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.
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.
The zebrafish (Danio rerio) is a popular vertebrate model organism to investigate molecular mechanisms driving development and disease. Due to its transparency at embryonic and larval stages, investigations in the living organism are possible with subcellular resolution using intravital microscopy. The beneficial optical characteristics of zebrafish not only allow for passive observation, but also active manipulation of proteins and cells by light using optogenetic tools. Initially, photosensitive ion channels have been applied for neurobiological studies in zebrafish to dissect complex behaviors on a cellular level. More recently, exciting non-neural optogenetic tools have been established to control gene expression or protein localization and activity, allowing for unprecedented non-invasive and precise manipulation of various aspects of cellular physiology. Zebrafish will likely be a vertebrate model organism at the forefront of in vivo application of non-neural optogenetic tools and pioneering work has already been performed. In this review, we provide an overview of non-neuromodulatory optogenetic tools successfully applied in zebrafish to control gene expression, protein localization, cell signaling, migration and cell ablation.
Selective targeting of DNA by means of fluorescent labeling has become a mainstay in the life sciences. While genetic engineering serves as a powerful technique and allows the visualization of nucleic acid by using DNA‐targeting fluorescent fusion proteins in a cell‐type‐ and subcellular‐specific manner, it relies on the introduction of foreign genes. On the other hand, DNA‐binding small fluorescent molecules can be used without genetic engineering, but they are not spatially restricted. Herein, we report a photocaged version of the DNA dye Hoechst33342 (pcHoechst), which can be uncaged by using UV to blue light for the selective staining of chromosomal DNA in subnuclear regions of live cells. Expanding its application to a vertebrate model organism, we demonstrate uncaging in epithelial cells and short‐term cell tracking in vivo in zebrafish. We envision pcHoechst as a valuable tool for targeting and interrogating DNA with precise spatiotemporal resolution in living cells and wild‐type organisms.
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