In vivo photocontrol of microtubule dynamics and integrity, migration and mitosis, by the potent GFP-imaging-compatible photoswitchable reagents SBTubA4P and SBTub2M

Abstract

Photoswitchable reagents to modulate microtubule stability and dynamics are an exciting tool approach towards micron- and millisecond-scale control over endogenous cytoskeleton-dependent processes. When these reagents are globally administered yet locally photoactivated in 2D cell culture, they can exert precise biological control that would have great potential for in vivo translation across a variety of research fields and for all eukaryotes. However, photopharmacology's reliance on the azobenzene photoswitch scaffold has been accompanied by a failure to translate this temporally- and cellularly-resolved control to 3D models or to in vivo applications in multi-organ animals, which we attribute substantially to the metabolic liabilities of azobenzenes. Here, we optimised the potency and solubility of metabolically stable, druglike colchicinoid microtubule inhibitors based instead on the styrylbenzothiazole (SBT) photoswitch scaffold, that are non-responsive to the major fluorescent protein imaging channels and so enable multiplexed imaging studies. We applied these reagents to 3D systems (organoids, tissue explants) and classic model organisms (zebrafish, clawed frog) with one- and two-protein imaging experiments. We successfully used systemic treatment plus spatiotemporally-localised illuminations in vivo to photocontrol microtubule dynamics, network architecture, and microtubule-dependent processes in these systems with cellular precision and second-level resolution. These nanomolar, in vivo-capable photoswitchable reagents can prove a game-changer for high-precision cytoskeleton research in cargo transport, cell motility, cell division and development. More broadly, their straightforward design can also inspire the development of similarly capable optical reagents for a range of protein targets, so bringing general in vivo photopharmacology one step closer to productive realisation.

Photocontrolling Microtubule Dynamics with Photoswitchable Chemical Reagents

“…The most powerful SBTubs are the highly potent cosolvent-requiring SBTub2M, and slightly less potent but buffersoluble SBTubA4P [9]. These replace first generation SBTub3 and SBTub3P [7].…”

“…Drug Scaffolds: Pharmaceuticals that have already been used as part of MT-inhibiting photopharmaceuticals include taxol [14] and epothilone [13] for MT-stabilising photopharmaceuticals AzTax and STEpo; and colchicinoids (analogues of colchicine-binding-site drugs like colchicine, colcemid, nocodazole or combretastatin) have been used in MT-destabilising photopharmaceuticals e.g. PST, SBTub and PHTub (Fig 5) [2,7,9,15,23,24]. where the photoswitchable bond is inside the overall binding motif.…”

“…5-10 min presumably due to hindered access to the medium. SBTubs have been used also for temporally precise blockage of development in frog; and cell-precise targeting of anti-migration and antimitotic effects over 24 hours in organoid cultures in a matrix [9]. SBTubs have shown some ability to apply subcellularly-localised effects in large neurons [7].…”

Photoswitchable epothilone-based microtubule stabilisers allow GFP-imaging-compatible, optical control over the microtubule cytoskeleton

“…GFP-orthogonality is also needed to keep imaging channels free for the multiplexed readouts that are increasingly standard in cutting-edge research. 23 (2) The SBT's C=C chromophore is ideally situated for efficient photoswitching at 405 nm or 442 nm, so matching the typical photoactivation lasers installed in microscope -again a crucial feature for practical impact. (3) SBT is highly metabolically stable, and avoids biochemical photoswitch scission.…”

“…Taken together, these advantages of the SBTs proved crucial in allowing SBTubs to succeed as MT photoswitches in tissue and animal applications across a variety of animal models, while photopharmaceuticals based on the other photoswitches have not generally succeeded in moving beyond 2D cell culture or early embryo applications. 23 We were therefore motivated to bring the powerful advantages of the SBT photoswitch to bear on the attractive class of microtubule stabilisers, to generate novel optically-controlled MT-stabilisers with improved practical applicability and scope. We now report the development of such GFP-orthogonal photopharmaceutical MT stabilisers, based on epothilone (Fig 1b).…”