI: 10.1002/elan.200603855
      Google Scholar
      68
      Label-free impedance biosensors: opportunities and challenges
      Daniels, Jonathan S.; Pourmand, Nader
      Electroanalysis (2007), 19 (12), 1239-1257CODEN: ELANEU; ISSN:1040-0397. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. Impedance biosensors are a class of elec. biosensors that show promise for point-of-care and other applications due to low cost, ease of miniaturization, and label-free operation. Unlabeled DNA and protein targets can be detected by monitoring changes in surface impedance when a target mol. binds to an immobilized probe. The affinity capture step leads to challenges shared by all label-free affinity biosensors; these challenges are discussed along with others unique to impedance readout. Various possible mechanisms for impedance change upon target binding are discussed. The authors critically summarize accomplishments of past label-free impedance biosensors and identify areas for future research.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXntlCgtLg%253D&md5=ff30599e2516efceaf1d69a839aa5fe8
    70. 70
      Lukyanov, P.; Furtak, V.; Ochieng, J. Galectin-3 Interacts with Membrane Lipids and Penetrates the Lipid Bilayer. Biochem. Biophys. Res. Commun. 2005, 338, 1031– 1036,  DOI: 10.1016/j.bbrc.2005.10.033
      Google Scholar
      69
      Galectin-3 interacts with membrane lipids and penetrates the lipid bilayer
      Lukyanov, Pavel; Furtak, Vyacheslav; Ochieng, Josiah
      Biochemical and Biophysical Research Communications (2005), 338 (2), 1031-1036CODEN: BBRCA9; ISSN:0006-291X. (Elsevier)
      The precise mechanism by which galectin-3 and other cytosolic proteins that lack signal peptides are secreted is yet to be elucidated. In the present analyses, we detd. that galectin-3, a β-galactoside binding protein, can interact directly with membrane lipids in solid phase binding assays. More interestingly, we detd. by spectrophotometric methods that it can spontaneously penetrate the lipid bilayer of liposomes in either direction. These findings suggest that galectin-3 on its own has the capacity to traverse the lipid bilayer. Whereas the situation is rather simplified in liposomes, the interaction of galectin-3 with the plasma membrane may involve cholesterol-rich membrane domains where galectin-3 can be concd. and form multimers or interact covalently with other proteins.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXht1WjurrO&md5=61474ea225fb5a6798a4c57d015b043c
    71. 71
      Sarangi, N. K.; Prabhakaran, A.; Keyes, T. E. Multimodal Investigation into the Interaction of Quinacrine with Microcavity-Supported Lipid Bilayers. Langmuir 2022, 38, 6411– 6424,  DOI: 10.1021/acs.langmuir.2c00524
      Google Scholar
      70
      Multimodal Investigation into the Interaction of Quinacrine with Microcavity-Supported Lipid Bilayers
      Sarangi, Nirod Kumar; Prabhakaran, Amrutha; Keyes, Tia E.
      Langmuir (2022), 38 (20), 6411-6424CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      Quinacrine is a versatile drug that is widely recognized for its antimalarial action through its inhibition of the phospholipase enzyme. It also has antianthelmintic and antiprotozoan activities and is a strong DNA binder that may be used to combat multidrug resistance in cancer. Despite extensive cell-based studies, a detailed understanding of quinacrine's influence on the cell membrane, including permeability, binding, and rearrangement at the mol. level, is lacking. Herein, we apply microcavity-suspended lipid bilayers (MSLBs) as in vitro models of the cell membrane comprising DOPC, DOPC:Chol(3:1), and DOPC:SM:Chol(2:2:1) to investigate the influence of cholesterol and intrinsic phase heterogeneity induced by mixed-lipid compn. on the membrane interactions of quinacrine. Using electrochem. impedance spectroscopy (EIS) and surface-enhanced Raman spectroscopy (SERS) as label-free surface-sensitive techniques, we have studied quinacrine interaction and permeability across the different MSLBs. Our EIS data reveal that the drug is permeable through ternary DOPC:SM:Chol and DOPC-only bilayer compns. In contrast, the binary cholesterol/DOPC membrane arrested permeation, yet the drug binds or intercalates at this membrane as reflected by an increase in membrane impedance. SERS supported the EIS data, which was utilized to gain structural insights into the drug-membrane interaction. Our SERS data also provides a simple but powerful label-free assessment of drug permeation because a significant SERS enhancement of the drug's Raman signature was obsd. only if the drug accessed the plasmonic interior of the pore cavity passing through the membrane. Fluorescent lifetime correlation spectroscopy (FLCS) provides further biophys. insight, revealing that quinacrine binding increases the lipid diffusivity of DOPC and the ternary membrane while remarkably decreasing the lipid diffusivity of the DOPC:Chol membrane. Overall, because of its adaptability to multimodal approaches, the MSLB platform provides rich and detailed insights into drug-membrane interactions, making it a powerful tool for in vitro drug screening.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xht1KrurjJ&md5=d2e7575d74217350051f227b00705c3f
    72. 72
      Chung, M.; Lowe, R. D.; Chan, Y.-H. M.; Ganesan, P. V.; Boxer, S. G. DNA-Tethered Membranes Formed by Giant Vesicle Rupture. J. Struct. Biol. 2009, 168, 190– 199,  DOI: 10.1016/j.jsb.2009.06.015
      Google Scholar
      71
      DNA-tethered membranes formed by giant vesicle rupture
      Chung, Minsub; Lowe, Randall D.; Chan, Yee-Hung M.; Ganesan, Prasad V.; Boxer, Steven G.
      Journal of Structural Biology (2009), 168 (1), 190-199CODEN: JSBIEM; ISSN:1047-8477. (Elsevier B.V.)
      We have developed a strategy for prepg. tethered lipid bilayer membrane patches on solid surfaces by DNA hybridization. In this way, the tethered membrane patch is held at a controllable distance from the surface by varying the length of the DNA used. Two basic strategies are described. In the first, single-stranded DNA strands are immobilized by click chem. to a silica surface, whose remaining surface is passivated to prevent direct assembly of a solid supported bilayer. Then giant unilamellar vesicles (GUVs) displaying the antisense strand, using a DNA-lipid conjugate developed in an earlier work. Lipid-anchored DNA mediates vesicle fusion as obsd. by lipid and content mixing are allowed to tether, spread and rupture to form tethered bilayer patches. In the second, a supported lipid bilayer displaying DNA using the DNA-lipid conjugate is first assembled on the surface. Then GUVs displaying the antisense strand are allowed to tether, spread and rupture to form tethered bilayer patches. The essential difference between these methods is that the tethering hybrid DNA is immobile in the first, while it is mobile in the second. Both strategies are successful; however, with mobile DNA hybrids as tethers, the patches are unstable, while in the first strategy stable patches can be formed. In the case of mobile tethers, if different length DNA hybrids are present, lateral segregation by length occurs and can be visualized by fluorescence interference contrast microscopy making this an interesting model for interactions that occur in cell junctions. In both cases, lipid mobility is high and there is a negligible immobile fraction. Thus, these architectures offer a flexible platform for the assembly of lipid bilayers at a well-defined distance from a solid support.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtVKkur7O&md5=c3667ad44f1bbe7e08d63aec16da9882
    73. 73
      Weng, K. C.; Kanter, J. L.; Robinson, W. H.; Frank, C. W. Fluid Supported Lipid Bilayers Containing Monosialoganglioside GM1: A QCM-D and FRAP Study. Colloids Surf., B 2006, 50, 76– 84,  DOI: 10.1016/j.colsurfb.2006.03.010
      Google Scholar
      72
      Fluid supported lipid bilayers containing monosialoganglioside GM1: A QCM-D and FRAP study
      Weng, Kevin C.; Kanter, Jennifer L.; Robinson, William H.; Frank, Curtis W.
      Colloids and Surfaces, B: Biointerfaces (2006), 50 (1), 76-84CODEN: CSBBEQ; ISSN:0927-7765. (Elsevier B.V.)
      In an effort to use model fluid membranes for immunol. studies, the authors compared the formation of planar phospholipid bilayers supported on silicon dioxide surfaces with and without incorporation of glycolipids as the antigen for in situ antibody binding. Dynamic light scattering measurements did not differentiate the hydrodynamic vols. of extruded small unilamellar vesicles (E-SUVs) contg. physiol. relevant concns. (0.5-5 mol%) of monosialoganglioside GM1 from exclusive egg yolk L-α-phosphatidylcholine (egg PC) E-SUVs. However, quantifiable differences in deposition mass and dissipative energy loss emerged in the transformation of 5 mol% GM1/95 mol% egg PC E-SUVs to planar supported lipid bilayers (PSLBs) by vesicle fusion on thermally evapd. SiO2, as monitored by the quartz crystal microbalance with dissipation (QCM-D) technique. Compared to the 100 mol% egg PC bilayers on the same surface, E-SUVs contg. 5 mol% GM1 reached a ∼12% higher mass and a lower dissipative energy loss during bilayer transformation. PSLBs with 5 mol% GM1 are ∼18% heavier than 100 mol% egg PC and ∼11% smaller in projected area per lipid, indicating an increased rigidity and a tighter packing. Subsequent binding of polyclonal IgG anti-GM1 to the PSLBs was performed in situ and showed specificity. The anti-GM1 to GM1 ratios at equil. were roughly proportional to the concns. of anti-GM1 administered in the soln. Fluorescence recovery after photobleaching was utilized to verify the retained, albeit reduced lateral fluidity of the supported membranes. Five moles percentage of GM1 membranes (GM1 to PC ratio ∼1:19) decorated with 1 mol% N-(Texas Red sulfonyl)-1,2-dihexadecanoyl-sn-glycerol-3-phosphoethanolamine (Texas Red DHPE) exhibited an approx. 16% lower diffusion coeff. of 1.32±0.06 μM2/s, compared to 1.58±0.04 μM2/s for egg PC membranes without GM1 (p < 0.01). The changes in vesicle properties and membrane lateral fluidity are attributed to the interactions of GM1 with itself and GM1 with other membrane lipids. This system allows for mols. of interest such as GM1 to exist on a more biol. relevant surface than those used in conventional methods such as ELISA. The authors' anal. of rabbit serum antibodies binding to GM1 demonstrates this platform can be used to test for the presence of anti-lipid antibodies in serum.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XltlKisro%253D&md5=20a14c6720e4797318b7391a6528c9cf
    74. 74
      Johnson, M. A.; Seifert, S.; Petrache, H. I.; Kimble-Hill, A. C. Phase Coexistence in Single-Lipid Membranes Induced by Buffering Agents. Langmuir 2014, 30, 9880– 9885,  DOI: 10.1021/la5018938
      Google Scholar
      73
      Phase coexistence in single-lipid membranes induced by buffering agents
      Johnson, Merrell A.; Seifert, Soenke; Petrache, Horia I.; Kimble-Hill, Ann C.
      Langmuir (2014), 30 (33), 9880-9885CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      Recent literature has shown that buffers affect the interaction between lipid bilayers through a mechanism that involves van der Waals forces, electrostatics, hydration forces, and membrane bending rigidity. Here, the authors show an addnl. peculiar effect of buffers on mixed-chain POPC lipid bilayers, namely phase coexistence similar to what was previously reported for alkali chlorides. The data presented suggested that one phase appeared to dehydrate below the value in pure water, while the other phase swelled as the concn. of buffer was increased. However, since the 2 phases must be in osmotic equil. with one another, this behavior challenges theor. models of lipid interactions.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhtlaisLnN&md5=270c8a26f3919cc62e9db4a492051ab4
    75. 75
      Gaul, V.; Lopez, S. G.; Lentz, B. R.; Moran, N.; Forster, R. J.; Keyes, T. E. The Lateral Diffusion and Fibrinogen Induced Clustering of Platelet Integrin ΑiIbβ3 Reconstituted into Physiologically Mimetic GUVs. Integr. Biol. 2015, 7, 402– 411,  DOI: 10.1039/c5ib00003c
      Google Scholar
      74
      The lateral diffusion and fibrinogen induced clustering of platelet integrin αIIbβ3 reconstituted into physiologically mimetic GUVs
      Gaul, Vinnie; Lopez, Sergio G.; Lentz, Barry R.; Moran, Niamh; Forster, Robert J.; Keyes, Tia E.
      Integrative Biology (2015), 7 (4), 402-411CODEN: IBNIFL; ISSN:1757-9694. (Royal Society of Chemistry)
      Platelet integrin αIIbβ3 is a key mediator of platelet activation and thrombosis. Upon activation αIIbβ3 undergoes significant conformational rearrangement, inducing complex bidirectional signalling and protein recruitment leading to platelet activation. Reconstituted lipid models of the integrin can enhance our understanding of the structural and mechanistic details of αIIbβ3 behavior away from the complexity of the platelet machinery. Here, a novel method of αIIbβ3 insertion into Giant Unilamellar Vesicles (GUVs) is described that allows for effective integrin reconstitution unrestricted by lipid compn. αIIbβ3 was inserted into two GUV lipid compns. that seek to better mimic the platelet membrane. First, "nature's own", comprising 32% DOPC, 25% DOPE, 20% CH, 15% SM and 8% DOPS, intended to mimic the platelet cell membrane. Fluorescence Lifetime Correlation Spectroscopy (FLCS) reveals that exposure of the integrin to the activators Mn2+ or DTT does not influence the diffusion coeff. of αIIbβ3. Similarly, exposure to αIIbβ3's primary ligand fibrinogen (Fg) alone does not affect αIIbβ3's diffusion coeff. However, addn. of Fg with either activator reduces the integrin diffusion coeff. from 2.52 ± 0.29 to μm2 s-1 to 1.56 ± 0.26 (Mn2+) or 1.49 ± 0.41 μm2 s-1 (DTT) which is consistent with aggregation of activated αIIbβ3 induced by fibrinogen binding. The Multichannel Scaler (MCS) trace shows that the integrin-Fg complex diffuses through the confocal vol. in clusters. Using the Saffman-Delbr.ovrddot.uck model as a first approxn., the diffusion coeff. of the complex suggests at least a 20-fold increase in the radius of membrane bound protein, consistent with integrin clustering. Second, αIIbβ3 was also reconstituted into a "raft forming" GUV with well defined liq. disordered (Ld) and liq. ordered (Lo) phases. Using confocal microscopy and lipid partitioning dyes, αIIbβ3 showed an affinity for the DOPC rich Ld phase of the raft forming GUVs, and was effectively excluded from the cholesterol and sphingomyelin rich Lo phase. Activation and Fg binding of the integrin did not alter the distribution of αIIbβ3 between the lipid phases. This observation suggests partitioning of the activated fibrinogen bound αIIbβ3 into cholesterol rich domains is not responsible for the integrin clustering obsd.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXivFKitLg%253D&md5=989f815fb1f24c2aabbaeb83db55a787
    76. 76
      Yang, R.-Y.; Hill, P. N.; Hsu, D. K.; Liu, F.-T. Role of the Carboxyl-Terminal Lectin Domain in Self-Association of Galectin-3. Biochemistry 1998, 37, 4086– 4092,  DOI: 10.1021/bi971409c
      Google Scholar
      75
      Role of the carboxyl-terminal lectin domain in self-association of galectin 3
      Yang, Ri-Yao; Hill, Paul N.; Hsu, Daniel K.; Liu, Fu-Tong
      Biochemistry (1998), 37 (12), 4086-4092CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      Galectin 3 is a member of a large family of β-galactoside-binding animal lectins and is composed of a C-terminal lectin domain connected to an N-terminal nonlectin part. Previous exptl. results suggested that when bound to multivalent glycoconjugates, galectin 3 self-assocs. through intermol. interactions involving the N-terminal domain. Here, the authors obtained evidence suggesting that the protein self-assocs. in the absence of its saccharide ligands, in a manner that is dependent on the C-terminal domain. This mode of self-assocn. was inhibitable by the lectin's saccharide ligands. Specifically, recombinant human galectin 3 was found to bind to galectin 3C (the C-terminal domain fragment) conjugated to Sepharose 4B and the binding was inhibitable by lactose. In addn., biotinylated galectin 3 bound to galectin 3 immobilized on plastic surfaces and the binding could also be inhibited by various saccharide ligands of the lectin. A mutant with a Trp → Leu replacement in the C-terminal domain, which exhibited diminished carbohydrate-binding activity, did not bind to galectin 3C-Sepharose 4B. Furthermore, galectin 3C formed covalent homodimers when it was treated with a chem. crosslinker and the dimer formation was completely inhibited by lactose. Therefore, galectin 3 can self-assoc. through intermol. interactions involving both the N- and the C-terminal domains and the relative contribution of each depends on whether the lectin is bound to its saccharide ligands.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXhsVOksLY%253D&md5=e70c4a4d3c309ec89ab1288029a6b76c
    77. 77
      Lepur, A.; Salomonsson, E.; Nilsson, U. J.; Leffler, H. Ligand Induced Galectin-3 Protein Self-Association. J. Biol. Chem. 2012, 287, 21751– 21756,  DOI: 10.1074/jbc.C112.358002
      Google Scholar
      76
      Ligand induced galectin-3 protein self-association
      Lepur, Adriana; Salomonsson, Emma; Nilsson, Ulf J.; Leffler, Hakon
      Journal of Biological Chemistry (2012), 287 (26), 21751-21756CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Many functions of galectin-3 entail binding of its carbohydrate recognition site to glycans of a glycoprotein, resulting in crosslinking thought to be mediated by its N-terminal noncarbohydrate-binding domain. Here, the authors studied the interaction of galectin-3 with the model glycoprotein, asialofetuin (ASF), using a fluorescence anisotropy assay to measure the concn. of free galectin carbohydrate recognition sites in soln. Surprisingly, in the presence of ASF, this remained low even at high galectin-3 concns., showing that many more galectin-3 mols. were engaged than expected due to the ∼9 known glycan-based binding sites per ASF mol. This suggested that after ASF-induced nucleation, galectin-3 assocd. with itself by the carbohydrate recognition site binding to another galectin-3 mol., possibly forming oligomers. The authors named this type-C self-assocn. to distinguish it from the previously proposed models (type-N) where galectin-3 mols. bind to each other through the N-terminal domain, and all carbohydrate recognition sites are available for binding glycans. Both types of self-assocn. could result in ppts., as measured here by turbidimetry and dynamic light scattering. Type-C self-assocn. and pptn. occurred even with a galectin-3 mutant (R186S) that bound poorly to ASF but required a much higher concn. (∼50 μM) as compared with wild type (∼1 μM). ASF also induced weaker type-C self-assocn. of galectin-3 lacking its N-terminal domains, but as expected, no pptn. Neither a monovalent nor a divalent N-acetyl-D-lactosamine-contg. glycan induced type-C self-assocn., even if the latter gave ppts. with high concns. of galectin-3 (> ∼50 μM) in agreement with published results and perhaps due to type-N self-assocn.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XptVWrtro%253D&md5=3bba1bbe7b159bdea7ae0f1dee647ef5
    78. 78
      Vaz, W. L. C.; Goodsaid-Zalduondo, F.; Jacobson, K. Lateral Diffusion of Lipids and Proteins in Bilayer Membranes. FEBS Lett. 1984, 174, 199– 207,  DOI: 10.1016/0014-5793(84)81157-6
      Google Scholar
      77
      Lateral diffusion of lipids and proteins in bilayer membranes
      Vaz, Winchil L. C.; Goodsaid-Zalduondo, Federico; Jacobson, Ken
      FEBS Letters (1984), 174 (2), 199-207CODEN: FEBLAL; ISSN:0014-5793.
      A review with 37 refs.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXls1GlsbY%253D&md5=ef8b7dc46e215bae9a0d3ea2a1b53e05
    79. 79
      Saxton, M. J. Lateral Diffusion in an Archipelago. The Effect of Mobile Obstacles. Biophys. J. 1987, 52, 989– 997,  DOI: 10.1016/S0006-3495(87)83291-5
      Google Scholar
      78
      Lateral diffusion in an archipelago. The effect of mobile obstacles
      Saxton, Michael J.
      Biophysical Journal (1987), 52 (6), 989-97CODEN: BIOJAU; ISSN:0006-3495.
      Lateral diffusion of mobile proteins and lipids (tracers) in a membrane is hindered by the presence of proteins (obstacles) in the membrane. If the obstacles are immobile, their effect may be described by percolation theory, which states that the long-range diffusion const. of the tracers goes to zero when the area fraction of obstacles is greater than the percolation threshold. If the obstacles are themselves mobile, the diffusion const. of the tracers depends on the area fraction of obstacles and the relative jump rate of tracers and obstacles. Monte Carlo calcns. are presented of diffusion consts. on square and triangular lattices as a function of the concn. of obstacles and the relative jump rate. The diffusion const. for particles of various sizes is also obtained. Calcd. values of the concn.-dependent diffusion const. are compared with obsd. values for gramicidin and bacteriorhodopsin. The effect of the proteins as inert obstacles is significant, but other factors, such as protein-protein interactions and perturbation of lipid viscosity by proteins, are of comparable importance. Potential applications include the diffusion of proteins at high concns. (such as rhodopsin in rod outer segments), the modulation of diffusion by release of membrane proteins from cytoskeletal attachment, and the diffusion of mobile redox carriers in mitochondria, chloroplasts, and endoplasmic reticulum.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXptVWgsg%253D%253D&md5=8737507970a84533ffa5b30975d5caeb
    80. 80
      Pearce, K. H.; Hof, M.; Lentz, B. R.; Thompson, N. L. Comparison of the Membrane Binding Kinetics of Bovine Prothrombin and Its Fragment 1. J. Biol. Chem. 1993, 268, 22984– 22991,  DOI: 10.1016/S0021-9258(19)49415-5
      Google Scholar
      79
      Comparison of the membrane binding kinetics of bovine prothrombin and its fragment 1
      Pearce, Kenneth H.; Hof, Martin; Lentz, Barry R.; Thompson, Nancy L.
      Journal of Biological Chemistry (1993), 268 (31), 22984-91CODEN: JBCHA3; ISSN:0021-9258.
      Total internal reflection fluorescence microscopy was used to compare the membrane-binding characteristics of fluorescein-labeled bovine prothrombin and fluorescein-labeled bovine prothrombin fragment 1. The Ca2+-dependent assocn. of these proteins with quartz-supported planar membranes composed of mixts. of phosphatidylserine (2-10 mol %) and phosphatidylcholine was examd. Equil. binding measurements showed that the apparent equil. dissocn. consts. increased with decreasing molar fractions of phosphatidylserine and that the dissocn. consts. were somewhat lower for intact prothrombin. Kinetic measurements, using fluorescence photobleaching recovery, showed that the measured dissocn. rates were approx. equiv. for prothrombin and fragment 1 and did not change with the protein soln. concn. or the molar fraction of phosphatidylserine. The kinetic data also implied that the surface-binding mechanism for both proteins is more complex than a simple reversible reaction between monovalent proteins and monovalent surface sites. Measured equil. and kinetic consts. are reported and compared for prothrombin and fragment 1 on planar membranes.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXlslyhs7Y%253D&md5=ded44c3c88afdc71e4abbb8ba93dfda2
    81. 81
      Yang, E. H.; Rode, J.; Howlader, M. A.; Eckermann, M.; Santos, J. T.; Hernandez Armada, D.; Zheng, R.; Zou, C.; Cairo, C. W. Galectin-3 Alters the Lateral Mobility and Clustering of Β1-Integrin Receptors. PLoS One 2017, 12, e0184378  DOI: 10.1371/journal.pone.0184378
      Google Scholar
      80
      Galectin-3 alters the lateral mobility and clustering of β1-integrin receptors
      Yang, Esther H.; Rode, Julia; Howlader, Md. Amran; Eckermann, Marina; Santos, Jobette T.; Armada, Daniel Hernandez; Zheng, Ruixiang; Zou, Chunxia; Cairo, Christopher W.
      PLoS One (2017), 12 (10), e0184378/1-e0184378/17CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Glycoprotein receptors are influenced by myriad intermol. interactions at the cell surface. Specific glycan structures may interact with endogenous lectins that enforce or disrupt receptor-receptor interactions. Glycoproteins bound by multivalent lectins may form extended oligomers or lattices, altering the lateral mobility of the receptor and influencing its function through endocytosis or changes in activation. In this study, we have examd. the interaction of Galectin-3 (Gal-3), a human lectin, with adhesion receptors. We measured the effect of recombinant Gal-3 added exogenously on the lateral mobility of the α5β1 integrin on HeLa cells. Using single-particle tracking (SPT) we detected increased lateral mobility of the integrin in the presence of Gal-3, while its truncated C-terminal domain (Gal-3C) showed only minor redns. in lateral mobility. Treatment of cells with Gal-3 increased β1-integrin mediated migration with no apparent changes in viability. In contrast, Gal-3C decreased both cell migration and viability. Fluorescence microscopy allowed us to confirm that exogenous Gal-3 resulted in reorganization of the integrin into larger clusters. We used a proteomics anal. to confirm that cells expressed endogenous Gal-3, and found that addn. of competitive oligosaccharide ligands for the lectin altered the lateral mobility of the integrin. Together, our results are consistent with a Gal-3-integrin lattice model of binding and confirm that the lateral mobility of integrins is natively regulated, in part, by galectins.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXpvVOktbc%253D&md5=5399528cf1dc4c395ae87febc6548e51

  • Supporting Information

    Supporting Information

    ARTICLE SECTIONS
    Jump To

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpcb.2c05717.

    • Fabrication of gold microcavity array electrodes, DLS, EIS, FLIM, AFM, and FCS data (PDF)


    Terms & Conditions

    Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

PDF [ 10MB]

Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

You’ve supercharged your research process with ACS and Mendeley!

STEP 1:
Click to create an ACS ID

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

MENDELEY PAIRING EXPIRED
Your Mendeley pairing has expired. Please reconnect