Photocontrolling Microtubule Dynamics with Photoswitchable Chemical Reagents

Abstract

Microtubule dynamics can be inhibited with sub-second temporal resolution and cellular-scale spatial resolution, by using precise illuminations to optically pattern where and when photoswitchable microtubule-inhibiting chemical reagents exert their latent bioactivity. The recently-available reagents (SBTub, PST, STEpo, AzTax, PHTub) now enable researchers to use light to reversibly modulate microtubule-dependent processes in eukaryotes, in 2D and 3D cell culture as well as in vivo, across a variety of model organisms: with applications in fields from cargo transport to cell migration, cell division, and embryonic development.<br><br>However, a wide knowledge gap has remained in the literature, which has blocked further translation of these and many other classes of photopharmaceuticals. No generally-applicable procedures or workflows to establish biological assays using photopharmaceuticals have been published. Accordingly, the rate of adoption of photopharmaceutical tools in the broader chemical biology community (beyond the original chemical developers of the tools) has remained very low. Vital information about assay benchmarking for photoconversion, testing for isomer solubility, proving the retention of mechanism of action, estimating the limits of phototoxicity etc has either simply not been formalised in the literature, or has remained buried in diverse reports without being unified and codified for an audience beyond that of synthetic organic chemists.<br><br>Here we have developed a robust four-step assay establishment procedure to optimise assay parameters for achieving reliable photocontrol over microtubule dynamics, that is applicable to diverse families of photoswitchable inhibitors. This procedure also controls for these common sources of irreproducibility and includes numerous troubleshooting steps. We also collect together the relevant information for non-chemist "users" such as microscopists and biologists, to introduce the theory of small molecule photoswitching; the unique features, usage requirements, and limitations that photoswitchable chemical reagents have; and the specific performance features of the major classes of photoswitchable microtubule inhibitors that are currently available; to highlight their properties that suit them to different applications. The generally-applicable workflows that we present allow establishing cellular assays optically controlling microtubule dynamics in a temporally reversible fashion with spatial specificity down to a single selected cell within a field of view. These workflows and methods also equip the reader to tackle advanced uses of photoswitchable chemical reagents for general protein targets, in 3D culture and in vivo, and can represent an important bridge to reach the high-value biological applications that photopharmacology can promise.<br>

Pyrrole Hemithioindigo Antimitotics with Near‐Quantitative Bidirectional Photoswitching that Photocontrol Cellular Microtubule Dynamics with Single‐Cell Precision**

“…[1,9,40] Photopharmaceuticals for the cytoskeleton have transitioned rapidly beyond their cellular proofs of concept showing noninvasive,r eversible,c ellularly resolved optical control over MT dynamics and network structure. [41] Forexample,despite the relatively poor functional dynamic range of the azobenzene PSTs( 3-fold at 405/514 nm), they have found highprecision uses in embryonic fruit fly D. melanogaster, [42] worm C. elegans, [3] zebrafish D. rerio, [43] and mouse M. musculus, [44] where they have helped to resolve questions in development and in neuroscience. [45,46] Such uses illustrate the power of photopharmacology to enable previously inaccessible studies of spatiotemporally complex processes.…”

“…In this work we have applied the pyrrole hemithioindigo (PHT) photoswitch scaffold to develop ar ationally designed series of photopharmaceutical reagents for in situ-photoswitching-based optical control over the cellular microtubule (MT) cytoskeleton:acritical biological target in urgent need of reagents enabling high-resolution modulation assays. [41] Thec hoice of the PHT scaffold was driven by its superior photoswitching performance at microscopy laser lines.W e have demonstrated these PHTub reagents capacity to apply optical control over MT network integrity,c ell division, and cell death in long-term assays.I ns hort-term assays,t hey achieve temporally resolved, cell-specific,optical modulation of MT dynamics in live cells.T oour knowledge this is the first high-resolution live cell application of any hemithioindigo as aphotopharmaceutical pharmacophore;and its success highlights both the PHTubs and the PHT scaffoldsp romise for ar ange of high-spatiotemporal-precision biological studies. We therefore believe that PHTub-7 in particular will be av aluable photopharmaceutical for high-precision cytoskeleton research, with promising applications in the fields of cellular transport, mechanostasis,migration, cell division, and embryonic development.…”

BTDAzo - a photoswitchable TRPC5 channel activator

“…Firstly, the potency of Z-BTDAzo (EC50 1.5 µM, Fig 3b ) was as good as that of its parent molecule BTD (EC50 1.4 µM) [18] ; potency matching is only very rarely achieved in photopharmacology. [27] Secondly, not only was all-E-BTDAzo at 50 µM fully inactive on TRPC5, and nearly nonresponsive to 470 nm imaging (initial 60 s of Fig 3a), but photoswitching of 50 µM mostly-Z-BTDAzo with 440 nm reduced channel currents to below those seen with mostly-Z BTDAzo at just 1.5 µM. It is BTDAzo's combination of activity exclusively in the Z-isomer, with remarkably efficient Z→E photoswitching at 440 nm in cells, that gives it such effective bidirectional photoswitching of bioactivity.…”

Self-reporting styrylthiazolium photopharmaceuticals: mitochondrial localisation as well as SAR drive biological activity

“…1,2 They may even be irreplaceable as tools to properly address systems in which spatiotemporal patterning of biological activity on the subcellular/cellular or tissue level is crucial: e.g. microtubule and actin dynamics in cytoskeleton-supported motion (subcellular/cellular level) 3,4 , or firing of retinal neurons for vision restoration (tissue level) 5,6 .…”

“…Several photoswitchable tubulin inhibitors allowing spatiotemporal control over microtubule (MT) dynamics and network architecture have been developed. 4 The most biologically-used are Z-active colchicine-site reagents inspired by Z-active stilbene combretastatin A4 (CA4) 19 : PSTs 20 at 405 nm, Z→E at 514 nm), 22,23 but their Z-isomers are rather rapidly metabolised by cellular thiol machinery preventing later-stage in vivo uses 21 . Heterostilbene SBTubs have better E→Z photoswitching at 405 nm, are optically transparent above 480 nm to allow GFP-imaging, and are metabolically robust, so they have succeeded in various in vivo applications 15 (Fig 1b).…”