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Scale up of Transmembrane NADH Oxidation in Synthetic Giant Vesicles

  • MinHui Wang
    MinHui Wang
    Process Systems Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany
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  • , 
  • André Weber
    André Weber
    Combinatorial Neuroimaging Core Facility, Leibniz Institute for Neurobiology, Brenneckestrasse 6, 39118 Magdeburg, Germany
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  • , 
  • Roland Hartig
    Roland Hartig
    Institute of Molecular and Clinical Immunology, Otto-von-Guericke University Magdeburg, Leipziger Strasse 44, 39120 Magdeburg, Germany
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  • , 
  • Yiran Zheng
    Yiran Zheng
    Process Systems Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany
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  • , 
  • Dorothee Krafft
    Dorothee Krafft
    Process Systems Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany
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  • , 
  • Tanja Vidaković-Koch
    Tanja Vidaković-Koch
    Electrochemical Energy Conversion, Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany
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    Orcidhttp://orcid.org/0000-0003-3896-3585
  • , 
  • Werner Zuschratter
    Werner Zuschratter
    Combinatorial Neuroimaging Core Facility, Leibniz Institute for Neurobiology, Brenneckestrasse 6, 39118 Magdeburg, Germany
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  • , 
  • Ivan Ivanov*
    Ivan Ivanov
    Process Systems Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany
    *Email: [email protected]. Phone: +49 391 6110 805.
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    Orcidhttp://orcid.org/0000-0002-4675-5287
  • , and 
  • Kai Sundmacher
    Kai Sundmacher
    Process Systems Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany
    Department of Process Systems Engineering, Otto-von-Guericke University Magdeburg, Universitätsplatz 2, 39106 Magdeburg, Germany
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    Orcidhttp://orcid.org/0000-0003-3251-0593
Cite this: Bioconjugate Chem. 2021, 32, 5, 897–903
Publication Date (Web):April 27, 2021
https://doi.org/10.1021/acs.bioconjchem.1c00096

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Abstract

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The transfer of electrons across and along biological membranes drives the cellular energetics. In the context of artificial cells, it can be mimicked by minimal means, while using synthetic alternatives of the phospholipid bilayer and the electron-transducing proteins. Furthermore, the scaling up to biologically relevant and optically accessible dimensions may provide further insight and allow assessment of individual events but has been rarely attempted so far. Here, we visualized the mediated transmembrane oxidation of encapsulated NADH in giant unilamellar vesicles via confocal laser scanning and time-correlated single photon counting wide-field microscopy. To this end, we first augmented phospholipid membranes with an amphiphilic copolymer in order to check its influence on the oxidation kinetics spectrophotometrically. Then, we scaled up the compartments and followed the process microscopically.

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Membrane phenomena play a crucial role in living cells and, among other functions, enable out-of-equilibrium states, which drive the cellular metabolism. E.g., the proton gradient generated by oxidation of NADH and other reduced substrates in the electron transport chain drives ATP synthesis. (1) In the origin-of-life conundrum, the universality of these pathways contradicts the complexity of the energy-transducing machinery, which is addressed in minimal configurations of simpler and plausible catalysts. (2) On the other side, bottom-up synthetic biology is not burdened by evolutionary constraints and offers the possibility for reproduction of fundamental mechanisms by the use of highly evolved molecules or even fully synthetic alternatives. (3) In line with these motivations, we have assembled a minimal oxidative phosphorylation system in polymersomes (4) and transmembrane cofactor oxidation in liposomes, mediated by the electron shuttle tetracyanoquinodimethane (TCNQ). (5) Regarding the latter, electron transfer across vesicle bilayers has been almost exclusively studied in nanocompartments, (6) which restricts the assessment to bulk methods and may necessitate additional controls, e.g., to ensure compartment integrity. Thus, there is an incentive to employ objects, compatible with optical methods, in order to provide straightforward confirmation and access individual events. Furthermore, microcompartments such as giant unilamellar vesicles (GUVs) are established workhorses for mimicking cellular functions due to the structural and dimensional similarity to modern cells. (7)

In the present work, we spectroscopically tested the influence of the synthetic amphiphile poly(dimethylsiloxane)-graft-poly(ethylene oxide) (PDMS-g-PEO) on the interfacial oxidation of encapsulated NADH at the nanoscale, seeking improved performance. Next, we scaled up the transmembrane setup to micrometer vesicles. During the microscopic monitoring of the NADH fluorescence, we faced a bleaching issue due to the low quantum efficiency of NADH (1.9% in aqueous solution (8)). This shortcoming was amplified at high intensity or point scanning illumination, and it is expected to appear not only in minimal systems but also in living cells, provided the established role of NADH as intrinsic bioenergetic marker, (9,10) and altogether underlines the importance of sensitive low-noise imaging techniques for long-term monitoring of NADH kinetics.

Liposomes are simplified membrane models, which use the building blocks of living cells but lack sugar and protein ornaments. With respect to cellular mimicking, there is a parallel search for versatile and easily obtainable models with alternative chemistry that can provide favorable attributes for application. (11) Polymer vesicles (polymersomes) typically have enhanced stability and lower permeability, while the trade-offs between natural and synthetic membranes can be alleviated by the use of hybrid systems. (12) The latter approach is particularly relevant for the reconstitution of complex proteins when the supramolecular polymer arrangement does not correspond well to the natural phospholipid environment. PDMS-g-PEO, however, forms layers of similar thickness to lipid membranes and accommodates the bacterial proton pump cytochrome bo3 ubiquinol oxidase with fully retained activity. (4) In addition to the matching dimensions, the protein compatibility is associated with the 20-fold higher fluidity of the latter polymer membrane, compared to the canonical poly(butadiene)-block-poly(ethylene oxide) (PBd-b-PEO) (4.1 ± 0.9 vs 0.22 ± 0.06 μm2 s–1). (13,14) Since the diffusion of artificial mediators in bilayers is rate-limiting for the electron transfer, (15) we hypothesized that the increased fluidity of PDMS-g-PEO (in the order of natural lipids, e.g., 11.3 ± 1.5 μm2 s–1 for soy phosphatidylcholine (16)) will qualify it as a suitable interface for transmembrane NADH oxidation by embedded TCNQ. Therefore, we first spectrophotometrically assessed the transmembrane electron transfer in ∼150 nm large unilamellar vesicles (LUVs) made of different amphiphiles. Thereby, in addition to the pure polymer, we tested hybrid membranes with different molar ratios of PDMS-g-PEO and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and benchmarked them against lipid vesicles. (5)

The extrusion resulted in uniform size distributions (Figure S1), matching the pore size of the filter (<200 nm), and the polydispersity index was lower than 0.2. There was an overall decrease in the hydrodynamic diameter with an increasing polymer content, which may be associated with lower lysis tension, (17) but since the extrusion was done manually and the applied pressure was not controlled, we did not investigate this further. Similar stochastic dependence on the polymer content was shown for PBd-b-PEO/POPC blends. (18) TCNQ incorporation on the other side showed no significant effect on the LUV size.

For the spectrophotometric assessment of the transmembrane electron transfer in different membranes, we first performed the following control experiments for each composition in triplicates: blank vesicles, vesicles with membrane-incorporated TCNQ, and vesicles with encapsulated NADH in the presence of 400 μM ferricyanide but lacking the mediator (examples in Figures 1a and S2). Thereby, the first two controls served to validate the baseline from background fluorescence. The third control without the mediator assessed the physical separation between NADH and the external oxidant. It also allowed discriminating the initial transmembrane kinetics from the oxidation of small amounts of nonencapsulated NADH. The NADH fluorescence alone decreased by ∼10% within 3 h, but the kinetics in the presence of TCNQ was not corrected by this decay because it was not clear whether it was caused by degradation or photobleaching and if the resulting products could participate in the redox process.

Figure 1

Figure 1. a) Schematic representations of the experiments shown in b) incl. chemical structures of the amphiphiles and the mediator. Color code corresponds to the respective traces. b) Spectrophotometric fluorescence profiles of hybrid vesicles composed of 20 mol % POPC/80 mol % PDMS-g-PEO with varying inner and outer membrane compositions. c) Normalized fluorescence profiles of NADH encapsulated in different types of LUVs with embedded TCNQ upon addition of 400 μm outer ferricyanide. The schematic inset shows a possible interpretation of the interplay between membrane order and fluidity.

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The transmembrane electron transfer proceeded in vesicles with membrane-incorporated TCNQ and encapsulated NADH in the presence of 400 μM external ferricyanide (Figure 1a). We simplified the kinetic analysis (5) by fitting a single exponential decay curve to obtain a lumped rate constant (Figure S2). The fitting protocol excluded the initial steep drop originating from the oxidation of small amounts of nonencapsulated NADH, which, in turn, may have led to a slight underestimation of the rates. Altogether, the hybrids containing 20 mol % POPC surpassed the other membranes with a rate constant of 7.4 × 10–4 s–1, followed by the pure polymer (5.7 × 10–4 s–1), and were nearly two times faster than pure POPC (the respective profiles, normalized to the starting fluorescence value and after extraction of the baseline, are shown in Figure 1b). We do not expect deviations in the LUV concentrations for different amphiphiles, because we followed identical preparation protocols.

In the case of the 50 mol % polymer/lipid blend, the ferricyanide control lacking mediator exhibited exponential decay, similar to the actual test with TCNQ, while the rest of the compositions showed a fairly linear response (Figure S2). Although bleaching and degradation may also proceed exponentially, this suggested possibly compromised compartment integrity. Therefore, the 50 mol % blend was excluded from further tests. In the case of hybrid LUVs containing ≥30 mol % POPC, the potential leakage can be ascribed to increased phase separation, as observed previously, (19) which may cause multiple membrane defects at the phase junctions. In parallel, above 30 mol % POPC the demixing of polymer and lipid could result in distinct vesicle populations, which are not discernible in the present nanoscale experiments. Therefore, the membrane containing 80 mol % POPC was disregarded for further tests too, although it did not exhibit dubious behavior in the control experiments.

Considering a potential oxidant leak, the membrane integrity of the hybrids containing 20 mol % POPC was further tested by prolonged incubation in concentrated ferricyanide in order to rule out its penetration to the compartment interior. The oxidant was added to the vesicle suspensions in 50-fold concentration compared to the standard experimental conditions, and after overnight incubation, it was removed by gel filtration. No characteristic absorption at 420 nm was detected (Figure S3), and the size of the vesicles remained unchanged. While hybrid LUVs containing 30 mol % POPC previously exhibited nanodomains and enhanced proton permeability to a certain extent, full demixing to separate polymer and lipid populations was never observed via cryo electron microscopy. (16) The lower amount of lipid in the present case suggests its more uniform distribution in the PDMS-g-PEO monolayer, hence we consider the hybrid vesicles homogeneous, while virtually impermeable to larger molecules like NADH and ferricyanide. The hybrid membrane containing 20 mol % POPC and 80 mol % PDMS-g-PEO accommodates both membrane integrity and enhanced electron transfer. The origin of the latter merit cannot be correlated with the membrane fluidity because hybrid and polymer membranes exhibited lower lateral diffusion coefficients in fluorescence recovery after photobleaching (FRAP) experiments. (16) However, the increasing polymer content was also associated with a growing membrane disorder, evidenced by the bilayer probe Laurdan. Thus, we suggest that the optimal activity of hybrids originates from the trade-off between the mobility of the hydrophobic mediator and its exposure to the polar ferricyanide (and NADH), see the inset in Figure 1b. A similar effect of more pronounced quenching of photoexcited chlorophyll by ferricyanide has been ascribed to perturbation of the polar/nonpolar border and display of the chlorin ring to the water phase upon addition of cholesterol. (20)

Electroformation or electroswelling (21) is one of the most widely used techniques for making micrometer vesicles due to the simple experimental setup, short duration, and attained quality (unilamellarity) of the membrane. (7) Although tailored protocols for the growth of GUVs under physiological conditions exist, (22,23) electroformation with high salt is generally considered difficult. Therefore, we modified the composition of the NADH solution and formed an ample amount of 10–30 μm hybrid and lipid GUVs in the presence of 1 mM NADH and sucrose (Figure S4). Higher NADH concentration or the presence of buffer resulted in lower GUV yield and diameter. Dilution of the resulting suspension with isosmotic glucose settled the GUVs to the bottom of the observation slide and provided sufficient contrast between the lumen and the exterior so that the separate vesicles could be easily identified by conventional epifluorescence microscopy.

Addition of 10 mM ferricyanide significantly decreased the blue fluorescence throughout the sample (Figure S4), whereby the nonencapsulated cofactor was oxidized first. A small portion of the compartments remained unaffected, which was ascribed to the presence of multivesicular vesicles and the potentially nonuniform distribution of TCNQ. Overall, the kinetics of transmembrane oxidation could not be resolved due to the severe bleaching of NADH (the weak intrinsic fluorescence disappeared even after prolonged focusing over tens of seconds). Therefore, the utility of epifluorescence microscopy with a standard camera in the present approach remained in the initial assessment of successful GUV formation and NADH encapsulation.

Confocal laser scanning microscopy (CLSM) is an established technique to observe membrane phenomena in GUVs, and we next used it to follow the NADH oxidation. The hybrid GUVs did not exhibit membrane protrusions, and the encapsulation was more uniform in comparison to pure POPC (Figure 2). The formation of buds and tubes in lipid bilayers is due to the excess area and the spontaneous curvature, which, in turn, is affected by solute asymmetry (24) among other factors. Different salt and sugar concentrations across the membrane are present in the current system as well, but the predominant component of hybrids (i.e., PDMS-g-PEO) self-assembles into a soft monolayer, which appears to be less responsive to the unbalanced conditions. On the other side, the low encapsulation efficiency of large and charged molecules is a known drawback of both the spontaneous and the electrically assisted swelling of lipid films (7) (note the absence of a cofactor in lipid GUVs with excess surface). In the case of hybrids though, NADH evidently better penetrates the amphiphile film during the formation, possibly due to less organized multilayers. In both types of GUVs, the intensity of the membrane fluorescence varied significantly. This can be partially ascribed to positioning out of the focal plane or focus instabilities during image acquisition between channels (tiny focus drift), but the potentially nonuniform distribution of the lipid dye and TCNQ should not be discounted either.

Figure 2

Figure 2. Confocal images of 100 mol % POPC GUVs (upper panel) and 20 mol % POPC/80 mol % PDMS-g-PEO GUVs (lower panel) with embedded TCNQ and encapsulated NADH. Left: liss-labeled membrane (red); middle: encapsulated NADH (cyan); right: bright-field channel. The red signal associated with the lipid vesicles (perceived as interior) is due to membrane protrusions.

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CLSM indeed allowed monitoring of the NADH fluorescence, and the transmembrane oxidation proceeded faster (within a few minutes) compared to the nanometer scale due to the different ratios of ferricyanide to NADH. The amount of GUV-encapsulated cofactor (1 mM) was limited by the experimental procedure, and we employed higher oxidant concentration (10 mM) to accelerate the process and avoid long imaging. However, the resulting narrow time window for locating the GUVs and adjustment of imaging parameters prevented the analysis of sufficient individual events, whereby lowering the oxidant concentration twice did not solve this issue. Altogether, the mass transport could not be controlled during pipetting of ferricyanide aliquots on the observation slide. This resulted in the exposure of the compartments to different ferricyanide concentrations at different times. Moreover, bleaching of NADH hampered kinetic quantification also in CLSM (although not as pronounced as with standard epifluorescence) by the inability to discriminate it from past oxidation events.

In some cases, we noticed that the spherical membrane assumed an irregular shape upon full NADH oxidation (Figure S5), which indicated adsorption of the GUV to the glass surface (deflation was unlikely due to the matching osmolarity). Vesicle fusion to surfaces is known to be favored by lower pH and high ionic strength. (25) In fact, upon direct oxidation of 1 mM NADH by a 20-fold excess of ferricyanide in bulk, the pH decreased from 5.5 to 4.2, and therefore, the observed adsorption was ascribed to acidification of the unbuffered solution. This experimental artifact actually demonstrates that a pH gradient can be achieved merely by the liberation of protons in the reaction, as previously shown with iron–sulfur peptide catalysts. (2)

To quantify the mediated NADH oxidation in GUVs, we made use of the higher signal-to-noise ratio of a novel time-correlated single-photon counting camera under very low illumination, which was developed for wide-field fluorescence lifetime imaging (FLIM). (26) The background photons during the experiment, acquired by blocking the laser, were below 60 per second. A similar setup was previously used to unveil the desynchronization of glycolytic oscillations in yeast. (27,28) The short time window after ferricyanide addition made it difficult to follow enough hybrid GUVs and prevented their analysis, but quantification of lipid GUVs was successful due to their slower transmembrane electron transfer kinetics. The time-resolved fluorescence of individual lipid vesicles varied with respect to the initial intensity (Figure 3a), which was due to the combination of different encapsulations and the integral photon detection (larger GUVs emitted more photons in sum, as evidenced by a positive linear correlation with the diameter: ρ = 0.76, Figure S6). Nevertheless, the vast majority of the compartments exhibited a common sigmoidal profile with a varying onset (Figure S7) upon addition of oxidant, while the GUVs lacking the mediator were unresponsive as expected (normalized traces without and with TCNQ in Figures 3b and 3c).

Figure 3

Figure 3. A) Time lapse of NADH oxidation in lipid (100% POPC) GUVs with embedded TCNQ monitored by time-correlated single photon counting (TCSPC) wide-field microscopy. Approximate time upon addition of ferricyanide is indicated in the upper right. B) Normalized NADH fluorescence of GUVs without TCNQ in the presence of ferricyanide. C) Selected profiles of the normalized NADH fluorescence of GUVs with embedded TCNQ in the presence of ferricyanide. Several traces are grouped by onset and designated by color code (orange, blue, green, red). Inset shows the respective position of the GUVs with the same color code.

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The mild excitation by short laser pulses (averaged laser power <5 mW cm–2) resulted in negligible bleaching. This was tested before the addition of ferricyanide over several hours (time course within test window in Figure S8). The stability of the NADH signal allowed for extraction of the lumped electron transfer rate constants by following a manual workflow. Toward this end, the fluorescence intensity before the decrease was averaged, and the value was used to normalize the respective signal for more convenient comparison (Figure S9). The fluorescence profiles were then fitted by an exponential function, starting from the highest decrease rate, and GUVs that did not exhibit a clearly defined sigmoidal profile (<30%) were excluded. This resulted in an apparent electron transfer rate constant of 2.8 ± 1.8 × 10–2 s–1 for POPC GUVs. The slower decrease at the beginning and the delayed onsets of NADH oxidation were ascribed to the highly irregular ferricyanide diffusion front (onsets could not be correlated to the position of the GUVs, Figure 3c).

Unlike the bulk analysis at the nanoscale, which determines the behavior of the entire population, monitoring individual GUVs may provide a finer level of detail. In fact, observation of single nanovesicles has been reported several times, e.g., for deeper analysis of proton permeation by total internal reflection fluorescence (TIRF) microscopy. (29) However, the optical access at the microscale potentially enables the correlation of activity with additional factors such as size, number of lamellae, membrane protrusions, etc. For this reason though, the ferricyanide supply would need to be controlled, e.g., by the use of microfluidic devices. (30)

The NAD(P)/NAD(P)H ratio is a hallmark for many cellular processes, and the NAD(P)H signal is widely used as a readout for enzymatic fluorescent assays. Precise NADH analysis is required for the design of minimal systems in the context of bottom-up synthetic biology as well. (31,32) In the present study, we showed that synthetic augmentation may improve the transmembrane electron transfer by introducing favorable membrane properties, while the scaling up to micrometer vesicles provided an unequivocal demonstration of the process at biologically relevant dimensions and NADH concentrations. Thereby, the analysis of the membrane and oxidation kinetics required the use of CLSM and time-correlated single photon counting (TCSPC) wide-field microscopy. The latter imaging method accounts for sample heterogeneity and can be easily extrapolated to the investigation of passive or facilitated membrane transport, when using other sensitive fluorophores.

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.bioconjchem.1c00096.

  • Materials and methods; size distribution of LUVs (DLS); fluorescent/UV–vis spectrophotometric data for electron transfer/ferricyanide leakage; epifluorescence/confocal images of GUVs; TCSPC-based fluorescence profiles of GUVs; and workflow for extraction of electron transfer rate constants (PDF)

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  • Corresponding Author
  • Authors
    • MinHui Wang - Process Systems Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany
    • André Weber - Combinatorial Neuroimaging Core Facility, Leibniz Institute for Neurobiology, Brenneckestrasse 6, 39118 Magdeburg, Germany
    • Roland Hartig - Institute of Molecular and Clinical Immunology, Otto-von-Guericke University Magdeburg, Leipziger Strasse 44, 39120 Magdeburg, Germany
    • Yiran Zheng - Process Systems Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany
    • Dorothee Krafft - Process Systems Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany
    • Tanja Vidaković-Koch - Electrochemical Energy Conversion, Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany;  Orcidhttp://orcid.org/0000-0003-3896-3585
    • Werner Zuschratter - Combinatorial Neuroimaging Core Facility, Leibniz Institute for Neurobiology, Brenneckestrasse 6, 39118 Magdeburg, Germany
    • Kai Sundmacher - Process Systems Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstrasse 1, 39106 Magdeburg, Germany;  Department of Process Systems Engineering, Otto-von-Guericke University Magdeburg, Universitätsplatz 2, 39106 Magdeburg, Germany;  Orcidhttp://orcid.org/0000-0003-3251-0593
  • Notes
    The authors declare no competing financial interest.

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This work is part of the MaxSynBio consortium, which is jointly funded by the Federal Ministry of Education and Research of Germany (BMBF) and the Max Planck Society (MPG). A.W. and W.Z. acknowledge funding by DFG SFB 854 TP Z01.

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    The design of efficient schemes for NAD regeneration is essential for the development of enzymic biotechnol. processes in order to sustain continuous prodn. In line with our motivation for encapsulation of redox cascades in liposomes to serve as microbioreactors, we developed a straightforward strategy for interfacial oxidn. of entrapped NADH by ferricyanide as external electron acceptor. Instead of the commonly applied enzymic regeneration methods, we employed hydrophobic redox shuttle embedded in the liposome bilayer. Tetracyanoquinodimethane (TCNQ) mediated electron transfer across the membrane and thus allowed us to shortcut and to emulate part of the electron transfer chain functionality without the involvement of membrane proteins. To describe the exptl. system we developed a math. model, which allowed for detn. of electron transfer rate consts. and exhibited handy predictive utility.
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    Robinson, J. N. and Colehamilton, D. J. (1991) Electron-Transfer across Vesicle Bilayers. Chem. Soc. Rev. 20 (1), 49– 94,  DOI: 10.1039/cs9912000049
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    Electron transfer across vesicle bilayers
    Robinson, Julian N.; Cole-Hamilton, David J.
    Chemical Society Reviews (1991), 20 (1), 49-94CODEN: CSRVBR; ISSN:0306-0012.
    A review with many refs. on electron transfer reactions in vesicle systems, including ground state electron transfer reactions across phospholipid-based vesicle bilayers and excited state electron transfer reactions in predominately (a) natural product (phospholipid) and (b) synthetic (surfactant) vesicle assemblies, in each case considering charge sepn. phenomena localized at one or both bilayer surfaces before examg. transmembrane redox reactions.
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    Walde, P., Cosentino, K., Engel, H., and Stano, P. (2010) Giant vesicles: preparations and applications. ChemBioChem 11 (7), 848– 65,  DOI: 10.1002/cbic.201000010
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    Giant Vesicles: preparations and Applications
    Walde, Peter; Cosentino, Katia; Engel, Helen; Stano, Pasquale
    ChemBioChem (2010), 11 (7), 848-865CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. There is considerable interest in prepg. cell-sized giant unilamellar vesicles from natural or nonnatural amphiphiles because a giant vesicle membrane resembles the self-closed lipid matrix of the plasma membrane of all biol. cells. Currently, giant vesicles are applied to investigate certain aspects of biomembranes. Examples include lateral lipid heterogeneities, membrane budding and fission, activities of reconstituted membrane proteins, or membrane permeabilization caused by added chem. compds. One of the challenging applications of giant vesicles include gene expressions inside the vesicles with the ultimate goal of constructing a dynamic artificial cell-like system that is endowed with all those essential features of living cells that distinguish them from the nonliving form of matter. Although this goal still seems to be far away and currently difficult to reach, it is expected that progress in this and other fields of giant vesicle research strongly depend on whether reliable methods for the reproducible prepn. of giant vesicles are available. The key concepts of currently known methods for prepg. giant unilamellar vesicles are summarized, and advantages and disadvantages of the main methods are compared and critically discussed.
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  8. 8
    Scott, T. G., Spencer, R. D., Leonard, N. J., and Weber, G. (1970) Synthetic spectroscopic models related to coenzymes and base pairs. V. Emission properties of NADH. Studies of fluorescence lifetimes and quantum efficiencies of NADH, AcPyADH, [reduced acetylpyridineadenine dinucleotide] and simplified synthetic models. J. Am. Chem. Soc. 92 (3), 687– 695,  DOI: 10.1021/ja00706a043
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    8
    Synthetic spectroscopic models related to coenzymes and base pairs. V. Emission properties of NADH. Studies of fluorescence lifetimes and quantum efficiencies of NADH, AcPyADH, [reduced acetylpyridineadenine dinucleotide] and simplified synthetic models
    Scott, T. Gordon; Spencer, Richard D.; Leonard, Nelson J.; Weber, Gregorio
    Journal of the American Chemical Society (1970), 92 (3), 687-95CODEN: JACSAT; ISSN:0002-7863.
    The fluorescence lifetimes τ and quantum efficiencies -q of NADH, AcPyADH, and of the model compds. Ad-C3-NicH (1-[3-(aden-9-yl)-propyl]-3-carbamoyl-1,4-dihydropyridine) and Ad-C6-NicH (1-[6-(aden-9-yl)-hexyl]-3-carbamoyl-1,4-dihydropyridine) have been measured in water and in 1,2-propanediol soln. at 0-30°. For Ad-C3-NicH, NADH, and Ad-C6-NicH at 25° in water the abs. quantum efficiencies are 0.035, 0.019, and 0.017, resp., to a precision of a few percent, and the lifetimes are 0.70, 0.40, and 0.28 ± 0.03 to 0.05 nsec. The contribution of the ground to lowest singlet transition to the absorption spectrum has been evaluated by fluorescence polarization observations, and from these and the mol. fluorescence spectra, emissive lifetimes τe have been calcd. by the equation of Strickler and Berg. For NADH and AcPyADH under various conditions, the relationτ = τe-q is followed rigorously over a 30-fold change in quantum efficiency. The abs. efficiency of quinine sulfate measured either by comparison with these derivs. or by the relation ‾q = τ/τe is 0.70 ± 0.02 rather than the often quoted 0.55. The obsd. quantum efficiencies of energy transfer from the adenine to the dihydronicotinamide moiety were, for Ad-C3-NicH, NADH, and Ad-C6-NicH at 25° in aq. soln. 0.44, 0.34, and 0.10, resp. For these 3 compds., comparison of the rates and of the energies of activation for radiationless transitions calcd. from the temp. dependence of the lifetimes shows that the quenching processes are essentially identical in 1,2-propanediol but different in aq.soln., indicating that interactions in water, but not in 1,2-propanediol, are characteristic and specific for each compd. The simplified spectroscopic models, Ad-C3-NicH and Ad-C6-NicH, which were designed to incorporate the absorption-emission chromophores of NADH by linking the adenyl and dihydronicotinamide moieties of NADH with triand hexamethylene chains, were prepd. by dithionite redn. of the corresponding precursors possessing the nicotinamide ring in oxidized form.
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  9. 9
    Kolenc, O. I. and Quinn, K. P. (2019) Evaluating Cell Metabolism Through Autofluorescence Imaging of NAD(P)H and FAD. Antioxid. Redox Signaling 30 (6), 875– 889,  DOI: 10.1089/ars.2017.7451
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    9
    Evaluating Cell Metabolism Through Autofluorescence Imaging of NAD(P)H and FAD
    Kolenc, Olivia I.; Quinn, Kyle P.
    Antioxidants & Redox Signaling (2019), 30 (6), 875-889CODEN: ARSIF2; ISSN:1523-0864. (Mary Ann Liebert, Inc.)
    Significance: Optical imaging using the endogenous fluorescence of metabolic cofactors has enabled nondestructive examn. of dynamic changes in cell and tissue function both in vitro and in vivo. Quantifying NAD(P)H and FAD fluorescence through an optical redox ratio and fluorescence lifetime imaging (FLIM) provides sensitivity to the relative balance between oxidative phosphorylation and glucose catabolism. Since its introduction decades ago, the use of NAD(P)H imaging has expanded to include applications involving almost every major tissue type and a variety of pathologies. Recent Advances: This review focuses on the use of two-photon excited fluorescence and NAD(P)H fluorescence lifetime techniques in cancer, neuroscience, tissue engineering, and other biomedical applications over the last 5 years. In a variety of cancer models, NAD(P)H fluorescence intensity and lifetime measurements demonstrate a sensitivity to the Warburg effect, suggesting potential for early detection or high-throughput drug screening. The sensitivity to the biosynthetic demands of stem cell differentiation and tissue repair processes indicates the range of applications for this imaging technol. may be broad. Crit. Issues: As the no. of applications for these fluorescence imaging techniques expand, identifying and characterizing addnl. intrinsic fluorophores and chromophores present in vivo will be vital to accurately measure and interpret metabolic outcomes. Understanding the full capabilities and limitations of FLIM will also be key to future advances. Future Directions: Future work is needed to evaluate whether a combination of different biochem. and structural outcomes using these imaging techniques can provide complementary information regarding the utilization of specific metabolic pathways.
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  10. 10
    Schaefer, P. M., Kalinina, S., Rueck, A., von Arnim, C. A. F., and von Einem, B. (2019) NADH Autofluorescence-A Marker on its Way to Boost Bioenergetic Research. Cytometry, Part A 95 (1), 34– 46,  DOI: 10.1002/cyto.a.23597
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    10
    NADH Autofluorescence-A Marker on its Way to Boost Bioenergetic Research
    Schaefer Patrick M; von Arnim Christine A F; von Einem Bjoern; Kalinina Sviatlana; Rueck Angelika; von Arnim Christine A F
    Cytometry. Part A : the journal of the International Society for Analytical Cytology (2019), 95 (1), 34-46 ISSN:.
    More than 60 years ago, the idea was introduced that NADH autofluorescence could be used as a marker of cellular redox state and indirectly also of cellular energy metabolism. Fluorescence lifetime imaging microscopy of NADH autofluorescence offers a marker-free readout of the mitochondrial function of cells in their natural microenvironment and allows different pools of NADH to be distinguished within a cell. Despite its many advantages in terms of spatial resolution and in vivo applicability, this technique still requires improvement in order to be fully useful in bioenergetics research. In the present review, we give a summary of technical and biological challenges that have so far limited the spread of this powerful technology. To help overcome these challenges, we provide a comprehensible overview of biological applications of NADH imaging, along with a detailed summary of valid imaging approaches that may be used to tackle many biological questions. This review is meant to provide all scientists interested in bioenergetics with support on how to embed successfully NADH imaging in their research. © 2018 International Society for Advancement of Cytometry.
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    Rideau, E., Dimova, R., Schwille, P., Wurm, F. R., and Landfester, K. (2018) Liposomes and polymersomes: a comparative review towards cell mimicking. Chem. Soc. Rev. 47 (23), 8572,  DOI: 10.1039/C8CS00162F
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    Liposomes and polymersomes: a comparative review towards cell mimicking
    Rideau, Emeline; Dimova, Rumiana; Schwille, Petra; Wurm, Frederik R.; Landfester, Katharina
    Chemical Society Reviews (2018), 47 (23), 8572-8610CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    Cells are integral to all forms of life due to their compartmentalization by the plasma membrane. However, living organisms are immensely complex. Thus there is a need for simplified and controllable models of life for a deeper understanding of fundamental biol. processes and man-made applications. This is where the bottom-up approach of synthetic biol. comes from: a stepwise assembly of biomimetic functionalities ultimately into a protocell. A fundamental feature of such an endeavor is the generation and control of model membranes such as liposomes and polymersomes. We compare and contrast liposomes and polymersomes for a better a priori choice and design of vesicles and try to understand the advantages and shortcomings assocd. with using one or the other in many different aspects (properties, synthesis, self-assembly, applications) and which aspects have been studied and developed with each type and update the current development in the field.
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    Le Meins, J. F., Schatz, C., Lecommandoux, S., and Sandre, O. (2013) Hybrid polymer/lipid vesicles: state of the art and future perspectives. Mater. Today 16 (10), 397– 402,  DOI: 10.1016/j.mattod.2013.09.002
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    Hybrid polymer/lipid vesicles: state of the art and future perspectives
    Le Meins, J-F.; Schatz, C.; Lecommandoux, S.; Sandre, O.
    Materials Today (Oxford, United Kingdom) (2013), 16 (10), 397-402CODEN: MTOUAN; ISSN:1369-7021. (Elsevier Ltd.)
    A review. Hybrid vesicles resulting from the combined self-assembly of both amphiphilic copolymers and lipids have attracted particular interest from chemists and (bio)physicists over the last five years. Such assemblies may be viewed as an advanced vesicular structure compared to their liposome and polymersome forerunners as the best characteristics from the two different systems can be integrated in a new, single vesicle. To afford such a design, the different parameters controlling both self-assembly and membrane structure must be tuned. This highlight aims to present a comprehensive overview of the fundamental aspects related to these structures, and discuss emerging developments and future applications in this field of research.
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    Dao, T. P. T., Fernandes, F., Fauquignon, M., Ibarboure, E., Prieto, M., and Le Meins, J. F. (2018) The combination of block copolymers and phospholipids to form giant hybrid unilamellar vesicles (GHUVs) does not systematically lead to ″intermediate’’ membrane properties. Soft Matter 14 (31), 6476– 6484,  DOI: 10.1039/C8SM00547H
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    13
    The combination of block copolymers and phospholipids to form giant hybrid unilamellar vesicles (GHUVs) does not systematically lead to "intermediate" membrane properties
    Dao, T. P. T.; Fernandes, F.; Fauquignon, M.; Ibarboure, E.; Prieto, M.; Le Meins, J. F.
    Soft Matter (2018), 14 (31), 6476-6484CODEN: SMOABF; ISSN:1744-6848. (Royal Society of Chemistry)
    In this work, the elasticity under stretching as well as the fluidity of Giant Hybrid Unilamellar Vesicles (GHUV) has been studied. The membrane structuration of these GHUVs has already been studied at the micro and nanoscale in a previous study of the team. These GHUVs were obtained by the assocn. of a fluid phospholipid (POPC) and a triblock copolymer, poly(ethyleneoxide)-b-poly(dimethylsiloxane)-b-poly(ethyleneoxide). Although the architecture of triblock copolymers can facilitate vesicle formation, they have been scarcely used to generate GHUVs. We show, through micropipette aspiration and FRAP expts., that the incorporation of a low amt. of lipids in the polymer membrane leads to a significant loss of the toughness of the vesicle and subtle modification of the lateral diffusion of polymer chains. We discuss the results within the framework of the conformation of the triblock copolymer chain in the membrane and in the presence of lipid nanodomains.
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    Nam, J., Beales, P. A., and Vanderlick, T. K. (2011) Giant Phospholipid/Block Copolymer Hybrid Vesicles: Mixing Behavior and Domain Formation. Langmuir 27 (1), 1– 6,  DOI: 10.1021/la103428g
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    14
    Giant Phospholipid/Block Copolymer Hybrid Vesicles: Mixing Behavior and Domain Formation
    Nam, Jin; Beales, Paul A.; Vanderlick, T. Kyle
    Langmuir (2011), 27 (1), 1-6CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
    Lipids and block copolymers can be individually assembled into unsupported, spherical membranes (liposomes or polymersomes), each having their own particular benefits and limitations. Here the authors demonstrate the prepn. of microscale, hybrid "lipopolymersomes" composed of the common lipid POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine) and the com. available copolymer PBd-b-PEO (polybutadiene-b-poly(ethylene oxide)) with the goal of incorporating the advantageous qualities of the unitary systems into mixed-membrane capsules. The authors investigate the lipopolymersomes using confocal fluorescence microscopy and demonstrate that these hybrid membranes are well mixed on nanoscopic length scales within the permittable compositional windows for hybrid vesicle formation. The authors measure the intramembrane dynamics and mech. properties of these hybrid membranes by fluorescence recovery after photobleaching (FRAP) and micropipet aspiration, resp. For the first time, the authors demonstrate the demixing of lipid-rich and polymer-rich membrane domains within the same vesicle membrane. This is achieved by the biotinylation of one of the constituent species and cross linking with the protein NeutrAvidin. The resultant domain patterning is dependent upon which component carries the biotin functionality: cross linking of the copolymer species results in domains that ripen into a single, large, copolymer-rich island, and cross linking of the lipids yields many small, "spot-like", lipid-rich domains within a copolymer-rich matrix. The authors discuss these morphol. differences in terms of the fluidity and mech. properties of the membrane phases and the possible resultant interdomain interactions within the membrane. These heterogeneous hybrid lipopolymersomes could find applications in fields such as targeted delivery, controlled release, and environmental detection assays where these capsules possess the characteristics of biocompatible lipid membranes combined with enhanced mech. strength and stability from the copolymer matrix.
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    Hammarstrom, L., Almgren, M., Lind, J., Merenyi, G., Norrby, T., and Akermark, B. (1993) Mechanisms of Transmembrane Electron-Transfer - Diffusion of Uncharged Redox Forms of Viologen, 4,4’-Bipyridine, and Nicotinamide with Long Alkyl Chains. J. Phys. Chem. 97 (39), 10083– 10091,  DOI: 10.1021/j100141a031
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    Marušič, N., Otrin, L., Zhao, Z., Lira, R. B., Kyrilis, F. L., Hamdi, F., Kastritis, P. L., Vidaković-Koch, T., Ivanov, I., and Sundmacher, K. (2020) Constructing artificial respiratory chain in polymer compartments: insights into the interplay between bo3 oxidase and the membrane. Proc. Natl. Acad. Sci. U. S. A. 117, 15006,  DOI: 10.1073/pnas.1919306117
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    16
    Constructing artificial respiratory chain in polymer compartments: Insights into the interplay between bo3 oxidase and the membrane
    Marusic Nika; Ivanov Ivan; Sundmacher Kai; Otrin Lado; Vidakovic-Koch Tanja; Zhao Ziliang; Lira Rafael B; Dimova Rumiana; Kyrilis Fotis L; Hamdi Farzad; Kastritis Panagiotis L; Kyrilis Fotis L; Hamdi Farzad; Kastritis Panagiotis L
    Proceedings of the National Academy of Sciences of the United States of America (2020), 117 (26), 15006-15017 ISSN:.
    Cytochrome bo3 ubiquinol oxidase is a transmembrane protein, which oxidizes ubiquinone and reduces oxygen, while pumping protons. Apart from its combination with F1Fo-ATPase to assemble a minimal ATP regeneration module, the utility of the proton pump can be extended to other applications in the context of synthetic cells such as transport, signaling, and control of enzymatic reactions. In parallel, polymers have been speculated to be phospholipid mimics with respect to their ability to self-assemble in compartments with increased stability. However, their usability as interfaces for complex membrane proteins has remained questionable. In the present work, we optimized a fusion/electroformation approach to reconstitute bo3 oxidase in giant unilamellar vesicles made of PDMS-g-PEO and/or phosphatidylcholine (PC). This enabled optical access, while microfluidic trapping allowed for online analysis of individual vesicles. The tight polymer membranes and the inward oriented enzyme caused 1 pH unit difference in 30 min, with an initial rate of 0.35 pH·min(-1) To understand the interplay in these composite systems, we studied the relevant mechanical and rheological membrane properties. Remarkably, the proton permeability of polymer/lipid hybrids decreased after protein insertion, while the latter also led to a 20% increase of the polymer diffusion coefficient in polymersomes. In addition, PDMS-g-PEO increased the activity lifetime and the resistance to free radicals. These advantageous properties may open diverse applications, ranging from cell-free biotechnology to biomedicine. Furthermore, the presented study serves as a comprehensive road map for studying the interactions between membrane proteins and synthetic membranes, which will be fundamental for the successful engineering of such hybrid systems.
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    Patty, P. J. and Frisken, B. J. (2003) The pressure-dependence of the size of extruded vesicles. Biophys. J. 85 (2), 996– 1004,  DOI: 10.1016/S0006-3495(03)74538-X
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    17
    The pressure-dependence of the size of extruded vesicles
    Patty, Philipus J.; Frisken, Barbara J.
    Biophysical Journal (2003), 85 (2), 996-1004CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
    Variations in the size of vesicles formed by extrusion through small pores are discussed in terms of a simple model. The authors' model predicts that the radius should decrease as the square root of the applied pressure, consistent with data for vesicles extruded under various conditions. The model also predicts dependencies on the pore size used and on the lysis tension of the vesicles being extruded that are consistent with the authors' data. The pore size was varied by using track-etched polycarbonate membranes with av. pore diams. ranging from 50 to 200 nm. To vary the lysis tension, vesicles made from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine), mixts. of POPC and cholesterol, and mixts. of POPC and C16-ceramide were studied. The lysis tension, as measured by an extrusion-based technique, of POPC:cholesterol vesicles is higher than that of pure POPC vesicles, whereas POPC:ceramide vesicles have lower lysis tensions than POPC vesicles.
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    Lim, S. K., de Hoog, H. P., Parikh, A. N., Nallani, M., and Liedberg, B. (2013) Hybrid, Nanoscale Phospholipid/Block Copolymer Vesicles. Polymers 5 (3), 1102– 1114,  DOI: 10.3390/polym5031102
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    18
    Hybrid, nanoscale phospholipid/block copolymer vesicles
    Lim, Seng Koon; de Hoog, Hans-Peter; Parikh, Atul N.; Nallani, Madhavan; Liedberg, Bo
    Polymers (Basel, Switzerland) (2013), 5 (3), 1102-1114, 13 pp.CODEN: POLYCK; ISSN:2073-4360. (MDPI AG)
    Hybrid phospholipid/block copolymer vesicles, in which the polymeric membrane is blended with phospholipids, display interesting self-assembly behavior, incorporating the robustness and chem. versatility of polymersomes with the softness and biocompatibility of liposomes. Such structures can be conveniently characterized by prepg. giant unilamellar vesicles (GUVs) via electroformation. Here, we are interested in exploring the self-assembly and properties of the analogous nanoscale hybrid vesicles (ca. 100 nm in diam.) of the same compn. prepd. by film-hydration and extrusion. We show that the self-assembly and content-release behavior of nanoscale polybutadiene-b-poly(ethylene oxide) (PB-PEO)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) hybrid phospholipid/block copolymer vesicles can be tuned by the mixing ratio of the amphiphiles. In brief, these hybrids may provide alternative tools for drug delivery purposes and mol. imaging/sensing applications and clearly open up new avenues for further investigation.
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    Chemin, M., Brun, P. M., Lecommandoux, S., Sandre, O., and Le Meins, J. F. (2012) Hybrid polymer/lipid vesicles: fine control of the lipid and polymer distribution in the binary membrane. Soft Matter 8 (10), 2867– 2874,  DOI: 10.1039/c2sm07188f
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    19
    Hybrid polymer/lipid vesicles: fine control of the lipid and polymer distribution in the binary membrane
    Chemin, Maud; Brun, Pierre-Marie; Lecommandoux, Sebastien; Sandre, Olivier; Le Meins, Jean-Francois
    Soft Matter (2012), 8 (10), 2867-2874CODEN: SMOABF; ISSN:1744-683X. (Royal Society of Chemistry)
    Hybrid polymer/lipid giant unilamellar vesicles (GUVs) were developed using lipids of resp. low and high melting transition temp. (DPPC: 1,2-dipalmitoyl-sn-glycero-3 phosphocholine, Tm = 41, and POPC: palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; Tm = -2) and a copolymer poly(dimethylsiloxane)-graft-poly(ethylene oxide) (PDMS-g-PEO) well known to self-assemble into vesicular structures. Using epifluorescence microscopy as well as differential scanning calorimetry (DSC), different structures have been identified depending on the molar compn. and on the fluid or gel state of the lipid used. The most promising objects are hybrid vesicles with copolymer as the major component, in which lipids are either randomly distributed or present "raft-like" domains in the polymer-rich membrane. The results are discussed on the basis of the fluidity of the different components and of their resp. membrane thicknesses.
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    Ford, W. E. and Tollin, G. (1984) Chlorophyll Photosensitized Electron-Transfer in Phospholipid-Bilayer Vesicle Systems - Effects of Cholesterol on Radical Yields and Kinetic-Parameters. Photochem. Photobiol. 40 (2), 249– 259,  DOI: 10.1111/j.1751-1097.1984.tb04583.x
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    20
    Chlorophyll photosensitized electron transfer in phospholipid bilayer vesicle systems: effects of cholesterol on radical yields and kinetic parameters
    Ford, William E.; Tollin, Gordon
    Photochemistry and Photobiology (1984), 40 (2), 249-59CODEN: PHCBAP; ISSN:0031-8655.
    The quenching of the triplet state of chlorophyll a (Chl) by asym. located electron acceptors was examd. in vesicle systems contg. egg yolk phosphatidylcholine and 0-50 mol% cholesterol. The incorporation of cholesterol had 2 main effects: (1) the distribution of Chl within the vesicle wall shifted from one favoring the inner monolayer to one favoring the outer monolayer, and (2) the Chl mols. (both ground and excited states) became more accessible to water and to the quencher mols. This latter property was probably due to the creation of space between the phospholipid head groups by insertion of cholesterol. These phenomena required cholesterol concns. >15 mol%. In general, the addn. of cholesterol caused increases in the apparent bimol. rate const. for triplet quenching, in the probability that quenching produced radicals, and in the rate of radical recombination. Some of the specific effects of cholesterol depended upon whether or not the quencher mols. were amphiphilic.
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    Angelova, M. I. and Dimitrov, D. S. (1986) Liposome Electroformation. Faraday Discuss. Chem. Soc. 81, 303,  DOI: 10.1039/dc9868100303
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    Liposome electroformation
    Angelova, M.; Dimitrov, D.
    Faraday Discussions of the Chemical Society (1986), 81 (1), 303-11CODEN: FDCSB7; ISSN:0301-7249.
    Liposome formation and lipid swelling on Pt electrodes in distd. water and water solns. in d.c. elec. fields were investigated for different amts. of a neg. charged lipid (mixt. from 71% phosphatidylcholines, 21.5% phosphatidylethanolamines and 7.5% phosphatidylserines), and a neutral lipid (dimyristoylphosphatidylcholine, DMPC). Neg. charged lipids do not form liposomes without fields when the thickness of the dried lipid layer is ≤90 bilayers. The rate and extent of swelling of layers thicker than 90 bilayers is largest on the cathode, smaller without fields and smallest on the anode. The theory, based on the assumption that osmotic and electrostatic forces drive lipid swelling and liposome formation. is in semi-quant. agreement with the exptl. data; in particular, it gives the obsd. linear dependence of the rate of swelling on the inverse lipid layer thickness. To induce liposome formation for layers thinner than 90 bilayers it was necessary to apply a neg. potential which is proportional to the logarithm of the inverse layer thickness. The characteristic crit. potential is proportional to RTk/F; R being the gas const., Tk the abs. temp., and F the Faraday const. This indicates that redistribution of counterions may be the cause which increases the repulsive electrostatic intermembrane forces to overcome van der Waals attraction. For thicknesses <10 bilayers, formation of very thin-walled liposomes of narrow size distribution and mean diam. of ∼30 μm was obsd. These liposomes grow in size before detachment, and a formula for the kinetics of growth was derived, which is in very good agreement with the exptl. data. The effects of d.c. field on DMPC swelling are smaller and lead to formation of liposome-like structures of different appearance. Bilayer sepn. and bending are prerequisites for liposome formation from hydrating lipids. Therefore, a possible mol. mechanism is that membranes should be destabilized to bend and fuse to form liposomes. This requires the right proportion between structured regions, in the form of bilayers, and defects and (or) nonbilayer structures, and in many cases external constraints, in particular, elec. fields.
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    Li, Q., Wang, X., Ma, S., Zhang, Y., and Han, X. (2016) Electroformation of giant unilamellar vesicles in saline solution. Colloids Surf., B 147, 368– 375,  DOI: 10.1016/j.colsurfb.2016.08.018
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    Electroformation of giant unilamellar vesicles in saline solution
    Li, Qingchuan; Wang, Xuejing; Ma, Shenghua; Zhang, Ying; Han, Xiaojun
    Colloids and Surfaces, B: Biointerfaces (2016), 147 (), 368-375CODEN: CSBBEQ; ISSN:0927-7765. (Elsevier B.V.)
    Giant unilamellar vesicle (GUV) formation on indium tin oxide (ITO) electrodes in saline soln. and from charged lipids has proven to be difficult in the past. Yet the best cell membrane models contain charged lipids and require physiol. conditions. GUVs from zwitterionic lipids, lipid mixts. and even pure charged lipids could be electroformed under physiol. conditions and even higher concns. of NaCl. The hydrophilic ITO surface may facilitate the hydration of the solid lipid film and the formation of lipid bilayers that subsequently bend and form vesicles. The formation of GUVs in saline soln. is influenced by different parameters. The influences of the amplitude and frequency of the used AC field, the NaCl concn., and the temp. were investigated. Finite element anal. simulating the effect of the elec. field on GUV formation in saline soln. could well explain the exptl. results. Frequencies in the kHz-range favored for GUVs formation in saline soln., as they suppress the formation of elec. double layer, while higher frequencies could again impair the effect of elec. field and impede GUV formation. The diams. of the GUVs increased gradually with NaCl concn. from 0 mM to 200 mM and subsequently decreased from 200 mM to 2 M. High yields of GUVs were also formed in PBS soln. and cell culture medium, which indicates this method is a promising way to prep. GUVs on a large scale in physiol. relevant conditions.
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    Zong, W., Ma, S., Zhang, X., Wang, X., Li, Q., and Han, X. (2017) A Fissionable Artificial Eukaryote-like Cell Model. J. Am. Chem. Soc. 139 (29), 9955– 9960,  DOI: 10.1021/jacs.7b04009
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    The use of artificial cells has attracted considerable attention in various fields from biotechnol. to medicine. Here, the authors develop a cell-sized vesicle-in-vesicle (VIV) structure contg. a sep. inner vesicle (IV) that can be loaded with DNA. The authors use polymerase chain reaction (PCR) to successfully amplify the amt. of DNA confined to the IV. Subsequent osmotic stress-induced fission of a mother VIV into two daughter VIVs successfully divides the IV content while keeping it confined to the IV of the daughter VIVs. The fission rate was estd. to be ∼20% quantified by fluorescence microscope. The VIV structure represents a step forward toward construction of an advanced, fissionable cell model.
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    Karimi, M., Steinkuhler, J., Roy, D., Dasgupta, R., Lipowsky, R., and Dimova, R. (2018) Asymmetric Ionic Conditions Generate Large Membrane Curvatures. Nano Lett. 18 (12), 7816– 7821,  DOI: 10.1021/acs.nanolett.8b03584
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    Asymmetric ionic conditions generate large membrane curvatures
    Karimi, Marzieh; Steinkuehler, Jan; Roy, Debjit; Dasgupta, Raktim; Lipowsky, Reinhard; Dimova, Rumiana
    Nano Letters (2018), 18 (12), 7816-7821CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
    Biol. membranes possess intrinsic asymmetry. This asymmetry is assocd. not only with leaflet compn. in terms of membrane species, but also with differences in the cytosolic and periplasmic solns. contg. macromols. and ions. There has been a long quest for understanding the effect of ions on the phys. and morphol. properties of membranes. Here, we elucidated the changes in the mech. properties of membranes exposed to asym. buffer conditions and the assocd. curvature generation. As a model system, we used giant unilamellar vesicles (GUVs) with asym. salt and sugar solns. on the 2 sides of the membrane. We aspirated the GUVs into micropipettes and attached small beads to their membranes. An optical tweezer was used to exert a local force on a bead, thereby pulling out a membrane tube from the vesicle. The assay allowed us to measure the spontaneous curvature and the bending rigidity of the bilayer in the presence of different ions and sugar. At low sugar/salt (inside/out) concns., the membrane spontaneous curvature generated by NaCl and KCl was close to zero, but neg. in the presence of LiCl. In the latter case, the membrane bulged away from the salt soln. At high sugar/salt conditions, the membranes were obsd. to become more flexible and the spontaneous curvature was enhanced to even more neg. values, comparable to those generated by some proteins. These findings revealed the reshaping role of alkali chlorides on biomembranes.
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    Formation and Spreading of Lipid Bilayers on Planar Glass Supports
    Cremer, Paul S.; Boxer, Steven G.
    Journal of Physical Chemistry B (1999), 103 (13), 2554-2559CODEN: JPCBFK; ISSN:1089-5647. (American Chemical Society)
    The fusion and spreading of phospholipid bilayers on glass surfaces was investigated as a function of pH and ionic strength. Membrane fusion to the support was favorable at high ionic strength and low pH for vesicles contg. a net neg. charge; however, neutral and pos. charged vesicles fused under all conditions attempted. This result suggests that van der Waals and electrostatic interactions govern the fusion process. Membrane spreading over a planar surface was favorable at low pH regardless of the net charge on the bilayer, and the process is driven by van der Waals forces. On the other hand membrane propagation is impeded at high pH or on highly curved surfaces. In this case a combination of hydration and bending interactions is primarily responsible for arresting the spreading process. These results provide a framework for understanding many of the factors that influence the effectiveness of scratches on planar supported bilayers as barriers to lateral diffusion and lead to a simple method to heal these scratches.
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    Weber, A., Prokazov, Y., Zuschratter, W., and Hauser, M. J. B. (2012) Desynchronisation of Glycolytic Oscillations in Yeast Cell Populations. PLoS One 7 (9), e43276,  DOI: 10.1371/journal.pone.0043276
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    Desynchronisation of glycolytic oscillations in yeast cell populations
    Weber, Andre; Prokazov, Yury; Zuschratter, Werner; Hauser, Marcus J. B.
    PLoS One (2012), 7 (9), e43276CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
    Glycolytic oscillations of intact yeast cells of the strain Saccharomyces carlsbergensis were investigated at both the levels of cell populations and of individual cells. Individual cells showed glycolytic oscillations even at very low cell densities (e.g. 1.0 × 105 cells/mL). By contrast, the collective behavior on the population level was cell d.-dependent: at high cell densities it is oscillatory, but below the threshold d. of 1.0 × 106 cells/mL the collective dynamics becomes quiescent. We demonstrate that the transition in the collective dynamics is caused by the desynchronization of the oscillations of individual cells. This is characteristic for a Kuramoto transition. Spatially resolved measurements at low cell densities revealed that even cells that adhere to their neighbors oscillated with their own, independent frequencies and phases.
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    Weber, A., Zuschratter, W., and Hauser, M. J. B. (2020) Partial synchronisation of glycolytic oscillations in yeast cell populations. Sci. Rep. 10 (1), 19714,  DOI: 10.1038/s41598-020-76242-8
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    Partial synchronisation of glycolytic oscillations in yeast cell populations
    Weber, Andre; Zuschratter, Werner; Hauser, Marcus J. B.
    Scientific Reports (2020), 10 (1), 19714CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
    Abstr.: The transition between synchronized and asynchronous behavior of immobilized yeast cells of the strain Saccharomyces carlsbergensis was investigated by monitoring the autofluorescence of the coenzyme NADH. In populations of intermediate cell densities the individual cells remained oscillatory, whereas on the level of the cell population both a partially synchronized and an asynchronous state were accessible for exptl. studies. In the partially synchronized state, the mean oscillatory frequency was larger than that of the cells in the asynchronous state. This suggests that synchronisation occurred due to entrainment by the cells that oscillated more rapidly. This is typical for synchronisation due to phase advancement. Furthermore, the synchronisation of the frequency of the glycolytic oscillations preceded the synchronisation of their phases. However, the cells did not synchronize completely, as the distribution of the oscillatory frequencies only narrowed but did not collapse to a unique frequency. Cells belonging to spatially denser clusters showed a slightly enhanced local synchronisation during the episode of partial synchronisation. Neither the clusters nor a transition from partially synchronized glycolytic oscillations to travelling glycolytic waves did substantially affect the degree of partial synchronisation. Chimera states, i.e., the coexistence of a synchronized and an asynchronous part of the population, could not be found.
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    Kuyper, C. L., Kuo, J. S., Mutch, S. A., and Chiu, D. T. (2006) Proton permeation into single vesicles occurs via a sequential two-step mechanism and is heterogeneous. J. Am. Chem. Soc. 128 (10), 3233– 3240,  DOI: 10.1021/ja057349c
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    Proton Permeation into Single Vesicles Occurs via a Sequential Two-Step Mechanism and Is Heterogeneous
    Kuyper, Christopher L.; Kuo, Jason S.; Mutch, Sarah A.; Chiu, Daniel T.
    Journal of the American Chemical Society (2006), 128 (10), 3233-3240CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    This article describes the first single-vesicle study of proton permeability across the lipid membrane of small (∼100 nm) uni- and multilamellar vesicles, which were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). To follow proton permeation into the internal vol. of each vesicle, we encapsulated carboxyfluorescein, a pH-sensitive dye whose fluorescence was quenched in the presence of excess protons. A microfluidic platform was used for easy exchange of high- and low-pH solns., and fluorescence quenching of single vesicles was detected with single-mol. total internal reflection fluorescence (TIRF) microscopy. Upon soln. exchange and acidification of the extravesicular soln. (from pH 9 to 3.5), we obsd. for each vesicle a biphasic decay in fluorescence. Through single-vesicle anal., we found that rate consts. for the first decay followed a Poisson distribution, whereas rate consts. for the second decay followed a normal distribution. We propose that proton permeation into each vesicle first arose from formation of transient pores and then transitioned into the second decay phase, which occurred by the soly.-diffusion mechanism. Furthermore, for the bulk population of vesicles, the decay rate const. and vesicle intensity (dependent on size) correlated to give an av. permeability coeff.; however, for individual vesicles, we found little correlation, which suggested that proton permeability among single vesicles was heterogeneous in our expts.
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    Yandrapalli, N., Seemann, T., and Robinson, T. (2020) On-Chip Inverted Emulsion Method for Fast Giant Vesicle Production, Handling, and Analysis. Micromachines 11 (3), 285,  DOI: 10.3390/mi11030285
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    Beneyton, T., Krafft, D., Bednarz, C., Kleineberg, C., Woelfer, C., Ivanov, I., Vidaković-Koch, T., Sundmacher, K., and Baret, J.-C. (2018) Out-of-equilibrium microcompartments for the bottom-up integration of metabolic functions. Nat. Commun. 9 (1), 2391,  DOI: 10.1038/s41467-018-04825-1
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    Out-of-equilibrium microcompartments for the bottom-up integration of metabolic functions
    Beneyton Thomas; Baret Jean-Christophe; Krafft Dorothee; Bednarz Claudia; Kleineberg Christin; Woelfer Christian; Ivanov Ivan; Vidakovic-Koch Tanja; Sundmacher Kai; Sundmacher Kai
    Nature communications (2018), 9 (1), 2391 ISSN:.
    Self-sustained metabolic pathways in microcompartments are the corner-stone for living systems. From a technological viewpoint, such pathways are a mandatory prerequisite for the reliable design of artificial cells functioning out-of-equilibrium. Here we develop a microfluidic platform for the miniaturization and analysis of metabolic pathways in man-made microcompartments formed of water-in-oil droplets. In a modular approach, we integrate in the microcompartments a nicotinamide adenine dinucleotide (NAD)-dependent enzymatic reaction and a NAD-regeneration module as a minimal metabolism. We show that the microcompartments sustain a metabolically active state until the substrate is fully consumed. Reversibly, the external addition of the substrate reboots the metabolic activity of the microcompartments back to an active state. We therefore control the metabolic state of thousands of independent monodisperse microcompartments, a step of relevance for the construction of large populations of metabolically active artificial cells.
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    Biner, O., Fedor, J. G., Yin, Z., and Hirst, J. (2020) Bottom-Up Construction of a Minimal System for Cellular Respiration and Energy Regeneration. ACS Synth. Biol. 9 (6), 1450– 1459,  DOI: 10.1021/acssynbio.0c00110
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    Bottom-Up Construction of a Minimal System for Cellular Respiration and Energy Regeneration
    Biner, Olivier; Fedor, Justin G.; Yin, Zhan; Hirst, Judy
    ACS Synthetic Biology (2020), 9 (6), 1450-1459CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
    ATP, the cellular energy currency, is essential for life. The ability to provide a const. supply of ATP is therefore crucial for the construction of artificial cells in synthetic biol. Here, the authors describe the bottom-up assembly and characterization of a minimal respiratory system that uses NADH as a fuel to produce ATP from ADP and inorg. phosphate, and is thus capable of sustaining both upstream metabolic processes that rely on NAD+, and downstream energy-demanding processes that are powered by ATP hydrolysis. A detergent-mediated approach was used to coreconstitute respiratory mitochondrial complex I and an F-type ATP synthase into nanosized liposomes. Addn. of the alternative oxidase to the resulting proteoliposomes produced a minimal artificial "organelle" that reproduces the energy-converting catalytic reactions of the mitochondrial respiratory chain: NADH oxidn., ubiquinone cycling, oxygen redn., proton pumping, and ATP synthesis. As a proof-of-principle, the authors demonstrate that the nanovesicles are capable of using an NAD+-linked substrate to drive cell-free protein expression. The nanovesicles are both efficient and durable and may be applied to sustain artificial cells in future work.
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Cited By

This article is cited by 2 publications.

  1. Camila Guindani, Lucas Caire da Silva, Shoupeng Cao, Tsvetomir Ivanov, Katharina Landfester. Synthetic Cells: From Simple Bio‐Inspired Modules to Sophisticated Integrated Systems. Angewandte Chemie 2022, 134 (16) https://doi.org/10.1002/ange.202110855
  2. Camila Guindani, Lucas Caire da Silva, Shoupeng Cao, Tsvetomir Ivanov, Katharina Landfester. Synthetic Cells: From Simple Bio‐Inspired Modules to Sophisticated Integrated Systems. Angewandte Chemie International Edition 2022, 61 (16) https://doi.org/10.1002/anie.202110855
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  • Abstract

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    Figure 1

    Figure 1. a) Schematic representations of the experiments shown in b) incl. chemical structures of the amphiphiles and the mediator. Color code corresponds to the respective traces. b) Spectrophotometric fluorescence profiles of hybrid vesicles composed of 20 mol % POPC/80 mol % PDMS-g-PEO with varying inner and outer membrane compositions. c) Normalized fluorescence profiles of NADH encapsulated in different types of LUVs with embedded TCNQ upon addition of 400 μm outer ferricyanide. The schematic inset shows a possible interpretation of the interplay between membrane order and fluidity.

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    Figure 2

    Figure 2. Confocal images of 100 mol % POPC GUVs (upper panel) and 20 mol % POPC/80 mol % PDMS-g-PEO GUVs (lower panel) with embedded TCNQ and encapsulated NADH. Left: liss-labeled membrane (red); middle: encapsulated NADH (cyan); right: bright-field channel. The red signal associated with the lipid vesicles (perceived as interior) is due to membrane protrusions.

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    Figure 3

    Figure 3. A) Time lapse of NADH oxidation in lipid (100% POPC) GUVs with embedded TCNQ monitored by time-correlated single photon counting (TCSPC) wide-field microscopy. Approximate time upon addition of ferricyanide is indicated in the upper right. B) Normalized NADH fluorescence of GUVs without TCNQ in the presence of ferricyanide. C) Selected profiles of the normalized NADH fluorescence of GUVs with embedded TCNQ in the presence of ferricyanide. Several traces are grouped by onset and designated by color code (orange, blue, green, red). Inset shows the respective position of the GUVs with the same color code.

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    This article references 32 other publications.

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      Nature Catalysis (2018), 1 (8), 616-623CODEN: NCAACP; ISSN:2520-1158. (Nature Research)
      Prebiotic chem. was likely mediated by metals, but how such prebiotic chem. progressed into the metabolic-like networks needed to sustain life remains unclear. Here we exptl. delineate a potential path from prebiotically plausible iron-sulfur peptide catalysts to the types of pH gradients exploited by all known living organisms. Iron-sulfur peptides cooperatively accept electrons from NADH in a manner that is only partially mediated by ionic interactions. The electrons are then either passed to a terminal electron acceptor, such as hydrogen peroxide, or to an intermediate carrier, such as ubiquinone. The redn. of hydrogen peroxide leads to the prodn. of hydroxide, which then contributes to the formation of a pH gradient across late protocell membranes. The data are consistent with the activity of prebiotic iron-sulfur peptide catalysts providing a selective advantage by equipping protocells with a pathway that connects catabolism to anabolism.
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      A review. A large German research consortium mainly within the Max Planck Society ("MaxSynBio") was formed to investigate living systems from a fundamental perspective. The research program of MaxSynBio relies solely on the bottom-up approach to synthetic biol. MaxSynBio focuses on the detailed anal. and understanding of essential processes of life through modular reconstitution in minimal synthetic systems. The ultimate goal is to construct a basic living unit entirely from non-living components. The fundamental insights gained from the activities in MaxSynBio could eventually be utilized for establishing a new generation of biotechnol. processes, which would be based on synthetic cell constructs that replace the natural cells currently used in conventional biotechnol.
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      Otrin, Lado; Marusic, Nika; Bednarz, Claudia; Vidakovic-Koch, Tanja; Lieberwirth, Ingo; Landfester, Katharina; Sundmacher, Kai
      Nano Letters (2017), 17 (11), 6816-6821CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
      For energy supply to biomimetic constructs, a complex chem. energy-driven ATP-generating artificial system was built. The system was assembled with bottom-up detergent-mediated reconstitution of an ATP synthase and a terminal oxidase into two types of novel nanocontainers, built from either graft copolymer membranes or from hybrid graft copolymer/lipid membranes. The versatility and biocompatibility of the proposed nanocontainers was demonstrated through convenient system assembly and through high retained activity of both membrane-embedded enzymes. In the future, the nanocontainers might be used as a platform for the functional reconstitution of other complex membrane proteins and could considerably expedite the design of nanoreactors, biosensors, and artificial organelles.
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    5. 5
      Wang, M. H., Woelfer, C., Otrin, L., Ivanov, I., Vidakovic-Koch, T., and Sundmacher, K. (2018) Transmembrane NADH Oxidation with Tetracyanoquinodimethane. Langmuir 34 (19), 5435– 5443,  DOI: 10.1021/acs.langmuir.8b00443
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      Transmembrane NADH Oxidation with Tetracyanoquinodimethane
      Wang, Min Hui; Woelfer, Christian; Otrin, Lado; Ivanov, Ivan; Vidakovic-Koch, Tanja; Sundmacher, Kai
      Langmuir (2018), 34 (19), 5435-5443CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      The design of efficient schemes for NAD regeneration is essential for the development of enzymic biotechnol. processes in order to sustain continuous prodn. In line with our motivation for encapsulation of redox cascades in liposomes to serve as microbioreactors, we developed a straightforward strategy for interfacial oxidn. of entrapped NADH by ferricyanide as external electron acceptor. Instead of the commonly applied enzymic regeneration methods, we employed hydrophobic redox shuttle embedded in the liposome bilayer. Tetracyanoquinodimethane (TCNQ) mediated electron transfer across the membrane and thus allowed us to shortcut and to emulate part of the electron transfer chain functionality without the involvement of membrane proteins. To describe the exptl. system we developed a math. model, which allowed for detn. of electron transfer rate consts. and exhibited handy predictive utility.
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      Robinson, J. N. and Colehamilton, D. J. (1991) Electron-Transfer across Vesicle Bilayers. Chem. Soc. Rev. 20 (1), 49– 94,  DOI: 10.1039/cs9912000049
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      Electron transfer across vesicle bilayers
      Robinson, Julian N.; Cole-Hamilton, David J.
      Chemical Society Reviews (1991), 20 (1), 49-94CODEN: CSRVBR; ISSN:0306-0012.
      A review with many refs. on electron transfer reactions in vesicle systems, including ground state electron transfer reactions across phospholipid-based vesicle bilayers and excited state electron transfer reactions in predominately (a) natural product (phospholipid) and (b) synthetic (surfactant) vesicle assemblies, in each case considering charge sepn. phenomena localized at one or both bilayer surfaces before examg. transmembrane redox reactions.
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      Walde, P., Cosentino, K., Engel, H., and Stano, P. (2010) Giant vesicles: preparations and applications. ChemBioChem 11 (7), 848– 65,  DOI: 10.1002/cbic.201000010
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      Giant Vesicles: preparations and Applications
      Walde, Peter; Cosentino, Katia; Engel, Helen; Stano, Pasquale
      ChemBioChem (2010), 11 (7), 848-865CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. There is considerable interest in prepg. cell-sized giant unilamellar vesicles from natural or nonnatural amphiphiles because a giant vesicle membrane resembles the self-closed lipid matrix of the plasma membrane of all biol. cells. Currently, giant vesicles are applied to investigate certain aspects of biomembranes. Examples include lateral lipid heterogeneities, membrane budding and fission, activities of reconstituted membrane proteins, or membrane permeabilization caused by added chem. compds. One of the challenging applications of giant vesicles include gene expressions inside the vesicles with the ultimate goal of constructing a dynamic artificial cell-like system that is endowed with all those essential features of living cells that distinguish them from the nonliving form of matter. Although this goal still seems to be far away and currently difficult to reach, it is expected that progress in this and other fields of giant vesicle research strongly depend on whether reliable methods for the reproducible prepn. of giant vesicles are available. The key concepts of currently known methods for prepg. giant unilamellar vesicles are summarized, and advantages and disadvantages of the main methods are compared and critically discussed.
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    8. 8
      Scott, T. G., Spencer, R. D., Leonard, N. J., and Weber, G. (1970) Synthetic spectroscopic models related to coenzymes and base pairs. V. Emission properties of NADH. Studies of fluorescence lifetimes and quantum efficiencies of NADH, AcPyADH, [reduced acetylpyridineadenine dinucleotide] and simplified synthetic models. J. Am. Chem. Soc. 92 (3), 687– 695,  DOI: 10.1021/ja00706a043
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      Synthetic spectroscopic models related to coenzymes and base pairs. V. Emission properties of NADH. Studies of fluorescence lifetimes and quantum efficiencies of NADH, AcPyADH, [reduced acetylpyridineadenine dinucleotide] and simplified synthetic models
      Scott, T. Gordon; Spencer, Richard D.; Leonard, Nelson J.; Weber, Gregorio
      Journal of the American Chemical Society (1970), 92 (3), 687-95CODEN: JACSAT; ISSN:0002-7863.
      The fluorescence lifetimes τ and quantum efficiencies -q of NADH, AcPyADH, and of the model compds. Ad-C3-NicH (1-[3-(aden-9-yl)-propyl]-3-carbamoyl-1,4-dihydropyridine) and Ad-C6-NicH (1-[6-(aden-9-yl)-hexyl]-3-carbamoyl-1,4-dihydropyridine) have been measured in water and in 1,2-propanediol soln. at 0-30°. For Ad-C3-NicH, NADH, and Ad-C6-NicH at 25° in water the abs. quantum efficiencies are 0.035, 0.019, and 0.017, resp., to a precision of a few percent, and the lifetimes are 0.70, 0.40, and 0.28 ± 0.03 to 0.05 nsec. The contribution of the ground to lowest singlet transition to the absorption spectrum has been evaluated by fluorescence polarization observations, and from these and the mol. fluorescence spectra, emissive lifetimes τe have been calcd. by the equation of Strickler and Berg. For NADH and AcPyADH under various conditions, the relationτ = τe-q is followed rigorously over a 30-fold change in quantum efficiency. The abs. efficiency of quinine sulfate measured either by comparison with these derivs. or by the relation ‾q = τ/τe is 0.70 ± 0.02 rather than the often quoted 0.55. The obsd. quantum efficiencies of energy transfer from the adenine to the dihydronicotinamide moiety were, for Ad-C3-NicH, NADH, and Ad-C6-NicH at 25° in aq. soln. 0.44, 0.34, and 0.10, resp. For these 3 compds., comparison of the rates and of the energies of activation for radiationless transitions calcd. from the temp. dependence of the lifetimes shows that the quenching processes are essentially identical in 1,2-propanediol but different in aq.soln., indicating that interactions in water, but not in 1,2-propanediol, are characteristic and specific for each compd. The simplified spectroscopic models, Ad-C3-NicH and Ad-C6-NicH, which were designed to incorporate the absorption-emission chromophores of NADH by linking the adenyl and dihydronicotinamide moieties of NADH with triand hexamethylene chains, were prepd. by dithionite redn. of the corresponding precursors possessing the nicotinamide ring in oxidized form.
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    9. 9
      Kolenc, O. I. and Quinn, K. P. (2019) Evaluating Cell Metabolism Through Autofluorescence Imaging of NAD(P)H and FAD. Antioxid. Redox Signaling 30 (6), 875– 889,  DOI: 10.1089/ars.2017.7451
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      Evaluating Cell Metabolism Through Autofluorescence Imaging of NAD(P)H and FAD
      Kolenc, Olivia I.; Quinn, Kyle P.
      Antioxidants & Redox Signaling (2019), 30 (6), 875-889CODEN: ARSIF2; ISSN:1523-0864. (Mary Ann Liebert, Inc.)
      Significance: Optical imaging using the endogenous fluorescence of metabolic cofactors has enabled nondestructive examn. of dynamic changes in cell and tissue function both in vitro and in vivo. Quantifying NAD(P)H and FAD fluorescence through an optical redox ratio and fluorescence lifetime imaging (FLIM) provides sensitivity to the relative balance between oxidative phosphorylation and glucose catabolism. Since its introduction decades ago, the use of NAD(P)H imaging has expanded to include applications involving almost every major tissue type and a variety of pathologies. Recent Advances: This review focuses on the use of two-photon excited fluorescence and NAD(P)H fluorescence lifetime techniques in cancer, neuroscience, tissue engineering, and other biomedical applications over the last 5 years. In a variety of cancer models, NAD(P)H fluorescence intensity and lifetime measurements demonstrate a sensitivity to the Warburg effect, suggesting potential for early detection or high-throughput drug screening. The sensitivity to the biosynthetic demands of stem cell differentiation and tissue repair processes indicates the range of applications for this imaging technol. may be broad. Crit. Issues: As the no. of applications for these fluorescence imaging techniques expand, identifying and characterizing addnl. intrinsic fluorophores and chromophores present in vivo will be vital to accurately measure and interpret metabolic outcomes. Understanding the full capabilities and limitations of FLIM will also be key to future advances. Future Directions: Future work is needed to evaluate whether a combination of different biochem. and structural outcomes using these imaging techniques can provide complementary information regarding the utilization of specific metabolic pathways.
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    10. 10
      Schaefer, P. M., Kalinina, S., Rueck, A., von Arnim, C. A. F., and von Einem, B. (2019) NADH Autofluorescence-A Marker on its Way to Boost Bioenergetic Research. Cytometry, Part A 95 (1), 34– 46,  DOI: 10.1002/cyto.a.23597
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      NADH Autofluorescence-A Marker on its Way to Boost Bioenergetic Research
      Schaefer Patrick M; von Arnim Christine A F; von Einem Bjoern; Kalinina Sviatlana; Rueck Angelika; von Arnim Christine A F
      Cytometry. Part A : the journal of the International Society for Analytical Cytology (2019), 95 (1), 34-46 ISSN:.
      More than 60 years ago, the idea was introduced that NADH autofluorescence could be used as a marker of cellular redox state and indirectly also of cellular energy metabolism. Fluorescence lifetime imaging microscopy of NADH autofluorescence offers a marker-free readout of the mitochondrial function of cells in their natural microenvironment and allows different pools of NADH to be distinguished within a cell. Despite its many advantages in terms of spatial resolution and in vivo applicability, this technique still requires improvement in order to be fully useful in bioenergetics research. In the present review, we give a summary of technical and biological challenges that have so far limited the spread of this powerful technology. To help overcome these challenges, we provide a comprehensible overview of biological applications of NADH imaging, along with a detailed summary of valid imaging approaches that may be used to tackle many biological questions. This review is meant to provide all scientists interested in bioenergetics with support on how to embed successfully NADH imaging in their research. © 2018 International Society for Advancement of Cytometry.
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    11. 11
      Rideau, E., Dimova, R., Schwille, P., Wurm, F. R., and Landfester, K. (2018) Liposomes and polymersomes: a comparative review towards cell mimicking. Chem. Soc. Rev. 47 (23), 8572,  DOI: 10.1039/C8CS00162F
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      Liposomes and polymersomes: a comparative review towards cell mimicking
      Rideau, Emeline; Dimova, Rumiana; Schwille, Petra; Wurm, Frederik R.; Landfester, Katharina
      Chemical Society Reviews (2018), 47 (23), 8572-8610CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      Cells are integral to all forms of life due to their compartmentalization by the plasma membrane. However, living organisms are immensely complex. Thus there is a need for simplified and controllable models of life for a deeper understanding of fundamental biol. processes and man-made applications. This is where the bottom-up approach of synthetic biol. comes from: a stepwise assembly of biomimetic functionalities ultimately into a protocell. A fundamental feature of such an endeavor is the generation and control of model membranes such as liposomes and polymersomes. We compare and contrast liposomes and polymersomes for a better a priori choice and design of vesicles and try to understand the advantages and shortcomings assocd. with using one or the other in many different aspects (properties, synthesis, self-assembly, applications) and which aspects have been studied and developed with each type and update the current development in the field.
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    12. 12
      Le Meins, J. F., Schatz, C., Lecommandoux, S., and Sandre, O. (2013) Hybrid polymer/lipid vesicles: state of the art and future perspectives. Mater. Today 16 (10), 397– 402,  DOI: 10.1016/j.mattod.2013.09.002
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      Hybrid polymer/lipid vesicles: state of the art and future perspectives
      Le Meins, J-F.; Schatz, C.; Lecommandoux, S.; Sandre, O.
      Materials Today (Oxford, United Kingdom) (2013), 16 (10), 397-402CODEN: MTOUAN; ISSN:1369-7021. (Elsevier Ltd.)
      A review. Hybrid vesicles resulting from the combined self-assembly of both amphiphilic copolymers and lipids have attracted particular interest from chemists and (bio)physicists over the last five years. Such assemblies may be viewed as an advanced vesicular structure compared to their liposome and polymersome forerunners as the best characteristics from the two different systems can be integrated in a new, single vesicle. To afford such a design, the different parameters controlling both self-assembly and membrane structure must be tuned. This highlight aims to present a comprehensive overview of the fundamental aspects related to these structures, and discuss emerging developments and future applications in this field of research.
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      Dao, T. P. T., Fernandes, F., Fauquignon, M., Ibarboure, E., Prieto, M., and Le Meins, J. F. (2018) The combination of block copolymers and phospholipids to form giant hybrid unilamellar vesicles (GHUVs) does not systematically lead to ″intermediate’’ membrane properties. Soft Matter 14 (31), 6476– 6484,  DOI: 10.1039/C8SM00547H
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      13
      The combination of block copolymers and phospholipids to form giant hybrid unilamellar vesicles (GHUVs) does not systematically lead to "intermediate" membrane properties
      Dao, T. P. T.; Fernandes, F.; Fauquignon, M.; Ibarboure, E.; Prieto, M.; Le Meins, J. F.
      Soft Matter (2018), 14 (31), 6476-6484CODEN: SMOABF; ISSN:1744-6848. (Royal Society of Chemistry)
      In this work, the elasticity under stretching as well as the fluidity of Giant Hybrid Unilamellar Vesicles (GHUV) has been studied. The membrane structuration of these GHUVs has already been studied at the micro and nanoscale in a previous study of the team. These GHUVs were obtained by the assocn. of a fluid phospholipid (POPC) and a triblock copolymer, poly(ethyleneoxide)-b-poly(dimethylsiloxane)-b-poly(ethyleneoxide). Although the architecture of triblock copolymers can facilitate vesicle formation, they have been scarcely used to generate GHUVs. We show, through micropipette aspiration and FRAP expts., that the incorporation of a low amt. of lipids in the polymer membrane leads to a significant loss of the toughness of the vesicle and subtle modification of the lateral diffusion of polymer chains. We discuss the results within the framework of the conformation of the triblock copolymer chain in the membrane and in the presence of lipid nanodomains.
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      Nam, J., Beales, P. A., and Vanderlick, T. K. (2011) Giant Phospholipid/Block Copolymer Hybrid Vesicles: Mixing Behavior and Domain Formation. Langmuir 27 (1), 1– 6,  DOI: 10.1021/la103428g
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      14
      Giant Phospholipid/Block Copolymer Hybrid Vesicles: Mixing Behavior and Domain Formation
      Nam, Jin; Beales, Paul A.; Vanderlick, T. Kyle
      Langmuir (2011), 27 (1), 1-6CODEN: LANGD5; ISSN:0743-7463. (American Chemical Society)
      Lipids and block copolymers can be individually assembled into unsupported, spherical membranes (liposomes or polymersomes), each having their own particular benefits and limitations. Here the authors demonstrate the prepn. of microscale, hybrid "lipopolymersomes" composed of the common lipid POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine) and the com. available copolymer PBd-b-PEO (polybutadiene-b-poly(ethylene oxide)) with the goal of incorporating the advantageous qualities of the unitary systems into mixed-membrane capsules. The authors investigate the lipopolymersomes using confocal fluorescence microscopy and demonstrate that these hybrid membranes are well mixed on nanoscopic length scales within the permittable compositional windows for hybrid vesicle formation. The authors measure the intramembrane dynamics and mech. properties of these hybrid membranes by fluorescence recovery after photobleaching (FRAP) and micropipet aspiration, resp. For the first time, the authors demonstrate the demixing of lipid-rich and polymer-rich membrane domains within the same vesicle membrane. This is achieved by the biotinylation of one of the constituent species and cross linking with the protein NeutrAvidin. The resultant domain patterning is dependent upon which component carries the biotin functionality: cross linking of the copolymer species results in domains that ripen into a single, large, copolymer-rich island, and cross linking of the lipids yields many small, "spot-like", lipid-rich domains within a copolymer-rich matrix. The authors discuss these morphol. differences in terms of the fluidity and mech. properties of the membrane phases and the possible resultant interdomain interactions within the membrane. These heterogeneous hybrid lipopolymersomes could find applications in fields such as targeted delivery, controlled release, and environmental detection assays where these capsules possess the characteristics of biocompatible lipid membranes combined with enhanced mech. strength and stability from the copolymer matrix.
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    15. 15
      Hammarstrom, L., Almgren, M., Lind, J., Merenyi, G., Norrby, T., and Akermark, B. (1993) Mechanisms of Transmembrane Electron-Transfer - Diffusion of Uncharged Redox Forms of Viologen, 4,4’-Bipyridine, and Nicotinamide with Long Alkyl Chains. J. Phys. Chem. 97 (39), 10083– 10091,  DOI: 10.1021/j100141a031
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      There is no corresponding record for this reference.
    16. 16
      Marušič, N., Otrin, L., Zhao, Z., Lira, R. B., Kyrilis, F. L., Hamdi, F., Kastritis, P. L., Vidaković-Koch, T., Ivanov, I., and Sundmacher, K. (2020) Constructing artificial respiratory chain in polymer compartments: insights into the interplay between bo3 oxidase and the membrane. Proc. Natl. Acad. Sci. U. S. A. 117, 15006,  DOI: 10.1073/pnas.1919306117
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      16
      Constructing artificial respiratory chain in polymer compartments: Insights into the interplay between bo3 oxidase and the membrane
      Marusic Nika; Ivanov Ivan; Sundmacher Kai; Otrin Lado; Vidakovic-Koch Tanja; Zhao Ziliang; Lira Rafael B; Dimova Rumiana; Kyrilis Fotis L; Hamdi Farzad; Kastritis Panagiotis L; Kyrilis Fotis L; Hamdi Farzad; Kastritis Panagiotis L
      Proceedings of the National Academy of Sciences of the United States of America (2020), 117 (26), 15006-15017 ISSN:.
      Cytochrome bo3 ubiquinol oxidase is a transmembrane protein, which oxidizes ubiquinone and reduces oxygen, while pumping protons. Apart from its combination with F1Fo-ATPase to assemble a minimal ATP regeneration module, the utility of the proton pump can be extended to other applications in the context of synthetic cells such as transport, signaling, and control of enzymatic reactions. In parallel, polymers have been speculated to be phospholipid mimics with respect to their ability to self-assemble in compartments with increased stability. However, their usability as interfaces for complex membrane proteins has remained questionable. In the present work, we optimized a fusion/electroformation approach to reconstitute bo3 oxidase in giant unilamellar vesicles made of PDMS-g-PEO and/or phosphatidylcholine (PC). This enabled optical access, while microfluidic trapping allowed for online analysis of individual vesicles. The tight polymer membranes and the inward oriented enzyme caused 1 pH unit difference in 30 min, with an initial rate of 0.35 pH·min(-1) To understand the interplay in these composite systems, we studied the relevant mechanical and rheological membrane properties. Remarkably, the proton permeability of polymer/lipid hybrids decreased after protein insertion, while the latter also led to a 20% increase of the polymer diffusion coefficient in polymersomes. In addition, PDMS-g-PEO increased the activity lifetime and the resistance to free radicals. These advantageous properties may open diverse applications, ranging from cell-free biotechnology to biomedicine. Furthermore, the presented study serves as a comprehensive road map for studying the interactions between membrane proteins and synthetic membranes, which will be fundamental for the successful engineering of such hybrid systems.
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    17. 17
      Patty, P. J. and Frisken, B. J. (2003) The pressure-dependence of the size of extruded vesicles. Biophys. J. 85 (2), 996– 1004,  DOI: 10.1016/S0006-3495(03)74538-X
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      17
      The pressure-dependence of the size of extruded vesicles
      Patty, Philipus J.; Frisken, Barbara J.
      Biophysical Journal (2003), 85 (2), 996-1004CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
      Variations in the size of vesicles formed by extrusion through small pores are discussed in terms of a simple model. The authors' model predicts that the radius should decrease as the square root of the applied pressure, consistent with data for vesicles extruded under various conditions. The model also predicts dependencies on the pore size used and on the lysis tension of the vesicles being extruded that are consistent with the authors' data. The pore size was varied by using track-etched polycarbonate membranes with av. pore diams. ranging from 50 to 200 nm. To vary the lysis tension, vesicles made from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine), mixts. of POPC and cholesterol, and mixts. of POPC and C16-ceramide were studied. The lysis tension, as measured by an extrusion-based technique, of POPC:cholesterol vesicles is higher than that of pure POPC vesicles, whereas POPC:ceramide vesicles have lower lysis tensions than POPC vesicles.
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    18. 18
      Lim, S. K., de Hoog, H. P., Parikh, A. N., Nallani, M., and Liedberg, B. (2013) Hybrid, Nanoscale Phospholipid/Block Copolymer Vesicles. Polymers 5 (3), 1102– 1114,  DOI: 10.3390/polym5031102
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      18
      Hybrid, nanoscale phospholipid/block copolymer vesicles
      Lim, Seng Koon; de Hoog, Hans-Peter; Parikh, Atul N.; Nallani, Madhavan; Liedberg, Bo
      Polymers (Basel, Switzerland) (2013), 5 (3), 1102-1114, 13 pp.CODEN: POLYCK; ISSN:2073-4360. (MDPI AG)
      Hybrid phospholipid/block copolymer vesicles, in which the polymeric membrane is blended with phospholipids, display interesting self-assembly behavior, incorporating the robustness and chem. versatility of polymersomes with the softness and biocompatibility of liposomes. Such structures can be conveniently characterized by prepg. giant unilamellar vesicles (GUVs) via electroformation. Here, we are interested in exploring the self-assembly and properties of the analogous nanoscale hybrid vesicles (ca. 100 nm in diam.) of the same compn. prepd. by film-hydration and extrusion. We show that the self-assembly and content-release behavior of nanoscale polybutadiene-b-poly(ethylene oxide) (PB-PEO)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) hybrid phospholipid/block copolymer vesicles can be tuned by the mixing ratio of the amphiphiles. In brief, these hybrids may provide alternative tools for drug delivery purposes and mol. imaging/sensing applications and clearly open up new avenues for further investigation.
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    19. 19
      Chemin, M., Brun, P. M., Lecommandoux, S., Sandre, O., and Le Meins, J. F. (2012) Hybrid polymer/lipid vesicles: fine control of the lipid and polymer distribution in the binary membrane. Soft Matter 8 (10), 2867– 2874,  DOI: 10.1039/c2sm07188f
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      19
      Hybrid polymer/lipid vesicles: fine control of the lipid and polymer distribution in the binary membrane
      Chemin, Maud; Brun, Pierre-Marie; Lecommandoux, Sebastien; Sandre, Olivier; Le Meins, Jean-Francois
      Soft Matter (2012), 8 (10), 2867-2874CODEN: SMOABF; ISSN:1744-683X. (Royal Society of Chemistry)
      Hybrid polymer/lipid giant unilamellar vesicles (GUVs) were developed using lipids of resp. low and high melting transition temp. (DPPC: 1,2-dipalmitoyl-sn-glycero-3 phosphocholine, Tm = 41, and POPC: palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; Tm = -2) and a copolymer poly(dimethylsiloxane)-graft-poly(ethylene oxide) (PDMS-g-PEO) well known to self-assemble into vesicular structures. Using epifluorescence microscopy as well as differential scanning calorimetry (DSC), different structures have been identified depending on the molar compn. and on the fluid or gel state of the lipid used. The most promising objects are hybrid vesicles with copolymer as the major component, in which lipids are either randomly distributed or present "raft-like" domains in the polymer-rich membrane. The results are discussed on the basis of the fluidity of the different components and of their resp. membrane thicknesses.
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    20. 20
      Ford, W. E. and Tollin, G. (1984) Chlorophyll Photosensitized Electron-Transfer in Phospholipid-Bilayer Vesicle Systems - Effects of Cholesterol on Radical Yields and Kinetic-Parameters. Photochem. Photobiol. 40 (2), 249– 259,  DOI: 10.1111/j.1751-1097.1984.tb04583.x
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      20
      Chlorophyll photosensitized electron transfer in phospholipid bilayer vesicle systems: effects of cholesterol on radical yields and kinetic parameters
      Ford, William E.; Tollin, Gordon
      Photochemistry and Photobiology (1984), 40 (2), 249-59CODEN: PHCBAP; ISSN:0031-8655.
      The quenching of the triplet state of chlorophyll a (Chl) by asym. located electron acceptors was examd. in vesicle systems contg. egg yolk phosphatidylcholine and 0-50 mol% cholesterol. The incorporation of cholesterol had 2 main effects: (1) the distribution of Chl within the vesicle wall shifted from one favoring the inner monolayer to one favoring the outer monolayer, and (2) the Chl mols. (both ground and excited states) became more accessible to water and to the quencher mols. This latter property was probably due to the creation of space between the phospholipid head groups by insertion of cholesterol. These phenomena required cholesterol concns. >15 mol%. In general, the addn. of cholesterol caused increases in the apparent bimol. rate const. for triplet quenching, in the probability that quenching produced radicals, and in the rate of radical recombination. Some of the specific effects of cholesterol depended upon whether or not the quencher mols. were amphiphilic.
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    21. 21
      Angelova, M. I. and Dimitrov, D. S. (1986) Liposome Electroformation. Faraday Discuss. Chem. Soc. 81, 303,  DOI: 10.1039/dc9868100303
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      21
      Liposome electroformation
      Angelova, M.; Dimitrov, D.
      Faraday Discussions of the Chemical Society (1986), 81 (1), 303-11CODEN: FDCSB7; ISSN:0301-7249.
      Liposome formation and lipid swelling on Pt electrodes in distd. water and water solns. in d.c. elec. fields were investigated for different amts. of a neg. charged lipid (mixt. from 71% phosphatidylcholines, 21.5% phosphatidylethanolamines and 7.5% phosphatidylserines), and a neutral lipid (dimyristoylphosphatidylcholine, DMPC). Neg. charged lipids do not form liposomes without fields when the thickness of the dried lipid layer is ≤90 bilayers. The rate and extent of swelling of layers thicker than 90 bilayers is largest on the cathode, smaller without fields and smallest on the anode. The theory, based on the assumption that osmotic and electrostatic forces drive lipid swelling and liposome formation. is in semi-quant. agreement with the exptl. data; in particular, it gives the obsd. linear dependence of the rate of swelling on the inverse lipid layer thickness. To induce liposome formation for layers thinner than 90 bilayers it was necessary to apply a neg. potential which is proportional to the logarithm of the inverse layer thickness. The characteristic crit. potential is proportional to RTk/F; R being the gas const., Tk the abs. temp., and F the Faraday const. This indicates that redistribution of counterions may be the cause which increases the repulsive electrostatic intermembrane forces to overcome van der Waals attraction. For thicknesses <10 bilayers, formation of very thin-walled liposomes of narrow size distribution and mean diam. of ∼30 μm was obsd. These liposomes grow in size before detachment, and a formula for the kinetics of growth was derived, which is in very good agreement with the exptl. data. The effects of d.c. field on DMPC swelling are smaller and lead to formation of liposome-like structures of different appearance. Bilayer sepn. and bending are prerequisites for liposome formation from hydrating lipids. Therefore, a possible mol. mechanism is that membranes should be destabilized to bend and fuse to form liposomes. This requires the right proportion between structured regions, in the form of bilayers, and defects and (or) nonbilayer structures, and in many cases external constraints, in particular, elec. fields.
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    22. 22
      Li, Q., Wang, X., Ma, S., Zhang, Y., and Han, X. (2016) Electroformation of giant unilamellar vesicles in saline solution. Colloids Surf., B 147, 368– 375,  DOI: 10.1016/j.colsurfb.2016.08.018
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      22
      Electroformation of giant unilamellar vesicles in saline solution
      Li, Qingchuan; Wang, Xuejing; Ma, Shenghua; Zhang, Ying; Han, Xiaojun
      Colloids and Surfaces, B: Biointerfaces (2016), 147 (), 368-375CODEN: CSBBEQ; ISSN:0927-7765. (Elsevier B.V.)
      Giant unilamellar vesicle (GUV) formation on indium tin oxide (ITO) electrodes in saline soln. and from charged lipids has proven to be difficult in the past. Yet the best cell membrane models contain charged lipids and require physiol. conditions. GUVs from zwitterionic lipids, lipid mixts. and even pure charged lipids could be electroformed under physiol. conditions and even higher concns. of NaCl. The hydrophilic ITO surface may facilitate the hydration of the solid lipid film and the formation of lipid bilayers that subsequently bend and form vesicles. The formation of GUVs in saline soln. is influenced by different parameters. The influences of the amplitude and frequency of the used AC field, the NaCl concn., and the temp. were investigated. Finite element anal. simulating the effect of the elec. field on GUV formation in saline soln. could well explain the exptl. results. Frequencies in the kHz-range favored for GUVs formation in saline soln., as they suppress the formation of elec. double layer, while higher frequencies could again impair the effect of elec. field and impede GUV formation. The diams. of the GUVs increased gradually with NaCl concn. from 0 mM to 200 mM and subsequently decreased from 200 mM to 2 M. High yields of GUVs were also formed in PBS soln. and cell culture medium, which indicates this method is a promising way to prep. GUVs on a large scale in physiol. relevant conditions.
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    23. 23
      Zong, W., Ma, S., Zhang, X., Wang, X., Li, Q., and Han, X. (2017) A Fissionable Artificial Eukaryote-like Cell Model. J. Am. Chem. Soc. 139 (29), 9955– 9960,  DOI: 10.1021/jacs.7b04009
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      23
      A Fissionable Artificial Eukaryote-like Cell Model
      Zong, Wei; Ma, Shenghua; Zhang, Xunan; Wang, Xuejing; Li, Qingchuan; Han, Xiaojun
      Journal of the American Chemical Society (2017), 139 (29), 9955-9960CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The use of artificial cells has attracted considerable attention in various fields from biotechnol. to medicine. Here, the authors develop a cell-sized vesicle-in-vesicle (VIV) structure contg. a sep. inner vesicle (IV) that can be loaded with DNA. The authors use polymerase chain reaction (PCR) to successfully amplify the amt. of DNA confined to the IV. Subsequent osmotic stress-induced fission of a mother VIV into two daughter VIVs successfully divides the IV content while keeping it confined to the IV of the daughter VIVs. The fission rate was estd. to be ∼20% quantified by fluorescence microscope. The VIV structure represents a step forward toward construction of an advanced, fissionable cell model.
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    24. 24
      Karimi, M., Steinkuhler, J., Roy, D., Dasgupta, R., Lipowsky, R., and Dimova, R. (2018) Asymmetric Ionic Conditions Generate Large Membrane Curvatures. Nano Lett. 18 (12), 7816– 7821,  DOI: 10.1021/acs.nanolett.8b03584
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      24
      Asymmetric ionic conditions generate large membrane curvatures
      Karimi, Marzieh; Steinkuehler, Jan; Roy, Debjit; Dasgupta, Raktim; Lipowsky, Reinhard; Dimova, Rumiana
      Nano Letters (2018), 18 (12), 7816-7821CODEN: NALEFD; ISSN:1530-6984. (American Chemical Society)
      Biol. membranes possess intrinsic asymmetry. This asymmetry is assocd. not only with leaflet compn. in terms of membrane species, but also with differences in the cytosolic and periplasmic solns. contg. macromols. and ions. There has been a long quest for understanding the effect of ions on the phys. and morphol. properties of membranes. Here, we elucidated the changes in the mech. properties of membranes exposed to asym. buffer conditions and the assocd. curvature generation. As a model system, we used giant unilamellar vesicles (GUVs) with asym. salt and sugar solns. on the 2 sides of the membrane. We aspirated the GUVs into micropipettes and attached small beads to their membranes. An optical tweezer was used to exert a local force on a bead, thereby pulling out a membrane tube from the vesicle. The assay allowed us to measure the spontaneous curvature and the bending rigidity of the bilayer in the presence of different ions and sugar. At low sugar/salt (inside/out) concns., the membrane spontaneous curvature generated by NaCl and KCl was close to zero, but neg. in the presence of LiCl. In the latter case, the membrane bulged away from the salt soln. At high sugar/salt conditions, the membranes were obsd. to become more flexible and the spontaneous curvature was enhanced to even more neg. values, comparable to those generated by some proteins. These findings revealed the reshaping role of alkali chlorides on biomembranes.
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    25. 25
      Cremer, P. S. and Boxer, S. G. (1999) Formation and spreading of lipid bilayers on planar glass supports. J. Phys. Chem. B 103 (13), 2554– 2559,  DOI: 10.1021/jp983996x
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      25
      Formation and Spreading of Lipid Bilayers on Planar Glass Supports
      Cremer, Paul S.; Boxer, Steven G.
      Journal of Physical Chemistry B (1999), 103 (13), 2554-2559CODEN: JPCBFK; ISSN:1089-5647. (American Chemical Society)
      The fusion and spreading of phospholipid bilayers on glass surfaces was investigated as a function of pH and ionic strength. Membrane fusion to the support was favorable at high ionic strength and low pH for vesicles contg. a net neg. charge; however, neutral and pos. charged vesicles fused under all conditions attempted. This result suggests that van der Waals and electrostatic interactions govern the fusion process. Membrane spreading over a planar surface was favorable at low pH regardless of the net charge on the bilayer, and the process is driven by van der Waals forces. On the other hand membrane propagation is impeded at high pH or on highly curved surfaces. In this case a combination of hydration and bending interactions is primarily responsible for arresting the spreading process. These results provide a framework for understanding many of the factors that influence the effectiveness of scratches on planar supported bilayers as barriers to lateral diffusion and lead to a simple method to heal these scratches.
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    26. 26
      Prokazov, Y., Turbin, E., Weber, A., Hartig, R., and Zuschratter, W. (2014) Position sensitive detector for fluorescence lifetime imaging. J. Instrum. 9, C12015,  DOI: 10.1088/1748-0221/9/12/C12015
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      Weber, A., Prokazov, Y., Zuschratter, W., and Hauser, M. J. B. (2012) Desynchronisation of Glycolytic Oscillations in Yeast Cell Populations. PLoS One 7 (9), e43276,  DOI: 10.1371/journal.pone.0043276
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      Desynchronisation of glycolytic oscillations in yeast cell populations
      Weber, Andre; Prokazov, Yury; Zuschratter, Werner; Hauser, Marcus J. B.
      PLoS One (2012), 7 (9), e43276CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)
      Glycolytic oscillations of intact yeast cells of the strain Saccharomyces carlsbergensis were investigated at both the levels of cell populations and of individual cells. Individual cells showed glycolytic oscillations even at very low cell densities (e.g. 1.0 × 105 cells/mL). By contrast, the collective behavior on the population level was cell d.-dependent: at high cell densities it is oscillatory, but below the threshold d. of 1.0 × 106 cells/mL the collective dynamics becomes quiescent. We demonstrate that the transition in the collective dynamics is caused by the desynchronization of the oscillations of individual cells. This is characteristic for a Kuramoto transition. Spatially resolved measurements at low cell densities revealed that even cells that adhere to their neighbors oscillated with their own, independent frequencies and phases.
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    28. 28
      Weber, A., Zuschratter, W., and Hauser, M. J. B. (2020) Partial synchronisation of glycolytic oscillations in yeast cell populations. Sci. Rep. 10 (1), 19714,  DOI: 10.1038/s41598-020-76242-8
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      28
      Partial synchronisation of glycolytic oscillations in yeast cell populations
      Weber, Andre; Zuschratter, Werner; Hauser, Marcus J. B.
      Scientific Reports (2020), 10 (1), 19714CODEN: SRCEC3; ISSN:2045-2322. (Nature Research)
      Abstr.: The transition between synchronized and asynchronous behavior of immobilized yeast cells of the strain Saccharomyces carlsbergensis was investigated by monitoring the autofluorescence of the coenzyme NADH. In populations of intermediate cell densities the individual cells remained oscillatory, whereas on the level of the cell population both a partially synchronized and an asynchronous state were accessible for exptl. studies. In the partially synchronized state, the mean oscillatory frequency was larger than that of the cells in the asynchronous state. This suggests that synchronisation occurred due to entrainment by the cells that oscillated more rapidly. This is typical for synchronisation due to phase advancement. Furthermore, the synchronisation of the frequency of the glycolytic oscillations preceded the synchronisation of their phases. However, the cells did not synchronize completely, as the distribution of the oscillatory frequencies only narrowed but did not collapse to a unique frequency. Cells belonging to spatially denser clusters showed a slightly enhanced local synchronisation during the episode of partial synchronisation. Neither the clusters nor a transition from partially synchronized glycolytic oscillations to travelling glycolytic waves did substantially affect the degree of partial synchronisation. Chimera states, i.e., the coexistence of a synchronized and an asynchronous part of the population, could not be found.
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    29. 29
      Kuyper, C. L., Kuo, J. S., Mutch, S. A., and Chiu, D. T. (2006) Proton permeation into single vesicles occurs via a sequential two-step mechanism and is heterogeneous. J. Am. Chem. Soc. 128 (10), 3233– 3240,  DOI: 10.1021/ja057349c
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      29
      Proton Permeation into Single Vesicles Occurs via a Sequential Two-Step Mechanism and Is Heterogeneous
      Kuyper, Christopher L.; Kuo, Jason S.; Mutch, Sarah A.; Chiu, Daniel T.
      Journal of the American Chemical Society (2006), 128 (10), 3233-3240CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      This article describes the first single-vesicle study of proton permeability across the lipid membrane of small (∼100 nm) uni- and multilamellar vesicles, which were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). To follow proton permeation into the internal vol. of each vesicle, we encapsulated carboxyfluorescein, a pH-sensitive dye whose fluorescence was quenched in the presence of excess protons. A microfluidic platform was used for easy exchange of high- and low-pH solns., and fluorescence quenching of single vesicles was detected with single-mol. total internal reflection fluorescence (TIRF) microscopy. Upon soln. exchange and acidification of the extravesicular soln. (from pH 9 to 3.5), we obsd. for each vesicle a biphasic decay in fluorescence. Through single-vesicle anal., we found that rate consts. for the first decay followed a Poisson distribution, whereas rate consts. for the second decay followed a normal distribution. We propose that proton permeation into each vesicle first arose from formation of transient pores and then transitioned into the second decay phase, which occurred by the soly.-diffusion mechanism. Furthermore, for the bulk population of vesicles, the decay rate const. and vesicle intensity (dependent on size) correlated to give an av. permeability coeff.; however, for individual vesicles, we found little correlation, which suggested that proton permeability among single vesicles was heterogeneous in our expts.
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    30. 30
      Yandrapalli, N., Seemann, T., and Robinson, T. (2020) On-Chip Inverted Emulsion Method for Fast Giant Vesicle Production, Handling, and Analysis. Micromachines 11 (3), 285,  DOI: 10.3390/mi11030285
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      Beneyton, T., Krafft, D., Bednarz, C., Kleineberg, C., Woelfer, C., Ivanov, I., Vidaković-Koch, T., Sundmacher, K., and Baret, J.-C. (2018) Out-of-equilibrium microcompartments for the bottom-up integration of metabolic functions. Nat. Commun. 9 (1), 2391,  DOI: 10.1038/s41467-018-04825-1
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      31
      Out-of-equilibrium microcompartments for the bottom-up integration of metabolic functions
      Beneyton Thomas; Baret Jean-Christophe; Krafft Dorothee; Bednarz Claudia; Kleineberg Christin; Woelfer Christian; Ivanov Ivan; Vidakovic-Koch Tanja; Sundmacher Kai; Sundmacher Kai
      Nature communications (2018), 9 (1), 2391 ISSN:.
      Self-sustained metabolic pathways in microcompartments are the corner-stone for living systems. From a technological viewpoint, such pathways are a mandatory prerequisite for the reliable design of artificial cells functioning out-of-equilibrium. Here we develop a microfluidic platform for the miniaturization and analysis of metabolic pathways in man-made microcompartments formed of water-in-oil droplets. In a modular approach, we integrate in the microcompartments a nicotinamide adenine dinucleotide (NAD)-dependent enzymatic reaction and a NAD-regeneration module as a minimal metabolism. We show that the microcompartments sustain a metabolically active state until the substrate is fully consumed. Reversibly, the external addition of the substrate reboots the metabolic activity of the microcompartments back to an active state. We therefore control the metabolic state of thousands of independent monodisperse microcompartments, a step of relevance for the construction of large populations of metabolically active artificial cells.
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    32. 32
      Biner, O., Fedor, J. G., Yin, Z., and Hirst, J. (2020) Bottom-Up Construction of a Minimal System for Cellular Respiration and Energy Regeneration. ACS Synth. Biol. 9 (6), 1450– 1459,  DOI: 10.1021/acssynbio.0c00110
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      32
      Bottom-Up Construction of a Minimal System for Cellular Respiration and Energy Regeneration
      Biner, Olivier; Fedor, Justin G.; Yin, Zhan; Hirst, Judy
      ACS Synthetic Biology (2020), 9 (6), 1450-1459CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)
      ATP, the cellular energy currency, is essential for life. The ability to provide a const. supply of ATP is therefore crucial for the construction of artificial cells in synthetic biol. Here, the authors describe the bottom-up assembly and characterization of a minimal respiratory system that uses NADH as a fuel to produce ATP from ADP and inorg. phosphate, and is thus capable of sustaining both upstream metabolic processes that rely on NAD+, and downstream energy-demanding processes that are powered by ATP hydrolysis. A detergent-mediated approach was used to coreconstitute respiratory mitochondrial complex I and an F-type ATP synthase into nanosized liposomes. Addn. of the alternative oxidase to the resulting proteoliposomes produced a minimal artificial "organelle" that reproduces the energy-converting catalytic reactions of the mitochondrial respiratory chain: NADH oxidn., ubiquinone cycling, oxygen redn., proton pumping, and ATP synthesis. As a proof-of-principle, the authors demonstrate that the nanovesicles are capable of using an NAD+-linked substrate to drive cell-free protein expression. The nanovesicles are both efficient and durable and may be applied to sustain artificial cells in future work.
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    • Materials and methods; size distribution of LUVs (DLS); fluorescent/UV–vis spectrophotometric data for electron transfer/ferricyanide leakage; epifluorescence/confocal images of GUVs; TCSPC-based fluorescence profiles of GUVs; and workflow for extraction of electron transfer rate constants (PDF)


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