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>
Stu2p binds tubulin and undergoes an open-to-closed conformational change