ACS Publications. Most Trusted. Most Cited. Most Read
In Vivo Photocontrol of Microtubule Dynamics and Integrity, Migration and Mitosis, by the Potent GFP-Imaging-Compatible Photoswitchable Reagents SBTubA4P and SBTub2M
More

OR SEARCH CITATIONS

My Activity
Publications

Figure 1Loading Img
Download Hi-Res ImageDownload to MS-PowerPointCite This:J. Am. Chem. Soc. 2022, 144, 12, 5614-5628
  • Open Access
Article

In Vivo Photocontrol of Microtubule Dynamics and Integrity, Migration and Mitosis, by the Potent GFP-Imaging-Compatible Photoswitchable Reagents SBTubA4P and SBTub2M
Click to copy article linkArticle link copied!

  • Li Gao
    Li Gao
    Department of Pharmacy, Ludwig-Maximilians University of Munich, Munich 81377, Germany
    More by Li Gao
  • Joyce C. M. Meiring
    Joyce C. M. Meiring
    Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht CH 3584, Netherlands
    More by Joyce C. M. Meiring
  • Adam Varady
    Adam Varady
    St. Anna Children’s Cancer Research Institute (CCRI), Vienna 1090, Austria
    More by Adam Varady
  • Iris E. Ruider
    Iris E. Ruider
    Physics Department and Center for Protein Assemblies CPA, Technical University of Munich, Garching 85747, Germany
    More by Iris E. Ruider
  • Constanze Heise
    Constanze Heise
    Department of Pharmacy, Ludwig-Maximilians University of Munich, Munich 81377, Germany
    More by Constanze Heise
  • Maximilian Wranik
    Maximilian Wranik
    Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, Villigen 5232, Switzerland
    More by Maximilian Wranik
  • Cecilia D. Velasco
    Cecilia D. Velasco
    Laboratory of Neurobiology, Department of Pathology and Experimental Therapy, Institute of Neurosciences, University of Barcelona, L’Hospitalet de Llobregat, Barcelona 08907, Spain
    Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet de Llobregat, Barcelona 08907, Spain
    More by Cecilia D. Velasco
    Orcidhttps://orcid.org/0000-0003-1527-607X
  • Jennifer A. Taylor
    Jennifer A. Taylor
    Department of Biology, University of Washington, Seattle, Washington 98195, United States
    More by Jennifer A. Taylor
  • Beatrice Terni
    Beatrice Terni
    Laboratory of Neurobiology, Department of Pathology and Experimental Therapy, Institute of Neurosciences, University of Barcelona, L’Hospitalet de Llobregat, Barcelona 08907, Spain
    Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet de Llobregat, Barcelona 08907, Spain
    More by Beatrice Terni
  • Tobias Weinert
    Tobias Weinert
    Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, Villigen 5232, Switzerland
    More by Tobias Weinert
  • Jörg Standfuss
    Jörg Standfuss
    Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, Villigen 5232, Switzerland
    More by Jörg Standfuss
  • Clemens C. Cabernard
    Clemens C. Cabernard
    Department of Biology, University of Washington, Seattle, Washington 98195, United States
    More by Clemens C. Cabernard
  • Artur Llobet
    Artur Llobet
    Laboratory of Neurobiology, Department of Pathology and Experimental Therapy, Institute of Neurosciences, University of Barcelona, L’Hospitalet de Llobregat, Barcelona 08907, Spain
    Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet de Llobregat, Barcelona 08907, Spain
    More by Artur Llobet
  • Michel O. Steinmetz
    Michel O. Steinmetz
    Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, Villigen 5232, Switzerland
    Biozentrum, University of Basel, Basel 4056, Switzerland
    More by Michel O. Steinmetz
    Orcidhttps://orcid.org/0000-0001-6157-3687
  • Andreas R. Bausch
    Andreas R. Bausch
    Physics Department and Center for Protein Assemblies CPA, Technical University of Munich, Garching 85747, Germany
    More by Andreas R. Bausch
  • Martin Distel
    Martin Distel
    St. Anna Children’s Cancer Research Institute (CCRI), Vienna 1090, Austria
    Zebrafish Platform Austria for Preclinical Drug Screening (ZANDR), Vienna 1090, Austria
    More by Martin Distel
  • Julia Thorn-Seshold
    Julia Thorn-Seshold
    Department of Pharmacy, Ludwig-Maximilians University of Munich, Munich 81377, Germany
    More by Julia Thorn-Seshold
    Orcidhttps://orcid.org/0000-0002-4879-4159
  • Anna Akhmanova
    Anna Akhmanova
    Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht CH 3584, Netherlands
    More by Anna Akhmanova
  • Oliver Thorn-Seshold*
    Oliver Thorn-Seshold
    Department of Pharmacy, Ludwig-Maximilians University of Munich, Munich 81377, Germany
    *Email: [email protected]
    More by Oliver Thorn-Seshold
    Orcidhttps://orcid.org/0000-0003-3981-651X
Open PDFSupporting Information (17)

Journal of the American Chemical Society

Cite this: J. Am. Chem. Soc. 2022, 144, 12, 5614–5628
Click to copy citationCitation copied!
https://doi.org/10.1021/jacs.2c01020
Published March 15, 2022

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

CC-BY-NC-ND 4.0 .

Abstract

Click to copy section linkSection link copied!
High Resolution Image
Download MS PowerPoint Slide

Photoswitchable reagents are powerful tools for high-precision studies in cell biology. When these reagents are globally administered yet locally photoactivated in two-dimensional (2D) cell cultures, they can exert micron- and millisecond-scale biological control. This gives them great potential for use in biologically more relevant three-dimensional (3D) models and in vivo, particularly for studying systems with inherent spatiotemporal complexity, such as the cytoskeleton. However, due to a combination of photoswitch isomerization under typical imaging conditions, metabolic liabilities, and insufficient water solubility at effective concentrations, the in vivo potential of photoswitchable reagents addressing cytosolic protein targets remains largely unrealized. Here, we optimized the potency and solubility of metabolically stable, druglike colchicinoid microtubule inhibitors based on the styrylbenzothiazole (SBT) scaffold that are nonresponsive to typical fluorescent protein imaging wavelengths and so enable multichannel imaging studies. We applied these reagents both to 3D organoids and tissue explants and to classic model organisms (zebrafish, clawed frog) in one- and two-protein imaging experiments, in which spatiotemporally localized illuminations allowed them to photocontrol microtubule dynamics, network architecture, and microtubule-dependent processes in vivo with cellular precision and second-level resolution. These nanomolar, in vivo capable photoswitchable reagents should open up new dimensions for high-precision cytoskeleton research in cargo transport, cell motility, cell division, and development. More broadly, their design can also inspire similarly capable optical reagents for a range of cytosolic protein targets, thus bringing in vivo photopharmacology one step closer to general realization.

This publication is licensed under

CC-BY-NC-ND 4.0 .
  • cc licence
  • by licence
  • nc licence
  • nd licence
Copyright © 2022 The Authors. Published by American Chemical Society

Subjects

what are subjects

1. Introduction

Click to copy section linkSection link copied!
Biological methods and instrumentation now allow us to observe cellular structures and dynamics on the micron scale, with dynamics resolved to the scale of milliseconds, in settings from in vitro cell culture (1) to in vivo (multiorgan) animal models. (2) However, the development of tools to manipulate cellular processes with matching micrometer spatial precision and millisecond temporal precision has lagged far behind, despite its value for research. (3,4)
One biological system with particularly urgent need of such tools is the microtubule (MT) cytoskeleton. (5,6) MTs are giant tubelike noncovalent polymers of α/β-tubulin protein heterodimers that extend throughout the cytosol. They are centrally organized and can be rapidly remodeled to support hundreds of spatiotemporally regulated functions in the life of a cell. The most visible roles of MTs include force generation to maintain and change cell shape and position, and scaffolding the transport of cargos by motor proteins, including chromosomes during cell division. Tools to noninvasively manipulate MT network structure and remodeling dynamics with high spatiotemporal precision have great potential to drive biological research. However, to fulfill this potential, tools must succeed in settings from two-dimensional (2D) cell culture (e.g., cell migration and division) through to in vivo, three-dimensional (3D) systems (embryonic development and neuroscience). It is also important that tools be easily transferrable across different models and organisms. (7,8)
Optogenetics has been extremely successful in patterning ion currents and cell signaling with high spatiotemporal precision. However, until recently, only two optogenetic tools to modulate MT dynamics and network architecture were known: Wittmann’s photo-inactivatable MT-polymerizing tool π-EB1, (9) and Slep’s photoactivated plus tip recruitment system. (10) Two further optogenetic tools to photocontrol enzymatic severing of MTs have recently appeared as preprints, (11,12) highlighting the interest in methods to photocontrol microtubule dynamics, network organization, and interaction partners.
By comparison, drugs to modulate MT structure and dynamics reliably across all eukaryotes are very well studied, with their ease of being translated across models and species (no time needed for breeding and validating transgenic lines, optimizing expression systems, etc.) being a strong practical advantage over genetic approaches. Taxanes, epothilones, colchicine analogues, and vinca alkaloids are all used for nonspecific suppression of MT-dependent cellular processes, in settings from single-cell studies through to in vivo therapeutic use in humans.
Much effort has been invested to develop light-triggered analogues of these drugs, to improve the spatiotemporal precision with which their MT-inhibiting activity can be applied (toward the scale of μm and ms). Photouncaging approaches have been known for some decades; (13) while more recently, photoisomerization-based drug analogues or “photopharmaceuticals” have been developed, to elegantly avoid many of the drawbacks that limited the in vivo use of typical photouncaging methods, (14) such as toxic and/or phototoxic byproducts, requirements for <360 nm wavelengths and high light intensities, slow post-illumination fragmentation, irreversibly photosensitive stock solutions, and non-optical drug release mechanisms (e.g., enzymatic hydrolysis of cages). In the MT field, recent photoswitchable analogues of taxane, (15) epothilone, (16) and colchicinoid (5,17−22) MT inhibitors have all been applied to cellular studies. These photopharmaceuticals have enabled noninvasive, reversible optical control over MT dynamics and MT-associated downstream effects, with cell-specific spatial precision and subsecond-scale temporal precision, and have been brought to bear on research in embryology, (23) neuroscience, (24) and cytoskeleton. (5)
However, the typical photoswitch scaffolds introduce problems particularly for in vivo application, three of which this paper will focus on: (1) Metabolic stability: azobenzenes, particularly as their Z-isomers, can be reductively degraded by cellular glutathione (GSH), although the substituent-dependency of this degradation has not yet been elucidated. (25−27) This degradation reduces reagent photoswitchability as well as potency, and on timescales typical for in vivo studies (>hours) the degradation byproducts are likely to give off-target effects, particularly in metabolically active later-stage animals (tested below, and also discussed elsewhere (18)). (2) Orthogonal photocontrol and imaging: under the high intensities of focussed lasers in microscopy, azobenzenes and hemithioindigos are isomerized by excitation of the typical fluorescent proteins or labels imaged in biological studies (GFP/fluorescein at 490 nm, YFP at 514 nm; some also isomerize with RFP/rhodamine imaging at 561 nm). (18,28) Typically this leaves only the 647 nm laser channel available for orthogonal imaging during photoswitching, yet this laser typically only addresses small-molecule probes. These scaffolds’ inability to allow multichannel protein imaging during photocontrol is particularly problematic for in vivo research that extensively relies on two- or three-channel imaging with fluorescent protein fusions to resolve processes with biological specificity. (3) Practical applicability: in vivo studies must ensure sufficient delivery of photopharmaceuticals to target tissues without organic cosolvents, which are less tolerated by embryos than they are in cell culture. This requires either high-potency compounds or effective solubilizing strategies: neither of which are often seen with typically hydrophobic photoswitchable drug analogues. A separate concern is to minimize the illumination needed to photoswitch an effective amount of reagent (another problem that is more urgent for in vivo than cellular studies). Photoswitch performance at optimal but unavailable wavelengths (e.g., 260–380 nm) is irrelevant for this: tuning the scaffold’s photoresponse at the laser wavelength actually used in biological studies (18) is required, to reach both high efficiency of isomerization E(λ) (5) as well as a high photostationary state (PSS).
Novel photoswitches addressing these issues are recently gaining attention to expand the biological scope of photopharmacology. (29−31) To tackle them in the context of microtubule photocontrol, we previously introduced styrylbenzothiazoles SBTub2/3 (Figure 1a) as highly metabolically stable, fully GFP/YFP/RFP-orthogonal photoswitchable tubulin inhibitors (Figure 1b,c), with excellent photoresponse to the 405 nm laser line that is standard in confocal microscopy. SBTub3 could photocontrol MT dynamics, organization, and MT-dependent processes in live cells with reversible temporal patterning, and with cellular and even subcellular spatial precision. (18)

Figure 1

Figure 1. Design and synthesis. (a) The colchicinoid pharmacophore (gray shaded trimethoxyphenyl south ring and isovanillyl north ring) can be applied to various scaffolds, giving photoswitchable azobenzene-based PST and SBT-based SBTub antimitotics. Previously published SBTub2/3 lacked key interaction residues (red shaded sites). (b) Z-SBTub3 inhibits tubulin polymerization and MT-dependent processes. (c) X-ray structure of tubulin:Z-SBTub3 complex (carbons as purple spheres). The south ring is buried in β-tubulin (green); only the north ring interacts with α-tubulin at the α-T5 loop (cyan). (d) Evolved SBTub compound library used in this paper. (e) Typical synthesis of SBTubs proceeds by acetanilide sulfurization, Jacobson cyclization, and basic condensation. Phosphate prodrug SBTubA4P is further accessed by phosphoester formation and deprotection.

High Resolution Image
Download MS PowerPoint Slide
The metabolic stability and imaging-orthogonal photocontrol of SBTub3 were excellent features toward in vivo use, but practical limitations remained, which we determined to address in this study. Primarily, while their parent colchicinoid inhibitor combretastatin A-4 (CA4) has ca. 20 nM cellular potency, the slightly larger Z-SBTub2/3 gave only micromolar bioactivity (though note that the CA4-isosteric azobenzene analogue Z-PST-1 has only ca. 500 nM potency; Figure 1a). The lower the potency, the more compound and the more cosolvent are needed, which we found blocked animal model applications of both SBTub2/3 and PST-1 (see below). Thus, we prioritized a structure–activity relationship (SAR) study to improve the potency of SBTubs (Figure 1d). Second, the SBTubs are hydrophobic, so we prioritized fully water-soluble prodrugs for in vivo use without cosolvents. Third, while the SBT scaffold is essentially a unidirectional (E → Z) photoswitch in the biological range because its isomers’ absorption bands overlap, we were curious if tautomerizable electron-donating substituents could accelerate its spontaneous thermal Z → E relaxation, as they do for azobenzenes, toward deriving SBT-based photopharmaceuticals with appreciable rates of spontaneous “bioactivity switch-off”. (28)
Our goal was then to test whether suitably potent and soluble SBT derivatives could be used for photocontrol not only in cell culture settings where many photoswitches succeed, but over a range of in vivo multi-organ animal models, with temporally specific and cell-precise in situ photoswitching after systemic administration: settings in which no other photoswitchable reagents have so far succeeded. We now report the development of these potent, soluble, metabolically stable, GFP-orthogonal SBT-based photopharmaceuticals, and characterize the optimal ligands SBTubA4 and with tubulin:SBT X-ray crystal structures. We then showcase the unprecedented success of the fully water-soluble prodrug SBTubA4P in allowing systemic administration but local photoactivations to achieve (i) spatiotemporally specific control over MT dynamics in cell culture, (ii) long-term spatially specific control over development and migration in 3D organoid culture, (iii) short-term temporally resolved photocontrol of neural development in fly brain explant, (iv) photocontrol of embryonic development in the clawed frog, and (v) temporally reversible photocontrol of microtubule dynamics in zebrafish.

2. Results

Click to copy section linkSection link copied!

2.1. Cellular Structure–Activity Optimization of SBTubs

For a colchicinoid SBTub to be cellularly effective, its Z-isomer should bind tubulin, (5,32) halting cell proliferation and inducing apoptosis; (33) while the E-isomer should have negligible tubulin binding and no other significant toxicity mechanisms over a wide concentration range where the Z-isomer is bioactive, resulting in a high lit/dark bioactivity ratio (“photoswitchability of bioactivity”). (18) Thus, we performed cellular structure–activity optimization by comparing the proliferation of cells treated with SBTubs (Table 1 and Figure S5) under in situ pulsed illuminations with near-UV light (“lit”: mostly-Z-isomer; aim: nanomolar EC50), to their proliferation without illumination (“dark”: all-E-isomer; aim: no antiproliferative effects at concentrations of up to 100 times the lit EC50).
Table 1. Cytotoxicity EC50 Values [mostly-Z lit (hν 360–450 nm), all-E dark] and Dark/Lit EC50 Ratio; and Standard Photoactivation Working Concentration [I]WC [all in HeLa cell line; na: analysis not applicable (see Supporting Information)]
 EC50 (μM)EC50 ratio[I]WC
compoundhνdarkdark/lit(μM)
SBTub21.4>25>186.0
SBTub31.2>25>213.2
SBTubA40.123.8310.21
20.52>100>1901.6
30.2015750.65
40.679.3141.5
51.418133.2
62327nana
72.6>35>13na
81.2>100>833.0
91.048482.6
103.0>100>3310
111.7>100>584.3
120.7764831.6
130.428.8211.8
141.0>15>142.7
SBTub2M0.0357.02000.06
160.5248921.1
170.23401740.65
183.0>15>510
SBTub3P3.4>20>57.5
SBTubA4P0.0520.458.70.10
We furthermore introduce a single metric, the “typically useful working concentration for cell culture” [I]WC, by which to rank photopharmaceuticals for their practical utility (Table 1) and so guide optimization. [I]WC is derived by examining the dose–response curves (Figures 2a and S5) to find the lowest concentration at which illuminated cells experience strong inhibition while nonilluminated cells experience insignificant inhibition. We systematically define [I]WC as either the lowest concentration where the lit efficacy reaches >80% of its plateau, or the highest concentration where treatment in the dark causes <10% difference of biological effect compared to the untreated control: whichever is the lower (Figure 2a). Szymanski has previously proposed an alternative working concentration metric [I]opt, which emphasizes maximal illuminated efficacy, and is based on idealized dose–response curves with high Hill coefficients (4) that are rarely obtained in practice. Our empirical [I]WC instead emphasizes our experience that baseline/background inhibition of biological systems must be minimal for photopharmaceutical control to be biologically useful. For example, even if IC50 values are well separated, [I]WC values are undefined if background bioactivity is too strong for the reagent to be useful (e.g., 7, Table 1). We believe that ranking by [I]WC is the most useful systematic single-value method for early-stage optimization of photopharmaceuticals’ performance and that [I]WC will find traction in the community.

Figure 2

Figure 2. (a) Leads SBTub2M and SBTubA4 have highly nonlinear dose–response profiles, high lit/dark ratio of bioactivity, and mid-nanomolar [I]WC values. (b, c) Photocharacterization: (b) SBTub2M is not isomerized from its all-E dark state by 488 nm illumination, but is photoswitched to majority-Z lit states by UV/violet light (78% Z at 405 nm by NMR; 1:1 phosphate-buffered saline/dimethylsulfoxide, PBS:DMSO). (c) Comparison of absorbance spectra of SBTubA4 and azobenzene PST-1 illustrates the SBT’s ideal match to 405 nm photoactivation, combining stronger 405 nm absorption, with sharper absorption cutoff above 405 nm, which makes it orthogonal to GFP (488 nm), YFP (514 nm), and RFP (561 nm) imaging.

High Resolution Image
Download MS PowerPoint Slide
Our first priority for in vivo use was to increase Z-SBTub potency. We first tested whether restoring the methoxy groups of the CA4 pharmacophore would increase potency despite the extra size, with SBTubA4 (1). This, like most SBTubs, was synthesized in a short sequence (Figure 1e) via basic aldol condensation of the derivatized 2-methylbenzothiazole with the corresponding benzaldehyde, with the 2-methylbenzothiazole being obtained via Jacobson cyclization (34) of the thioacetanilide using potassium ferricyanide (exceptions were para-aniline 7 which was obtained by Horner-Wadsworth-Emmons olefination; and 9 and 11 where 2,5-/2,7-dimethylbenzothiazoles were synthesized by Ullmann-type coupling according to Ma; (35) see the Supporting Information).
SBTubA4 was an early hit, with a ca. 10-fold more potent Z-isomer than the previous best SBTub3 and with an excellent dark/lit ratio of 30 (Figure 2a). We then tested if the Z-SBTubs obey similar structure–activity relationships (SAR) as known for CA4. First, we rearranged the methoxy groups in 2, reducing the Z-potency, which matched literature expectations (36) as the middle methoxy group on the south ring otherwise accepts a hydrogen bond from β-Cys239. We also flipped the SBT scaffold orientation in analogues 3–5. This was not convenient for installing the hydroxy/methoxy north ring substituent pair, so in 3 and 4 we retained only one of these groups and the compounds suffered a predictable (36) though a small loss of potency. The 7-fold potency loss when moving the methoxy group from the space-filling 6-position to the 5-position which is best occupied by a small polar substituent (compound 5) was striking and expected. We concluded that indeed Z-SBTubs obey similar SAR as CA4, which could guide further development.
At this stage, we wished to test if common strategies to accelerate thermal Z → E relaxation and redshift spectral response in azobenzenes could also be applied to SBTubs. We created close steric matches of bioactive SBTub3 but with tautomerizable ortho-catechol 6 and para-amino 7 groups. Since o-hydroxy derivatives of CA4 have entered clinical trials, (36) we were surprised that 6 gave negligible potency under lit (and dark) conditions; however, 7 retained similar light-specific bioactivity as its isostere SBTub3 (see Section 2.2).
Continuing potency optimization, we also tested whether retaining the CA4 methoxy groups on a reduced-size photoswitch would be beneficial. We created styrylthiazole (ST) 8 which can be considered as a near-perfect isostere of CA4/PST-1 minus the small polar hydroxyl group, or as a shrunken analogue of 3. As far as we know, STs have never been used in photopharmacology before. We considered them interesting to the field as we expected them to retain the metabolic stability and GFP orthogonality of SBT, while additionally being isosteric to the better-known azobenzene: which offers attractive possibilities for adapting known azobenzenes (against other targets) into potentially biologically more applicable ST-based photopharmaceuticals. 8 was accessed by closing the thiazole, starting from a methyl cinnamoylglycinate (see the Supporting Information). Pleasingly, 8 gave strong Z-specific cellular bioactivity with ca. 100-fold photoswitchability, although its 10-fold loss of potency compared to 1 suggested that for the colchicine site, we should explore SBTubs intermediate in size between SBTub3 and 1.
Therefore, for potency optimization, we tested both adding methyl groups to SBTub3 and “walking” them around the scaffold south ring (9–13); and also deleting the north ring hydroxy/methoxy substituent pair from 1 and replacing them with smaller methyls (14–18). Since SBT Z-isomers orient their sulfur toward the inner face of the molecule, (18) we did not rely on rotations around the alkene─benzothiazole single bond to “reflect” substituents into similar positions (e.g., 9 vs 12, or 10 vs 11).
South ring derivatives 9–13 did not approach the potency of 1–5, although 10–11 still have excellent performance (∼100-fold photoswitchability of bioactivity, ∼1 μM Z-potency). However, north ring derivative SBTub2M (15) where a methyl group takes the space-filling position, had excellent Z-potency (Z-IC50 35 nM) while also having excellent photoswitchability of bioactivity (ca. 200-fold; Figure 2a). This makes it the most potent and the most photoswitchable of the photoswitchable antimitotics known. 16 (methyl at the small polar position) and 14 and 18 (methyl projecting to inner face, clashes with protein) were predictably weaker than SBTub2M (though 16 still has submicromolar Z activity and ca. 100-fold switching), while 17 that projects the methyl to the outer face (toward the exit tunnel) was well tolerated (0.2 μM) and retained nearly 200-fold switching. It was pleasing that this SAR also followed the SAR known for CA4, as this supports that their target and binding site are conserved.
Our second priority was to develop soluble SBTubs for in vivo use in multiorgan animal models, which do not easily tolerate even low amounts of organic cosolvents, and which may require temperatures below those of typical 2D cell cultures (e.g., <30 °C for zebrafish). We aimed to formulate water-soluble SBTub prodrugs by phenol phosphorylation─an approach that has been successful for other colchicinoid inhibitors (including clinically advanced CA4P, BNC105P, etc). (37) We had initially produced SBTub3P, the phosphate prodrug of previous lead compound SBTub3, however, its aqueous solubility was only moderate (<2 mg/mL) potentially due to aggregate formation by π-stacking of the planar compound. We synthesized SBTubA4P hoping that the out-of-plane central methoxy group would reduce π-stacking, which combined with the hydrophilicity of the three extra methoxy groups would give better solubility, as we have seen in other contexts. (6,19) Indeed, SBTA4P dissolved to at least 10 mM in water at 25 °C, which was to prove important in later assays. Both prodrugs SBTubA4P and SBTub3P had similar cellular Z/E potencies as their drug forms.
In summary, we had developed SBTubA4 and SBTub2M as mid-nanomolar antimitotics with 30–200-fold photoswitchability of bioactivity for cell culture studies (both cross-validated on A549 lung carcinoma cell line, Figure S5); and we had further developed SBTubA4P as a convenient fully water-soluble prodrug of SBTubA4 for in vivo applications. These became the focus of our further biological evaluations.

2.2. SBTub Photoswitch Performance Studies

The photoresponses of most SBTubs (1–5 and 9–20) were similar to previously reported SBTs SBTub2/3, (18) with absorption maxima and absorption cut-offs being excellently balanced for both efficient E → Z photoswitching with the common 405 nm microscopy laser, and for full orthogonality to GFP imaging (ca. 488 nm). The separated π → π* absorption maxima for E- (∼360 nm) and Z-isomers (∼330 nm) enable efficient directional E → Z photoisomerization at 360–420 nm reaching ca. 80% Z (Figure 2b), and extinction coefficients at 405 nm were up to twice those of similar azobenzenes, promising high-efficiency photoactivation on the confocal microscope. Importantly, E- and Z-SBT absorptions drop sharply toward zero above 410 nm (Figures 2c, S1, and S2) which is crucial for avoiding photoresponse to 488 nm GFP imaging under high-intensity focussed lasers, as well as with broader filtered excitation sources e.g., 490 ± 25 nm: since absorption “tails” extending far beyond band maxima can otherwise cause substantial photoswitching. (For example, 561 nm RFP imaging on the confocal microscope photoisomerizes azobenzene PST-1 despite its extinction coefficients being less than 30 cm–1 M–1. (24)) However, the SBTubs’ cutoff suggested they would indeed be GFP-orthogonal, which was later confirmed and found to be crucial for in vivo use (see below).
In biological settings (>360 nm) and on the population level, these SBTs act similarly to photoactivation probes: (i) illuminations all give similar majority-Z equilibrium photostationary states (PSSs) in the photoresponsive spectral region (360 to ca. <440 nm), and (ii) they show no significant (<2%) Z → E thermal relaxation after hours in physiological buffers at pH ∼ 7 at 25 °C (Figures S3 and S4). However, they have all of the other practical advantages of photoswitches that are relevant to most research uses (38) (no toxic/phototoxic byproducts, fast illumination response, no nonoptical drug activation mechanisms, and stocks can be quantitatively relaxed to E by warming to 60 °C, which is advantageous for stock handling over sequential assays). Their photostability to continuous illumination at >380 nm was excellent. These features are shared with previously reported SBTub2/3. (18)
We used ortho-hydroxy 6 and para-amino 7 to test if strong electron donor groups that can tautomerize to freely rotatable quinoids (C–C single bond instead of C═C), can accelerate thermal Z → E relaxation to make better-reversible SBTubs; and if these would induce spectral red-shifting. However, in a cuvette, Z-6/7 thermally relaxed only slowly (half-lives ≫ hours), and since Z-7 gave similar cytotoxicity in the long-term cellular assay as its isostere SBTub3, we concluded that its cellular relaxation was not fast on a biological timescale either. The spectra of 7 were red-shifted by nearly 60 nm compared to typical SBTs putting the E-7 absorption maximum exactly at the 405 nm laser line (Figure S1); however, as neither 6/7 brought relaxation rate benefits, we did not pursue them in this study.
Styrylthiazoles as in 8 have not yet been studied as scaffolds for photopharmaceuticals. (16) We were pleased that its isomers’ spectra were only ca. 25 nm blue-shifted compared to the SBTs (giving less efficient isomerization at 405 nm), and all other properties, such as its completeness of E → Z photoswitching, were similar to the SBTs. We believe this opens up new possibilities for GFP-orthogonal, metabolically resistant photoswitches that are isosteric to azobenzenes, and so can replace them for applications where these biologically advantageous properties are required.
For all further details, including full discussion of photoswitch performance of 6/7 and of E-SBTub toxicity, see the Supporting Information including Figures S1–S5.

2.3. SBTubs Isomer-Dependently Target Tubulin in Cells

To test their cellular mechanism of bioactivity, we first examined the SBTubs’ isomer-dependent inhibition of polymerization of purified tubulin protein in a cell-free assay. Both leads SBTubA4 and SBTub2M were noninhibiting in the all-E dark state, allowing tubulin to polymerize as in the cosolvent control (Figure 3a). However, in the majority-Z illuminated state, they were potent inhibitors that suppressed polymerization entirely to baseline readout levels, comparable to the archetypical colchicinoid nocodazole. Matching the antiproliferation assays, they were significantly stronger inhibitors than first-generation SBTub3 (Figure 3a).

Figure 3

Figure 3. Tubulin-specific cellular mechanism. (a) SBTubs light-dependently inhibit tubulin polymerization (turbidimetric cell-free assay; absorbance mirrors the extent of polymerization; lit indicates majority-Z-SBTub (20 μM) preisomerized to PSS at 405 nm; nocodazole control at 10 μM). (b) Cell cycle analysis of Jurkat cells treated with SBTub2M/SBTubA4P matches photoswitchable reference PST-1: with significant G2/M arrest under 405 nm pulsing (lit), but without cell cycle effects in the dark (matching cosolvent controls). (c) Immunofluorescence imaging of cells treated with SBTubA4P under pulsed 405 nm illuminations (lit, mostly-Z) and in the dark (all-E), compared to cosolvent control (HeLa cells, 20 h incubation; α-tubulin in green, DNA (stained with 4′,6-diamidino-2-phenylindole, DAPI) in blue). (d) Close-up views at the colchicine-binding site of X-ray co-crystal structures (PDB 7Z01, 7Z02) of Z-SBTubA4 (green) and E-SBTub2M (orange) bound to the Darpin D1:tubulin complex (dark gray α-tubulin, light gray β-tubulin in cartoon representation; Z-SBTubA4 and Z-SBTub2M in stick representation, oxygens red, nitrogen blue, and sulfur yellow). (e) Superimposition of tubulin:CA4 (white carbons; PDB 5LYJ) and TD1:Z-SBTubA4 (PDB 7Z01) shows that lead SBTubs share the same binding site as the parent natural product CA4 (see also Figure S11).

High Resolution Image
Download MS PowerPoint Slide
If microtubule inhibition is also the main cellular mechanism of action of Z-SBTubs, we would expect Z-SBTub-treated cells to show G2/M-phase cell cycle arrest due to mitotic checkpoint failure. (15) We therefore used flow cytometry-based analysis to quantify cell cycle repartition. E-SBTubs caused either no change or small changes compared to controls, whereas G2/M arrest was strongly induced by in situ lit (mostly-Z) SBTubA4P and SBTub2M (Figures 3b and S6).
As G2/M-phase arrest is necessary but insufficient to conclude on cellular tubulin inhibition being their major mechanism of action, we next performed confocal microscopy imaging of the MT network architecture in immunofluorescently stained cells, to directly observe tubulin-inhibiting effects. In situ illuminated SBTubA4P caused microtubules to curve and MT architecture to break down, (39) but E-SBTubs caused no disorganization at corresponding concentrations in the dark, matching cosolvent controls (Figure 3c). This matches the assumption that their photoswitchable cytotoxicity arises from their Z-isomers potently inhibiting MT dynamics and stability in cells. While microscopy is a qualitative method that can misrepresent population-level statistics, the quantitative cell cycle data from flow cytometry (104 cells per datapoint) as well as the qualitative match to previous SBTub work (18) give confidence to this result.
Finally, to check our design that Z-SBTubs act as colchicinoids, we crystallized the Z-SBTubA4 and Z-SBTub2M:tubulin DARPin D1 (TD1) complexes. X-ray diffraction studies showed that both Z-SBTubA4 and Z-SBTub2M indeed directly bind tubulin at the colchicine site very similarly to CA4, even with the same orientation of its substituents (Figures 3d,e and S11; data deposited as PDB 7Z01 and 7Z02). This explains why the SBTub SAR determined in this study (Table 1) mirrors that known for CA4. It also highlights the plasticity of the binding site, which accepts such differently sized inhibitors.
Taken together, these cell-free and cellular assays support that the SBTubs act as light-dependent tubulin inhibitors in cells, with their Z-isomers binding potently at the colchicine-binding site and their E-isomers having no effects at the corresponding concentrations.

2.4. SBTub Photocontrol Enables Cell-Precise, Temporally Reversible MT Inhibition in 2D Cell Culture

Aiming later to apply SBTubs to photocontrol MT dynamics in complex models and in vivo, we now switched to using the fully water-soluble prodrug SBTubA4P. This is an important step: (i) it avoids cosolvents that can be problematic for in vivo toxicity and (ii) it prevents hydrophobic adsorption onto the matrix materials (PDMS, collagen, agarose) that are used in 2D structured surfaces, 3D cell culture/organoid models, and for embedding motile animals during long-term imaging. Avoiding adsorption is important in our experience, as hydrophobic compounds can exhibit irreproducible apparent potencies or effects in these settings, which is timewise- and ethically prohibitive for resource-intensive low-throughput animal studies.
Before performing animal work, we probed the spatiotemporal resolution that SBTubs could achieve for in situphotoswitching-based control over MT dynamics, in 2D cell culture. Using spinning disk confocal live-cell microscopy, we imaged SBTubA4P-treated cells transfected with a fluorescently labeled fusion of the MT end binding protein EB3, to directly monitor MT polymerization dynamics during photoswitching. (18) This is possible since EB3 labels the GTP-cap of MTs, so EB3-tdTomato acts as a fluorescent marker revealing the tips of polymerizing MT plus ends in cells as hundreds of dynamic “comets” that cascade through the cell at significant velocities (tdTomato excitation at 561 nm). (40) Our protocol for cell-precise photoswitching of MT dynamics, with internal controls for compound application and for photobleaching, was as follows. We imaged transfected cells before SBTubA4P application to establish untreated MT dynamics baselines, simultaneously controlling for effects of 405 nm laser pulses on single selected cells [targeted by region of interest (ROI) illumination]; then, we added E-SBTubA4P to these same cells and continued imaging the whole field of view while applying targeted pulses of the 405 nm laser to a single ROI-selected cell.
SBTubA4P enabled repeatable cycles of temporally reversible, photoswitching-induced inhibition of MT dynamics in live cells, with single-cell spatial targeting precision and second-scale onset time precision (Figure 4a,b and Movie S1). In SBTubA4P-treated ROI cells, within seconds upon each single-frame 405 nm pulse, polymerizing MT tips stop moving and disappear, then more slowly reappear and resume movement (best seen in Movie S1). Statistics collected over multiple independent experiments showed these inhibition spikes are highly reproducible; recovery toward uninhibited baseline has a half-life of ca. 25 s, which we attribute to the diffusion of Z-SBTub out of the ROI cell (Figure 4b). There were minor effects on MT dynamics in treated non-ROI neighbor cells compared to pre-treatment controls, and the 405 nm pulsing protocol alone did not cause any readout changes (Figure 4a).

Figure 4

Figure 4. Spatiotemporal control over MT dynamics in 2D-cultured HeLa cells. (a, b) MT inhibition in SBTubA4P-treated cells is initiated only upon 405 nm illumination pulses and only in ROI-targeted cells (data related to Movie S1; live-cell EB3-tdTomato comets quantify polymerizing MTs). (a) Comet count statistics are similar to cosolvent-only baseline in both ROI-pulsed-cosolvent and non-ROI-SBTubA4P conditions; ROI-SBTubA4P statistics show inhibition spikes. (b) Stills from Movie S1 at the times indicated in (a), initially during the untreated timecourse, then during the SBTubA4P-treated timecourse on the same cells. Purple arrowhead indicates the ROI cell; purple dotted circle indicates where the 405 nm ROI is applied at times 26, 88, and 148 s; and white arrowhead indicates the non-ROI cell quantified as the internal control (scale bar 15 μm). (c) EB3 comet counts of cells imaged at 561 nm only (dark, gray), with 47 frames at 487 nm applied to full field of view during the time span indicated with dashed lines (“487”, cyan) and SBTubA4P (6 μM), or with single-frame 405 nm pulses, SBTubA4P (0.6 μM), applied to full field of view at times indicated with dashed lines (“405”, violet) (n = 3 cells). Temporally precise onset and full-field diffusional reversibility are shown (data related to Movies S2 and S3). [(a, c) Mean ± standard error of the mean (SEM) EB3 comet counts as normalized to the means of the first five time points; 405 nm ROIs applied at indicated times; for further details, see the Supporting Information].

High Resolution Image
Download MS PowerPoint Slide
We also modified this protocol to apply single-frame 405 nm pulses to the whole field of view instead (Figure 4c and Movie S2). As expected, this confirmed the temporal precision of onset and the temporal reversibility of the spiking seen with the single-cell-resolved studies; though the video data are easier to interpret as they are even more visually impressive (Movie S2). Finally, we performed full-field-of-view imaging while applying a train of 47 frames of 487 nm pulses, to test whether SBTubA4P can be used orthogonally to GFP imaging wavelengths. Indeed, even at this high concentration (6 μM, 40× [I]WC), there was no induction of MT inhibition, so we concluded that SBTubA4P is indeed GFP-orthogonal, matching our design (Movie S3 and Figure 4c).

2.5. From Cell Culture to MT Photocontrol in 3D Models, Tissue Explants, and Animals

By now we had optimized the potency of metabolically stable, GFP-orthogonal photoswitchable SBTub2M and fully water-soluble analogue prodrug SBTubA4P, clarified the SBTubs’ tubulin-specific cellular mechanism of action, and shown high-spatiotemporal-resolution photocontrol of MT dynamics in 2D cell culture. We were now primed to tackle the central photopharmacology research challenge, which has so far frustrated essentially all prior approaches: in vivo translation using systemic administration but local photocontrol, that clearly and usefully retains a defined cellular mechanism of action. We set out to test if SBTubs’ performance features would allow this operation across a range of complex models from 3D culture, to 3D tissue explant, to two in vivo animal models: with spatiotemporally localized illuminations photocontrolling the full sequence of their bioactivity (from suppressing MT polymerization to altering/depolymerizing MT network structures and to reducing/stopping microtubule-dependent downstream processes), where appropriate with second-level resolution, but all with cellular precision.

2.6. SBTub Photocontrol in 3D Organoids Enables Spatially Targeted Blockade of Migration and Mitosis in the Long Term

We first tested SBTubA4P in 3D human mammary gland organoids grown from isolated patient tissue embedded in collagen gels. These resemble miniaturized and simplified organs with realistic micro-anatomy, and feature collective motility/invasion behavior directing cells to migrate and proliferate to form ordered, branched structures. (41,42) Controlling organoid morphology is a sought-after goal, which has been mostly interpreted as requiring spatiotemporal control of gene expression, for which optogenetic approaches have been suggested. (43) Yet, the spatiotemporally localized application of photochemical compounds offers an alternative, in which the possibility of instantaneous cellular response to stimulus is highly attractive for temporally precise control. Based on the good performance of SBTubA4P in 2D cell culture, we assessed whether SBTubA4P can be controlled similarly precisely in a 3D organoid model, aiming to manipulate organoid development by locally interfering with the invasion of individual branches during the elongation phase. (42)
Though the actin cytoskeleton is the major driver of cell migration, MTs are integral to directional migration and leading-edge stabilization (24,44,45) (as well as to proliferation): so we expected the single-cell motility as well as cell division rates could be locally affected. Therefore, we wished to test if repeated localized photoactivations of SBTubA4P (every 7 min) could be used over long timescales (>24 h) to inhibit outgrowth of light-targeted organoid branches, while leaving other branches of the same organoids to develop: so shaping and modulating cell migration and invasion with spatiotemporal control. This aim brings conceptual challenges for photopharmaceuticals: since all cells are exposed to the same drug concentration, and over long timescales the cumulative impacts of scattered photoactivation light, of the diffused isomerized compound, and of imaging light itself, may build a spatially nonspecific background pattern of bioactivity. Organoid morphology does not tolerate >0.1% organic cosolvent either, making full solubility important.
We first determined a suitable working concentration for SBTubA4P without spatially resolved activation, by monitoring organoid areas for morphological disruption under lit/dark conditions (Figure S7a). We determined an [I]WC of 200 nM for preventing organoid growth over 24 h with UV-lit SBTubA4P, whereas organoids treated at 400 nM but kept in the dark grew healthily with no antiproliferative/branch-retracting effects (Figures 5a and S7a,b).

Figure 5

Figure 5. Spatiotemporal control over MT architecture, migration, and mitosis in 3D culture and tissue explant. (a) 3D human mammary gland organoids embedded in collagen gels only have inhibited branch outgrowth when treated with both SBTubA4P and UV pulses. (b) Local applications of UV light to ROItarg regions of SBTubA4P-treated organoids (blue box, one ca. 450 ms pulse per 7 min per z-stack) stops branch proliferation and outgrowth (red outline), while branches in untargeted ROIctrl regions develop dramatically (start: solid green line, final: dotted green line) (related to Movie S4). (c) Radial progress of branch tip fronts (directed and collective behavior) in ROItarg and ROIctrl regions. (d) Still image timecourse, zoomed on a branch tip in the ROIctrl (blue box) region, showing cell proliferation (yellow arrowhead) and matrix invasion (one representative of the migrating cells is tracked over time with green arrows), while branch tip of ROItarg region has static non-proliferating cells and even slight branch retraction (red arrows) (data related to Movie S5). (e) Branch progression and proliferation are unimpeded and continuous in ROIctrl regions, while ROItarg regions are static, and branches growing into the ROItarg stop their growth (color code as in (e), data related to Movie S6). [(a–e) Cell location in organoids tracked with nuclear stain SiR-DNA imaged at 647 nm]. (f) Whole-field-of-view 405 nm photoactivation of SBTub2M-treated intact 3D brain explants of larval Drosophila melanogaster (bottom row) causes neuroblast centrosomes (red arrows) to rapidly shrink in size and signal intensity (45 s and 3 min) and prevents the cell from progressing through division (13 min). Some MT signal accumulates at mid-cell at later time points (purple arrow) (data related to Movie S8). In DMSO-only controls (top row), centrosome integrity (white arrows, 45 s and 3 min) and progression through the cell cycle (13 min) are unaffected, indicated by myosin accumulation at the cleavage furrow (cyan arrows) (data related to Movie S9). [MTs in white (Jupiter::mCherry imaged at 561 nm), myosin in green (Squash::GFP imaged at 488 nm)]. (g) Relative mCherry fluorescence intensity of centrosomal ROIs in SBTub2M-treated prophase neuroblasts (red) after activation at 405 nm drops notably during the approximately 45 s activation period (blue box) compared to the DMSO control prophase neuroblasts (black). Signal intensities are shown as the proportion of the per-cell maximum preactivation signal intensity (shading indicates ±1 standard deviation, 1–2 centrosomes quantified from a total of five neuroblasts from three different animals). For details, see the Supporting Information.

High Resolution Image
Download MS PowerPoint Slide
Then, we applied localized UV ROI illuminations to selected organoid branches (ROItarg) every 7 min, comparing to non-UV-illuminated internal control branches (ROIctrl). This allowed us to noninvasively block cell migration/invasion and proliferation with striking spatial resolution and long-term persistence. With SBTubA4P, the development of ROItarg branches was totally blocked over >1 day (Figure 5b–d, Movies S4 and S5), while no-compound controls showed no photoinhibition of branch development (Movie S7 and Figure S7c). Control branches displayed high motility and matrix invasion (green outline, Figure 5b) and their cells freely proliferated (yellow arrowhead, Figure 5d), resulting in considerable branch development. The out-directed motion of the collective branch fronts was continuous in ROIctrl regions (ca. 4.5 μm/h) but was almost completely stopped in ROItarg (ca. 0.2 μm/h; Figure 5c). Overall development of branches, only entering ROIctrl areas, was however clearly visible without any statistical analysis (Figure 5e and Movie S6). Thus, SBTubA4P can be used in 3D matrix cell culture settings to noninvasively control cell motility, invasion, and proliferation, allowing photopatterning of branch growth and organoid development down to the spatial scale of individual cells.

2.7. SBTub Photocontrol in Intact 3D Tissue Explants Allows Temporally Precise MT Depolymerization and Mitotic Control in the Short Term

We next tested SBTub performance and tubulin-specific mechanism of action when directed against the more complex 3D environment of live intact brain lobes of early third instar larval D. melanogaster (fruit fly). Larvae are too motile for long-term imaging and the larval cuticle is largely impermeable, so neurodevelopment studies explant the whole brain. As the explant tolerates cosolvent, we took the opportunity to use SBTub2M in these assays to test the broader applicability of the SBTub design. Both the whole-organ and explant aspects bring significant challenges. (1) Compounds must permeate through two glia cell layers to reach mitotically active neural stem cells (neuroblasts). This forces the use of high bath concentrations and potent compounds: however, since surface and surrounding cells are exposed to far higher concentrations than central cells, only potent compounds with extremely high photoswitchability of bioactivity (“FDR”; discussed in ref (19)) can be used: otherwise, outer cells die, and morphology and physiology are lost. Indeed, we could use high concentrations of high-FDR SBTub2M (30 μM) in brain explants without noticeable toxicity. (2) Using multiple fluorescent protein labels for multiplexed imaging is a typical requirement to achieve useful readouts in biology, but can block chemical photoswitch applications. The most common long-wavelength fluorescent proteins for animal work are excited at 561 nm, which forces the use of GFP (488 nm) or YFP (514 nm) fusions as the next-longest-wavelength markers. For example, to image both MTs and cellular structural elements, we used animals expressing the microtubule-binding protein mCherry::jupiter, (46,47) and cortical structure marker sqh::GFP (spaghetti squash, the regulatory light chain of the non-muscle type 2 myosin, fused to eGFP (48)). With hemithioindigo or azobenzene reagents, such two-channel FP imaging would isomerize the photoswitch throughout the sample due to photoresponse at ≤530 nm (Figure 2b), so destroying spatiotemporal specificity in the study zone. In contrast, the nonresponse of the GFP-orthogonal SBT to 488 nm imaging avoids any photoisomerization during typical two-channel FP imaging, allowing precise temporal control of activation in our experiments.
We transferred freshly dissected brain explants into 30 μM SBTub2M and started imaging after 30 min loading (Figure S8). We imaged in both mCherry (ex 561 nm) “red channel” and GFP (ex 488 nm) “green channel” for 15 min to establish baseline, then photoactivated SBTub2M throughout the imaging stack volume with 405 nm. Photoactivated SBTub2M depolymerized centrosome microtubules within 60 s (Figure 5f, Movies S8, and S9, MTs shown in white). To control for target specificity, we also used mCherry::tubulin (49) for imaging MTs, and observed similar behavior (Movies S10 and S11). As microtubules are rapidly nucleated in prophase centrosomes, we quantified the loss of centrosomal fluorescence signal after activation as a highly conservative estimate of centrosome MT depolymerization. We saw dramatic, temporally resolved signal reduction at the approx. 45 s activation period, while SBTub controls were unaffected (Figure 5g). We used the second FP channel to image Sqh::GFP, a marker of the cell actomyosin cortex, which plays a key role in neuroblast asymmetric division. (50,51) Normally dividing neuroblasts accumulate Sqh::GFP at the cell cleavage furrow during anaphase. Neuroblasts in which SBTub2M was photoactivated retain uniform cortical myosin, indicating mitotic arrest in the absence of mitotic spindles (50) (cyan arrows, Figure 5f).
Previous short-term results imaging EBs at low SBTub concentrations in 2D cell culture had illustrated only its capacity to spatiotemporally block MT polymerization (Figure 4). Now, these useful results in the intact brain underlined that SBTub2M maintains its Z-isomer-specific, MT-depolymerizing mechanism of action in live tissue explant, casting SBTubs as flexible and powerful tools for cytoskeleton photomanipulation in complex 3D settings.

2.8. SBTub Photocontrol in Live Animals Enables Targeted Blockade of Embryonic Development

Encouraged by performance in 3D models, we evaluated using SBTubs for in vivo photocontrol in intact animals. First, we studied the effects of in situ photoactivations of SBTubA4P on the development of Xenopus tropicalis clawed frog embryos. During the initial 48 h of development, embryos normally transition over many division cycles from cell spheres through the blastula stage through to multi-organ tadpoles (Figure S9a). Initially, we tested the effects of SBTubA4P in the earliest stages of development, just after embryonic divisions had started, by treating 2-cell stage embryos with E-SBTubA4P (1 h loading, then medium exchange and optional in situ embryo-localized 410 nm photoactivation pulse during washout; note that this transient exposure to the SBTub parallels what could be expected for e.g., systemic i.v. administration in mammalian models─further discussion in the Supporting Information). Embryos only failed to develop morphologically over the subsequent 2 days if they had received the 410 nm photoactivation; embryos without photoactivation developed normally (Figure 6a,b). We also tested interfering with development at the later blastula stage (>64 cells) by a similar protocol. While subsequent morphological development was normal under lit and dark conditions (Figure S9b,c), sensorimotor responses to mechanical stimulation (52) were suppressed by lit SBTubA4P only (Figure S9e and Movie S12). Interestingly, the tubulin-inhibiting azobenzene photoswitch PST-1P (Figure S9d) light-independently suppresses sensorimotor responses. We believe the SBT’s success may reflect its greater metabolic robustness (see the Supporting Information); and at any rate, it indicated that SBTubs are suitable for in vivo use.

Figure 6

Figure 6. Photoinhibition of X. tropicalis development, and in vivo photocontrol of MT dynamics in Danio rerio. (a, b) Xenopus embryos incubated with compounds for 1 h at the two-cell stage, before medium exchange optionally with 410 nm photoactivation. Embryos show irreversible development inhibition by in situ formed Z-SBTubA4 in lit conditions but had no effects in the dark or with a low concentration of SBTubA4P [(a) SBTubA4P at 5 μM; (b) development quantified by the ratio of major to minor embryo axis lengths, six embryos per condition, mean ± SEM]. (c) Development of D. rerio treated at the indicated stages for 24 h with SBTubA4P or control compounds under dark or pulsed lit (1 s/5 min) conditions. SBTubA4P (1 or 25 μM) causes morphological abnormalities only in the lit state, showing that it remains effective in vivo. (d, e) Reversible modulation of MT dynamics in 48 hpf zebrafish embryo (25 μM). (EB3-GFP in green, histone H2B in red). (data related to Movies S12–S16; see the Supporting Information).

High Resolution Image
Download MS PowerPoint Slide

2.9. SBTub Photocontrol in Live Animals Enables Cell-Precise Temporally Reversible Inhibition of MT Dynamics

Finally, we switched to highly spatiotemporally resolved in vivo MT-imaging studies that would test SBTubA4P’s mechanism of action and applicability in the zebrafish D. rerio, when systemically applied and maintained in the bath medium.
We first determined useful working concentrations in zebrafish, incubating 24 and 48 hpf (hours post fertilization) embryos in SBTubA4P under lit and dark conditions for 24 h. While zebrafish morphology remained unaltered in all dark SBTub treatments, 24 hpf embryos treated with lit SBTub showed major morphological changes even down to 1 μM, whereas more developed 48 hpf embryos showed similar morphological changes only at higher SBTubA4P concentrations e.g., 25 μM (Figures 6c and S10). Again, we compared these effects to those of azobenzene reagent PST-1P, now observing a dramatic difference: even 25 μM lit PST-1P did not interfere with development at either the 24 hpf or 48 hpf stage (Figures 6c and S10). This argues still more conclusively than Figure 6b, that the SBTub scaffold is uniquely suitable for light-controlled biological effects, compared to the previously known azobenzene scaffold. Finally, we checked the lower-potency soluble prodrug SBTub3P in the same assay; matching expectations, it caused only weak changes at 24 hpf and no visible changes at 48 hpf (Figure S10), showing the necessity of the potency optimizations we performed in this study.
Aiming to test the MT-modulating effects of SBTubs in a challenging live animal system, we therefore decided to proceed with 48 hpf zebrafish embryos, and an SBTubA4P working concentration of 25 μM. We took 48 hpf embryos coexpressing EB3-GFP and histone H2B-mRFP as a nuclear marker, (53,54) loaded them with 25 μM E-SBTubA4P for 4 h, then washed and embedded them in agarose. Imaging at 488 nm caused no suppression of EB3 comets, confirming SBTubA4P’s GFP orthogonality in vivo. However, photoactivation with the 405 nm laser at a single point caused EB3 comets to vanish rapidly in cells around the targeted region, recovering over ca. 10 min. Cells further from the targeted region were predictably less inhibited than those with direct contact to the photoactivation region. The photoactivation-recovery cycle could be repeated multiple times during imaging (Figure 6d,e and Movies S13–S16). Not only microtubule polymerization dynamics but also mitotic progression, could be stopped by spatiotemporally localized SBTubA4P photoactivation in vivo (Movie S16). These experiments confirm that the SBT scaffold, in general, is viable for light-triggered in vivo studies, and that SBTubA4P when applied in vivo retains its mechanism of action as a potent, light-dependent MT inhibitor with excellent spatial specificity and satisfying temporal reversibility.

3. Conclusions

Click to copy section linkSection link copied!
Noninvasive optical tools to modulate microtubule dynamics, structure, and function with high precision offer unique potential in the many fields of biology impacted by the spatiotemporally resolved processes that MTs support, such as cargo transport, cell motility, cell division, development, and neuroscience. Photopharmaceutical chemical reagents are conceptually elegant optical tools in that they can be rapidly transitioned across models and settings and that they can be rationally designed for photoresponse patterns that interfere minimally with imaging while maximizing optical response to a chosen photoactivation wavelength. Locally applied photopharmaceuticals, particularly intraocularly applied reagents for action potential control in the retina, have made great progress in adult mammalian disease models. (55,56) However, reaching general uses of photopharmacology involving precise targeting of drug activity in vivo by localized in situ photoactivations following systemic administration remains an unsolved challenge for photopharmaceuticals with cytosolic target proteins. This would require combining high photoswitchability of bioactivity, high potency, metabolic robustness, aqueous solubility, and imaging orthogonality. Indeed, very few systemic in vivo applications of photopharmaceuticals have been made, and to date, no in vivo studies have combined testing a defined mechanism of drug action, with exploring the potential for cell-scale spatially resolved targeting following systemic administration. (57−59) Thus, the ability of photopharmacology to contribute useful systemically applicable reagents for organism studies has remained unclear.
In this work, we develop highly light-specific tubulin polymerization inhibitors with unprecedented applicability from 2D cell culture, through 3D culture and whole-organ explant, to systemic in vivo administration with local photoactivation. Realizing that the metabolic robustness and imaging orthogonality of a photopharmaceutical largely depend on its photoswitch scaffold, we consciously selected the recently developed SBT photoswitch and screened a SAR panel of 20 SBTubs. We optimized the potency and photoswitchability of bioactivity in lead SBTub2M, and we solubilized another lead to create SBTubA4P, which does not require organic cosolvents. SBTubs are efficiently photoactivated with the common 405 nm laser, but their sharp absorption cutoff leaves GFP, YFP, and RFP channels free for multiplexed imaging of fusion protein markers without risking compound photoactivation. This is a highly desirable feature for areas of research where photopharmacology’s optical precision can best contribute unique solutions on the cellular spatial scale (although this practical use logic does run counter to the goal of “photoswitch red-shifting” that is often cited in chemical design). We cross-validated the SBTubs’ molecular mechanism of action in cell-free, cellular, and in vivo settings. SBTubs light-dependently interfere with mitosis in cell culture, and in vivo they depolymerize mitotic spindles, ultimately blocking development. They can be optically patterned to control motility and branch development in 3D organoid cultures, and their photocontrol allows rapid response, cell-specific inhibition of microtubule dynamics in cell culture and in vivo.
These consistent results across a range of models at different scales of time, length, and biological complexity recommend the SBTubs as excellent and unique general-purpose tools for optically manipulating microtubule dynamics, microtubule structure, and microtubule-dependent processes with high spatiotemporal precision.
The proof-of-concept biological performance of the SBTubs has been very satisfying. We believe that the most valuable improvement to this system will now be to extend the temporal reversibility of inhibition (seen in 2D cell culture by diffusion to the medium with ca. 20 s half-time, Figure 4) to whole-organ/whole-animal settings. In these settings, diffusion is slower to achieve reversibility (ca. 10 min), so we seek techniques for in situ bidirectional isomerization of SBTubs in our ongoing research. We believe that bidirectional photoswitching may be difficult within the biologically compatible wavelength range. (16) However, accelerating thermal relaxation to the minute scale, which is probably the most appropriate scale for 3D/in vivo applications of interest, may be feasible, and efforts are underway.
In conclusion, the SBTubs are excellent photoswitchable microtubule-depolymerizing reagents for use in cell culture, 3D culture, small explant, and early-stage animals. Their potency, flexibility, and ease of use recommend them for high-spatiotemporal-precision research across cytoskeleton biology; particularly, we feel, for cell-specific applications to motility and development, but they will also be of great interest in cargo transport, biophysics, cell polarity, neurodegeneration, and cell division. Finally, we expect that by supporting conceptual innovations in photoswitch scaffold chemistry and rational photopharmaceutical design, and particularly by starting to unlock the applications promise of photopharmacology for globally administered, locally targeted in vivo use, this SBTub research represents a promising advance for high-performance photopharmacology against other protein targets in general, beyond their immediate impact on microtubule biology.

Supporting Information

Click to copy section linkSection link copied!

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.2c01020.

  • Temporally precise and cell-precise inhibitions of cellular MT polymerization dynamics by photoactivations of E-SBTubA4P at 405 nm (Movie S1) (MP4)

  • Temporally precise full-field-of-view inhibitions of cellular MT polymerization dynamics by photoactivations of E-SBTubA4P at 405 nm (Movie S2) (MP4)

  • No inhibition of cellular MT polymerization dynamics by illumination of E-SBTubA4P at 487 nm (Movie S3) (MP4)

  • SBTubA4P blocks branch development in primary human mammary gland organoids, light-dependently and with spatiotemporal precision (Movies S4−S6) (MP4, MP4, MP4)

  • No-compound control shows no photoinhibition of organoid branch development in both ROItarg and ROIctrl (Movie S7) (MP4)

  • Temporally precise depolymerization of the mitotic spindle in a prophase Drosophila neuroblast by photoactivation of E-SBTub2M (30 μM) at 405 nm (Movie S8) (MP4)

  • DMSO-only control to Movie S8 shows prophase Drosophila neuroblast undergoing normal mitosis after 405 nm illumination (Movie S9) (MP4)

  • Temporally precise depolymerization of the mitotic spindle in a prophase Drosophila neuroblast by photoactivation of E-SBTub2M (30 μM) at 405 nm (Movie S10) (MP4)

  • DMSO-only control to Movie S10 shows prophase Drosophila neuroblast undergoing normal mitosis after 405 nm illumination (Movie S11) (MP4)

  • Reaction of hatchling frog X. tropicalis embryos to mechanical stimulus depends on the temporally precise application of Z-SBTubA4 during prior development (Movie S12) (MP4)

  • Photoactivation of SBTubA4P (25 μM) in a live zebrafish embryo shows temporally reversible inhibitions of EB3 dynamics over several cycles (Movies S13 and S14) (MP4, MP4)

  • Inhibition of MT polymerization dynamics in zebrafish embryo when SBTubA4P (25 μM) is photoactivated (Movie S15) (MP4)

  • Photoactivation of SBTubA4P (25 μM) stops both EB3 dynamics and cell division in the developing zebrafish embryo (Movie S16) (MP4)

  • Chemical synthesis, photocharacterization, biological data, protein crystallization, and NMR spectra (PDF)

Terms & Conditions

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

Author Information

Click to copy section linkSection link copied!
  • Corresponding Author
  • Authors
    • Li Gao - Department of Pharmacy, Ludwig-Maximilians University of Munich, Munich 81377, Germany
    • Joyce C. M. Meiring - Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht CH 3584, Netherlands
    • Adam Varady - St. Anna Children’s Cancer Research Institute (CCRI), Vienna 1090, Austria
    • Iris E. Ruider - Physics Department and Center for Protein Assemblies CPA, Technical University of Munich, Garching 85747, Germany
    • Constanze Heise - Department of Pharmacy, Ludwig-Maximilians University of Munich, Munich 81377, Germany
    • Maximilian Wranik - Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, Villigen 5232, Switzerland
    • Cecilia D. Velasco - Laboratory of Neurobiology, Department of Pathology and Experimental Therapy, Institute of Neurosciences, University of Barcelona, L’Hospitalet de Llobregat, Barcelona 08907, Spain;  Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet de Llobregat, Barcelona 08907, Spain;  Orcidhttps://orcid.org/0000-0003-1527-607X
    • Jennifer A. Taylor - Department of Biology, University of Washington, Seattle, Washington 98195, United States
    • Beatrice Terni - Laboratory of Neurobiology, Department of Pathology and Experimental Therapy, Institute of Neurosciences, University of Barcelona, L’Hospitalet de Llobregat, Barcelona 08907, Spain;  Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet de Llobregat, Barcelona 08907, Spain
    • Tobias Weinert - Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, Villigen 5232, Switzerland
    • Jörg Standfuss - Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, Villigen 5232, Switzerland
    • Clemens C. Cabernard - Department of Biology, University of Washington, Seattle, Washington 98195, United States
    • Artur Llobet - Laboratory of Neurobiology, Department of Pathology and Experimental Therapy, Institute of Neurosciences, University of Barcelona, L’Hospitalet de Llobregat, Barcelona 08907, Spain;  Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet de Llobregat, Barcelona 08907, Spain
    • Michel O. Steinmetz - Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, Villigen 5232, Switzerland;  Biozentrum, University of Basel, Basel 4056, Switzerland;  Orcidhttps://orcid.org/0000-0001-6157-3687
    • Andreas R. Bausch - Physics Department and Center for Protein Assemblies CPA, Technical University of Munich, Garching 85747, Germany
    • Martin Distel - St. Anna Children’s Cancer Research Institute (CCRI), Vienna 1090, Austria;  Zebrafish Platform Austria for Preclinical Drug Screening (ZANDR), Vienna 1090, Austria
    • Julia Thorn-Seshold - Department of Pharmacy, Ludwig-Maximilians University of Munich, Munich 81377, Germany;  Orcidhttps://orcid.org/0000-0002-4879-4159
    • Anna Akhmanova - Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht CH 3584, Netherlands
  • Funding

    This research was supported by funds from the German Research Foundation (DFG: Emmy Noether grant number 400324123 to O.T.-S.; SFB 1032 number 201269156 project B09 to O.T.-S. and project A10 to A.R.B.; SFB TRR 152 number 239283807 project P24 to O.T.-S.; and SPP 1926 number 426018126 project XVIII to O.T.-S.). J.C.M.M. acknowledges support from an EMBO Long Term Fellowship (ALTF 261-2019). A.V. acknowledges support by a DOC fellowship of the Austrain Academy of Sciences (ÖAW). J.T.-S. acknowledges support from a Joachim Herz Foundation Stipend. A.R.B. gratefully acknowledges the financial support of the European Research Council (ERC) through the funding of the grant Principles of Integrin Mechanics and Adhesion (PoINT). A.L. acknowledges funding from the Spanish government (Ministerio de Ciencia e Innovación), grant RTI2018-096948-B-100 (A.L.), co-funded by the European Regional Development Fund (ERDF). M.D. acknowledges funding from the Austrian Research Promotion Agency (FFG) project 7940628 (Danio4Can). C.C.C. is supported by NIH grant 1R01GM126029.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

Click to copy section linkSection link copied!

We are grateful to Henrietta Lacks, now deceased, and to her surviving family members for their contributions to biomedical research. We thank Monique Preusse for early cell viability testing, and Rebekkah Hammar for performing the tubulin polymerization assay. We thank Christian Gabka from the Nymphenburg Clinic for Plastic and Aesthetic Surgery, Munich 80637, Germany, for providing primary human mammary gland tissue.

References

Click to copy section linkSection link copied!

This article references 59 other publications.

  1. 1
    Stepanova, T.; Slemmer, J.; Hoogenraad, C. C.; Lansbergen, G.; Dortland, B.; De Zeeuw, C. I.; Grosveld, F.; van Cappellen, G.; Akhmanova, A.; Galjart, N. Visualization of Microtubule Growth in Cultured Neurons via the Use of EB3-GFP (End-Binding Protein 3-Green Fluorescent Protein). J. Neurosci. 2003, 23, 2655– 2664,  DOI: 10.1523/JNEUROSCI.23-07-02655.2003
    Google Scholar
    1
    Visualization of microtubule growth in cultured neurons via the use of EB3-GFP (end-binding protein 3-green fluorescent protein)
    Stepanova, Tatiana; Slemmer, Jenny; Hoogenraad, Casper C.; Lansbergen, Gideon; Dortland, Bjorn; De Zeeuw, Chris I.; Grosveld, Frank; van Cappellen, Gert; Akhmanova, Anna; Galjart, Niels
    Journal of Neuroscience (2003), 23 (7), 2655-2664CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
    Several microtubule binding proteins, including CLIP-170 (cytoplasmic linker protein-170), CLIP-115, and EB1 (end-binding protein 1), have been shown to assoc. specifically with the ends of growing microtubules in non-neuronal cells, thereby regulating microtubule dynamics and the binding of microtubules to protein complexes, organelles, and membranes. When fused to GFP (green fluorescent protein), these proteins, which collectively are called +TIPs (plus end tracking proteins), also serve as powerful markers for visualizing microtubule growth events. Here we demonstrate that endogenous +TIPs are present at distal ends of microtubules in fixed neurons. Using EB3-GFP as a marker of microtubule growth in live cells, we subsequently analyze microtubule dynamics in neurons. Our results indicate that microtubules grow slower in neurons than in glia and COS-1 cells. The av. speed and length of EB3-GFP movements are comparable in cell bodies, dendrites, axons, and growth cones. In the proximal region of differentiated dendrites ∼65% of EB3-GFP movements are directed toward the distal end, whereas 35% are directed toward the cell body. In more distal dendritic regions and in axons most EB3-GFP dots move toward the growth cone. This difference in directionality of EB3-GFP movements in dendrites and axons reflects the highly specific microtubule organization in neurons. Together, these results suggest that local microtubule polymn. contributes to the formation of the microtubule network in all neuronal compartments. We propose that similar mechanisms underlie the specific assocn. of CLIPs and EB1-related proteins with the ends of growing microtubules in non-neuronal and neuronal cells.
  2. 2
    Kleele, T.; Marinković, P.; Williams, P. R.; Stern, S.; Weigand, E. E.; Engerer, P.; Naumann, R.; Hartmann, J.; Karl, R. M.; Bradke, F.; Bishop, D.; Herms, J.; Konnerth, A.; Kerschensteiner, M.; Godinho, L.; Misgeld, T. An Assay to Image Neuronal Microtubule Dynamics in Mice. Nat. Commun. 2014, 5, 4827  DOI: 10.1038/ncomms5827
    Google Scholar
    2
    An assay to image neuronal microtubule dynamics in mice
    Kleele, Tatjana; Marinkovic, Petar; Williams, Philip R.; Stern, Sina; Weigand, Emily E.; Engerer, Peter; Naumann, Ronald; Hartmann, Jana; Karl, Rosa M.; Bradke, Frank; Bishop, Derron; Herms, Jochen; Konnerth, Arthur; Kerschensteiner, Martin; Godinho, Leanne; Misgeld, Thomas
    Nature Communications (2014), 5 (), 4827CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
    Microtubule dynamics in neurons play crit. roles in physiol., injury and disease and det. microtubule orientation, the cell biol. correlate of neurite polarization. Several microtubule binding proteins, including end-binding protein 3 (EB3), specifically bind to the growing plus tip of microtubules. In the past, fluorescently tagged end-binding proteins have revealed microtubule dynamics in vitro and in non-mammalian model organisms. Here, we devise an imaging assay based on transgenic mice expressing yellow fluorescent protein-tagged EB3 to study microtubules in intact mammalian neurites. Our approach allows measurement of microtubule dynamics in vivo and ex vivo in peripheral nervous system and central nervous system neurites under physiol. conditions and after exposure to microtubule-modifying drugs. We find an increase in dynamic microtubules after injury and in neurodegenerative disease states, before axons show morphol. indications of degeneration or regrowth. Thus increased microtubule dynamics might serve as a general indicator of neurite remodelling in health and disease.
  3. 3
    Goglia, A. G.; Toettcher, J. E. A Bright Future: Optogenetics to Dissect the Spatiotemporal Control of Cell Behavior. Curr. Opin. Chem. Biol. 2019, 48, 106– 113,  DOI: 10.1016/j.cbpa.2018.11.010
    Google Scholar
    3
    A bright future: optogenetics to dissect the spatiotemporal control of cell behavior
    Goglia, Alexander G.; Toettcher, Jared E.
    Current Opinion in Chemical Biology (2019), 48 (), 106-113CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
    Cells sense, process, and respond to extracellular information using signaling networks: collections of proteins that act as precise biochem. sensors. These protein networks are characterized by both complex temporal organization, such as pulses of signaling activity, and by complex spatial organization, where proteins assemble structures at particular locations and times within the cell. Yet despite their ubiquity, studying these spatial and temporal properties has remained challenging because they emerge from the entire protein network rather than a single node, and cannot be easily tuned by drugs or mutations. These challenges are being met by a new generation of optogenetic tools capable of directly controlling the activity of individual signaling nodes over time and the assembly of protein complexes in space. Here, we outline how these recent innovations are being used in conjunction with engineering-influenced exptl. design to address longstanding questions in signaling biol.
  4. 4
    Hoorens, M. W. H.; Szymanski, W. Reversible, Spatial and Temporal Control over Protein Activity Using Light. Trends Biochem. Sci. 2018, 43, 567– 575,  DOI: 10.1016/j.tibs.2018.05.004
    Google Scholar
    4
    Reversible, Spatial and Temporal Control over Protein Activity Using Light
    Hoorens, Mark W. H.; Szymanski, Wiktor
    Trends in Biochemical Sciences (2018), 43 (8), 567-575CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Ltd.)
    A review. In biomedical sciences, the function of a protein of interest is investigated by altering its net activity and assessing the consequences for the cell or organism. To change the activity of a protein, a wide variety of chem. and genetic tools have been developed. The drawback of most of these tools is that they do not allow for reversible, spatial and temporal control. Here, we describe selected developments in photopharmacol. that aim at establishing such control over protein activity through bioactive mols. with photo-controlled potency. We also discuss why such control is desired and what challenges still need to be overcome for photopharmacol. to reach its maturity as a chem. biol. research tool.
  5. 5
    Borowiak, M.; Nahaboo, W.; Reynders, M.; Nekolla, K.; Jalinot, P.; Hasserodt, J.; Rehberg, M.; Delattre, M.; Zahler, S.; Vollmar, A.; Trauner, D.; Thorn-Seshold, O. Photoswitchable Inhibitors of Microtubule Dynamics Optically Control Mitosis and Cell Death. Cell 2015, 162, 403– 411,  DOI: 10.1016/j.cell.2015.06.049
    Google Scholar
    5
    Photoswitchable Inhibitors of Microtubule Dynamics Optically Control Mitosis and Cell Death
    Borowiak, Malgorzata; Nahaboo, Wallis; Reynders, Martin; Nekolla, Katharina; Jalinot, Pierre; Hasserodt, Jens; Rehberg, Markus; Delattre, Marie; Zahler, Stefan; Vollmar, Angelika; Trauner, Dirk; Thorn-Seshold, Oliver
    Cell (Cambridge, MA, United States) (2015), 162 (2), 403-411CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    Small mols. that interfere with microtubule dynamics, such as Taxol and the Vinca alkaloids, are widely used in cell biol. research and as clin. anticancer drugs. However, their activity cannot be restricted to specific target cells, which also causes severe side effects in chemotherapy. Here, the authors introduce the photostatins, inhibitors that can be switched on and off in vivo by visible light, to optically control microtubule dynamics. Photostatins modulate microtubule dynamics with a subsecond response time and control mitosis in living organisms with single-cell spatial precision. In longer-term applications in cell culture, photostatins are up to 250 times more cytotoxic when switched on with blue light than when kept in the dark. Therefore, photostatins are both valuable tools for cell biol., and are promising as a new class of precision chemotherapeutics whose toxicity may be spatiotemporally constrained using light.
  6. 6
    Borowiak, M.; Küllmer, F.; Gegenfurtner, F.; Peil, S.; Nasufovic, V.; Zahler, S.; Thorn-Seshold, O.; Trauner, D.; Arndt, H.-D. Optical Manipulation of F-Actin with Photoswitchable Small Molecules. J. Am. Chem. Soc. 2020, 142, 9240– 9249,  DOI: 10.1021/jacs.9b12898
    Google Scholar
    6
    Optical Manipulation of F-Actin with Photoswitchable Small Molecules
    Borowiak, Malgorzata; Kuellmer, Florian; Gegenfurtner, Florian; Peil, Sebastian; Nasufovic, Veselin; Zahler, Stefan; Thorn-Seshold, Oliver; Trauner, Dirk; Arndt, Hans-Dieter
    Journal of the American Chemical Society (2020), 142 (20), 9240-9249CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Cell-permeable photoswitchable small mols., termed optojasps, are introduced to optically control the dynamics of the actin cytoskeleton and cellular functions that depend on it. These light-dependent effectors were designed from the F-actin-stabilizing marine depsipeptide jasplakinolide by functionalizing them with azobenzene photoswitches. As demonstrated, optojasps can be employed to control cell viability, cell motility, and cytoskeletal signaling with the high spatial and temporal resoln. that light affords. Optojasps can be expected to find applications in diverse areas of cell biol. research. They may also provide a template for photopharmacol. targeting the ubiquitous actin cytoskeleton with precision control in the micrometer range.
  7. 7
    Glotzer, M. The 3Ms of Central Spindle Assembly: Microtubules, Motors and MAPs. Nat. Rev. Mol. Cell Biol. 2009, 10, 9– 20,  DOI: 10.1038/nrm2609
    Google Scholar
    7
    The 3Ms of central spindle assembly: microtubules, motors and MAPs
    Glotzer, Michael
    Nature Reviews Molecular Cell Biology (2009), 10 (1), 9-20CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
    A review. During metaphase, sister chromatids are positioned at the midpoint of the microtubule-based mitotic spindle in prepn. for their segregation. The onset of anaphase triggers inactivation of the key mitotic kinase, cyclin-dependent kinase 1 (CDK1), and the poleward movement of sister chromatids. During anaphase, the mitotic spindle reorganizes in prepn. for cytokinesis. Kinesin motor proteins and microtubule-assocd. proteins bundle the plus ends of interpolar microtubules and generate the central spindle, which regulates cleavage furrow initiation and the completion of cytokinesis. Complementary approaches, including cell biol., genetics, and computational modeling, have provided new insights into the mechanism and regulation of central spindle assembly.
  8. 8
    Kapitein, L. C.; Hoogenraad, C. C. Building the Neuronal Microtubule Cytoskeleton. Neuron 2015, 87, 492– 506,  DOI: 10.1016/j.neuron.2015.05.046
    Google Scholar
    8
    Building the Neuronal Microtubule Cytoskeleton
    Kapitein, Lukas C.; Hoogenraad, Casper C.
    Neuron (2015), 87 (3), 492-506CODEN: NERNET; ISSN:0896-6273. (Cell Press)
    Microtubules are one of the major cytoskeletal components of neurons, essential for many fundamental cellular and developmental processes, such as neuronal migration, polarity, and differentiation. Microtubules have been regarded as crit. structures for stable neuronal morphol. because they serve as tracks for long-distance transport, provide dynamic and mech. functions, and control local signaling events. Establishment and maintenance of the neuronal microtubule architecture requires tight control over different dynamic parameters, such as microtubule no., length, distribution, orientations, and bundling. Recent genetic studies have identified mutations in a wide variety of tubulin isotypes and microtubule-related proteins in many of the major neurodevelopmental and neurodegenerative diseases. Here, we highlight the functions of the neuronal microtubule cytoskeleton, its architecture, and the way its organization and dynamics are shaped by microtubule-related proteins.
  9. 9
    van Haren, J.; Charafeddine, R. A.; Ettinger, A.; Wang, H.; Hahn, K. M.; Wittmann, T. Local Control of Intracellular Microtubule Dynamics by EB1 Photodissociation. Nat. Cell Biol. 2018, 20, 252– 261,  DOI: 10.1038/s41556-017-0028-5
    Google Scholar
    9
    Local control of intracellular microtubule dynamics by EB1 photodissociation
    van Haren, Jeffrey; Charafeddine, Rabab A.; Ettinger, Andreas; Wang, Hui; Hahn, Klaus M.; Wittmann, Torsten
    Nature Cell Biology (2018), 20 (3), 252-261CODEN: NCBIFN; ISSN:1465-7392. (Nature Research)
    End-binding proteins (EBs) are adaptors that recruit functionally diverse microtubule plus-end-tracking proteins (+TIPs) to growing microtubule plus ends. To test with high spatial and temporal accuracy how, when and where +TIP complexes contribute to dynamic cell biol., it developed a photo-inactivated EB1 variant (.product. -EB1) by inserting a blue-light-sensitive protein-protein interaction module between the microtubule-binding and +TIP-binding domains of EB1. .product. -EB1 replaces endogenous EB1 function in the absence of blue light. By contrast, blue-light-mediated .product. -EB1 photodissocn. results in rapid +TIP complex disassembly, and acutely and reversibly attenuates microtubule growth independent of microtubule end assocn. of the microtubule polymerase CKAP5 (also known as ch-TOG and XMAP215). Local .product. -EB1 photodissocn. allows subcellular control of microtubule dynamics at the second and micrometer scale, and elicits aversive turning of migrating cancer cells. Importantly, light-mediated domain splitting can serve as a template to optically control other intracellular protein activities.
  10. 10
    Adikes, R. C.; Hallett, R. A.; Saway, B. F.; Kuhlman, B.; Slep, K. C. Control of Microtubule Dynamics Using an Optogenetic Microtubule plus End–F-Actin Cross-Linker. J. Cell Biol. 2018, 217, 779– 793,  DOI: 10.1083/jcb.201705190
    Google Scholar
    10
    Control of microtubule dynamics using an optogenetic microtubule plus end-F-actin cross-linker
    Adikes, Rebecca C.; Hallett, Ryan A.; Saway, Brian F.; Kuhlman, Brian; Slep, Kevin C.
    Journal of Cell Biology (2018), 217 (2), 779-793CODEN: JCLBA3; ISSN:1540-8140. (Rockefeller University Press)
    We developed a novel optogenetic tool, SxIP-improved light-inducible dimer (iLID), to facilitate the reversible recruitment of factors to microtubule (MT) plus ends in an end-binding protein-dependent manner using blue light. We show that SxIP-iLID can track MT plus ends and recruit tgRFP-SspB upon blue light activation. We used this system to investigate the effects of crosslinking MT plus ends and F-actin in Drosophila melanogaster S2 cells to gain insight into spectraplakin function and mechanism. We show that SxIP-iLID can be used to temporally recruit an F-actin binding domain to MT plus ends and cross-link the MT and F-actin networks. Crosslinking decreases MT growth velocities and generates a peripheral MT exclusion zone. SxIP-iLID facilitates the general recruitment of specific factors to MT plus ends with temporal control enabling researchers to systematically regulate MT plus end dynamics and probe MT plus end function in many biol. processes.
  11. 11
    Meiring, J. C. M.; Grigoriev, I.; Nijenhuis, W.; Kapitein, L. C.; Akhmanova, A. Opto-Katanin: An Optogenetic Tool for Localized Microtubule Disassembly. bioRxiv 2021, 22, 473806  DOI: 10.1101/2021.12.22.473806
    Google Scholar
    There is no corresponding record for this reference.
  12. 12
    Liu, G. Y.; Chen, S.-C.; Shaiv, K.; Hong, S.-R.; Yang, W.-T.; Huang, S.-H.; Chang, Y.-C.; Cheng, H.; Lin, Y.-C. Precise Control of Microtubule Disassembly in Living Cells. bioRxiv 2021, 31, 463668  DOI: 10.1101/2021.10.08.463668
    Google Scholar
    There is no corresponding record for this reference.
  13. 13
    Wühr, M.; Tan, E. S.; Parker, S. K.; Detrich, H. W.; Mitchison, T. J. A Model for Cleavage Plane Determination in Early Amphibian and Fish Embryos. Curr. Biol. 2010, 20, 2040– 2045,  DOI: 10.1016/j.cub.2010.10.024
    Google Scholar
    13
    A Model for Cleavage Plane Determination in Early Amphibian and Fish Embryos
    Wuehr, Martin; Tan, Edwin S.; Parker, Sandra K.; Detrich, H. William, III; Mitchison, Timothy J.
    Current Biology (2010), 20 (22), 2040-2045CODEN: CUBLE2; ISSN:0960-9822. (Cell Press)
    Summary: Current models for cleavage plane detn. propose that metaphase spindles are positioned and oriented by interactions of their astral microtubules with the cellular cortex, followed by cleavage in the plane of the metaphase plate []. We show that in early frog and fish embryos, where cells are unusually large, astral microtubules in metaphase are too short to position and orient the spindle. Rather, the preceding interphase aster centers and orients a pair of centrosomes prior to nuclear envelope breakdown, and the spindle assembles between these prepositioned centrosomes. Interphase asters center and orient centrosomes with dynein-mediated pulling forces. These forces act before astral microtubules contact the cortex; thus, dynein must pull from sites in the cytoplasm, not the cell cortex as is usually proposed for smaller cells. Aster shape is detd. by interactions of the expanding periphery with the cell cortex or with an interaction zone that forms between sister-asters in telophase. We propose a model to explain cleavage plane geometry in which the length of astral microtubules is limited by interaction with these boundaries, causing length asymmetries. Dynein anchored in the cytoplasm then generates length-dependent pulling forces, which move and orient centrosomes.
  14. 14
    Josa-Culleré, L.; Llebaria, A. In the Search for Photocages Cleavable with Visible Light: An Overview of Recent Advances and Chemical Strategies. ChemPhotoChem 2021, 5, 296– 314,  DOI: 10.1002/cptc.202000253
    Google Scholar
    There is no corresponding record for this reference.
  15. 15
    Müller-Deku, A.; Meiring, J. C. M.; Loy, K.; Kraus, Y.; Heise, C.; Bingham, R.; Jansen, K. I.; Qu, X.; Bartolini, F.; Kapitein, L. C.; Akhmanova, A.; Ahlfeld, J.; Trauner, D.; Thorn-Seshold, O. Photoswitchable Paclitaxel-Based Microtubule Stabilisers Allow Optical Control over the Microtubule Cytoskeleton. Nat. Commun. 2020, 11, 4640  DOI: 10.1038/s41467-020-18389-6
    Google Scholar
    15
    Photoswitchable paclitaxel-based microtubule stabilisers allow optical control over the microtubule cytoskeleton
    Mueller-Deku, Adrian; Meiring, Joyce C. M.; Loy, Kristina; Kraus, Yvonne; Heise, Constanze; Bingham, Rebekkah; Jansen, Klara I.; Qu, Xiaoyi; Bartolini, Francesca; Kapitein, Lukas C.; Akhmanova, Anna; Ahlfeld, Julia; Trauner, Dirk; Thorn-Seshold, Oliver
    Nature Communications (2020), 11 (1), 4640CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Small mol. inhibitors are prime reagents for studies in microtubule cytoskeleton research, being applicable across a range of biol. models and not requiring genetic engineering. However, traditional chem. inhibitors cannot be exptl. applied with spatiotemporal precision suiting the length and time scales inherent to microtubule-dependent cellular processes. We have synthesized photoswitchable paclitaxel-based microtubule stabilizers, whose binding is induced by photoisomerisation to their metastable state. Photoisomerising these reagents in living cells allows optical control over microtubule network integrity and dynamics, cell division and survival, with biol. response on the timescale of seconds and spatial precision to the level of individual cells within a population. In primary neurons, they enable regulation of microtubule dynamics resolved to subcellular regions within individual neurites. These azobenzene-based microtubule stabilizers thus enable non-invasive, spatiotemporally precise modulation of the microtubule cytoskeleton in living cells, and promise new possibilities for studying intracellular transport, cell motility, and neuronal physiol.
  16. 16
    Gao, L.; Meiring, J. C. M.; Heise, C.; Rai, A.; Müller-Deku, A.; Akhmanova, A.; Thorn-Seshold, J.; Thorn-Seshold, O. Photoswitchable Epothilone-Based Microtubule Stabilisers Allow GFP-Imaging-Compatible, Optical Control over the Microtubule Cytoskeleton. Angew. Chem., Int. Ed. 2021, 48, e202114614  DOI: 10.1002/anie.202114614
    Google Scholar
    There is no corresponding record for this reference.
  17. 17
    Rastogi, S. K.; Zhao, Z.; Gildner, M. B.; Shoulders, B. A.; Velasquez, T. L.; Blumenthal, M. O.; Wang, L.; Li, X.; Hudnall, T. W.; Betancourt, T.; Du, L.; Brittain, W. J. Synthesis, Optical Properties and in Vitro Cell Viability of Novel Spiropyrans and Their Photostationary States. Tetrahedron 2021, 80, 131854  DOI: 10.1016/j.tet.2020.131854
    Google Scholar
    17
    Synthesis, optical properties and in vitro cell viability of novel spiropyrans and their photostationary states
    Rastogi, Shiva K.; Zhao, Zhenze; Gildner, M. Brenton; Shoulders, Ben A.; Velasquez, Tara L.; Blumenthal, Madeleine O.; Wang, Lei; Li, Xiaopeng; Hudnall, Todd W.; Betancourt, Tania; Du, Liqin; Brittain, William J.
    Tetrahedron (2021), 80 (), 131854CODEN: TETRAB; ISSN:0040-4020. (Elsevier Ltd.)
    A novel class of spiropyrans I (R = H, OEt, OMe; R1 = R2 = R3 = H, OMe; R4 = H, NO2) was synthesized using a multistep process that involves three key intermediates: (a) diazonium-tetrafluoroborate, (b) hydrazine and (c) indolium iodide. The SP analogs I were confirmed as being able to isomerize and attain a photostationary state (PSS) upon irradn. with UV light (UV, 365 nm) in an aq. environment. UV-visible absorption spectra were recorded to confirm the isomerization properties. The ability of the synthesized compds. I to induce growth inhibition of HeLa cervical cancer cells was assessed via the MTT assay after incubation with either the SP or their PSS. The IC50 values of two PSS II (R = H; R1 = R2 = R3 = OMe; R4 = nitro, R = R1 = R2 = R3 = OMe; R4 = H), were obsd. to be around 14 ± 4 fold lower (26 ± 3μM) than their corresponding SPs. The most cytotoxic compds. I [R = R1 = R2 = R3 = R4 = H (III)] and II [R = R1 = R2 = R3 = R4 = H (IV)] showed the lowest IC50 values (12μM). An in vitro tubulin polymn. assay showed that III and IV exhibited the greatest difference in tubulin inhibition relative to unirradiated I (R = R1 = R2 = R3 = H, R4 = NO2; R = R1 = R2 = R3 = OMe, R4 = H).
  18. 18
    Gao, L.; Meiring, J. C. M.; Kraus, Y.; Wranik, M.; Weinert, T.; Pritzl, S. D.; Bingham, R.; Ntouliou, E.; Jansen, K. I.; Olieric, N.; Standfuss, J.; Kapitein, L. C.; Lohmüller, T.; Ahlfeld, J.; Akhmanova, A.; Steinmetz, M. O.; Thorn-Seshold, O. A Robust, GFP-Orthogonal Photoswitchable Inhibitor Scaffold Extends Optical Control over the Microtubule Cytoskeleton. Cell Chem. Biol. 2021, 28, 228– 241,  DOI: 10.1016/j.chembiol.2020.11.007
    Google Scholar
    18
    A Robust, GFP-Orthogonal Photoswitchable Inhibitor Scaffold Extends Optical Control over the Microtubule Cytoskeleton
    Gao, Li; Meiring, Joyce C. M.; Kraus, Yvonne; Wranik, Maximilian; Weinert, Tobias; Pritzl, Stefanie D.; Bingham, Rebekkah; Ntouliou, Evangelia; Jansen, Klara I.; Olieric, Natacha; Standfuss, Joerg; Kapitein, Lukas C.; Lohmueller, Theobald; Ahlfeld, Julia; Akhmanova, Anna; Steinmetz, Michel O.; Thorn-Seshold, Oliver
    Cell Chemical Biology (2021), 28 (2), 228-241.e6CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)
    Optically controlled chem. reagents, termed photopharmaceuticals, are powerful tools for precise spatiotemporal control of proteins particularly when genetic methods, such as knockouts or optogenetics are not viable options. However, current photopharmaceutical scaffolds, such as azobenzenes are intolerant of GFP/YFP imaging and are metabolically labile, posing severe limitations for biol. use. We rationally designed a photoswitchable SBT scaffold to overcome these problems, then derivatized it to create exceptionally metabolically robust and fully GFP/YFP-orthogonal SBTub photopharmaceutical tubulin inhibitors. Lead compd. SBTub3 allows temporally reversible, cell-precise, and even subcellularly precise photomodulation of microtubule dynamics, organization, and microtubule-dependent processes. By overcoming the previous limitations of microtubule photopharmaceuticals, SBTubs offer powerful applications in cell biol., and their robustness and druglikeness are favorable for intracellular biol. control in in vivo applications. We furthermore expect that the robustness and imaging orthogonality of the SBT scaffold will inspire other derivatizations directed at extending the photocontrol of a range of other biol. targets.
  19. 19
    Sailer, A.; Meiring, J. C. M.; Heise, C.; Pettersson, L. N.; Akhmanova, A.; Thorn-Seshold, J.; Thorn-Seshold, O. Pyrrole Hemithioindigo Antimitotics with Near-Quantitative Bidirectional Photoswitching That Photocontrol Cellular Microtubule Dynamics with Single-Cell Precision. Angew. Chem., Int. Ed. 2021, 60, 23695– 23704,  DOI: 10.1002/anie.202104794
    Google Scholar
    19
    Pyrrole Hemithioindigo Antimitotics with Near-Quantitative Bidirectional Photoswitching that Photocontrol Cellular Microtubule Dynamics with Single-Cell Precision
    Sailer, Alexander; Meiring, Joyce C. M.; Heise, Constanze; Pettersson, Linda N.; Akhmanova, Anna; Thorn-Seshold, Julia; Thorn-Seshold, Oliver
    Angewandte Chemie, International Edition (2021), 60 (44), 23695-23704CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    We report the first cellular application of the emerging near-quant. photoswitch pyrrole hemithioindigo, by rationally designing photopharmaceutical PHTub inhibitors of the cytoskeletal protein tubulin. PHTubs allow simultaneous visible-light imaging and photoswitching in live cells, delivering cell-precise photomodulation of microtubule dynamics, and photocontrol over cell cycle progression and cell death. This is the first acute use of a hemithioindigo photopharmaceutical for high-spatiotemporal-resoln. biol. control in live cells. It addnl. demonstrates the utility of near-quant. photoswitches, by enabling a dark-active design to overcome residual background activity during cellular photopatterning. This work opens up new horizons for high-precision microtubule research using PHTubs and shows the cellular applicability of pyrrole hemithioindigo as a valuable scaffold for photocontrol of a range of other biol. targets.
  20. 20
    Sailer, A.; Ermer, F.; Kraus, Y.; Lutter, F. H.; Donau, C.; Bremerich, M.; Ahlfeld, J.; Thorn-Seshold, O. Hemithioindigos for Cellular Photopharmacology: Desymmetrised Molecular Switch Scaffolds Enabling Design Control over the Isomer-Dependency of Potent Antimitotic Bioactivity. ChemBioChem 2019, 20, 1305– 1314,  DOI: 10.1002/cbic.201800752
    Google Scholar
    20
    Hemithioindigos for Cellular Photopharmacology: Desymmetrised Molecular Switch Scaffolds Enabling Design Control over the Isomer-Dependency of Potent Antimitotic Bioactivity
    Sailer, Alexander; Ermer, Franziska; Kraus, Yvonne; Lutter, Ferdinand H.; Donau, Carsten; Bremerich, Maximilian; Ahlfeld, Julia; Thorn-Seshold, Oliver
    ChemBioChem (2019), 20 (10), 1305-1314CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Druglike small mols. with photoswitchable bioactivity-photopharmaceuticals-allow biologists to perform studies with exquisitely precise and reversible, spatial and temporal control over crit. biol. systems inaccessible to genetic manipulation. The photoresponsive pharmacophores disclosed have been almost exclusively azobenzenes, which has limited the structural and substituent scope of photopharmacol. More detrimentally, for azobenzene reagents, it is not researchers' needs for adapted exptl. tools, but rather protein binding site sterics, that typically force whether the trans (dark) or cis (lit) isomer is the more bioactive. We now present the rational design of HOTubs, the first hemithioindigo-based pharmacophores enabling photoswitchable control over endogenous biol. activity in cellulo. HOTubs optically control microtubule depolymn. and cell death in unmodified mammalian cells. Notably, we show how the asymmetry of hemithioindigos allows a priori design of either Z- or E- (dark- or lit)-toxic antimitotics, whereas the corresponding azobenzenes are exclusively lit-toxic. We thus demonstrate that hemithioindigos enable an important expansion of the substituent and design scope of photopharmacol. interventions for biol. systems.
  21. 21
    Sailer, A.; Ermer, F.; Kraus, Y.; Bingham, R.; Lutter, F. H.; Ahlfeld, J.; Thorn-Seshold, O. Potent Hemithioindigo-Based Antimitotics Photocontrol the Microtubule Cytoskeleton in Cellulo. Beilstein J. Org. Chem. 2020, 16, 125– 134,  DOI: 10.3762/bjoc.16.14
    Google Scholar
    21
    Potent hemithioindigo-based antimitotics photocontrol the microtubule cytoskeleton in cellulo
    Sailer, Alexander; Ermer, Franziska; Kraus, Yvonne; Bingham, Rebekkah; Lutter, Ferdinand H.; Ahlfeld, Julia; Thorn-Seshold, Oliver
    Beilstein Journal of Organic Chemistry (2020), 16 (), 125-134CODEN: BJOCBH; ISSN:1860-5397. (Beilstein-Institut zur Foerderung der Chemischen Wissenschaften)
    Uniquely, in contrast to other photoswitches that have been applied to biol., the pseudosym. hemithioindigo scaffold has allowed the creation of both dark-active and lit-active photopharmaceuticals for the same binding site by a priori design. However, the potency of previous hemithioindigo photopharmaceuticals has not been optimal for their translation to other biol. models. Inspired by the structure of tubulin-inhibiting indanones, we created hemithioindigo-based indanone-like tubulin inhibitors (HITubs) and optimized their cellular potency as antimitotic photopharmaceuticals. The use of the hemithioindigo scaffold also permitted us to employ a parahydroxyhemistilbene motif, a structural feature which is denied to most azobenzenes due to the negligibly short lifetimes of their metastable Z-isomers, which proved crucial to enhancing the potency and photoswitchability. The HITubs were ten times more potent than previously reported hemithioindigo photopharmaceutical antimitotics in a series of cell-free and cellular assays, and allowed robust photocontrol over tubulin polymn., microtubule (MT) network structure, cell cycle, and cell survival. Addnl., as the hemithioindigo scaffold allows photoswitchable bioactivity for substituent patterns inaccessible to the majority of current photopharmaceuticals, wider adoption of the hemithioindigo scaffold may significantly expand the scope of cellular and in vivo targets addressable by photopharmacol.
  22. 22
    Engdahl, A. J.; Torres, E. A.; Lock, S. E.; Engdahl, T. B.; Mertz, P. S.; Streu, C. N. Synthesis, Characterization, and Bioactivity of the Photoisomerizable Tubulin Polymerization Inhibitor Azo-Combretastatin A4. Org. Lett. 2015, 17, 4546– 4549,  DOI: 10.1021/acs.orglett.5b02262
    Google Scholar
    22
    Synthesis, Characterization, and Bioactivity of the Photoisomerizable Tubulin Polymerization Inhibitor azo-Combretastatin A4
    Engdahl, Ashton J.; Torres, Edith A.; Lock, Sarah E.; Engdahl, Taylor B.; Mertz, Pamela S.; Streu, Craig N.
    Organic Letters (2015), 17 (18), 4546-4549CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)
    Combretastatin A4 is a stilbenoid tubulin binding mitotic inhibitor whose conformation greatly influences its potency, making it an excellent candidate for adaptation as a photoactivatable tool. Herein, the authors report a novel synthesis, the facile isomerization with com. grade equipment, and biol. activity of azo-combretastatin A4 in vitro and in human cancer cells. Photoisomerized azo-combretastatin A4 is at least 200-fold more potent in cellular culture, making it a promising phototherapeutic and biomedical research tool.
  23. 23
    Zenker, J.; White, M. D.; Gasnier, M.; Alvarez, Y. D.; Lim, H. Y. G.; Bissiere, S.; Biro, M.; Plachta, N. Expanding Actin Rings Zipper the Mouse Embryo for Blastocyst Formation. Cell 2018, 173, 776– 791,  DOI: 10.1016/j.cell.2018.02.035
    Google Scholar
    23
    Expanding Actin Rings Zipper the Mouse Embryo for Blastocyst Formation
    Zenker, Jennifer; White, Melanie D.; Gasnier, Maxime; Alvarez, Yanina D.; Lim, Hui Yi Grace; Bissiere, Stephanie; Biro, Mate; Plachta, Nicolas
    Cell (Cambridge, MA, United States) (2018), 173 (3), 776-791.e17CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    Transformation from morula to blastocyst is a defining event of preimplantation embryo development. During this transition, the embryo must establish a paracellular permeability barrier to enable expansion of the blastocyst cavity. Here, using live imaging of mouse embryos, we reveal an actin-zippering mechanism driving this embryo sealing. Preceding blastocyst stage, a cortical F-actin ring assembles at the apical pole of the embryo's outer cells. The ring structure forms when cortical actin flows encounter a network of polar microtubules that exclude F-actin. Unlike stereotypical actin rings, the actin rings of the mouse embryo are not contractile, but instead, they expand to the cell-cell junctions. Here, they couple to the junctions by recruiting and stabilizing adherens and tight junction components. Coupling of the actin rings triggers localized myosin II accumulation, and it initiates a tension-dependent zippering mechanism along the junctions that is required to seal the embryo for blastocyst formation.
  24. 24
    Theisen, U.; Ernst, A. U.; Heyne, R. L. S.; Ring, T. P.; Thorn-Seshold, O.; Köster, R. W. Microtubules and Motor Proteins Support Zebrafish Neuronal Migration by Directing Cargo. J. Cell Biol. 2020, 219, e201908040  DOI: 10.1083/jcb.201908040
    Google Scholar
    24
    Microtubules and motor proteins support zebrafish neuronal migration by directing cargo
    Theisen, Ulrike; Ernst, Alexander U.; Heyne, Ronja L. S.; Ring, Tobias P.; Thorn-Seshold, Oliver; Koester, Reinhard W.
    Journal of Cell Biology (2020), 219 (10), e201908040CODEN: JCLBA3; ISSN:1540-8140. (Rockefeller University Press)
    Neuronal migration during development is necessary to form an ordered and functional brain. Postmitotic neurons require microtubules and dynein to move, but the mechanisms by which they contribute to migration are not fully characterized. Using tegmental hindbrain nuclei neurons in zebrafish embryos together with subcellular imaging, optogenetics, and photopharmacol., we show that, in vivo, the centrosome's position relative to the nucleus is not linked to greatest motility in this cell type. Nevertheless, microtubules, dynein, and kinesin-1 are essential for migration, and we find that interference with endosome formation or the Golgi app. impairs migration to a similar extent as disrupting microtubules. In addn., an imbalance in the traffic of the model cargo Cadherin-2 also reduces neuronal migration. These results lead us to propose that microtubules act as cargo carriers to control spatiotemporal protein distribution, which in turn controls motility. This adds crucial insights into the variety of ways that microtubules can support successful neuronal migration in vivo.
  25. 25
    Gavin, J.; Ruiz, J. F. M.; Kedziora, K.; Windle, H.; Kelleher, D. P.; Gilmer, J. F. Structure Requirements for Anaerobe Processing of Azo Compounds: Implications for Prodrug Design. Bioorg. Med. Chem. Lett. 2012, 22, 7647– 7652,  DOI: 10.1016/j.bmcl.2012.10.014
    Google Scholar
    25
    Structure requirements for anaerobe processing of azo compounds: Implications for prodrug design
    Gavin, Jason; Ruiz, Juan F. Marquez; Kedziora, Kinga; Windle, Henry; Kelleher, Dermot P.; Gilmer, John F.
    Bioorganic & Medicinal Chemistry Letters (2012), 22 (24), 7647-7652CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)
    This Letter generalizes the metab. of the azo class of compds. by Clostridium perfringens, an anaerobe found in the human colon. A recently reported 5-aminosalicylic acid-based prednisolone prodrug was shown to release the drug when incubated with the bacteria, while the para-aminobenzoic acid (PABA) based analog did not. Instead, it showed a new HPLC peak with a relatively close retention time to the parent which was identified by LCMS as the partially reduced hydrazine product. This Letter investigates azoredn. across a panel of substrates with varying degrees of electronic and steric similarity to the PABA-based compd. Azo compds. with an electron donating group on the azo-contg. arom. ring showed immediate disproportionation to their parent amines without any detection of hydrazine intermediates by HPLC. Compds. contg. only electron withdrawing groups are partially and reversibly reduced to produce a stable detectable hydrazine. They do not disproportionate to their parent amines, but regenerate the parent azo compd. This incomplete redn. is relevant to the design of azo-based prodrugs and the toxicol. of azo-based dyes.
  26. 26
    Sheldon, J. E.; Dcona, M. M.; Lyons, C. E.; Hackett, J. C.; Hartman, M. C. T. Photoswitchable Anticancer Activity via Trans-Cis Isomerization of a Combretastatin A-4 Analog. Org. Biomol. Chem. 2016, 14, 40– 49,  DOI: 10.1039/c5ob02005k
    Google Scholar
    26
    Photoswitchable anticancer activity via trans-cis isomerization of a combretastatin A-4 analog
    Sheldon, Jonathon E.; Dcona, M. Michael; Lyons, Charles E.; Hackett, John C.; Hartman, Matthew C. T.
    Organic & Biomolecular Chemistry (2016), 14 (1), 40-49CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
    Combretastatin A-4 (CA4) is highly potent anticancer drug that acts as an inhibitor of tubulin polymn. The core of the CA4 structure contains a cis-stilbene, and it is known that the trans isomer is significantly less potent. The authors prepd. an azobenzene analog of CA4 (Azo-CA4) that shows 13-35-fold enhancement in potency upon illumination. EC50 values in the light were in the mid nM range. Due to its ability to thermally revert to less toxic trans form, Azo-CA4 also has the ability to automatically turn its activity off with time. Azo-CA4 is less potent than CA-4 because it degrades in the presence of glutathione as evidenced by UV-Vis spectroscopy and ESI-MS. Nevertheless, Azo-CA4 represents a promising strategy for switchable potency for treatment of cancer.
  27. 27
    An, Y.; Chen, C.; Zhu, J.; Dwivedi, P.; Zhao, Y.; Wang, Z. Hypoxia-Induced Activity Loss of a Photo-Responsive Microtubule Inhibitor Azobenzene Combretastatin A4. Front. Chem. Sci. Eng. 2020, 14, 880– 888,  DOI: 10.1007/s11705-019-1864-6
    Google Scholar
    27
    Hypoxia-induced activity loss of a photo-responsive microtubule inhibitor azobenzene combretastatin A4
    An, Yang; Chen, Chao; Zhu, Jundong; Dwivedi, Pankaj; Zhao, Yanjun; Wang, Zheng
    Frontiers of Chemical Science and Engineering (2020), 14 (5), 880-888CODEN: FCSEA3; ISSN:2095-0187. (Springer)
    The conformation-dependent activity of azobenzene combretastatin A4 (Azo-CA4) provides a unique approach to reduce the side-effects of chemotherapy, due to the light-triggered conformation transition of its azobenzene moiety. Under hypoxic tumor microenvironment, however, the high expression of azoreductase can reduce azobenzene to aniline. It was postulated that the Azo-CA4 might be degraded under hypoxia, resulting in the decrease of its anti-tumor activity. The aim of this study was to verify such hypothesis in HeLa cells in vitro. The quant. drug concn. anal. shows the ratio-metric formation of degrdn. end-products, confirming the bioredn. of Azo-CA4. The tubulin staining study indicates that Azo-CA4 loses the potency of switching off microtubule dynamics under hypoxia. Furthermore, the cell cycle anal. shows that the ability of Azo-CA4 to induce mitotic arrest is lost at low oxygen content. Therefore, the cytotoxicity of Azo-CA4 is compromised under hypoxia. In contrast, combretastatin A4 as a pos. control maintains the potency to inhibit tubulin polymn. and break down the nuclei irresp. of light irradn. and oxygen level. This work highlights the influence of hypoxic tumor microenvironment on the anti-tumor potency of Azo-CA4, which should be considered during the early stage of designing translational Azo-CA4 delivery systems.
  28. 28
    Hüll, K.; Morstein, J.; Trauner, D. In Vivo Photopharmacology. Chem. Rev. 2018, 118, 10710– 10747,  DOI: 10.1021/acs.chemrev.8b00037
    Google Scholar
    28
    In Vivo Photopharmacology
    Hull, Katharina; Morstein, Johannes; Trauner, Dirk
    Chemical Reviews (Washington, DC, United States) (2018), 118 (21), 10710-10747CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. Synthetic photoswitches have been known for many years, but their usefulness in biol., pharmacol., and medicine has only recently been systematically explored. Over the past decade photopharmacol. has grown into a vibrant field. As the photophys., pharmacodynamic, and pharmacokinetic properties of photoswitches, such as azobenzenes, have become established, they have been applied to a wide range of biol. targets. These include transmembrane proteins (ion channels, transporters, G protein-coupled receptors, receptor-linked enzymes), sol. proteins (kinases, proteases, factors involved in epigenetic regulation), lipid membranes, and nucleic acids. In this review, the authors provide an overview of photopharmacol. using synthetic switches that have been applied in vivo, i.e., in living cells and organisms. The authors discuss the scope and limitations of this approach to study biol. function and the challenges it faces in translational medicine. The relationships between synthetic photoswitches, natural chromophores used in optogenetics, and caged ligands are addressed.
  29. 29
    Lachmann, D.; Lahmy, R.; König, B. Fulgimides as Light-Activated Tools in Biological Investigations: Fulgimides as Light-Activated Tools in Biological Investigations. Eur. J. Org. Chem. 2019, 2019, 5018– 5024,  DOI: 10.1002/ejoc.201900219
    Google Scholar
    29
    Fulgimides as Light-Activated Tools in Biological Investigations
    Lachmann, D.; Lahmy, R.; Koenig, B.
    European Journal of Organic Chemistry (2019), 2019 (31-32), 5018-5024CODEN: EJOCFK; ISSN:1099-0690. (Wiley-VCH Verlag GmbH & Co. KGaA)
    A review. With high spatiotemporal control, the conformation, rigidity and electronics of photoresponsive bioactive mols. can be altered. This, in turn, allows for control over the biol. properties of these mols. Incorporation of a photoswitchable moiety into a no. of reported inhibitors, ligands and modulators has demonstrated the ability to modulate enzyme, receptor and ion channel responses using light. To date, the major classes of photoswitches explored in biol. applications have been the azobenzenes and diarylethenes. Even though the use of these photoswitches has established the value of photoresponsive mols. as biol. tools, several limitations have become apparent. Fulgimides represent a promising class of photoswitches that are not widely used for such biol. purposes. Their properties are similar to that of diarylethenes, as their photochromism is based on a 6π-electrocyclic rearrangement, however, fulgimides have the added advantage of thermal stability for both isomers. Fulgimides exhibit high photostationary states and fatigue resistance, with the ability to switch in aq. buffer solns. In this minireview, these advantageous photophys. properties will be discussed, as well as the use of fulgimides in biol. investigations.
  30. 30
    Fuchter, M. J. On the Promise of Photopharmacology Using Photoswitches: A Medicinal Chemist’s Perspective. J. Med. Chem. 2020, 63, 11436– 11447,  DOI: 10.1021/acs.jmedchem.0c00629
    Google Scholar
    30
    On the Promise of Photopharmacology Using Photoswitches: A Medicinal Chemist's Perspective
    Fuchter, Matthew J.
    Journal of Medicinal Chemistry (2020), 63 (20), 11436-11447CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    A review. Photopharmacol. is a growing area of endeavor that employs photoswitchable ligands to allow for light-dependent pharmacol. activity. By coupling light to therapeutic action, improved spatial and temporal selectivity can be achieved and subsequently harnessed for new concepts in therapy. Tremendous progress has already been made, with photopharmacol. agents now reported against a wide array of target classes and light-dependent results demonstrated in a range of live cell and animal models. Several challenges remain, however, esp. in order for photopharmacol. to truly impact the clin. management of disease. This Perspective aims to summarize these challenges, particularly with attention to the medicinal chem. that will be unavoidably required for the further translation of these agents/approaches. By clearly defining challenges for drug hunters, it is hoped that further research into the medicinal chem. of photopharmacol. agents will be stimulated, ultimately enabling full realization of the huge potential for this exciting field.
  31. 31
    Welleman, I. M.; Hoorens, M. W. H.; Feringa, B. L.; Boersma, H. H.; Szymański, W. Photoresponsive Molecular Tools for Emerging Applications of Light in Medicine. Chem. Sci. 2020, 11, 11672– 11691,  DOI: 10.1039/D0SC04187D
    Google Scholar
    31
    Photoresponsive molecular tools for emerging applications of light in medicine
    Welleman, Ilse M.; Hoorens, Mark W. H.; Feringa, Ben L.; Boersma, Hendrikus H.; Szymanski, Wiktor
    Chemical Science (2020), 11 (43), 11672-11691CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
    A review. Light-based therapeutic and imaging modalities, which emerge in clin. applications, rely on mol. tools, such as photocleavable protecting groups and photoswitches that respond to photonic stimulus and translate it into a biol. effect. However, optimization of their key parameters (activation wavelength, band sepn., fatigue resistance and half-life) is necessary to enable application in the medical field. In this perspective, we describe the applications scenarios that can be envisioned in clin. practice and then we use those scenarios to explain the necessary properties that the photoresponsive tools used to control biol. function should possess, highlighted by examples from medical imaging, drug delivery and photopharmacol. We then present how the (photo)chem. parameters are currently being optimized and an outlook is given on pharmacol. aspects (toxicity, soly., and stability) of light-responsive mols. With these interdisciplinary insights, we aim to inspire the future directions for the development of photocontrolled tools that will empower clin. applications of light.
  32. 32
    Gaspari, R.; Prota, A. E.; Bargsten, K.; Cavalli, A.; Steinmetz, M. O. Structural Basis of Cis- and Trans-Combretastatin Binding to Tubulin. Chem 2017, 2, 102– 113,  DOI: 10.1016/j.chempr.2016.12.005
    Google Scholar
    32
    Structural Basis of cis- and trans-Combretastatin Binding to Tubulin
    Gaspari, Roberto; Prota, Andrea E.; Bargsten, Katja; Cavalli, Andrea; Steinmetz, Michel O.
    Chem (2017), 2 (1), 102-113CODEN: CHEMVE; ISSN:2451-9294. (Cell Press)
    Combretastatin A4 (CA-4) derivs. are microtubule-destabilizing agents, some of which are in advanced clin. trials for cancer therapy. The active cis conformation of CA-4 can readily isomerize into a thermodynamically more stable but significantly less active trans form. Here, we solved the high-resoln. crystal structure of cis-CA-4 in complex with tubulin. The compd. binds to the colchicine site of tubulin and displays both common and distinct interaction points with colchicine. Using metadynamics simulations, we generated the trans form of the ligand within its binding site and computed the relative binding free energy of the cis-CA-4 and trans-CA-4 isomers via a thermodn. cycle. The calcns. suggest structural distortions of the bound trans-CA-4 mol. as the likely cause of its reduced activity in comparison with that of its cis isomer. Our findings could open up unique possibilities for structure-guided drug engineering with the aim of discovering combretastatin variants with improved chem. properties and pharmacol. profiles.
  33. 33
    Tarade, D.; Ma, D.; Pignanelli, C.; Mansour, F.; Simard, D.; Berg, S.; van den Gauld, J.; McNulty, J.; Pandey, S. Structurally Simplified Biphenyl Combretastatin A4 Derivatives Retain in Vitro Anti-Cancer Activity Dependent on Mitotic Arrest. PLoS One 2017, 12, e0171806  DOI: 10.1371/journal.pone.0171806
    Google Scholar
    There is no corresponding record for this reference.
  34. 34
    Jacobson, P. Ueber Bildung von Anhydroverbindungen Des Orthoamidophenylmercaptans Aus Thioaniliden. Ber. Dtsch. Chem. Ges. 1886, 19, 1067– 1077,  DOI: 10.1002/cber.188601901239
    Google Scholar
    There is no corresponding record for this reference.
  35. 35
    Ma, D.; Xie, S.; Xue, P.; Zhang, X.; Dong, J.; Jiang, Y. Efficient and Economical Access to Substituted Benzothiazoles: Copper-Catalyzed Coupling of 2-Haloanilides with Metal Sulfides and Subsequent Condensation. Angew. Chem., Int. Ed. 2009, 48, 4222– 4225,  DOI: 10.1002/anie.200900486
    Google Scholar
    35
    Efficient and economical access to substituted benzothiazoles: copper-catalyzed coupling of 2-haloanilides with metal sulfides and subsequent condensation
    Ma, Dawei; Xie, Siwei; Xue, Peng; Zhang, Xiaojing; Dong, Jinhua; Jiang, Yongwen
    Angewandte Chemie, International Edition (2009), 48 (23), 4222-4225, S4222/1-S4222/18CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The first metal-catalyzed direct coupling of metal sulfides with aryl halides and subsequent intramol. condensation provided substituted benzothiazoles. A wide range of functional groups are tolerated under the reaction conditions.
  36. 36
    Tron, G. C.; Pirali, T.; Sorba, G.; Pagliai, F.; Busacca, S.; Genazzani, A. A. Medicinal Chemistry of Combretastatin A4: Present and Future Directions. J. Med. Chem. 2006, 49, 3033– 3044,  DOI: 10.1021/jm0512903
    Google Scholar
    36
    Medicinal chemistry of combretastatin A4: Present and future directions
    Tron, Gian Cesare; Pirali, Tracey; Sorba, Giovanni; Pagliai, Francesca; Busacca, Sara; Genazzani, Armando A.
    Journal of Medicinal Chemistry (2006), 49 (11), 3033-3044CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    A review with refs. A review on the medicinal chem. of combretastatin A4 (CA-4), a compd. with antitumor properties. The mechanism of action of CA-4, including its ability to inhibit tubulin polymn., is discussed, along with selected analogs of CA-4 modified on the arom. rings A and B, substituted Ph rings, heterocycling rings, non-substituted arom. rings, modifications on the double bond, and analogs where the olefinic group is replaced by a ring. Some issues related to the pharmacol. of CA-4 and future directions in research are considered.
  37. 37
    Kraus, Y.; Glas, C.; Melzer, B.; Gao, L.; Heise, C.; Preuße, M.; Ahlfeld, J.; Bracher, F.; Thorn-Seshold, O. Isoquinoline-Based Biaryls as a Robust Scaffold for Microtubule Inhibitors. Eur. J. Med. Chem. 2020, 186, 111865  DOI: 10.1016/j.ejmech.2019.111865
    Google Scholar
    37
    Isoquinoline-based biaryls as a robust scaffold for microtubule inhibitors
    Kraus, Yvonne; Glas, Carina; Melzer, Benedikt; Gao, Li; Heise, Constanze; Preusse, Monique; Ahlfeld, Julia; Bracher, Franz; Thorn-Seshold, Oliver
    European Journal of Medicinal Chemistry (2020), 186 (), 111865CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
    We here report the discovery of isoquinoline-based biaryls as a new scaffold for colchicine domain tubulin inhibitors. Colchicinoid inhibitors offer highly desirable cytotoxic and vascular disrupting bioactivities, but their further development requires improving in vivo robustness and tolerability: properties that both depend on the scaffold structure employed. We have developed isoquinoline-based biaryls as a novel scaffold for high-potency tubulin inhibitors, with excellent robustness, druglikeness, and facile late-stage structural diversification, accessible through a tolerant synthetic route. We confirmed their bioactivity mechanism in vitro, developed sol. prodrugs, and established safe in vivo dosing in mice. By addressing several problems facing the current families of inhibitors, we expect that this new scaffold will find a range of in vivo applications towards translational use in cancer therapy.
  38. 38
    Thorn-Seshold, O.; Meiring, J. C. M. Photocontrolling Microtubule Dynamics with Photoswitchable Chemical Reagents. In Microtubules─Methods and Protocols; Springer International Publishing, 2022; Chapter 26.
    Google Scholar
    There is no corresponding record for this reference.
  39. 39
    Florian, S.; Mitchison, T. J. Anti-Microtubule Drugs. In The Mitotic Spindle: Methods and Protocols; Chang, P.; Ohi, R., Eds.; Springer: New York, NY, 2016; Vol. 1413, pp 403– 421.
    Google Scholar
    There is no corresponding record for this reference.
  40. 40
    Roostalu, J.; Thomas, C.; Cade, N. I.; Kunzelmann, S.; Taylor, I. A.; Surrey, T. The Speed of GTP Hydrolysis Determines GTP Cap Size and Controls Microtubule Stability. eLife 2020, 9, e51992  DOI: 10.7554/eLife.51992
    Google Scholar
    40
    The speed of GTP hydrolysis determines GTP cap size and controls microtubule stability
    Roostalu, Johanna; Thomas, Claire; Cade, Nicholas Ian; Kunzelmann, Simone; Taylor, Ian A.; Surrey, Thomas
    eLife (2020), 9 (), e51992CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)
    Microtubules are cytoskeletal polymers whose function depends on their property to switch between states of growth and shrinkage. Growing microtubules are thought to be stabilized by a GTP cap at their ends. The nature of this cap, however, is still poorly understood. End Binding proteins (EBs) recruit a diverse range of regulators of microtubule function to growing microtubule ends. Whether the EB binding region is identical to the GTP cap is unclear. Using mutated human tubulin with blocked GTP hydrolysis, we demonstrate that EBs bind with high affinity to the GTP conformation of microtubules. Slowing-down GTP hydrolysis leads to extended GTP caps. We find that cap length dets. microtubule stability and that the microtubule conformation changes gradually in the cap as GTP is hydrolyzed. These results demonstrate the crit. importance of the kinetics of GTP hydrolysis for microtubule stability and establish that the GTP cap coincides with the EB-binding region.
  41. 41
    Linnemann, J. R.; Miura, H.; Meixner, L. K.; Irmler, M.; Kloos, U. J.; Hirschi, B.; Bartsch, H. S.; Sass, S.; Beckers, J.; Theis, F. J.; Gabka, C.; Sotlar, K.; Scheel, C. H. Quantification of Regenerative Potential in Primary Human Mammary Epithelial Cells. Development 2015, 31, 3239– 3251,  DOI: 10.1242/dev.123554
    Google Scholar
    There is no corresponding record for this reference.
  42. 42
    Buchmann, B.; Engelbrecht, L. K.; Fernandez, P.; Hutterer, F. P.; Raich, M. K.; Scheel, C. H.; Bausch, A. R. Mechanical Plasticity of Collagen Directs Branch Elongation in Human Mammary Gland Organoids. Nat. Commun. 2021, 12, 2759  DOI: 10.1038/s41467-021-22988-2
    Google Scholar
    42
    Mechanical plasticity of collagen directs branch elongation in human mammary gland organoids
    Buchmann, B.; Engelbrecht, L. K.; Fernandez, P.; Hutterer, F. P.; Raich, M. K.; Scheel, C. H.; Bausch, A. R.
    Nature Communications (2021), 12 (1), 2759CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Epithelial branch elongation is a central developmental process during branching morphogenesis in diverse organs. This fundamental growth process into large arborized epithelial networks is accompanied by structural reorganization of the surrounding extracellular matrix (ECM), well beyond its mech. linear response regime. Here, we report that epithelial ductal elongation within human mammary organoid branches relies on the non-linear and plastic mech. response of the surrounding collagen. Specifically, we demonstrate that collective back-and-forth motion of cells within the branches generates tension that is strong enough to induce a plastic reorganization of the surrounding collagen network which results in the formation of mech. stable collagen cages. Such matrix encasing in turn directs further tension generation, branch outgrowth and plastic deformation of the matrix. The identified mech. tension equil. sets a framework to understand how mech. cues can direct ductal branch elongation.
  43. 43
    Hofer, M.; Lutolf, M. P. Engineering Organoids. Nat. Rev. Mater. 2021, 6, 402– 420,  DOI: 10.1038/s41578-021-00279-y
    Google Scholar
    43
    Engineering organoids
    Hofer, Moritz; Lutolf, Matthias P.
    Nature Reviews Materials (2021), 6 (5), 402-420CODEN: NRMADL; ISSN:2058-8437. (Nature Portfolio)
    A review. Abstr.: Organoids are in vitro miniaturized and simplified model systems of organs that have gained enormous interest for modeling tissue development and disease, and for personalized medicine, drug screening and cell therapy. Despite considerable success in culturing physiol. relevant organoids, challenges remain to achieve real-life applications. In particular, the high variability of self-organizing growth and restricted exptl. and anal. access hamper the translatability of organoid systems. In this Review, we argue that many limitations of traditional organoid culture can be addressed by engineering approaches at all levels of organoid systems. We investigate cell surface and genetic engineering approaches, and discuss stem cell niche engineering based on the design of matrixes that allow spatiotemporal control of organoid growth and shape-guided morphogenesis. We examine how microfluidic approaches and lessons learnt from organs-on-a-chip enable the integration of mechano-physiol. parameters and increase accessibility of organoids to improve functional readouts. Applying engineering principles to organoids increases reproducibility and provides exptl. control, which will, ultimately, be required to enable clin. translation.
  44. 44
    Kopf, A.; Renkawitz, J.; Hauschild, R.; Girkontaite, I.; Tedford, K.; Merrin, J.; Thorn-Seshold, O.; Trauner, D.; Häcker, H.; Fischer, K.-D.; Kiermaier, E.; Sixt, M. Microtubules Control Cellular Shape and Coherence in Amoeboid Migrating Cells. J. Cell Biol. 2020, 219, e201907154  DOI: 10.1083/jcb.201907154
    Google Scholar
    44
    Microtubules control cellular shape and coherence in amoeboid migrating cells
    Kopf, Aglaja; Renkawitz, Joerg; Hauschild, Robert; Girkontaite, Irute; Tedford, Kerry; Merrin, Jack; Thorn-Seshold, Oliver; Trauner, Dirk; Haecker, Hans; Fischer, Klaus-Dieter; Kiermaier, Eva; Sixt, Michael
    Journal of Cell Biology (2020), 219 (6), e201907154/1-e201907154/24CODEN: JCLBA3; ISSN:1540-8140. (Rockefeller University Press)
    Cells navigating through complex tissues face a fundamental challenge: while multiple protrusions explore different paths, the cell needs to avoid entanglement. How a cell surveys and then corrects its own shape is poorly understood. Here, we demonstrate that spatially distinct microtubule dynamics regulate amoeboid cell migration by locally promoting the retraction of protrusions. In migrating dendritic cells, local microtubule depolymn. within protrusions remote from the microtubule organizing center triggers actomyosin contractility controlled by RhoA and its exchange factor Lfc. Depletion of Lfc leads to aberrant myosin localization, thereby causing two effects that rate-limit locomotion: (1) impaired cell edge coordination during path finding and (2) defective adhesion resoln. Compromised shape control is particularly hindering in geometrically complex microenvironments, where it leads to entanglement and ultimately fragmentation of the cell body. We thus demonstrate that microtubules can act as a proprioceptive device: they sense cell shape and control actomyosin retraction to sustain cellular coherence.
  45. 45
    Vandestadt, C.; Vanwalleghem, G. C.; Castillo, H. A.; Li, M.; Schulze, K.; Khabooshan, M.; Don, E.; Anko, M.-L.; Scott, E. K.; Kaslin, J. Early Migration of Precursor Neurons Initiates Cellular and Functional Regeneration after Spinal Cord Injury in Zebrafish. bioRxiv 2019, 539940  DOI: 10.1101/539940
    Google Scholar
    There is no corresponding record for this reference.
  46. 46
    Cabernard, C.; Doe, C. Q. Apical/Basal Spindle Orientation Is Required for Neuroblast Homeostasis and Neuronal Differentiation in Drosophila. Dev. Cell 2009, 17, 134– 141,  DOI: 10.1016/j.devcel.2009.06.009
    Google Scholar
    46
    Apical/basal spindle orientation is required for neuroblast homeostasis and neuronal differentiation in Drosophila
    Cabernard, Clemens; Doe, Chris Q.
    Developmental Cell (2009), 17 (1), 134-141CODEN: DCEEBE; ISSN:1534-5807. (Cell Press)
    Precise regulation of stem cell self-renewal/differentiation is essential for embryogenesis and tumor suppression. Drosophila neural progenitors (neuroblasts) align their spindle along an apical/basal polarity axis to generate a self-renewed apical neuroblast and a differentiating basal cell. Here, the authors genetically disrupt spindle orientation without altering cell polarity to test the role of spindle orientation in self-renewal/differentiation. The authors perform correlative live imaging of polarity markers and spindle orientation over multiple divisions within intact brains, followed by mol. marker anal. of cell fate. The authors find that spindle alignment orthogonal to apical/basal polarity always segregates apical determinants into both siblings, which invariably assume a neuroblast identity. Basal determinants can all be localized into one sibling without inducing neuronal differentiation, but overexpression of the basal determinant Prospero can deplete neuroblasts. The authors conclude that the ratio of apical/basal determinants specifies neuroblast/GMC identity, and that apical/basal spindle orientation is required for neuroblast homeostasis and neuronal differentiation.
  47. 47
    Karpova, N.; Bobinnec, Y.; Fouix, S.; Huitorel, P.; Debec, A. Jupiter, a New Drosophila Protein Associated with Microtubules. Cell Motil. 2006, 63, 301– 312,  DOI: 10.1002/cm.20124
    Google Scholar
    47
    Jupiter, a new Drosophila protein associated with microtubules
    Karpova, Nina; Bobinnec, Yves; Fouix, Sylvaine; Huitorel, Philippe; Debec, Alain
    Cell Motility and the Cytoskeleton (2006), 63 (5), 301-312CODEN: CMCYEO; ISSN:0886-1544. (Wiley-Liss, Inc.)
    We describe a novel Drosophila protein Jupiter, which shares properties with several structural microtubule-assocd. proteins (MAPs) including TAU, MAP2, MAP4. Jupiter is a sol. unfolded mol. with the high net pos. charge, rich in glycine. It possesses 2 degenerated repeats around the sequence PPGG, sepd. by a serine-rich region. Jupiter assocs. with microtubules in vitro and, fused with the green fluorescent protein (GFP), is an excellent marker to follow microtubule dynamics in vivo. In a jupiter transgenic Drosophila strain generated by the protein-trap technique, Jupiter:GFP fusion protein localizes to the microtubule network through the cell cycle at the different stages of development. We found particularly high Jupiter:GFP concns. in the young embryo, larval nervous system, precursors of eye photoreceptors and adult ovary. Moreover, from jupiter:gfp embryos we have established 2 permanent cell lines presenting strongly fluorescent microtubules during the whole cell cycle. In these cells, the distribution of the Jupiter:GFP fusion protein reproduces microtubule behavior upon treatment by the drugs colchicine and taxol. The Jupiter cell lines and fly strain should be of wide interest for biologists interested in in vivo anal. of microtubule dynamics.
  48. 48
    Royou, A.; Sullivan, W.; Karess, R. Cortical Recruitment of Nonmuscle Myosin II in Early Syncytial Drosophila Embryos: Its Role in Nuclear Axial Expansion and Its Regulation by Cdc2 Activity. J. Cell Biol. 2002, 158, 127– 137,  DOI: 10.1083/jcb.200203148
    Google Scholar
    48
    Cortical recruitment of nonmuscle myosin II in early syncytial Drosophila embryos: its role in nuclear axial expansion and its regulation by Cdc2 activity
    Royou, Anne; Sullivan, William; Karess, Roger
    Journal of Cell Biology (2002), 158 (1), 127-137CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)
    The nuclei of early syncytial Drosophila embryos migrate dramatically toward the poles. The cellular mechanisms driving this process, called axial expansion, are unclear, but myosin II activity is required. By following regulatory myosin light chain (RLC)-green fluorescent protein dynamics in living embryos, we obsd. cycles of myosin recruitment to the cortex synchronized with mitotic cycles. Cortical myosin is first seen in a patch at the anterocentral part of the embryo at cycle 4. With each succeeding cycle, the patch expands poleward, dispersing at the beginning of each mitosis and reassembling at the end of telophase. Each cycle of actin and myosin recruitment is accompanied by a cortical contraction. The cortical myosin cycle does not require microtubules but correlates inversely with Cdc2/cyclin B (mitosis-promoting factor) activity. A mutant RLC lacking inhibitory phosphorylation sites was fully functional with no effect on the cortical myosin cycle, indicating that Cdc2 must be modulating myosin activity by some other mechanism. An inhibitor of Rho kinase blocks the cortical myosin recruitment cycles and provokes a concomitant failure of axial expansion. These studies suggest a model in which cycles of myosin-mediated contraction and relaxation, tightly linked to Cdc2 and Rho kinase activity, are directly responsible for the axial expansion of the syncytial nuclei.
  49. 49
    Rusan, N. M.; Peifer, M. A Role for a Novel Centrosome Cycle in Asymmetric Cell Division. J. Cell Biol. 2007, 177, 13– 20,  DOI: 10.1083/jcb.200612140
    Google Scholar
    49
    A role for a novel centrosome cycle in asymmetric cell division
    Rusan, Nasser M.; Peifer, Mark
    Journal of Cell Biology (2007), 177 (1), 13-20CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)
    Drosophila melanogaster central brain neuroblasts are excellent models for stem cell asym. division. Earlier work showed that their mitotic spindle orientation is established before spindle formation. We investigated the mechanism by which this occurs, revealing a novel centrosome cycle. In interphase, the 2 centrioles sep., but only 1 is active, retaining pericentriolar material and forming a dominant centrosome. This centrosome acts as a microtubule organizing center (MTOC) and remains stationary, forming 1 pole of the future spindle. The 2nd centriole is inactive and moves to the opposite side of the cell before being activated as a centrosome/MTOC. This is accompanied by asym. localization of Polo kinase, a key centrosome regulator. Disruption of centrosomes disrupts the high fidelity of asym. division. We propose a 2-step mechanism to ensure faithful spindle positioning: the novel centrosome cycle produces a single interphase MTOC, coarsely aligning the spindle, and spindle-cortex interactions refine this alignment.
  50. 50
    Cabernard, C.; Prehoda, K. E.; Doe, C. Q. A Spindle-Independent Cleavage Furrow Positioning Pathway. Nature 2010, 467, 91– 94,  DOI: 10.1038/nature09334
    Google Scholar
    50
    A spindle-independent cleavage furrow positioning pathway
    Cabernard, Clemens; Prehoda, Kenneth E.; Doe, Chris Q.
    Nature (London, United Kingdom) (2010), 467 (7311), 91-94CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    The mitotic spindle dets. the cleavage furrow site during metazoan cell division, but whether other mechanisms exist remains unknown. Here we identify a spindle-independent mechanism for cleavage furrow positioning in Drosophila neuroblasts. We show that early and late furrow proteins (Pavarotti, Anillin, and Myosin) are localized to the neuroblast basal cortex at anaphase onset by a Pins cortical polarity pathway, and can induce a basally displaced furrow even in the complete absence of a mitotic spindle. Rotation or displacement of the spindle results in 2 furrows: an early polarity-induced basal furrow and a later spindle-induced furrow. This spindle-independent cleavage furrow mechanism may be relevant to other highly polarized mitotic cells, such as mammalian neural progenitors.
  51. 51
    Connell, M.; Cabernard, C.; Ricketson, D.; Doe, C. Q.; Prehoda, K. E. Asymmetric Cortical Extension Shifts Cleavage Furrow Position in Drosophila Neuroblasts. Mol. Biol. Cell 2011, 22, 4220– 4226,  DOI: 10.1091/mbc.e11-02-0173
    Google Scholar
    51
    Asymmetric cortical extension shifts cleavage furrow position in Drosophila neuroblasts
    Connell, Marisa; Cabernard, Clemens; Ricketson, Derek; Doe, Chris Q.; Prehoda, Kenneth E.
    Molecular Biology of the Cell (2011), 22 (22), 4220-4226CODEN: MBCEEV; ISSN:1939-4586. (American Society for Cell Biology)
    The cytokinetic cleavage furrow is typically positioned sym. relative to the cortical cell boundaries, but it can also be asym. The mechanisms that control furrow site specification have been intensively studied, but how polar cortex movements influence ultimate furrow position remains poorly understood. We measured the position of the apical and the basal cortex in asym. dividing Drosophila neuroblasts and obsd. preferential displacement of the apical cortex that becomes the larger daughter cell during anaphase, effectively shifting the cleavage furrow toward the smaller daughter cell. Asym. cortical extension is correlated with the presence of cortical myosin II, which is polarized in neuroblasts. Loss of myosin II asymmetry by perturbing heterotrimeric G-protein signaling results in sym. extension and equal-sized daughter cells. We propose a model in which contraction-driven asym. polar extension of the neuroblast cortex during anaphase contributes to asym. furrow position and daughter cell size.
  52. 52
    Roberts, A.; Borisyuk, R.; Buhl, E.; Ferrario, A.; Koutsikou, S.; Li, W.-C.; Soffe, S. R. The Decision to Move: Response Times, Neuronal Circuits and Sensory Memory in a Simple Vertebrate. Proc. R. Soc. B 2019, 286, 20190297  DOI: 10.1098/rspb.2019.0297
    Google Scholar
    There is no corresponding record for this reference.
  53. 53
    Distel, M.; Hocking, J. C.; Volkmann, K.; Köster, R. W. The Centrosome Neither Persistently Leads Migration nor Determines the Site of Axonogenesis in Migrating Neurons in Vivo. J. Cell Biol. 2010, 191, 875– 890,  DOI: 10.1083/jcb.201004154
    Google Scholar
    53
    The centrosome neither persistently leads migration nor determines the site of axonogenesis in migrating neurons in vivo
    Distel, Martin; Hocking, Jennifer C.; Volkmann, Katrin; Koester, Reinhard W.
    Journal of Cell Biology (2010), 191 (4), 875-890CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)
    The position of the centrosome ahead of the nucleus has been considered crucial for coordinating neuronal migration in most developmental situations. The proximity of the centrosome has also been correlated with the site of axonogenesis in certain differentiating neurons. Despite these pos. correlations, accumulating exptl. findings appear to negate a universal role of the centrosome in detg. where an axon forms, or in leading the migration of neurons. To further examine this controversy in an in vivo setting, we have generated cell type-specific multi-cistronic gene expression to monitor subcellular dynamics in the developing zebrafish cerebellum. We show that migration of rhombic lip-derived neurons is characterized by a centrosome that does not persistently lead the nucleus, but which is instead regularly overtaken by the nucleus. In addn., axonogenesis is initiated during the onset of neuronal migration and occurs independently of centrosome proximity. These in vivo data reveal a new temporal orchestration of organelle dynamics and provide important insights into the variation in intracellular processes during vertebrate brain differentiation.
  54. 54
    Distel, M.; Wullimann, M. F.; Köster, R. W. Optimized Gal4 Genetics for Permanent Gene Expression Mapping in Zebrafish. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 13365– 13370,  DOI: 10.1073/pnas.0903060106
    Google Scholar
    54
    Optimized Gal4 genetics for permanent gene expression mapping in zebrafish
    Distel, Martin; Wullimann, Mario F.; Koster, Reinhard W.
    Proceedings of the National Academy of Sciences of the United States of America (2009), 106 (32), 13365-13370, S13365/1-S13365/9CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Combinatorial genetics for conditional transgene activation allows studying gene function with temporal and tissue specific control like the Gal4-UAS system, which has enabled sophisticated genetic studies in Drosophila. Recently this system was adapted for zebrafish and promising applications have been introduced. Here, we report a systematic optimization of zebrafish Gal4-UAS genetics by establishing an optimized Gal4-activator (KalTA4). We provide quant. data for KalTA4-mediated transgene activation in dependence of UAS copy nos. to allow for studying dosage effects of transgene expression. Employing a Tol2 transposon-mediated KalTA4 enhancer trap screen biased for central nervous system expression, we present a collection of self-reporting red fluorescent KalTA4 activator strains. These strains reliably transactivate UAS-dependent transgenes and can be rendered homozygous. Furthermore, we have characterized the transactivation kinetics of tissue-specific KalTA4 activation, which led to the development of a self-maintaining effector strain "Kaloop.". This strain relates transient KalTA4 expression during embryogenesis via a KalTA4-mediated autoregulatory mechanism to live adult structures. We demonstrate its use by showing that the secondary octaval nucleus in the adult hindbrain is likely derived from egr2b-expressing cells in rhombomere 5 during stages of early embryogenesis. These data demonstrate prolonged and maintained expression by Kalooping, a technique that can be used for permanent spatiotemporal genetic fate mapping and targeted transgene expression in zebrafish.
  55. 55
    Fortin, D. L.; Banghart, M. R.; Dunn, T. W.; Borges, K.; Wagenaar, D. A.; Gaudry, Q.; Karakossian, M. H.; Otis, T. S.; Kristan, W. B.; Trauner, D.; Kramer, R. H. Photochemical Control of Endogenous Ion Channels and Cellular Excitability. Nat. Methods 2008, 5, 331– 338,  DOI: 10.1038/nmeth.1187
    Google Scholar
    55
    Photochemical control of endogenous ion channels and cellular excitability
    Fortin, Doris L.; Banghart, Matthew R.; Dunn, Timothy W.; Borges, Katharine; Wagenaar, Daniel A.; Gaudry, Quentin; Karakossian, Movses H.; Otis, Thomas S.; Kristan, William B.; Trauner, Dirk; Kramer, Richard H.
    Nature Methods (2008), 5 (4), 331-338CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    Light-activated ion channels provide a precise and noninvasive optical means for controlling action potential firing, but the genes encoding these channels must first be delivered and expressed in target cells. Here we describe a method for bestowing light sensitivity onto endogenous ion channels that does not rely on exogenous gene expression. The method uses a synthetic photoisomerizable small mol., or photoswitchable affinity label (PAL), that specifically targets K+ channels. PALs contain a reactive electrophile, enabling covalent attachment of the photoswitch to naturally occurring nucleophiles in K+ channels. Ion flow through PAL-modified channels is turned on or off by photoisomerizing PAL with different wavelengths of light. We showed that PAL treatment confers light sensitivity onto endogenous K+ channels in isolated rat neurons and in intact neural structures from rat and leech, allowing rapid optical regulation of excitability without genetic modification.
  56. 56
    Laprell, L.; Tochitsky, I.; Kaur, K.; Manookin, M. B.; Stein, M.; Barber, D. M.; Schön, C.; Michalakis, S.; Biel, M.; Kramer, R. H.; Sumser, M. P.; Trauner, D.; Gelder, R. N. V. Photopharmacological Control of Bipolar Cells Restores Visual Function in Blind Mice. J. Clin. Invest. 2017, 127, 2598– 2611,  DOI: 10.1172/JCI92156
    Google Scholar
    56
    Photopharmacological control of bipolar cells restores visual function in blind mice
    Laprell Laura; Stein Marco; Barber David M; Sumser Martin P; Trauner Dirk; Laprell Laura; Kaur Kuldeep; Manookin Michael B; Van Gelder Russell N; Tochitsky Ivan; Kramer Richard H; Schon Christian; Michalakis Stylianos; Biel Martin; Van Gelder Russell N
    The Journal of clinical investigation (2017), 127 (7), 2598-2611 ISSN:.
    Photopharmacological control of neuronal activity using synthetic photochromic ligands, or photoswitches, is a promising approach for restoring visual function in patients suffering from degenerative retinal diseases. Azobenzene photoswitches, such as AAQ and DENAQ, have been shown to restore the responses of retinal ganglion cells to light in mouse models of retinal degeneration but do not recapitulate native retinal signal processing. Here, we describe diethylamino-azo-diethylamino (DAD), a third-generation photoswitch that is capable of restoring retinal ganglion cell light responses to blue or white light. In acute brain slices of murine layer 2/3 cortical neurons, we determined that the photoswitch quickly relaxes to its inactive form in the dark. DAD is not permanently charged, and the uncharged form enables the photoswitch to rapidly and effectively cross biological barriers and thereby access and photosensitize retinal neurons. Intravitreal injection of DAD restored retinal light responses and light-driven behavior to blind mice. Unlike DENAQ, DAD acts upstream of retinal ganglion cells, primarily conferring light sensitivity to bipolar cells. Moreover, DAD was capable of generating ON and OFF visual responses in the blind retina by utilizing intrinsic retinal circuitry, which may be advantageous for restoring visual function.
  57. 57
    Matera, C.; Gomila, A. M. J.; Camarero, N.; Libergoli, M.; Soler, C.; Gorostiza, P. Photoswitchable Antimetabolite for Targeted Photoactivated Chemotherapy. J. Am. Chem. Soc. 2018, 140, 15764– 15773,  DOI: 10.1021/jacs.8b08249
    Google Scholar
    57
    Photoswitchable Antimetabolite for Targeted Photoactivated Chemotherapy
    Matera, Carlo; Gomila, Alexandre M. J.; Camarero, Nuria; Libergoli, Michela; Soler, Concepcio; Gorostiza, Pau
    Journal of the American Chemical Society (2018), 140 (46), 15764-15773CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The efficacy and tolerability of systemically administered anticancer agents are limited by their off-target effects. Precise spatiotemporal control over their cytotoxic activity would allow improving chemotherapy treatments, and light-regulated drugs are well suited to this purpose. We have developed phototrexate, the first photoswitchable inhibitor of the human dihydrofolate reductase (DHFR), as a photochromic analog of methotrexate, a widely prescribed chemotherapeutic drug to treat cancer and psoriasis. Quantification of the light-regulated DHFR enzymic activity, cell proliferation, and in vivo effects in zebrafish show that phototrexate behaves as a potent antifolate in its photoactivated cis configuration and that it is nearly inactive in its dark-relaxed trans form. Thus, phototrexate constitutes a proof-of-concept to design light-regulated cytotoxic small mols. and a step forward to develop targeted anticancer photochemotherapies with localized efficacy and reduced adverse effects.
  58. 58
    Babii, O.; Afonin, S.; Schober, T.; Garmanchuk, L. V.; Ostapchenko, L. I.; Yurchenko, V.; Zozulya, S.; Tarasov, O.; Pishel, I.; Ulrich, A. S.; Komarov, I. V. Peptide Drugs for Photopharmacology: How Much of a Safety Advantage Can Be Gained by Photocontrol?. Future Drug Discovery 2020, 2, FDD28  DOI: 10.4155/fdd-2019-0033
    Google Scholar
    There is no corresponding record for this reference.
  59. 59
    Afonin, S.; Babii, O.; Reuter, A.; Middel, V.; Takamiya, M.; Strähle, U.; Komarov, I. V.; Ulrich, A. S. Light-Controllable Dithienylethene-Modified Cyclic Peptides: Photoswitching the in Vivo Toxicity in Zebrafish Embryos. Beilstein J. Org. Chem. 2020, 16, 39– 49,  DOI: 10.3762/bjoc.16.6
    Google Scholar
    59
    Light-controllable dithienylethene-modified cyclic peptides: photoswitching the in vivo toxicity in zebrafish embryos
    Afonin, Sergii; Babii, Oleg; Reuter, Aline; Middel, Volker; Takamiya, Masanari; Straehle, Uwe; Komarov, Igor V.; Ulrich, Anne S.
    Beilstein Journal of Organic Chemistry (2020), 16 (), 39-49CODEN: BJOCBH; ISSN:1860-5397. (Beilstein-Institut zur Foerderung der Chemischen Wissenschaften)
    This study evaluates the embryotoxicity of dithienylethene-modified peptides upon photoswitching, using 19 analogs based on the β-hairpin scaffold of the natural membranolytic peptide gramicidin S. We established an in vivo assay in two variations (with ex vivo and in situ photoisomerization), using larvae of the model organism Danio rerio, and detd. the toxicities of the peptides in terms of 50% LDs (LD50). This study allowed us to: (i) demonstrate the feasibility of evaluating peptide toxicity with D. rerio larvae at 3-4 days post fertilization, (ii) det. the phototherapeutic safety windows for all peptides, (iii) demonstrate photoswitching of the whole-body toxicity for the dithienylethene-modified peptides in vivo, (iv) re-analyze previous structure-toxicity relationship data, and (v) select promising candidates for potential clin. development.

Cited By

Click to copy section linkSection link copied!
Citation Statements
Explore this article's citation statements on scite.ai
powered by  Scite

This article is cited by 38 publications.

  1. Sudeep Koppayithodi, Palash Jana, Subhajit Bandyopadhyay. Heteroarylstilbenes: Visible-Light-Tunable Photochromic Systems in Water. The Journal of Organic Chemistry 2025, 90 (13) , 4518-4524. https://doi.org/10.1021/acs.joc.4c02844
  2. Atin Chatterjee, Sandip Sarkar, Sangheeta Bhattacharjee, Arpan Bhattacharyya, Surajit Barman, Uttam Pal, Raviranjan Pandey, Anitha Ethirajan, Batakrishna Jana, Benu Brata Das, Amitava Das. Microtubule-Targeting NAP Peptide-Ru(II)-polypyridyl Conjugate As a Bimodal Therapeutic Agent for Triple Negative Breast Carcinoma. Journal of the American Chemical Society 2025, 147 (1) , 532-547. https://doi.org/10.1021/jacs.4c11820
  3. Anaëlle Dumazer, Xavier Gómez-Santacana, Fanny Malhaire, Chris Jopling, Damien Maurel, Guillaume Lebon, Amadeu Llebaria, Cyril Goudet. Optical Control of Adenosine A2A Receptor Using Istradefylline Photosensitivity. ACS Chemical Neuroscience 2024, 15 (3) , 645-655. https://doi.org/10.1021/acschemneuro.3c00721
  4. Adrian Müller-Deku, Oliver Thorn-Seshold. Exhaustive Catalytic ortho-Alkoxylation of Azobenzenes: Flexible Access to Functionally Diverse Yellow-Light-Responsive Photoswitches. The Journal of Organic Chemistry 2022, 87 (24) , 16526-16531. https://doi.org/10.1021/acs.joc.2c02214
  5. James L. Rushworth, Aditya R. Thawani, Elena Fajardo-Ruiz, Joyce C. M. Meiring, Constanze Heise, Andrew J. P. White, Anna Akhmanova, Jochen R. Brandt, Oliver Thorn-Seshold, Matthew J. Fuchter. [5]-Helistatins: Tubulin-Binding Helicenes with Antimitotic Activity. JACS Au 2022, 2 (11) , 2561-2570. https://doi.org/10.1021/jacsau.2c00435
  6. Saugata Sahu, Ammathnadu S. Amrutha, Nobuyuki Tamaoki. Controlling Protein Functionalities With Temporal and Cellular/Subcellular Dimensions of Spatial Resolution With Molecular Photoswitches. Medicinal Research Reviews 2025, 52 https://doi.org/10.1002/med.22106
  7. Adam Varady, Sarah Grissenberger, Andrea Wenninger-Weinzierl, Hugo Poplimont, Caterina Sturtzel, Nicole Schmitner, Li Gao, Robin A. Kimmel, Martin Distel. Precise photopharmacological eradication of metastatic tumor cells. Disease Models & Mechanisms 2025, 18 (2) https://doi.org/10.1242/dmm.052016
  8. Anh-Khoa Nguyen, Sompop Khotwong, Sumrit Wacharasindhu, Preecha Phuwapraisirisan. Three new α-glucosidase inhibitors from aqueous extract of Cannabis sativa leaves: isolation, characterisation, and kinetic study. Natural Product Research 2025, 5 , 1-10. https://doi.org/10.1080/14786419.2024.2448841
  9. Martin Reynders, Sabine Willems, Julian A. Marschner, Thomas Wein, Daniel Merk, Oliver Thorn‐Seshold. Eine hochwertige photoschaltbare Sonde, die selektiv und wirksam den Transkriptionsfaktor RORγ moduliert. Angewandte Chemie 2024, 136 (49) https://doi.org/10.1002/ange.202410139
  10. Martin Reynders, Sabine Willems, Julian A. Marschner, Thomas Wein, Daniel Merk, Oliver Thorn‐Seshold. A High‐Quality Photoswitchable Probe that Selectively and Potently Regulates the Transcription Factor RORγ. Angewandte Chemie International Edition 2024, 63 (49) https://doi.org/10.1002/anie.202410139
  11. Carina Schmitt, Philipp Mauker, Nynke A. Vepřek, Carolin Gierse, Joyce C. M. Meiring, Jürgen Kuch, Anna Akhmanova, Leif Dehmelt, Oliver Thorn‐Seshold. A Photocaged Microtubule‐Stabilising Epothilone Allows Spatiotemporal Control of Cytoskeletal Dynamics. Angewandte Chemie 2024, 136 (43) https://doi.org/10.1002/ange.202410169
  12. Carina Schmitt, Philipp Mauker, Nynke A. Vepřek, Carolin Gierse, Joyce C. M. Meiring, Jürgen Kuch, Anna Akhmanova, Leif Dehmelt, Oliver Thorn‐Seshold. A Photocaged Microtubule‐Stabilising Epothilone Allows Spatiotemporal Control of Cytoskeletal Dynamics. Angewandte Chemie International Edition 2024, 219 https://doi.org/10.1002/anie.202410169
  13. Gulia Bikbaeva, Anna Pilip, Anastasiya Egorova, Vasiliy Medvedev, Daria Mamonova, Dmitrii Pankin, Alexey Kalinichev, Natalya Mayachkina, Lyudmila Bakina, Ilya Kolesnikov, Gerd Leuchs, Alina Manshina. Smart photopharmacological agents: LaVO 4 :Eu 3+ @vinyl phosphonate combining luminescence imaging and photoswitchable butyrylcholinesterase inhibition. Nanoscale Advances 2024, 6 (17) , 4417-4425. https://doi.org/10.1039/D4NA00389F
  14. Martin Reynders, Małgorzata Garścia, Adrian Müller-Deku, Maximilian Wranik, Kristina Krauskopf, Luis de la Osa de la Rosa, Konstantin Schaffer, Anna Jötten, Alexander Rode, Valentin Stierle, Yvonne Kraus, Benedikt Baumgartner, Ahmed Ali, Andrei Bubeneck, Trina Seal, Michel O. Steinmetz, Philipp Paulitschke, Oliver Thorn-Seshold. A photo-SAR study of photoswitchable azobenzene tubulin-inhibiting antimitotics identifying a general method for near-quantitative photocontrol. Chemical Science 2024, 15 (31) , 12301-12309. https://doi.org/10.1039/D4SC03072A
  15. Akshay Saroha, Monica Swetha Bosco, Sneha Menon, Pratibha Kumari, Tanmoy Maity, Subinoy Rana, Sachin Kotak, Jagannath Mondal, Sarit S. Agasti. Regulation of microtubule dynamics and function in living cells via cucurbit[7]uril host–guest assembly. Chemical Science 2024, 15 (30) , 11981-11994. https://doi.org/10.1039/D4SC00204K
  16. Charlotte N. Stindt, Stefano Crespi, Ben L. Feringa. Synthesis of Styrylbenzazole Photoswitches and Evaluation of their Photochemical Properties. Chemistry – A European Journal 2024, 30 (39) https://doi.org/10.1002/chem.202401409
  17. Yen-Ling Lian, Yu-Chun Lin. The emerging tools for precisely manipulating microtubules. Current Opinion in Cell Biology 2024, 88 , 102360. https://doi.org/10.1016/j.ceb.2024.102360
  18. Angelika Seliwjorstow, Masanari Takamiya, Sepand Rastegar, Zbigniew Pianowski. Reversible Influence of Hemipiperazine Photochromism on the Early Development of Zebrafish Embryo. ChemBioChem 2024, 25 (8) https://doi.org/10.1002/cbic.202400143
  19. Yu Zhao, Qingqing Huang, Qiushi Li, Zihan Chen, Yang Liu. Bidirectional Regulation of Intracellular Enzyme Activity Using Light‐Driven Nano‐Inhibitors. Angewandte Chemie 2024, 136 (8) https://doi.org/10.1002/ange.202318533
  20. Yu Zhao, Qingqing Huang, Qiushi Li, Zihan Chen, Yang Liu. Bidirectional Regulation of Intracellular Enzyme Activity Using Light‐Driven Nano‐Inhibitors. Angewandte Chemie International Edition 2024, 63 (8) https://doi.org/10.1002/anie.202318533
  21. Hiroshi Inaba, Kazunori Matsuura. Functionalization of microtubules by Tau-derived peptides: encapsulation, cell manipulation, and construction of superstructures. 2024, 27-44. https://doi.org/10.1039/BK9781839169328-00027
  22. Bo-Wen Wu, Wen-Jing Huang, Yun-He Liu, Qiu-Ge Liu, Jian Song, Tao Hu, Ping Chen, Sai-Yang Zhang. Design, synthesis and biological evaluation of 1,2,3-triazole benzothiazole derivatives as tubulin polymerization inhibitors with potent anti-esophageal cancer activities. European Journal of Medicinal Chemistry 2024, 265 , 116118. https://doi.org/10.1016/j.ejmech.2023.116118
  23. Hiroshi Inaba. Construction of functional microtubules and artificial motile systems based on peptide design. Polymer Journal 2023, 55 (12) , 1261-1274. https://doi.org/10.1038/s41428-023-00838-w
  24. Maximilian Wranik, Tobias Weinert, Chavdar Slavov, Tiziana Masini, Antonia Furrer, Natacha Gaillard, Dario Gioia, Marco Ferrarotti, Daniel James, Hannah Glover, Melissa Carrillo, Demet Kekilli, Robin Stipp, Petr Skopintsev, Steffen Brünle, Tobias Mühlethaler, John Beale, Dardan Gashi, Karol Nass, Dmitry Ozerov, Philip J. M. Johnson, Claudio Cirelli, Camila Bacellar, Markus Braun, Meitian Wang, Florian Dworkowski, Chris Milne, Andrea Cavalli, Josef Wachtveitl, Michel O. Steinmetz, Jörg Standfuss. Watching the release of a photopharmacological drug from tubulin using time-resolved serial crystallography. Nature Communications 2023, 14 (1) https://doi.org/10.1038/s41467-023-36481-5
  25. Maximilian Wranik, Michal W. Kepa, Emma V. Beale, Daniel James, Quentin Bertrand, Tobias Weinert, Antonia Furrer, Hannah Glover, Dardan Gashi, Melissa Carrillo, Yasushi Kondo, Robin T. Stipp, Georgii Khusainov, Karol Nass, Dmitry Ozerov, Claudio Cirelli, Philip J. M. Johnson, Florian Dworkowski, John H. Beale, Scott Stubbs, Thierry Zamofing, Marco Schneider, Kristina Krauskopf, Li Gao, Oliver Thorn-Seshold, Christoph Bostedt, Camila Bacellar, Michel O. Steinmetz, Christopher Milne, Jörg Standfuss. A multi-reservoir extruder for time-resolved serial protein crystallography and compound screening at X-ray free-electron lasers. Nature Communications 2023, 14 (1) https://doi.org/10.1038/s41467-023-43523-5
  26. Kazunori Matsuura, Hiroshi Inaba. Photoresponsive peptide materials: Spatiotemporal control of self-assembly and biological functions. Biophysics Reviews 2023, 4 (4) https://doi.org/10.1063/5.0179171
  27. Sergio Ramos, Alba Vicente-Blázquez, Marta López-Rubio, Laura Gallego-Yerga, Raquel Álvarez, Rafael Peláez. Frentizole, a Nontoxic Immunosuppressive Drug, and Its Analogs Display Antitumor Activity via Tubulin Inhibition. International Journal of Molecular Sciences 2023, 24 (24) , 17474. https://doi.org/10.3390/ijms242417474
  28. Matthias Gehringer, Felix Pape, María Méndez, Philipp Barbie, Andrea Unzue Lopez, Julien Lefranc, Franca‐Maria Klingler, Gerhard Hessler, Thierry Langer, Eleonora Diamanti, Matthias Schiedel. Back in Person: Frontiers in Medicinal Chemistry 2023. ChemMedChem 2023, 18 (19) https://doi.org/10.1002/cmdc.202300344
  29. Xingyu Wang, Benoît Gigant, Xi Zheng, Qiang Chen. Microtubule‐targeting agents for cancer treatment: Seven binding sites and three strategies. MedComm – Oncology 2023, 2 (3) https://doi.org/10.1002/mog2.46
  30. Ana Patricia Ramos, Alicja Szalapak, Lucrezia Camilla Ferme, Carl D. Modes. From cells to form: A roadmap to study shape emergence in vivo. Biophysical Journal 2023, 122 (18) , 3587-3599. https://doi.org/10.1016/j.bpj.2023.05.015
  31. Filip Borys, Piotr Tobiasz, Hanna Fabczak, Ewa Joachimiak, Hanna Krawczyk. First-in-Class Colchicine-Based Visible Light Photoswitchable Microtubule Dynamics Disrupting Agent. Cells 2023, 12 (14) , 1866. https://doi.org/10.3390/cells12141866
  32. Hiroshi Inaba, Minamo Sakaguchi, Soei Watari, Shigesaburo Ogawa, Arif Md. Rashedul Kabir, Akira Kakugo, Kazuki Sada, Kazunori Matsuura. Reversible Photocontrol of Microtubule Stability by Spiropyran‐Conjugated Tau‐Derived Peptides. ChemBioChem 2023, 24 (8) https://doi.org/10.1002/cbic.202200782
  33. Susanne Kirchner, Anna-Lena Leistner, Peter Gödtel, Angelika Seliwjorstow, Sven Weber, Johannes Karcher, Martin Nieger, Zbigniew Pianowski. Hemipiperazines as peptide-derived molecular photoswitches with low-nanomolar cytotoxicity. Nature Communications 2022, 13 (1) https://doi.org/10.1038/s41467-022-33750-7
  34. Eleonora Diamanti, María Méndez, Tatjana Ross, Christian A. Kuttruff, Julien Lefranc, Franca‐Maria Klingler, Franz von Nussbaum, Manfred Jung, Matthias Gehringer. Frontiers in Medicinal Chemistry 2022 Goes Virtual. ChemMedChem 2022, 17 (21) https://doi.org/10.1002/cmdc.202200419
  35. Yan Zhang, Shouguo Peng, Songwen Lin, Ming Ji, Tingting Du, Xiaoguang Chen, Heng Xu. Discovery of a novel photoswitchable PI3K inhibitor toward optically-controlled anticancer activity. Bioorganic & Medicinal Chemistry 2022, 72 , 116975. https://doi.org/10.1016/j.bmc.2022.116975
  36. Filip Borys, Piotr Tobiasz, Jakub Sobel, Hanna Krawczyk. Synthesis and Study of Dibenzo[b, f]oxepine Combined with Fluoroazobenzenes—New Photoswitches for Application in Biological Systems. Molecules 2022, 27 (18) , 5836. https://doi.org/10.3390/molecules27185836
  37. Soei Watari, Hiroshi Inaba, Tomonori Tamura, Arif Md. Rashedul Kabir, Akira Kakugo, Kazuki Sada, Itaru Hamachi, Kazunori Matsuura. Light-induced stabilization of microtubules by photo-crosslinking of a Tau-derived peptide. Chemical Communications 2022, 58 (66) , 9190-9193. https://doi.org/10.1039/D2CC01890J
  38. Susanne Kirchner, Zbigniew Pianowski. Photopharmacology of Antimitotic Agents. International Journal of Molecular Sciences 2022, 23 (10) , 5657. https://doi.org/10.3390/ijms23105657
Download PDF
Go to Journal of the American Chemical Society
Get e-Alerts
Get e-Alerts

Journal of the American Chemical Society

Cite this: J. Am. Chem. Soc. 2022, 144, 12, 5614–5628
Click to copy citationCitation copied!
https://doi.org/10.1021/jacs.2c01020
Published March 15, 2022

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

CC-BY-NC-ND 4.0 .

Article Views

7019

Altmetric

-

Citations

38
Learn about these metrics

Article Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.

Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.

The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated.

  • Abstract

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 1

    Figure 1. Design and synthesis. (a) The colchicinoid pharmacophore (gray shaded trimethoxyphenyl south ring and isovanillyl north ring) can be applied to various scaffolds, giving photoswitchable azobenzene-based PST and SBT-based SBTub antimitotics. Previously published SBTub2/3 lacked key interaction residues (red shaded sites). (b) Z-SBTub3 inhibits tubulin polymerization and MT-dependent processes. (c) X-ray structure of tubulin:Z-SBTub3 complex (carbons as purple spheres). The south ring is buried in β-tubulin (green); only the north ring interacts with α-tubulin at the α-T5 loop (cyan). (d) Evolved SBTub compound library used in this paper. (e) Typical synthesis of SBTubs proceeds by acetanilide sulfurization, Jacobson cyclization, and basic condensation. Phosphate prodrug SBTubA4P is further accessed by phosphoester formation and deprotection.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 2

    Figure 2. (a) Leads SBTub2M and SBTubA4 have highly nonlinear dose–response profiles, high lit/dark ratio of bioactivity, and mid-nanomolar [I]WC values. (b, c) Photocharacterization: (b) SBTub2M is not isomerized from its all-E dark state by 488 nm illumination, but is photoswitched to majority-Z lit states by UV/violet light (78% Z at 405 nm by NMR; 1:1 phosphate-buffered saline/dimethylsulfoxide, PBS:DMSO). (c) Comparison of absorbance spectra of SBTubA4 and azobenzene PST-1 illustrates the SBT’s ideal match to 405 nm photoactivation, combining stronger 405 nm absorption, with sharper absorption cutoff above 405 nm, which makes it orthogonal to GFP (488 nm), YFP (514 nm), and RFP (561 nm) imaging.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 3

    Figure 3. Tubulin-specific cellular mechanism. (a) SBTubs light-dependently inhibit tubulin polymerization (turbidimetric cell-free assay; absorbance mirrors the extent of polymerization; lit indicates majority-Z-SBTub (20 μM) preisomerized to PSS at 405 nm; nocodazole control at 10 μM). (b) Cell cycle analysis of Jurkat cells treated with SBTub2M/SBTubA4P matches photoswitchable reference PST-1: with significant G2/M arrest under 405 nm pulsing (lit), but without cell cycle effects in the dark (matching cosolvent controls). (c) Immunofluorescence imaging of cells treated with SBTubA4P under pulsed 405 nm illuminations (lit, mostly-Z) and in the dark (all-E), compared to cosolvent control (HeLa cells, 20 h incubation; α-tubulin in green, DNA (stained with 4′,6-diamidino-2-phenylindole, DAPI) in blue). (d) Close-up views at the colchicine-binding site of X-ray co-crystal structures (PDB 7Z01, 7Z02) of Z-SBTubA4 (green) and E-SBTub2M (orange) bound to the Darpin D1:tubulin complex (dark gray α-tubulin, light gray β-tubulin in cartoon representation; Z-SBTubA4 and Z-SBTub2M in stick representation, oxygens red, nitrogen blue, and sulfur yellow). (e) Superimposition of tubulin:CA4 (white carbons; PDB 5LYJ) and TD1:Z-SBTubA4 (PDB 7Z01) shows that lead SBTubs share the same binding site as the parent natural product CA4 (see also Figure S11).

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 4

    Figure 4. Spatiotemporal control over MT dynamics in 2D-cultured HeLa cells. (a, b) MT inhibition in SBTubA4P-treated cells is initiated only upon 405 nm illumination pulses and only in ROI-targeted cells (data related to Movie S1; live-cell EB3-tdTomato comets quantify polymerizing MTs). (a) Comet count statistics are similar to cosolvent-only baseline in both ROI-pulsed-cosolvent and non-ROI-SBTubA4P conditions; ROI-SBTubA4P statistics show inhibition spikes. (b) Stills from Movie S1 at the times indicated in (a), initially during the untreated timecourse, then during the SBTubA4P-treated timecourse on the same cells. Purple arrowhead indicates the ROI cell; purple dotted circle indicates where the 405 nm ROI is applied at times 26, 88, and 148 s; and white arrowhead indicates the non-ROI cell quantified as the internal control (scale bar 15 μm). (c) EB3 comet counts of cells imaged at 561 nm only (dark, gray), with 47 frames at 487 nm applied to full field of view during the time span indicated with dashed lines (“487”, cyan) and SBTubA4P (6 μM), or with single-frame 405 nm pulses, SBTubA4P (0.6 μM), applied to full field of view at times indicated with dashed lines (“405”, violet) (n = 3 cells). Temporally precise onset and full-field diffusional reversibility are shown (data related to Movies S2 and S3). [(a, c) Mean ± standard error of the mean (SEM) EB3 comet counts as normalized to the means of the first five time points; 405 nm ROIs applied at indicated times; for further details, see the Supporting Information].

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 5

    Figure 5. Spatiotemporal control over MT architecture, migration, and mitosis in 3D culture and tissue explant. (a) 3D human mammary gland organoids embedded in collagen gels only have inhibited branch outgrowth when treated with both SBTubA4P and UV pulses. (b) Local applications of UV light to ROItarg regions of SBTubA4P-treated organoids (blue box, one ca. 450 ms pulse per 7 min per z-stack) stops branch proliferation and outgrowth (red outline), while branches in untargeted ROIctrl regions develop dramatically (start: solid green line, final: dotted green line) (related to Movie S4). (c) Radial progress of branch tip fronts (directed and collective behavior) in ROItarg and ROIctrl regions. (d) Still image timecourse, zoomed on a branch tip in the ROIctrl (blue box) region, showing cell proliferation (yellow arrowhead) and matrix invasion (one representative of the migrating cells is tracked over time with green arrows), while branch tip of ROItarg region has static non-proliferating cells and even slight branch retraction (red arrows) (data related to Movie S5). (e) Branch progression and proliferation are unimpeded and continuous in ROIctrl regions, while ROItarg regions are static, and branches growing into the ROItarg stop their growth (color code as in (e), data related to Movie S6). [(a–e) Cell location in organoids tracked with nuclear stain SiR-DNA imaged at 647 nm]. (f) Whole-field-of-view 405 nm photoactivation of SBTub2M-treated intact 3D brain explants of larval Drosophila melanogaster (bottom row) causes neuroblast centrosomes (red arrows) to rapidly shrink in size and signal intensity (45 s and 3 min) and prevents the cell from progressing through division (13 min). Some MT signal accumulates at mid-cell at later time points (purple arrow) (data related to Movie S8). In DMSO-only controls (top row), centrosome integrity (white arrows, 45 s and 3 min) and progression through the cell cycle (13 min) are unaffected, indicated by myosin accumulation at the cleavage furrow (cyan arrows) (data related to Movie S9). [MTs in white (Jupiter::mCherry imaged at 561 nm), myosin in green (Squash::GFP imaged at 488 nm)]. (g) Relative mCherry fluorescence intensity of centrosomal ROIs in SBTub2M-treated prophase neuroblasts (red) after activation at 405 nm drops notably during the approximately 45 s activation period (blue box) compared to the DMSO control prophase neuroblasts (black). Signal intensities are shown as the proportion of the per-cell maximum preactivation signal intensity (shading indicates ±1 standard deviation, 1–2 centrosomes quantified from a total of five neuroblasts from three different animals). For details, see the Supporting Information.

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 6

    Figure 6. Photoinhibition of X. tropicalis development, and in vivo photocontrol of MT dynamics in Danio rerio. (a, b) Xenopus embryos incubated with compounds for 1 h at the two-cell stage, before medium exchange optionally with 410 nm photoactivation. Embryos show irreversible development inhibition by in situ formed Z-SBTubA4 in lit conditions but had no effects in the dark or with a low concentration of SBTubA4P [(a) SBTubA4P at 5 μM; (b) development quantified by the ratio of major to minor embryo axis lengths, six embryos per condition, mean ± SEM]. (c) Development of D. rerio treated at the indicated stages for 24 h with SBTubA4P or control compounds under dark or pulsed lit (1 s/5 min) conditions. SBTubA4P (1 or 25 μM) causes morphological abnormalities only in the lit state, showing that it remains effective in vivo. (d, e) Reversible modulation of MT dynamics in 48 hpf zebrafish embryo (25 μM). (EB3-GFP in green, histone H2B in red). (data related to Movies S12–S16; see the Supporting Information).

    High Resolution Image
    Download MS PowerPoint Slide

  • References

    This article references 59 other publications.

    1. 1
      Stepanova, T.; Slemmer, J.; Hoogenraad, C. C.; Lansbergen, G.; Dortland, B.; De Zeeuw, C. I.; Grosveld, F.; van Cappellen, G.; Akhmanova, A.; Galjart, N. Visualization of Microtubule Growth in Cultured Neurons via the Use of EB3-GFP (End-Binding Protein 3-Green Fluorescent Protein). J. Neurosci. 2003, 23, 2655– 2664,  DOI: 10.1523/JNEUROSCI.23-07-02655.2003
      1
      Visualization of microtubule growth in cultured neurons via the use of EB3-GFP (end-binding protein 3-green fluorescent protein)
      Stepanova, Tatiana; Slemmer, Jenny; Hoogenraad, Casper C.; Lansbergen, Gideon; Dortland, Bjorn; De Zeeuw, Chris I.; Grosveld, Frank; van Cappellen, Gert; Akhmanova, Anna; Galjart, Niels
      Journal of Neuroscience (2003), 23 (7), 2655-2664CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
      Several microtubule binding proteins, including CLIP-170 (cytoplasmic linker protein-170), CLIP-115, and EB1 (end-binding protein 1), have been shown to assoc. specifically with the ends of growing microtubules in non-neuronal cells, thereby regulating microtubule dynamics and the binding of microtubules to protein complexes, organelles, and membranes. When fused to GFP (green fluorescent protein), these proteins, which collectively are called +TIPs (plus end tracking proteins), also serve as powerful markers for visualizing microtubule growth events. Here we demonstrate that endogenous +TIPs are present at distal ends of microtubules in fixed neurons. Using EB3-GFP as a marker of microtubule growth in live cells, we subsequently analyze microtubule dynamics in neurons. Our results indicate that microtubules grow slower in neurons than in glia and COS-1 cells. The av. speed and length of EB3-GFP movements are comparable in cell bodies, dendrites, axons, and growth cones. In the proximal region of differentiated dendrites ∼65% of EB3-GFP movements are directed toward the distal end, whereas 35% are directed toward the cell body. In more distal dendritic regions and in axons most EB3-GFP dots move toward the growth cone. This difference in directionality of EB3-GFP movements in dendrites and axons reflects the highly specific microtubule organization in neurons. Together, these results suggest that local microtubule polymn. contributes to the formation of the microtubule network in all neuronal compartments. We propose that similar mechanisms underlie the specific assocn. of CLIPs and EB1-related proteins with the ends of growing microtubules in non-neuronal and neuronal cells.
    2. 2
      Kleele, T.; Marinković, P.; Williams, P. R.; Stern, S.; Weigand, E. E.; Engerer, P.; Naumann, R.; Hartmann, J.; Karl, R. M.; Bradke, F.; Bishop, D.; Herms, J.; Konnerth, A.; Kerschensteiner, M.; Godinho, L.; Misgeld, T. An Assay to Image Neuronal Microtubule Dynamics in Mice. Nat. Commun. 2014, 5, 4827  DOI: 10.1038/ncomms5827
      2
      An assay to image neuronal microtubule dynamics in mice
      Kleele, Tatjana; Marinkovic, Petar; Williams, Philip R.; Stern, Sina; Weigand, Emily E.; Engerer, Peter; Naumann, Ronald; Hartmann, Jana; Karl, Rosa M.; Bradke, Frank; Bishop, Derron; Herms, Jochen; Konnerth, Arthur; Kerschensteiner, Martin; Godinho, Leanne; Misgeld, Thomas
      Nature Communications (2014), 5 (), 4827CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)
      Microtubule dynamics in neurons play crit. roles in physiol., injury and disease and det. microtubule orientation, the cell biol. correlate of neurite polarization. Several microtubule binding proteins, including end-binding protein 3 (EB3), specifically bind to the growing plus tip of microtubules. In the past, fluorescently tagged end-binding proteins have revealed microtubule dynamics in vitro and in non-mammalian model organisms. Here, we devise an imaging assay based on transgenic mice expressing yellow fluorescent protein-tagged EB3 to study microtubules in intact mammalian neurites. Our approach allows measurement of microtubule dynamics in vivo and ex vivo in peripheral nervous system and central nervous system neurites under physiol. conditions and after exposure to microtubule-modifying drugs. We find an increase in dynamic microtubules after injury and in neurodegenerative disease states, before axons show morphol. indications of degeneration or regrowth. Thus increased microtubule dynamics might serve as a general indicator of neurite remodelling in health and disease.
    3. 3
      Goglia, A. G.; Toettcher, J. E. A Bright Future: Optogenetics to Dissect the Spatiotemporal Control of Cell Behavior. Curr. Opin. Chem. Biol. 2019, 48, 106– 113,  DOI: 10.1016/j.cbpa.2018.11.010
      3
      A bright future: optogenetics to dissect the spatiotemporal control of cell behavior
      Goglia, Alexander G.; Toettcher, Jared E.
      Current Opinion in Chemical Biology (2019), 48 (), 106-113CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)
      Cells sense, process, and respond to extracellular information using signaling networks: collections of proteins that act as precise biochem. sensors. These protein networks are characterized by both complex temporal organization, such as pulses of signaling activity, and by complex spatial organization, where proteins assemble structures at particular locations and times within the cell. Yet despite their ubiquity, studying these spatial and temporal properties has remained challenging because they emerge from the entire protein network rather than a single node, and cannot be easily tuned by drugs or mutations. These challenges are being met by a new generation of optogenetic tools capable of directly controlling the activity of individual signaling nodes over time and the assembly of protein complexes in space. Here, we outline how these recent innovations are being used in conjunction with engineering-influenced exptl. design to address longstanding questions in signaling biol.
    4. 4
      Hoorens, M. W. H.; Szymanski, W. Reversible, Spatial and Temporal Control over Protein Activity Using Light. Trends Biochem. Sci. 2018, 43, 567– 575,  DOI: 10.1016/j.tibs.2018.05.004
      4
      Reversible, Spatial and Temporal Control over Protein Activity Using Light
      Hoorens, Mark W. H.; Szymanski, Wiktor
      Trends in Biochemical Sciences (2018), 43 (8), 567-575CODEN: TBSCDB; ISSN:0968-0004. (Elsevier Ltd.)
      A review. In biomedical sciences, the function of a protein of interest is investigated by altering its net activity and assessing the consequences for the cell or organism. To change the activity of a protein, a wide variety of chem. and genetic tools have been developed. The drawback of most of these tools is that they do not allow for reversible, spatial and temporal control. Here, we describe selected developments in photopharmacol. that aim at establishing such control over protein activity through bioactive mols. with photo-controlled potency. We also discuss why such control is desired and what challenges still need to be overcome for photopharmacol. to reach its maturity as a chem. biol. research tool.
    5. 5
      Borowiak, M.; Nahaboo, W.; Reynders, M.; Nekolla, K.; Jalinot, P.; Hasserodt, J.; Rehberg, M.; Delattre, M.; Zahler, S.; Vollmar, A.; Trauner, D.; Thorn-Seshold, O. Photoswitchable Inhibitors of Microtubule Dynamics Optically Control Mitosis and Cell Death. Cell 2015, 162, 403– 411,  DOI: 10.1016/j.cell.2015.06.049
      5
      Photoswitchable Inhibitors of Microtubule Dynamics Optically Control Mitosis and Cell Death
      Borowiak, Malgorzata; Nahaboo, Wallis; Reynders, Martin; Nekolla, Katharina; Jalinot, Pierre; Hasserodt, Jens; Rehberg, Markus; Delattre, Marie; Zahler, Stefan; Vollmar, Angelika; Trauner, Dirk; Thorn-Seshold, Oliver
      Cell (Cambridge, MA, United States) (2015), 162 (2), 403-411CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      Small mols. that interfere with microtubule dynamics, such as Taxol and the Vinca alkaloids, are widely used in cell biol. research and as clin. anticancer drugs. However, their activity cannot be restricted to specific target cells, which also causes severe side effects in chemotherapy. Here, the authors introduce the photostatins, inhibitors that can be switched on and off in vivo by visible light, to optically control microtubule dynamics. Photostatins modulate microtubule dynamics with a subsecond response time and control mitosis in living organisms with single-cell spatial precision. In longer-term applications in cell culture, photostatins are up to 250 times more cytotoxic when switched on with blue light than when kept in the dark. Therefore, photostatins are both valuable tools for cell biol., and are promising as a new class of precision chemotherapeutics whose toxicity may be spatiotemporally constrained using light.
    6. 6
      Borowiak, M.; Küllmer, F.; Gegenfurtner, F.; Peil, S.; Nasufovic, V.; Zahler, S.; Thorn-Seshold, O.; Trauner, D.; Arndt, H.-D. Optical Manipulation of F-Actin with Photoswitchable Small Molecules. J. Am. Chem. Soc. 2020, 142, 9240– 9249,  DOI: 10.1021/jacs.9b12898
      6
      Optical Manipulation of F-Actin with Photoswitchable Small Molecules
      Borowiak, Malgorzata; Kuellmer, Florian; Gegenfurtner, Florian; Peil, Sebastian; Nasufovic, Veselin; Zahler, Stefan; Thorn-Seshold, Oliver; Trauner, Dirk; Arndt, Hans-Dieter
      Journal of the American Chemical Society (2020), 142 (20), 9240-9249CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Cell-permeable photoswitchable small mols., termed optojasps, are introduced to optically control the dynamics of the actin cytoskeleton and cellular functions that depend on it. These light-dependent effectors were designed from the F-actin-stabilizing marine depsipeptide jasplakinolide by functionalizing them with azobenzene photoswitches. As demonstrated, optojasps can be employed to control cell viability, cell motility, and cytoskeletal signaling with the high spatial and temporal resoln. that light affords. Optojasps can be expected to find applications in diverse areas of cell biol. research. They may also provide a template for photopharmacol. targeting the ubiquitous actin cytoskeleton with precision control in the micrometer range.
    7. 7
      Glotzer, M. The 3Ms of Central Spindle Assembly: Microtubules, Motors and MAPs. Nat. Rev. Mol. Cell Biol. 2009, 10, 9– 20,  DOI: 10.1038/nrm2609
      7
      The 3Ms of central spindle assembly: microtubules, motors and MAPs
      Glotzer, Michael
      Nature Reviews Molecular Cell Biology (2009), 10 (1), 9-20CODEN: NRMCBP; ISSN:1471-0072. (Nature Publishing Group)
      A review. During metaphase, sister chromatids are positioned at the midpoint of the microtubule-based mitotic spindle in prepn. for their segregation. The onset of anaphase triggers inactivation of the key mitotic kinase, cyclin-dependent kinase 1 (CDK1), and the poleward movement of sister chromatids. During anaphase, the mitotic spindle reorganizes in prepn. for cytokinesis. Kinesin motor proteins and microtubule-assocd. proteins bundle the plus ends of interpolar microtubules and generate the central spindle, which regulates cleavage furrow initiation and the completion of cytokinesis. Complementary approaches, including cell biol., genetics, and computational modeling, have provided new insights into the mechanism and regulation of central spindle assembly.
    8. 8
      Kapitein, L. C.; Hoogenraad, C. C. Building the Neuronal Microtubule Cytoskeleton. Neuron 2015, 87, 492– 506,  DOI: 10.1016/j.neuron.2015.05.046
      8
      Building the Neuronal Microtubule Cytoskeleton
      Kapitein, Lukas C.; Hoogenraad, Casper C.
      Neuron (2015), 87 (3), 492-506CODEN: NERNET; ISSN:0896-6273. (Cell Press)
      Microtubules are one of the major cytoskeletal components of neurons, essential for many fundamental cellular and developmental processes, such as neuronal migration, polarity, and differentiation. Microtubules have been regarded as crit. structures for stable neuronal morphol. because they serve as tracks for long-distance transport, provide dynamic and mech. functions, and control local signaling events. Establishment and maintenance of the neuronal microtubule architecture requires tight control over different dynamic parameters, such as microtubule no., length, distribution, orientations, and bundling. Recent genetic studies have identified mutations in a wide variety of tubulin isotypes and microtubule-related proteins in many of the major neurodevelopmental and neurodegenerative diseases. Here, we highlight the functions of the neuronal microtubule cytoskeleton, its architecture, and the way its organization and dynamics are shaped by microtubule-related proteins.
    9. 9
      van Haren, J.; Charafeddine, R. A.; Ettinger, A.; Wang, H.; Hahn, K. M.; Wittmann, T. Local Control of Intracellular Microtubule Dynamics by EB1 Photodissociation. Nat. Cell Biol. 2018, 20, 252– 261,  DOI: 10.1038/s41556-017-0028-5
      9
      Local control of intracellular microtubule dynamics by EB1 photodissociation
      van Haren, Jeffrey; Charafeddine, Rabab A.; Ettinger, Andreas; Wang, Hui; Hahn, Klaus M.; Wittmann, Torsten
      Nature Cell Biology (2018), 20 (3), 252-261CODEN: NCBIFN; ISSN:1465-7392. (Nature Research)
      End-binding proteins (EBs) are adaptors that recruit functionally diverse microtubule plus-end-tracking proteins (+TIPs) to growing microtubule plus ends. To test with high spatial and temporal accuracy how, when and where +TIP complexes contribute to dynamic cell biol., it developed a photo-inactivated EB1 variant (.product. -EB1) by inserting a blue-light-sensitive protein-protein interaction module between the microtubule-binding and +TIP-binding domains of EB1. .product. -EB1 replaces endogenous EB1 function in the absence of blue light. By contrast, blue-light-mediated .product. -EB1 photodissocn. results in rapid +TIP complex disassembly, and acutely and reversibly attenuates microtubule growth independent of microtubule end assocn. of the microtubule polymerase CKAP5 (also known as ch-TOG and XMAP215). Local .product. -EB1 photodissocn. allows subcellular control of microtubule dynamics at the second and micrometer scale, and elicits aversive turning of migrating cancer cells. Importantly, light-mediated domain splitting can serve as a template to optically control other intracellular protein activities.
    10. 10
      Adikes, R. C.; Hallett, R. A.; Saway, B. F.; Kuhlman, B.; Slep, K. C. Control of Microtubule Dynamics Using an Optogenetic Microtubule plus End–F-Actin Cross-Linker. J. Cell Biol. 2018, 217, 779– 793,  DOI: 10.1083/jcb.201705190
      10
      Control of microtubule dynamics using an optogenetic microtubule plus end-F-actin cross-linker
      Adikes, Rebecca C.; Hallett, Ryan A.; Saway, Brian F.; Kuhlman, Brian; Slep, Kevin C.
      Journal of Cell Biology (2018), 217 (2), 779-793CODEN: JCLBA3; ISSN:1540-8140. (Rockefeller University Press)
      We developed a novel optogenetic tool, SxIP-improved light-inducible dimer (iLID), to facilitate the reversible recruitment of factors to microtubule (MT) plus ends in an end-binding protein-dependent manner using blue light. We show that SxIP-iLID can track MT plus ends and recruit tgRFP-SspB upon blue light activation. We used this system to investigate the effects of crosslinking MT plus ends and F-actin in Drosophila melanogaster S2 cells to gain insight into spectraplakin function and mechanism. We show that SxIP-iLID can be used to temporally recruit an F-actin binding domain to MT plus ends and cross-link the MT and F-actin networks. Crosslinking decreases MT growth velocities and generates a peripheral MT exclusion zone. SxIP-iLID facilitates the general recruitment of specific factors to MT plus ends with temporal control enabling researchers to systematically regulate MT plus end dynamics and probe MT plus end function in many biol. processes.
    11. 11
      Meiring, J. C. M.; Grigoriev, I.; Nijenhuis, W.; Kapitein, L. C.; Akhmanova, A. Opto-Katanin: An Optogenetic Tool for Localized Microtubule Disassembly. bioRxiv 2021, 22, 473806  DOI: 10.1101/2021.12.22.473806
      There is no corresponding record for this reference.
    12. 12
      Liu, G. Y.; Chen, S.-C.; Shaiv, K.; Hong, S.-R.; Yang, W.-T.; Huang, S.-H.; Chang, Y.-C.; Cheng, H.; Lin, Y.-C. Precise Control of Microtubule Disassembly in Living Cells. bioRxiv 2021, 31, 463668  DOI: 10.1101/2021.10.08.463668
      There is no corresponding record for this reference.
    13. 13
      Wühr, M.; Tan, E. S.; Parker, S. K.; Detrich, H. W.; Mitchison, T. J. A Model for Cleavage Plane Determination in Early Amphibian and Fish Embryos. Curr. Biol. 2010, 20, 2040– 2045,  DOI: 10.1016/j.cub.2010.10.024
      13
      A Model for Cleavage Plane Determination in Early Amphibian and Fish Embryos
      Wuehr, Martin; Tan, Edwin S.; Parker, Sandra K.; Detrich, H. William, III; Mitchison, Timothy J.
      Current Biology (2010), 20 (22), 2040-2045CODEN: CUBLE2; ISSN:0960-9822. (Cell Press)
      Summary: Current models for cleavage plane detn. propose that metaphase spindles are positioned and oriented by interactions of their astral microtubules with the cellular cortex, followed by cleavage in the plane of the metaphase plate []. We show that in early frog and fish embryos, where cells are unusually large, astral microtubules in metaphase are too short to position and orient the spindle. Rather, the preceding interphase aster centers and orients a pair of centrosomes prior to nuclear envelope breakdown, and the spindle assembles between these prepositioned centrosomes. Interphase asters center and orient centrosomes with dynein-mediated pulling forces. These forces act before astral microtubules contact the cortex; thus, dynein must pull from sites in the cytoplasm, not the cell cortex as is usually proposed for smaller cells. Aster shape is detd. by interactions of the expanding periphery with the cell cortex or with an interaction zone that forms between sister-asters in telophase. We propose a model to explain cleavage plane geometry in which the length of astral microtubules is limited by interaction with these boundaries, causing length asymmetries. Dynein anchored in the cytoplasm then generates length-dependent pulling forces, which move and orient centrosomes.
    14. 14
      Josa-Culleré, L.; Llebaria, A. In the Search for Photocages Cleavable with Visible Light: An Overview of Recent Advances and Chemical Strategies. ChemPhotoChem 2021, 5, 296– 314,  DOI: 10.1002/cptc.202000253
      There is no corresponding record for this reference.
    15. 15
      Müller-Deku, A.; Meiring, J. C. M.; Loy, K.; Kraus, Y.; Heise, C.; Bingham, R.; Jansen, K. I.; Qu, X.; Bartolini, F.; Kapitein, L. C.; Akhmanova, A.; Ahlfeld, J.; Trauner, D.; Thorn-Seshold, O. Photoswitchable Paclitaxel-Based Microtubule Stabilisers Allow Optical Control over the Microtubule Cytoskeleton. Nat. Commun. 2020, 11, 4640  DOI: 10.1038/s41467-020-18389-6
      15
      Photoswitchable paclitaxel-based microtubule stabilisers allow optical control over the microtubule cytoskeleton
      Mueller-Deku, Adrian; Meiring, Joyce C. M.; Loy, Kristina; Kraus, Yvonne; Heise, Constanze; Bingham, Rebekkah; Jansen, Klara I.; Qu, Xiaoyi; Bartolini, Francesca; Kapitein, Lukas C.; Akhmanova, Anna; Ahlfeld, Julia; Trauner, Dirk; Thorn-Seshold, Oliver
      Nature Communications (2020), 11 (1), 4640CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Small mol. inhibitors are prime reagents for studies in microtubule cytoskeleton research, being applicable across a range of biol. models and not requiring genetic engineering. However, traditional chem. inhibitors cannot be exptl. applied with spatiotemporal precision suiting the length and time scales inherent to microtubule-dependent cellular processes. We have synthesized photoswitchable paclitaxel-based microtubule stabilizers, whose binding is induced by photoisomerisation to their metastable state. Photoisomerising these reagents in living cells allows optical control over microtubule network integrity and dynamics, cell division and survival, with biol. response on the timescale of seconds and spatial precision to the level of individual cells within a population. In primary neurons, they enable regulation of microtubule dynamics resolved to subcellular regions within individual neurites. These azobenzene-based microtubule stabilizers thus enable non-invasive, spatiotemporally precise modulation of the microtubule cytoskeleton in living cells, and promise new possibilities for studying intracellular transport, cell motility, and neuronal physiol.
    16. 16
      Gao, L.; Meiring, J. C. M.; Heise, C.; Rai, A.; Müller-Deku, A.; Akhmanova, A.; Thorn-Seshold, J.; Thorn-Seshold, O. Photoswitchable Epothilone-Based Microtubule Stabilisers Allow GFP-Imaging-Compatible, Optical Control over the Microtubule Cytoskeleton. Angew. Chem., Int. Ed. 2021, 48, e202114614  DOI: 10.1002/anie.202114614
      There is no corresponding record for this reference.
    17. 17
      Rastogi, S. K.; Zhao, Z.; Gildner, M. B.; Shoulders, B. A.; Velasquez, T. L.; Blumenthal, M. O.; Wang, L.; Li, X.; Hudnall, T. W.; Betancourt, T.; Du, L.; Brittain, W. J. Synthesis, Optical Properties and in Vitro Cell Viability of Novel Spiropyrans and Their Photostationary States. Tetrahedron 2021, 80, 131854  DOI: 10.1016/j.tet.2020.131854
      17
      Synthesis, optical properties and in vitro cell viability of novel spiropyrans and their photostationary states
      Rastogi, Shiva K.; Zhao, Zhenze; Gildner, M. Brenton; Shoulders, Ben A.; Velasquez, Tara L.; Blumenthal, Madeleine O.; Wang, Lei; Li, Xiaopeng; Hudnall, Todd W.; Betancourt, Tania; Du, Liqin; Brittain, William J.
      Tetrahedron (2021), 80 (), 131854CODEN: TETRAB; ISSN:0040-4020. (Elsevier Ltd.)
      A novel class of spiropyrans I (R = H, OEt, OMe; R1 = R2 = R3 = H, OMe; R4 = H, NO2) was synthesized using a multistep process that involves three key intermediates: (a) diazonium-tetrafluoroborate, (b) hydrazine and (c) indolium iodide. The SP analogs I were confirmed as being able to isomerize and attain a photostationary state (PSS) upon irradn. with UV light (UV, 365 nm) in an aq. environment. UV-visible absorption spectra were recorded to confirm the isomerization properties. The ability of the synthesized compds. I to induce growth inhibition of HeLa cervical cancer cells was assessed via the MTT assay after incubation with either the SP or their PSS. The IC50 values of two PSS II (R = H; R1 = R2 = R3 = OMe; R4 = nitro, R = R1 = R2 = R3 = OMe; R4 = H), were obsd. to be around 14 ± 4 fold lower (26 ± 3μM) than their corresponding SPs. The most cytotoxic compds. I [R = R1 = R2 = R3 = R4 = H (III)] and II [R = R1 = R2 = R3 = R4 = H (IV)] showed the lowest IC50 values (12μM). An in vitro tubulin polymn. assay showed that III and IV exhibited the greatest difference in tubulin inhibition relative to unirradiated I (R = R1 = R2 = R3 = H, R4 = NO2; R = R1 = R2 = R3 = OMe, R4 = H).
    18. 18
      Gao, L.; Meiring, J. C. M.; Kraus, Y.; Wranik, M.; Weinert, T.; Pritzl, S. D.; Bingham, R.; Ntouliou, E.; Jansen, K. I.; Olieric, N.; Standfuss, J.; Kapitein, L. C.; Lohmüller, T.; Ahlfeld, J.; Akhmanova, A.; Steinmetz, M. O.; Thorn-Seshold, O. A Robust, GFP-Orthogonal Photoswitchable Inhibitor Scaffold Extends Optical Control over the Microtubule Cytoskeleton. Cell Chem. Biol. 2021, 28, 228– 241,  DOI: 10.1016/j.chembiol.2020.11.007
      18
      A Robust, GFP-Orthogonal Photoswitchable Inhibitor Scaffold Extends Optical Control over the Microtubule Cytoskeleton
      Gao, Li; Meiring, Joyce C. M.; Kraus, Yvonne; Wranik, Maximilian; Weinert, Tobias; Pritzl, Stefanie D.; Bingham, Rebekkah; Ntouliou, Evangelia; Jansen, Klara I.; Olieric, Natacha; Standfuss, Joerg; Kapitein, Lukas C.; Lohmueller, Theobald; Ahlfeld, Julia; Akhmanova, Anna; Steinmetz, Michel O.; Thorn-Seshold, Oliver
      Cell Chemical Biology (2021), 28 (2), 228-241.e6CODEN: CCBEBM; ISSN:2451-9448. (Cell Press)
      Optically controlled chem. reagents, termed photopharmaceuticals, are powerful tools for precise spatiotemporal control of proteins particularly when genetic methods, such as knockouts or optogenetics are not viable options. However, current photopharmaceutical scaffolds, such as azobenzenes are intolerant of GFP/YFP imaging and are metabolically labile, posing severe limitations for biol. use. We rationally designed a photoswitchable SBT scaffold to overcome these problems, then derivatized it to create exceptionally metabolically robust and fully GFP/YFP-orthogonal SBTub photopharmaceutical tubulin inhibitors. Lead compd. SBTub3 allows temporally reversible, cell-precise, and even subcellularly precise photomodulation of microtubule dynamics, organization, and microtubule-dependent processes. By overcoming the previous limitations of microtubule photopharmaceuticals, SBTubs offer powerful applications in cell biol., and their robustness and druglikeness are favorable for intracellular biol. control in in vivo applications. We furthermore expect that the robustness and imaging orthogonality of the SBT scaffold will inspire other derivatizations directed at extending the photocontrol of a range of other biol. targets.
    19. 19
      Sailer, A.; Meiring, J. C. M.; Heise, C.; Pettersson, L. N.; Akhmanova, A.; Thorn-Seshold, J.; Thorn-Seshold, O. Pyrrole Hemithioindigo Antimitotics with Near-Quantitative Bidirectional Photoswitching That Photocontrol Cellular Microtubule Dynamics with Single-Cell Precision. Angew. Chem., Int. Ed. 2021, 60, 23695– 23704,  DOI: 10.1002/anie.202104794
      19
      Pyrrole Hemithioindigo Antimitotics with Near-Quantitative Bidirectional Photoswitching that Photocontrol Cellular Microtubule Dynamics with Single-Cell Precision
      Sailer, Alexander; Meiring, Joyce C. M.; Heise, Constanze; Pettersson, Linda N.; Akhmanova, Anna; Thorn-Seshold, Julia; Thorn-Seshold, Oliver
      Angewandte Chemie, International Edition (2021), 60 (44), 23695-23704CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      We report the first cellular application of the emerging near-quant. photoswitch pyrrole hemithioindigo, by rationally designing photopharmaceutical PHTub inhibitors of the cytoskeletal protein tubulin. PHTubs allow simultaneous visible-light imaging and photoswitching in live cells, delivering cell-precise photomodulation of microtubule dynamics, and photocontrol over cell cycle progression and cell death. This is the first acute use of a hemithioindigo photopharmaceutical for high-spatiotemporal-resoln. biol. control in live cells. It addnl. demonstrates the utility of near-quant. photoswitches, by enabling a dark-active design to overcome residual background activity during cellular photopatterning. This work opens up new horizons for high-precision microtubule research using PHTubs and shows the cellular applicability of pyrrole hemithioindigo as a valuable scaffold for photocontrol of a range of other biol. targets.
    20. 20
      Sailer, A.; Ermer, F.; Kraus, Y.; Lutter, F. H.; Donau, C.; Bremerich, M.; Ahlfeld, J.; Thorn-Seshold, O. Hemithioindigos for Cellular Photopharmacology: Desymmetrised Molecular Switch Scaffolds Enabling Design Control over the Isomer-Dependency of Potent Antimitotic Bioactivity. ChemBioChem 2019, 20, 1305– 1314,  DOI: 10.1002/cbic.201800752
      20
      Hemithioindigos for Cellular Photopharmacology: Desymmetrised Molecular Switch Scaffolds Enabling Design Control over the Isomer-Dependency of Potent Antimitotic Bioactivity
      Sailer, Alexander; Ermer, Franziska; Kraus, Yvonne; Lutter, Ferdinand H.; Donau, Carsten; Bremerich, Maximilian; Ahlfeld, Julia; Thorn-Seshold, Oliver
      ChemBioChem (2019), 20 (10), 1305-1314CODEN: CBCHFX; ISSN:1439-4227. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Druglike small mols. with photoswitchable bioactivity-photopharmaceuticals-allow biologists to perform studies with exquisitely precise and reversible, spatial and temporal control over crit. biol. systems inaccessible to genetic manipulation. The photoresponsive pharmacophores disclosed have been almost exclusively azobenzenes, which has limited the structural and substituent scope of photopharmacol. More detrimentally, for azobenzene reagents, it is not researchers' needs for adapted exptl. tools, but rather protein binding site sterics, that typically force whether the trans (dark) or cis (lit) isomer is the more bioactive. We now present the rational design of HOTubs, the first hemithioindigo-based pharmacophores enabling photoswitchable control over endogenous biol. activity in cellulo. HOTubs optically control microtubule depolymn. and cell death in unmodified mammalian cells. Notably, we show how the asymmetry of hemithioindigos allows a priori design of either Z- or E- (dark- or lit)-toxic antimitotics, whereas the corresponding azobenzenes are exclusively lit-toxic. We thus demonstrate that hemithioindigos enable an important expansion of the substituent and design scope of photopharmacol. interventions for biol. systems.
    21. 21
      Sailer, A.; Ermer, F.; Kraus, Y.; Bingham, R.; Lutter, F. H.; Ahlfeld, J.; Thorn-Seshold, O. Potent Hemithioindigo-Based Antimitotics Photocontrol the Microtubule Cytoskeleton in Cellulo. Beilstein J. Org. Chem. 2020, 16, 125– 134,  DOI: 10.3762/bjoc.16.14
      21
      Potent hemithioindigo-based antimitotics photocontrol the microtubule cytoskeleton in cellulo
      Sailer, Alexander; Ermer, Franziska; Kraus, Yvonne; Bingham, Rebekkah; Lutter, Ferdinand H.; Ahlfeld, Julia; Thorn-Seshold, Oliver
      Beilstein Journal of Organic Chemistry (2020), 16 (), 125-134CODEN: BJOCBH; ISSN:1860-5397. (Beilstein-Institut zur Foerderung der Chemischen Wissenschaften)
      Uniquely, in contrast to other photoswitches that have been applied to biol., the pseudosym. hemithioindigo scaffold has allowed the creation of both dark-active and lit-active photopharmaceuticals for the same binding site by a priori design. However, the potency of previous hemithioindigo photopharmaceuticals has not been optimal for their translation to other biol. models. Inspired by the structure of tubulin-inhibiting indanones, we created hemithioindigo-based indanone-like tubulin inhibitors (HITubs) and optimized their cellular potency as antimitotic photopharmaceuticals. The use of the hemithioindigo scaffold also permitted us to employ a parahydroxyhemistilbene motif, a structural feature which is denied to most azobenzenes due to the negligibly short lifetimes of their metastable Z-isomers, which proved crucial to enhancing the potency and photoswitchability. The HITubs were ten times more potent than previously reported hemithioindigo photopharmaceutical antimitotics in a series of cell-free and cellular assays, and allowed robust photocontrol over tubulin polymn., microtubule (MT) network structure, cell cycle, and cell survival. Addnl., as the hemithioindigo scaffold allows photoswitchable bioactivity for substituent patterns inaccessible to the majority of current photopharmaceuticals, wider adoption of the hemithioindigo scaffold may significantly expand the scope of cellular and in vivo targets addressable by photopharmacol.
    22. 22
      Engdahl, A. J.; Torres, E. A.; Lock, S. E.; Engdahl, T. B.; Mertz, P. S.; Streu, C. N. Synthesis, Characterization, and Bioactivity of the Photoisomerizable Tubulin Polymerization Inhibitor Azo-Combretastatin A4. Org. Lett. 2015, 17, 4546– 4549,  DOI: 10.1021/acs.orglett.5b02262
      22
      Synthesis, Characterization, and Bioactivity of the Photoisomerizable Tubulin Polymerization Inhibitor azo-Combretastatin A4
      Engdahl, Ashton J.; Torres, Edith A.; Lock, Sarah E.; Engdahl, Taylor B.; Mertz, Pamela S.; Streu, Craig N.
      Organic Letters (2015), 17 (18), 4546-4549CODEN: ORLEF7; ISSN:1523-7052. (American Chemical Society)
      Combretastatin A4 is a stilbenoid tubulin binding mitotic inhibitor whose conformation greatly influences its potency, making it an excellent candidate for adaptation as a photoactivatable tool. Herein, the authors report a novel synthesis, the facile isomerization with com. grade equipment, and biol. activity of azo-combretastatin A4 in vitro and in human cancer cells. Photoisomerized azo-combretastatin A4 is at least 200-fold more potent in cellular culture, making it a promising phototherapeutic and biomedical research tool.
    23. 23
      Zenker, J.; White, M. D.; Gasnier, M.; Alvarez, Y. D.; Lim, H. Y. G.; Bissiere, S.; Biro, M.; Plachta, N. Expanding Actin Rings Zipper the Mouse Embryo for Blastocyst Formation. Cell 2018, 173, 776– 791,  DOI: 10.1016/j.cell.2018.02.035
      23
      Expanding Actin Rings Zipper the Mouse Embryo for Blastocyst Formation
      Zenker, Jennifer; White, Melanie D.; Gasnier, Maxime; Alvarez, Yanina D.; Lim, Hui Yi Grace; Bissiere, Stephanie; Biro, Mate; Plachta, Nicolas
      Cell (Cambridge, MA, United States) (2018), 173 (3), 776-791.e17CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      Transformation from morula to blastocyst is a defining event of preimplantation embryo development. During this transition, the embryo must establish a paracellular permeability barrier to enable expansion of the blastocyst cavity. Here, using live imaging of mouse embryos, we reveal an actin-zippering mechanism driving this embryo sealing. Preceding blastocyst stage, a cortical F-actin ring assembles at the apical pole of the embryo's outer cells. The ring structure forms when cortical actin flows encounter a network of polar microtubules that exclude F-actin. Unlike stereotypical actin rings, the actin rings of the mouse embryo are not contractile, but instead, they expand to the cell-cell junctions. Here, they couple to the junctions by recruiting and stabilizing adherens and tight junction components. Coupling of the actin rings triggers localized myosin II accumulation, and it initiates a tension-dependent zippering mechanism along the junctions that is required to seal the embryo for blastocyst formation.
    24. 24
      Theisen, U.; Ernst, A. U.; Heyne, R. L. S.; Ring, T. P.; Thorn-Seshold, O.; Köster, R. W. Microtubules and Motor Proteins Support Zebrafish Neuronal Migration by Directing Cargo. J. Cell Biol. 2020, 219, e201908040  DOI: 10.1083/jcb.201908040
      24
      Microtubules and motor proteins support zebrafish neuronal migration by directing cargo
      Theisen, Ulrike; Ernst, Alexander U.; Heyne, Ronja L. S.; Ring, Tobias P.; Thorn-Seshold, Oliver; Koester, Reinhard W.
      Journal of Cell Biology (2020), 219 (10), e201908040CODEN: JCLBA3; ISSN:1540-8140. (Rockefeller University Press)
      Neuronal migration during development is necessary to form an ordered and functional brain. Postmitotic neurons require microtubules and dynein to move, but the mechanisms by which they contribute to migration are not fully characterized. Using tegmental hindbrain nuclei neurons in zebrafish embryos together with subcellular imaging, optogenetics, and photopharmacol., we show that, in vivo, the centrosome's position relative to the nucleus is not linked to greatest motility in this cell type. Nevertheless, microtubules, dynein, and kinesin-1 are essential for migration, and we find that interference with endosome formation or the Golgi app. impairs migration to a similar extent as disrupting microtubules. In addn., an imbalance in the traffic of the model cargo Cadherin-2 also reduces neuronal migration. These results lead us to propose that microtubules act as cargo carriers to control spatiotemporal protein distribution, which in turn controls motility. This adds crucial insights into the variety of ways that microtubules can support successful neuronal migration in vivo.
    25. 25
      Gavin, J.; Ruiz, J. F. M.; Kedziora, K.; Windle, H.; Kelleher, D. P.; Gilmer, J. F. Structure Requirements for Anaerobe Processing of Azo Compounds: Implications for Prodrug Design. Bioorg. Med. Chem. Lett. 2012, 22, 7647– 7652,  DOI: 10.1016/j.bmcl.2012.10.014
      25
      Structure requirements for anaerobe processing of azo compounds: Implications for prodrug design
      Gavin, Jason; Ruiz, Juan F. Marquez; Kedziora, Kinga; Windle, Henry; Kelleher, Dermot P.; Gilmer, John F.
      Bioorganic & Medicinal Chemistry Letters (2012), 22 (24), 7647-7652CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)
      This Letter generalizes the metab. of the azo class of compds. by Clostridium perfringens, an anaerobe found in the human colon. A recently reported 5-aminosalicylic acid-based prednisolone prodrug was shown to release the drug when incubated with the bacteria, while the para-aminobenzoic acid (PABA) based analog did not. Instead, it showed a new HPLC peak with a relatively close retention time to the parent which was identified by LCMS as the partially reduced hydrazine product. This Letter investigates azoredn. across a panel of substrates with varying degrees of electronic and steric similarity to the PABA-based compd. Azo compds. with an electron donating group on the azo-contg. arom. ring showed immediate disproportionation to their parent amines without any detection of hydrazine intermediates by HPLC. Compds. contg. only electron withdrawing groups are partially and reversibly reduced to produce a stable detectable hydrazine. They do not disproportionate to their parent amines, but regenerate the parent azo compd. This incomplete redn. is relevant to the design of azo-based prodrugs and the toxicol. of azo-based dyes.
    26. 26
      Sheldon, J. E.; Dcona, M. M.; Lyons, C. E.; Hackett, J. C.; Hartman, M. C. T. Photoswitchable Anticancer Activity via Trans-Cis Isomerization of a Combretastatin A-4 Analog. Org. Biomol. Chem. 2016, 14, 40– 49,  DOI: 10.1039/c5ob02005k
      26
      Photoswitchable anticancer activity via trans-cis isomerization of a combretastatin A-4 analog
      Sheldon, Jonathon E.; Dcona, M. Michael; Lyons, Charles E.; Hackett, John C.; Hartman, Matthew C. T.
      Organic & Biomolecular Chemistry (2016), 14 (1), 40-49CODEN: OBCRAK; ISSN:1477-0520. (Royal Society of Chemistry)
      Combretastatin A-4 (CA4) is highly potent anticancer drug that acts as an inhibitor of tubulin polymn. The core of the CA4 structure contains a cis-stilbene, and it is known that the trans isomer is significantly less potent. The authors prepd. an azobenzene analog of CA4 (Azo-CA4) that shows 13-35-fold enhancement in potency upon illumination. EC50 values in the light were in the mid nM range. Due to its ability to thermally revert to less toxic trans form, Azo-CA4 also has the ability to automatically turn its activity off with time. Azo-CA4 is less potent than CA-4 because it degrades in the presence of glutathione as evidenced by UV-Vis spectroscopy and ESI-MS. Nevertheless, Azo-CA4 represents a promising strategy for switchable potency for treatment of cancer.
    27. 27
      An, Y.; Chen, C.; Zhu, J.; Dwivedi, P.; Zhao, Y.; Wang, Z. Hypoxia-Induced Activity Loss of a Photo-Responsive Microtubule Inhibitor Azobenzene Combretastatin A4. Front. Chem. Sci. Eng. 2020, 14, 880– 888,  DOI: 10.1007/s11705-019-1864-6
      27
      Hypoxia-induced activity loss of a photo-responsive microtubule inhibitor azobenzene combretastatin A4
      An, Yang; Chen, Chao; Zhu, Jundong; Dwivedi, Pankaj; Zhao, Yanjun; Wang, Zheng
      Frontiers of Chemical Science and Engineering (2020), 14 (5), 880-888CODEN: FCSEA3; ISSN:2095-0187. (Springer)
      The conformation-dependent activity of azobenzene combretastatin A4 (Azo-CA4) provides a unique approach to reduce the side-effects of chemotherapy, due to the light-triggered conformation transition of its azobenzene moiety. Under hypoxic tumor microenvironment, however, the high expression of azoreductase can reduce azobenzene to aniline. It was postulated that the Azo-CA4 might be degraded under hypoxia, resulting in the decrease of its anti-tumor activity. The aim of this study was to verify such hypothesis in HeLa cells in vitro. The quant. drug concn. anal. shows the ratio-metric formation of degrdn. end-products, confirming the bioredn. of Azo-CA4. The tubulin staining study indicates that Azo-CA4 loses the potency of switching off microtubule dynamics under hypoxia. Furthermore, the cell cycle anal. shows that the ability of Azo-CA4 to induce mitotic arrest is lost at low oxygen content. Therefore, the cytotoxicity of Azo-CA4 is compromised under hypoxia. In contrast, combretastatin A4 as a pos. control maintains the potency to inhibit tubulin polymn. and break down the nuclei irresp. of light irradn. and oxygen level. This work highlights the influence of hypoxic tumor microenvironment on the anti-tumor potency of Azo-CA4, which should be considered during the early stage of designing translational Azo-CA4 delivery systems.
    28. 28
      Hüll, K.; Morstein, J.; Trauner, D. In Vivo Photopharmacology. Chem. Rev. 2018, 118, 10710– 10747,  DOI: 10.1021/acs.chemrev.8b00037
      28
      In Vivo Photopharmacology
      Hull, Katharina; Morstein, Johannes; Trauner, Dirk
      Chemical Reviews (Washington, DC, United States) (2018), 118 (21), 10710-10747CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
      A review. Synthetic photoswitches have been known for many years, but their usefulness in biol., pharmacol., and medicine has only recently been systematically explored. Over the past decade photopharmacol. has grown into a vibrant field. As the photophys., pharmacodynamic, and pharmacokinetic properties of photoswitches, such as azobenzenes, have become established, they have been applied to a wide range of biol. targets. These include transmembrane proteins (ion channels, transporters, G protein-coupled receptors, receptor-linked enzymes), sol. proteins (kinases, proteases, factors involved in epigenetic regulation), lipid membranes, and nucleic acids. In this review, the authors provide an overview of photopharmacol. using synthetic switches that have been applied in vivo, i.e., in living cells and organisms. The authors discuss the scope and limitations of this approach to study biol. function and the challenges it faces in translational medicine. The relationships between synthetic photoswitches, natural chromophores used in optogenetics, and caged ligands are addressed.
    29. 29
      Lachmann, D.; Lahmy, R.; König, B. Fulgimides as Light-Activated Tools in Biological Investigations: Fulgimides as Light-Activated Tools in Biological Investigations. Eur. J. Org. Chem. 2019, 2019, 5018– 5024,  DOI: 10.1002/ejoc.201900219
      29
      Fulgimides as Light-Activated Tools in Biological Investigations
      Lachmann, D.; Lahmy, R.; Koenig, B.
      European Journal of Organic Chemistry (2019), 2019 (31-32), 5018-5024CODEN: EJOCFK; ISSN:1099-0690. (Wiley-VCH Verlag GmbH & Co. KGaA)
      A review. With high spatiotemporal control, the conformation, rigidity and electronics of photoresponsive bioactive mols. can be altered. This, in turn, allows for control over the biol. properties of these mols. Incorporation of a photoswitchable moiety into a no. of reported inhibitors, ligands and modulators has demonstrated the ability to modulate enzyme, receptor and ion channel responses using light. To date, the major classes of photoswitches explored in biol. applications have been the azobenzenes and diarylethenes. Even though the use of these photoswitches has established the value of photoresponsive mols. as biol. tools, several limitations have become apparent. Fulgimides represent a promising class of photoswitches that are not widely used for such biol. purposes. Their properties are similar to that of diarylethenes, as their photochromism is based on a 6π-electrocyclic rearrangement, however, fulgimides have the added advantage of thermal stability for both isomers. Fulgimides exhibit high photostationary states and fatigue resistance, with the ability to switch in aq. buffer solns. In this minireview, these advantageous photophys. properties will be discussed, as well as the use of fulgimides in biol. investigations.
    30. 30
      Fuchter, M. J. On the Promise of Photopharmacology Using Photoswitches: A Medicinal Chemist’s Perspective. J. Med. Chem. 2020, 63, 11436– 11447,  DOI: 10.1021/acs.jmedchem.0c00629
      30
      On the Promise of Photopharmacology Using Photoswitches: A Medicinal Chemist's Perspective
      Fuchter, Matthew J.
      Journal of Medicinal Chemistry (2020), 63 (20), 11436-11447CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      A review. Photopharmacol. is a growing area of endeavor that employs photoswitchable ligands to allow for light-dependent pharmacol. activity. By coupling light to therapeutic action, improved spatial and temporal selectivity can be achieved and subsequently harnessed for new concepts in therapy. Tremendous progress has already been made, with photopharmacol. agents now reported against a wide array of target classes and light-dependent results demonstrated in a range of live cell and animal models. Several challenges remain, however, esp. in order for photopharmacol. to truly impact the clin. management of disease. This Perspective aims to summarize these challenges, particularly with attention to the medicinal chem. that will be unavoidably required for the further translation of these agents/approaches. By clearly defining challenges for drug hunters, it is hoped that further research into the medicinal chem. of photopharmacol. agents will be stimulated, ultimately enabling full realization of the huge potential for this exciting field.
    31. 31
      Welleman, I. M.; Hoorens, M. W. H.; Feringa, B. L.; Boersma, H. H.; Szymański, W. Photoresponsive Molecular Tools for Emerging Applications of Light in Medicine. Chem. Sci. 2020, 11, 11672– 11691,  DOI: 10.1039/D0SC04187D
      31
      Photoresponsive molecular tools for emerging applications of light in medicine
      Welleman, Ilse M.; Hoorens, Mark W. H.; Feringa, Ben L.; Boersma, Hendrikus H.; Szymanski, Wiktor
      Chemical Science (2020), 11 (43), 11672-11691CODEN: CSHCCN; ISSN:2041-6520. (Royal Society of Chemistry)
      A review. Light-based therapeutic and imaging modalities, which emerge in clin. applications, rely on mol. tools, such as photocleavable protecting groups and photoswitches that respond to photonic stimulus and translate it into a biol. effect. However, optimization of their key parameters (activation wavelength, band sepn., fatigue resistance and half-life) is necessary to enable application in the medical field. In this perspective, we describe the applications scenarios that can be envisioned in clin. practice and then we use those scenarios to explain the necessary properties that the photoresponsive tools used to control biol. function should possess, highlighted by examples from medical imaging, drug delivery and photopharmacol. We then present how the (photo)chem. parameters are currently being optimized and an outlook is given on pharmacol. aspects (toxicity, soly., and stability) of light-responsive mols. With these interdisciplinary insights, we aim to inspire the future directions for the development of photocontrolled tools that will empower clin. applications of light.
    32. 32
      Gaspari, R.; Prota, A. E.; Bargsten, K.; Cavalli, A.; Steinmetz, M. O. Structural Basis of Cis- and Trans-Combretastatin Binding to Tubulin. Chem 2017, 2, 102– 113,  DOI: 10.1016/j.chempr.2016.12.005
      32
      Structural Basis of cis- and trans-Combretastatin Binding to Tubulin
      Gaspari, Roberto; Prota, Andrea E.; Bargsten, Katja; Cavalli, Andrea; Steinmetz, Michel O.
      Chem (2017), 2 (1), 102-113CODEN: CHEMVE; ISSN:2451-9294. (Cell Press)
      Combretastatin A4 (CA-4) derivs. are microtubule-destabilizing agents, some of which are in advanced clin. trials for cancer therapy. The active cis conformation of CA-4 can readily isomerize into a thermodynamically more stable but significantly less active trans form. Here, we solved the high-resoln. crystal structure of cis-CA-4 in complex with tubulin. The compd. binds to the colchicine site of tubulin and displays both common and distinct interaction points with colchicine. Using metadynamics simulations, we generated the trans form of the ligand within its binding site and computed the relative binding free energy of the cis-CA-4 and trans-CA-4 isomers via a thermodn. cycle. The calcns. suggest structural distortions of the bound trans-CA-4 mol. as the likely cause of its reduced activity in comparison with that of its cis isomer. Our findings could open up unique possibilities for structure-guided drug engineering with the aim of discovering combretastatin variants with improved chem. properties and pharmacol. profiles.
    33. 33
      Tarade, D.; Ma, D.; Pignanelli, C.; Mansour, F.; Simard, D.; Berg, S.; van den Gauld, J.; McNulty, J.; Pandey, S. Structurally Simplified Biphenyl Combretastatin A4 Derivatives Retain in Vitro Anti-Cancer Activity Dependent on Mitotic Arrest. PLoS One 2017, 12, e0171806  DOI: 10.1371/journal.pone.0171806
      There is no corresponding record for this reference.
    34. 34
      Jacobson, P. Ueber Bildung von Anhydroverbindungen Des Orthoamidophenylmercaptans Aus Thioaniliden. Ber. Dtsch. Chem. Ges. 1886, 19, 1067– 1077,  DOI: 10.1002/cber.188601901239
      There is no corresponding record for this reference.
    35. 35
      Ma, D.; Xie, S.; Xue, P.; Zhang, X.; Dong, J.; Jiang, Y. Efficient and Economical Access to Substituted Benzothiazoles: Copper-Catalyzed Coupling of 2-Haloanilides with Metal Sulfides and Subsequent Condensation. Angew. Chem., Int. Ed. 2009, 48, 4222– 4225,  DOI: 10.1002/anie.200900486
      35
      Efficient and economical access to substituted benzothiazoles: copper-catalyzed coupling of 2-haloanilides with metal sulfides and subsequent condensation
      Ma, Dawei; Xie, Siwei; Xue, Peng; Zhang, Xiaojing; Dong, Jinhua; Jiang, Yongwen
      Angewandte Chemie, International Edition (2009), 48 (23), 4222-4225, S4222/1-S4222/18CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The first metal-catalyzed direct coupling of metal sulfides with aryl halides and subsequent intramol. condensation provided substituted benzothiazoles. A wide range of functional groups are tolerated under the reaction conditions.
    36. 36
      Tron, G. C.; Pirali, T.; Sorba, G.; Pagliai, F.; Busacca, S.; Genazzani, A. A. Medicinal Chemistry of Combretastatin A4: Present and Future Directions. J. Med. Chem. 2006, 49, 3033– 3044,  DOI: 10.1021/jm0512903
      36
      Medicinal chemistry of combretastatin A4: Present and future directions
      Tron, Gian Cesare; Pirali, Tracey; Sorba, Giovanni; Pagliai, Francesca; Busacca, Sara; Genazzani, Armando A.
      Journal of Medicinal Chemistry (2006), 49 (11), 3033-3044CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      A review with refs. A review on the medicinal chem. of combretastatin A4 (CA-4), a compd. with antitumor properties. The mechanism of action of CA-4, including its ability to inhibit tubulin polymn., is discussed, along with selected analogs of CA-4 modified on the arom. rings A and B, substituted Ph rings, heterocycling rings, non-substituted arom. rings, modifications on the double bond, and analogs where the olefinic group is replaced by a ring. Some issues related to the pharmacol. of CA-4 and future directions in research are considered.
    37. 37
      Kraus, Y.; Glas, C.; Melzer, B.; Gao, L.; Heise, C.; Preuße, M.; Ahlfeld, J.; Bracher, F.; Thorn-Seshold, O. Isoquinoline-Based Biaryls as a Robust Scaffold for Microtubule Inhibitors. Eur. J. Med. Chem. 2020, 186, 111865  DOI: 10.1016/j.ejmech.2019.111865
      37
      Isoquinoline-based biaryls as a robust scaffold for microtubule inhibitors
      Kraus, Yvonne; Glas, Carina; Melzer, Benedikt; Gao, Li; Heise, Constanze; Preusse, Monique; Ahlfeld, Julia; Bracher, Franz; Thorn-Seshold, Oliver
      European Journal of Medicinal Chemistry (2020), 186 (), 111865CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
      We here report the discovery of isoquinoline-based biaryls as a new scaffold for colchicine domain tubulin inhibitors. Colchicinoid inhibitors offer highly desirable cytotoxic and vascular disrupting bioactivities, but their further development requires improving in vivo robustness and tolerability: properties that both depend on the scaffold structure employed. We have developed isoquinoline-based biaryls as a novel scaffold for high-potency tubulin inhibitors, with excellent robustness, druglikeness, and facile late-stage structural diversification, accessible through a tolerant synthetic route. We confirmed their bioactivity mechanism in vitro, developed sol. prodrugs, and established safe in vivo dosing in mice. By addressing several problems facing the current families of inhibitors, we expect that this new scaffold will find a range of in vivo applications towards translational use in cancer therapy.
    38. 38
      Thorn-Seshold, O.; Meiring, J. C. M. Photocontrolling Microtubule Dynamics with Photoswitchable Chemical Reagents. In Microtubules─Methods and Protocols; Springer International Publishing, 2022; Chapter 26.
      There is no corresponding record for this reference.
    39. 39
      Florian, S.; Mitchison, T. J. Anti-Microtubule Drugs. In The Mitotic Spindle: Methods and Protocols; Chang, P.; Ohi, R., Eds.; Springer: New York, NY, 2016; Vol. 1413, pp 403– 421.
      There is no corresponding record for this reference.
    40. 40
      Roostalu, J.; Thomas, C.; Cade, N. I.; Kunzelmann, S.; Taylor, I. A.; Surrey, T. The Speed of GTP Hydrolysis Determines GTP Cap Size and Controls Microtubule Stability. eLife 2020, 9, e51992  DOI: 10.7554/eLife.51992
      40
      The speed of GTP hydrolysis determines GTP cap size and controls microtubule stability
      Roostalu, Johanna; Thomas, Claire; Cade, Nicholas Ian; Kunzelmann, Simone; Taylor, Ian A.; Surrey, Thomas
      eLife (2020), 9 (), e51992CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)
      Microtubules are cytoskeletal polymers whose function depends on their property to switch between states of growth and shrinkage. Growing microtubules are thought to be stabilized by a GTP cap at their ends. The nature of this cap, however, is still poorly understood. End Binding proteins (EBs) recruit a diverse range of regulators of microtubule function to growing microtubule ends. Whether the EB binding region is identical to the GTP cap is unclear. Using mutated human tubulin with blocked GTP hydrolysis, we demonstrate that EBs bind with high affinity to the GTP conformation of microtubules. Slowing-down GTP hydrolysis leads to extended GTP caps. We find that cap length dets. microtubule stability and that the microtubule conformation changes gradually in the cap as GTP is hydrolyzed. These results demonstrate the crit. importance of the kinetics of GTP hydrolysis for microtubule stability and establish that the GTP cap coincides with the EB-binding region.
    41. 41
      Linnemann, J. R.; Miura, H.; Meixner, L. K.; Irmler, M.; Kloos, U. J.; Hirschi, B.; Bartsch, H. S.; Sass, S.; Beckers, J.; Theis, F. J.; Gabka, C.; Sotlar, K.; Scheel, C. H. Quantification of Regenerative Potential in Primary Human Mammary Epithelial Cells. Development 2015, 31, 3239– 3251,  DOI: 10.1242/dev.123554
      There is no corresponding record for this reference.
    42. 42
      Buchmann, B.; Engelbrecht, L. K.; Fernandez, P.; Hutterer, F. P.; Raich, M. K.; Scheel, C. H.; Bausch, A. R. Mechanical Plasticity of Collagen Directs Branch Elongation in Human Mammary Gland Organoids. Nat. Commun. 2021, 12, 2759  DOI: 10.1038/s41467-021-22988-2
      42
      Mechanical plasticity of collagen directs branch elongation in human mammary gland organoids
      Buchmann, B.; Engelbrecht, L. K.; Fernandez, P.; Hutterer, F. P.; Raich, M. K.; Scheel, C. H.; Bausch, A. R.
      Nature Communications (2021), 12 (1), 2759CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Epithelial branch elongation is a central developmental process during branching morphogenesis in diverse organs. This fundamental growth process into large arborized epithelial networks is accompanied by structural reorganization of the surrounding extracellular matrix (ECM), well beyond its mech. linear response regime. Here, we report that epithelial ductal elongation within human mammary organoid branches relies on the non-linear and plastic mech. response of the surrounding collagen. Specifically, we demonstrate that collective back-and-forth motion of cells within the branches generates tension that is strong enough to induce a plastic reorganization of the surrounding collagen network which results in the formation of mech. stable collagen cages. Such matrix encasing in turn directs further tension generation, branch outgrowth and plastic deformation of the matrix. The identified mech. tension equil. sets a framework to understand how mech. cues can direct ductal branch elongation.
    43. 43
      Hofer, M.; Lutolf, M. P. Engineering Organoids. Nat. Rev. Mater. 2021, 6, 402– 420,  DOI: 10.1038/s41578-021-00279-y
      43
      Engineering organoids
      Hofer, Moritz; Lutolf, Matthias P.
      Nature Reviews Materials (2021), 6 (5), 402-420CODEN: NRMADL; ISSN:2058-8437. (Nature Portfolio)
      A review. Abstr.: Organoids are in vitro miniaturized and simplified model systems of organs that have gained enormous interest for modeling tissue development and disease, and for personalized medicine, drug screening and cell therapy. Despite considerable success in culturing physiol. relevant organoids, challenges remain to achieve real-life applications. In particular, the high variability of self-organizing growth and restricted exptl. and anal. access hamper the translatability of organoid systems. In this Review, we argue that many limitations of traditional organoid culture can be addressed by engineering approaches at all levels of organoid systems. We investigate cell surface and genetic engineering approaches, and discuss stem cell niche engineering based on the design of matrixes that allow spatiotemporal control of organoid growth and shape-guided morphogenesis. We examine how microfluidic approaches and lessons learnt from organs-on-a-chip enable the integration of mechano-physiol. parameters and increase accessibility of organoids to improve functional readouts. Applying engineering principles to organoids increases reproducibility and provides exptl. control, which will, ultimately, be required to enable clin. translation.
    44. 44
      Kopf, A.; Renkawitz, J.; Hauschild, R.; Girkontaite, I.; Tedford, K.; Merrin, J.; Thorn-Seshold, O.; Trauner, D.; Häcker, H.; Fischer, K.-D.; Kiermaier, E.; Sixt, M. Microtubules Control Cellular Shape and Coherence in Amoeboid Migrating Cells. J. Cell Biol. 2020, 219, e201907154  DOI: 10.1083/jcb.201907154
      44
      Microtubules control cellular shape and coherence in amoeboid migrating cells
      Kopf, Aglaja; Renkawitz, Joerg; Hauschild, Robert; Girkontaite, Irute; Tedford, Kerry; Merrin, Jack; Thorn-Seshold, Oliver; Trauner, Dirk; Haecker, Hans; Fischer, Klaus-Dieter; Kiermaier, Eva; Sixt, Michael
      Journal of Cell Biology (2020), 219 (6), e201907154/1-e201907154/24CODEN: JCLBA3; ISSN:1540-8140. (Rockefeller University Press)
      Cells navigating through complex tissues face a fundamental challenge: while multiple protrusions explore different paths, the cell needs to avoid entanglement. How a cell surveys and then corrects its own shape is poorly understood. Here, we demonstrate that spatially distinct microtubule dynamics regulate amoeboid cell migration by locally promoting the retraction of protrusions. In migrating dendritic cells, local microtubule depolymn. within protrusions remote from the microtubule organizing center triggers actomyosin contractility controlled by RhoA and its exchange factor Lfc. Depletion of Lfc leads to aberrant myosin localization, thereby causing two effects that rate-limit locomotion: (1) impaired cell edge coordination during path finding and (2) defective adhesion resoln. Compromised shape control is particularly hindering in geometrically complex microenvironments, where it leads to entanglement and ultimately fragmentation of the cell body. We thus demonstrate that microtubules can act as a proprioceptive device: they sense cell shape and control actomyosin retraction to sustain cellular coherence.
    45. 45
      Vandestadt, C.; Vanwalleghem, G. C.; Castillo, H. A.; Li, M.; Schulze, K.; Khabooshan, M.; Don, E.; Anko, M.-L.; Scott, E. K.; Kaslin, J. Early Migration of Precursor Neurons Initiates Cellular and Functional Regeneration after Spinal Cord Injury in Zebrafish. bioRxiv 2019, 539940  DOI: 10.1101/539940
      There is no corresponding record for this reference.
    46. 46
      Cabernard, C.; Doe, C. Q. Apical/Basal Spindle Orientation Is Required for Neuroblast Homeostasis and Neuronal Differentiation in Drosophila. Dev. Cell 2009, 17, 134– 141,  DOI: 10.1016/j.devcel.2009.06.009
      46
      Apical/basal spindle orientation is required for neuroblast homeostasis and neuronal differentiation in Drosophila
      Cabernard, Clemens; Doe, Chris Q.
      Developmental Cell (2009), 17 (1), 134-141CODEN: DCEEBE; ISSN:1534-5807. (Cell Press)
      Precise regulation of stem cell self-renewal/differentiation is essential for embryogenesis and tumor suppression. Drosophila neural progenitors (neuroblasts) align their spindle along an apical/basal polarity axis to generate a self-renewed apical neuroblast and a differentiating basal cell. Here, the authors genetically disrupt spindle orientation without altering cell polarity to test the role of spindle orientation in self-renewal/differentiation. The authors perform correlative live imaging of polarity markers and spindle orientation over multiple divisions within intact brains, followed by mol. marker anal. of cell fate. The authors find that spindle alignment orthogonal to apical/basal polarity always segregates apical determinants into both siblings, which invariably assume a neuroblast identity. Basal determinants can all be localized into one sibling without inducing neuronal differentiation, but overexpression of the basal determinant Prospero can deplete neuroblasts. The authors conclude that the ratio of apical/basal determinants specifies neuroblast/GMC identity, and that apical/basal spindle orientation is required for neuroblast homeostasis and neuronal differentiation.
    47. 47
      Karpova, N.; Bobinnec, Y.; Fouix, S.; Huitorel, P.; Debec, A. Jupiter, a New Drosophila Protein Associated with Microtubules. Cell Motil. 2006, 63, 301– 312,  DOI: 10.1002/cm.20124
      47
      Jupiter, a new Drosophila protein associated with microtubules
      Karpova, Nina; Bobinnec, Yves; Fouix, Sylvaine; Huitorel, Philippe; Debec, Alain
      Cell Motility and the Cytoskeleton (2006), 63 (5), 301-312CODEN: CMCYEO; ISSN:0886-1544. (Wiley-Liss, Inc.)
      We describe a novel Drosophila protein Jupiter, which shares properties with several structural microtubule-assocd. proteins (MAPs) including TAU, MAP2, MAP4. Jupiter is a sol. unfolded mol. with the high net pos. charge, rich in glycine. It possesses 2 degenerated repeats around the sequence PPGG, sepd. by a serine-rich region. Jupiter assocs. with microtubules in vitro and, fused with the green fluorescent protein (GFP), is an excellent marker to follow microtubule dynamics in vivo. In a jupiter transgenic Drosophila strain generated by the protein-trap technique, Jupiter:GFP fusion protein localizes to the microtubule network through the cell cycle at the different stages of development. We found particularly high Jupiter:GFP concns. in the young embryo, larval nervous system, precursors of eye photoreceptors and adult ovary. Moreover, from jupiter:gfp embryos we have established 2 permanent cell lines presenting strongly fluorescent microtubules during the whole cell cycle. In these cells, the distribution of the Jupiter:GFP fusion protein reproduces microtubule behavior upon treatment by the drugs colchicine and taxol. The Jupiter cell lines and fly strain should be of wide interest for biologists interested in in vivo anal. of microtubule dynamics.
    48. 48
      Royou, A.; Sullivan, W.; Karess, R. Cortical Recruitment of Nonmuscle Myosin II in Early Syncytial Drosophila Embryos: Its Role in Nuclear Axial Expansion and Its Regulation by Cdc2 Activity. J. Cell Biol. 2002, 158, 127– 137,  DOI: 10.1083/jcb.200203148
      48
      Cortical recruitment of nonmuscle myosin II in early syncytial Drosophila embryos: its role in nuclear axial expansion and its regulation by Cdc2 activity
      Royou, Anne; Sullivan, William; Karess, Roger
      Journal of Cell Biology (2002), 158 (1), 127-137CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)
      The nuclei of early syncytial Drosophila embryos migrate dramatically toward the poles. The cellular mechanisms driving this process, called axial expansion, are unclear, but myosin II activity is required. By following regulatory myosin light chain (RLC)-green fluorescent protein dynamics in living embryos, we obsd. cycles of myosin recruitment to the cortex synchronized with mitotic cycles. Cortical myosin is first seen in a patch at the anterocentral part of the embryo at cycle 4. With each succeeding cycle, the patch expands poleward, dispersing at the beginning of each mitosis and reassembling at the end of telophase. Each cycle of actin and myosin recruitment is accompanied by a cortical contraction. The cortical myosin cycle does not require microtubules but correlates inversely with Cdc2/cyclin B (mitosis-promoting factor) activity. A mutant RLC lacking inhibitory phosphorylation sites was fully functional with no effect on the cortical myosin cycle, indicating that Cdc2 must be modulating myosin activity by some other mechanism. An inhibitor of Rho kinase blocks the cortical myosin recruitment cycles and provokes a concomitant failure of axial expansion. These studies suggest a model in which cycles of myosin-mediated contraction and relaxation, tightly linked to Cdc2 and Rho kinase activity, are directly responsible for the axial expansion of the syncytial nuclei.
    49. 49
      Rusan, N. M.; Peifer, M. A Role for a Novel Centrosome Cycle in Asymmetric Cell Division. J. Cell Biol. 2007, 177, 13– 20,  DOI: 10.1083/jcb.200612140
      49
      A role for a novel centrosome cycle in asymmetric cell division
      Rusan, Nasser M.; Peifer, Mark
      Journal of Cell Biology (2007), 177 (1), 13-20CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)
      Drosophila melanogaster central brain neuroblasts are excellent models for stem cell asym. division. Earlier work showed that their mitotic spindle orientation is established before spindle formation. We investigated the mechanism by which this occurs, revealing a novel centrosome cycle. In interphase, the 2 centrioles sep., but only 1 is active, retaining pericentriolar material and forming a dominant centrosome. This centrosome acts as a microtubule organizing center (MTOC) and remains stationary, forming 1 pole of the future spindle. The 2nd centriole is inactive and moves to the opposite side of the cell before being activated as a centrosome/MTOC. This is accompanied by asym. localization of Polo kinase, a key centrosome regulator. Disruption of centrosomes disrupts the high fidelity of asym. division. We propose a 2-step mechanism to ensure faithful spindle positioning: the novel centrosome cycle produces a single interphase MTOC, coarsely aligning the spindle, and spindle-cortex interactions refine this alignment.
    50. 50
      Cabernard, C.; Prehoda, K. E.; Doe, C. Q. A Spindle-Independent Cleavage Furrow Positioning Pathway. Nature 2010, 467, 91– 94,  DOI: 10.1038/nature09334
      50
      A spindle-independent cleavage furrow positioning pathway
      Cabernard, Clemens; Prehoda, Kenneth E.; Doe, Chris Q.
      Nature (London, United Kingdom) (2010), 467 (7311), 91-94CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      The mitotic spindle dets. the cleavage furrow site during metazoan cell division, but whether other mechanisms exist remains unknown. Here we identify a spindle-independent mechanism for cleavage furrow positioning in Drosophila neuroblasts. We show that early and late furrow proteins (Pavarotti, Anillin, and Myosin) are localized to the neuroblast basal cortex at anaphase onset by a Pins cortical polarity pathway, and can induce a basally displaced furrow even in the complete absence of a mitotic spindle. Rotation or displacement of the spindle results in 2 furrows: an early polarity-induced basal furrow and a later spindle-induced furrow. This spindle-independent cleavage furrow mechanism may be relevant to other highly polarized mitotic cells, such as mammalian neural progenitors.
    51. 51
      Connell, M.; Cabernard, C.; Ricketson, D.; Doe, C. Q.; Prehoda, K. E. Asymmetric Cortical Extension Shifts Cleavage Furrow Position in Drosophila Neuroblasts. Mol. Biol. Cell 2011, 22, 4220– 4226,  DOI: 10.1091/mbc.e11-02-0173
      51
      Asymmetric cortical extension shifts cleavage furrow position in Drosophila neuroblasts
      Connell, Marisa; Cabernard, Clemens; Ricketson, Derek; Doe, Chris Q.; Prehoda, Kenneth E.
      Molecular Biology of the Cell (2011), 22 (22), 4220-4226CODEN: MBCEEV; ISSN:1939-4586. (American Society for Cell Biology)
      The cytokinetic cleavage furrow is typically positioned sym. relative to the cortical cell boundaries, but it can also be asym. The mechanisms that control furrow site specification have been intensively studied, but how polar cortex movements influence ultimate furrow position remains poorly understood. We measured the position of the apical and the basal cortex in asym. dividing Drosophila neuroblasts and obsd. preferential displacement of the apical cortex that becomes the larger daughter cell during anaphase, effectively shifting the cleavage furrow toward the smaller daughter cell. Asym. cortical extension is correlated with the presence of cortical myosin II, which is polarized in neuroblasts. Loss of myosin II asymmetry by perturbing heterotrimeric G-protein signaling results in sym. extension and equal-sized daughter cells. We propose a model in which contraction-driven asym. polar extension of the neuroblast cortex during anaphase contributes to asym. furrow position and daughter cell size.
    52. 52
      Roberts, A.; Borisyuk, R.; Buhl, E.; Ferrario, A.; Koutsikou, S.; Li, W.-C.; Soffe, S. R. The Decision to Move: Response Times, Neuronal Circuits and Sensory Memory in a Simple Vertebrate. Proc. R. Soc. B 2019, 286, 20190297  DOI: 10.1098/rspb.2019.0297
      There is no corresponding record for this reference.
    53. 53
      Distel, M.; Hocking, J. C.; Volkmann, K.; Köster, R. W. The Centrosome Neither Persistently Leads Migration nor Determines the Site of Axonogenesis in Migrating Neurons in Vivo. J. Cell Biol. 2010, 191, 875– 890,  DOI: 10.1083/jcb.201004154
      53
      The centrosome neither persistently leads migration nor determines the site of axonogenesis in migrating neurons in vivo
      Distel, Martin; Hocking, Jennifer C.; Volkmann, Katrin; Koester, Reinhard W.
      Journal of Cell Biology (2010), 191 (4), 875-890CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)
      The position of the centrosome ahead of the nucleus has been considered crucial for coordinating neuronal migration in most developmental situations. The proximity of the centrosome has also been correlated with the site of axonogenesis in certain differentiating neurons. Despite these pos. correlations, accumulating exptl. findings appear to negate a universal role of the centrosome in detg. where an axon forms, or in leading the migration of neurons. To further examine this controversy in an in vivo setting, we have generated cell type-specific multi-cistronic gene expression to monitor subcellular dynamics in the developing zebrafish cerebellum. We show that migration of rhombic lip-derived neurons is characterized by a centrosome that does not persistently lead the nucleus, but which is instead regularly overtaken by the nucleus. In addn., axonogenesis is initiated during the onset of neuronal migration and occurs independently of centrosome proximity. These in vivo data reveal a new temporal orchestration of organelle dynamics and provide important insights into the variation in intracellular processes during vertebrate brain differentiation.
    54. 54
      Distel, M.; Wullimann, M. F.; Köster, R. W. Optimized Gal4 Genetics for Permanent Gene Expression Mapping in Zebrafish. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 13365– 13370,  DOI: 10.1073/pnas.0903060106
      54
      Optimized Gal4 genetics for permanent gene expression mapping in zebrafish
      Distel, Martin; Wullimann, Mario F.; Koster, Reinhard W.
      Proceedings of the National Academy of Sciences of the United States of America (2009), 106 (32), 13365-13370, S13365/1-S13365/9CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Combinatorial genetics for conditional transgene activation allows studying gene function with temporal and tissue specific control like the Gal4-UAS system, which has enabled sophisticated genetic studies in Drosophila. Recently this system was adapted for zebrafish and promising applications have been introduced. Here, we report a systematic optimization of zebrafish Gal4-UAS genetics by establishing an optimized Gal4-activator (KalTA4). We provide quant. data for KalTA4-mediated transgene activation in dependence of UAS copy nos. to allow for studying dosage effects of transgene expression. Employing a Tol2 transposon-mediated KalTA4 enhancer trap screen biased for central nervous system expression, we present a collection of self-reporting red fluorescent KalTA4 activator strains. These strains reliably transactivate UAS-dependent transgenes and can be rendered homozygous. Furthermore, we have characterized the transactivation kinetics of tissue-specific KalTA4 activation, which led to the development of a self-maintaining effector strain "Kaloop.". This strain relates transient KalTA4 expression during embryogenesis via a KalTA4-mediated autoregulatory mechanism to live adult structures. We demonstrate its use by showing that the secondary octaval nucleus in the adult hindbrain is likely derived from egr2b-expressing cells in rhombomere 5 during stages of early embryogenesis. These data demonstrate prolonged and maintained expression by Kalooping, a technique that can be used for permanent spatiotemporal genetic fate mapping and targeted transgene expression in zebrafish.
    55. 55
      Fortin, D. L.; Banghart, M. R.; Dunn, T. W.; Borges, K.; Wagenaar, D. A.; Gaudry, Q.; Karakossian, M. H.; Otis, T. S.; Kristan, W. B.; Trauner, D.; Kramer, R. H. Photochemical Control of Endogenous Ion Channels and Cellular Excitability. Nat. Methods 2008, 5, 331– 338,  DOI: 10.1038/nmeth.1187
      55
      Photochemical control of endogenous ion channels and cellular excitability
      Fortin, Doris L.; Banghart, Matthew R.; Dunn, Timothy W.; Borges, Katharine; Wagenaar, Daniel A.; Gaudry, Quentin; Karakossian, Movses H.; Otis, Thomas S.; Kristan, William B.; Trauner, Dirk; Kramer, Richard H.
      Nature Methods (2008), 5 (4), 331-338CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      Light-activated ion channels provide a precise and noninvasive optical means for controlling action potential firing, but the genes encoding these channels must first be delivered and expressed in target cells. Here we describe a method for bestowing light sensitivity onto endogenous ion channels that does not rely on exogenous gene expression. The method uses a synthetic photoisomerizable small mol., or photoswitchable affinity label (PAL), that specifically targets K+ channels. PALs contain a reactive electrophile, enabling covalent attachment of the photoswitch to naturally occurring nucleophiles in K+ channels. Ion flow through PAL-modified channels is turned on or off by photoisomerizing PAL with different wavelengths of light. We showed that PAL treatment confers light sensitivity onto endogenous K+ channels in isolated rat neurons and in intact neural structures from rat and leech, allowing rapid optical regulation of excitability without genetic modification.
    56. 56
      Laprell, L.; Tochitsky, I.; Kaur, K.; Manookin, M. B.; Stein, M.; Barber, D. M.; Schön, C.; Michalakis, S.; Biel, M.; Kramer, R. H.; Sumser, M. P.; Trauner, D.; Gelder, R. N. V. Photopharmacological Control of Bipolar Cells Restores Visual Function in Blind Mice. J. Clin. Invest. 2017, 127, 2598– 2611,  DOI: 10.1172/JCI92156
      56
      Photopharmacological control of bipolar cells restores visual function in blind mice
      Laprell Laura; Stein Marco; Barber David M; Sumser Martin P; Trauner Dirk; Laprell Laura; Kaur Kuldeep; Manookin Michael B; Van Gelder Russell N; Tochitsky Ivan; Kramer Richard H; Schon Christian; Michalakis Stylianos; Biel Martin; Van Gelder Russell N
      The Journal of clinical investigation (2017), 127 (7), 2598-2611 ISSN:.
      Photopharmacological control of neuronal activity using synthetic photochromic ligands, or photoswitches, is a promising approach for restoring visual function in patients suffering from degenerative retinal diseases. Azobenzene photoswitches, such as AAQ and DENAQ, have been shown to restore the responses of retinal ganglion cells to light in mouse models of retinal degeneration but do not recapitulate native retinal signal processing. Here, we describe diethylamino-azo-diethylamino (DAD), a third-generation photoswitch that is capable of restoring retinal ganglion cell light responses to blue or white light. In acute brain slices of murine layer 2/3 cortical neurons, we determined that the photoswitch quickly relaxes to its inactive form in the dark. DAD is not permanently charged, and the uncharged form enables the photoswitch to rapidly and effectively cross biological barriers and thereby access and photosensitize retinal neurons. Intravitreal injection of DAD restored retinal light responses and light-driven behavior to blind mice. Unlike DENAQ, DAD acts upstream of retinal ganglion cells, primarily conferring light sensitivity to bipolar cells. Moreover, DAD was capable of generating ON and OFF visual responses in the blind retina by utilizing intrinsic retinal circuitry, which may be advantageous for restoring visual function.
    57. 57
      Matera, C.; Gomila, A. M. J.; Camarero, N.; Libergoli, M.; Soler, C.; Gorostiza, P. Photoswitchable Antimetabolite for Targeted Photoactivated Chemotherapy. J. Am. Chem. Soc. 2018, 140, 15764– 15773,  DOI: 10.1021/jacs.8b08249
      57
      Photoswitchable Antimetabolite for Targeted Photoactivated Chemotherapy
      Matera, Carlo; Gomila, Alexandre M. J.; Camarero, Nuria; Libergoli, Michela; Soler, Concepcio; Gorostiza, Pau
      Journal of the American Chemical Society (2018), 140 (46), 15764-15773CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The efficacy and tolerability of systemically administered anticancer agents are limited by their off-target effects. Precise spatiotemporal control over their cytotoxic activity would allow improving chemotherapy treatments, and light-regulated drugs are well suited to this purpose. We have developed phototrexate, the first photoswitchable inhibitor of the human dihydrofolate reductase (DHFR), as a photochromic analog of methotrexate, a widely prescribed chemotherapeutic drug to treat cancer and psoriasis. Quantification of the light-regulated DHFR enzymic activity, cell proliferation, and in vivo effects in zebrafish show that phototrexate behaves as a potent antifolate in its photoactivated cis configuration and that it is nearly inactive in its dark-relaxed trans form. Thus, phototrexate constitutes a proof-of-concept to design light-regulated cytotoxic small mols. and a step forward to develop targeted anticancer photochemotherapies with localized efficacy and reduced adverse effects.
    58. 58
      Babii, O.; Afonin, S.; Schober, T.; Garmanchuk, L. V.; Ostapchenko, L. I.; Yurchenko, V.; Zozulya, S.; Tarasov, O.; Pishel, I.; Ulrich, A. S.; Komarov, I. V. Peptide Drugs for Photopharmacology: How Much of a Safety Advantage Can Be Gained by Photocontrol?. Future Drug Discovery 2020, 2, FDD28  DOI: 10.4155/fdd-2019-0033
      There is no corresponding record for this reference.
    59. 59
      Afonin, S.; Babii, O.; Reuter, A.; Middel, V.; Takamiya, M.; Strähle, U.; Komarov, I. V.; Ulrich, A. S. Light-Controllable Dithienylethene-Modified Cyclic Peptides: Photoswitching the in Vivo Toxicity in Zebrafish Embryos. Beilstein J. Org. Chem. 2020, 16, 39– 49,  DOI: 10.3762/bjoc.16.6
      59
      Light-controllable dithienylethene-modified cyclic peptides: photoswitching the in vivo toxicity in zebrafish embryos
      Afonin, Sergii; Babii, Oleg; Reuter, Aline; Middel, Volker; Takamiya, Masanari; Straehle, Uwe; Komarov, Igor V.; Ulrich, Anne S.
      Beilstein Journal of Organic Chemistry (2020), 16 (), 39-49CODEN: BJOCBH; ISSN:1860-5397. (Beilstein-Institut zur Foerderung der Chemischen Wissenschaften)
      This study evaluates the embryotoxicity of dithienylethene-modified peptides upon photoswitching, using 19 analogs based on the β-hairpin scaffold of the natural membranolytic peptide gramicidin S. We established an in vivo assay in two variations (with ex vivo and in situ photoisomerization), using larvae of the model organism Danio rerio, and detd. the toxicities of the peptides in terms of 50% LDs (LD50). This study allowed us to: (i) demonstrate the feasibility of evaluating peptide toxicity with D. rerio larvae at 3-4 days post fertilization, (ii) det. the phototherapeutic safety windows for all peptides, (iii) demonstrate photoswitching of the whole-body toxicity for the dithienylethene-modified peptides in vivo, (iv) re-analyze previous structure-toxicity relationship data, and (v) select promising candidates for potential clin. development.

  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.2c01020.

    • Temporally precise and cell-precise inhibitions of cellular MT polymerization dynamics by photoactivations of E-SBTubA4P at 405 nm (Movie S1) (MP4)

    • Temporally precise full-field-of-view inhibitions of cellular MT polymerization dynamics by photoactivations of E-SBTubA4P at 405 nm (Movie S2) (MP4)

    • No inhibition of cellular MT polymerization dynamics by illumination of E-SBTubA4P at 487 nm (Movie S3) (MP4)

    • SBTubA4P blocks branch development in primary human mammary gland organoids, light-dependently and with spatiotemporal precision (Movies S4−S6) (MP4, MP4, MP4)

    • No-compound control shows no photoinhibition of organoid branch development in both ROItarg and ROIctrl (Movie S7) (MP4)

    • Temporally precise depolymerization of the mitotic spindle in a prophase Drosophila neuroblast by photoactivation of E-SBTub2M (30 μM) at 405 nm (Movie S8) (MP4)

    • DMSO-only control to Movie S8 shows prophase Drosophila neuroblast undergoing normal mitosis after 405 nm illumination (Movie S9) (MP4)

    • Temporally precise depolymerization of the mitotic spindle in a prophase Drosophila neuroblast by photoactivation of E-SBTub2M (30 μM) at 405 nm (Movie S10) (MP4)

    • DMSO-only control to Movie S10 shows prophase Drosophila neuroblast undergoing normal mitosis after 405 nm illumination (Movie S11) (MP4)

    • Reaction of hatchling frog X. tropicalis embryos to mechanical stimulus depends on the temporally precise application of Z-SBTubA4 during prior development (Movie S12) (MP4)

    • Photoactivation of SBTubA4P (25 μM) in a live zebrafish embryo shows temporally reversible inhibitions of EB3 dynamics over several cycles (Movies S13 and S14) (MP4, MP4)

    • Inhibition of MT polymerization dynamics in zebrafish embryo when SBTubA4P (25 μM) is photoactivated (Movie S15) (MP4)

    • Photoactivation of SBTubA4P (25 μM) stops both EB3 dynamics and cell division in the developing zebrafish embryo (Movie S16) (MP4)

    • Chemical synthesis, photocharacterization, biological data, protein crystallization, and NMR spectra (PDF)


    Terms & Conditions

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