Cytoskeleton Photocontrol
Eukaryotic life depends on the continuous operation of microtubules (MTs): gigantic, highly dynamic noncovalent protein assemblies that act as roads for cellular trafficking, parking lots for organising proteins, and girders to apply or sense forces. MTs are also critical to the separation of DNA and daughter cells during cell division, and many of the most powerful anticancer drugs (taxanes, vinca alkaloids, discodermolides) work by altering MT dynamics. These drugs were for a long time our only chemical toolset to probe MT biology - which was a clear problem, since MTs play so many roles across nearly all aspects of cell biology, all of which would be suppressed indiscriminately by drug treatment.
Therefore, we pioneered photoisomerisable MT reagents, whose activity can be targeted by light to precisely modulate MT-dependent processes in 2D and 3D cell culture, and in vivo across model organisms from worm and fly to fish, frog, and mouse. Our reagents have been applied to diverse biological studies, from cargo transport to cell migration, cell division, and embryonic development (see background). This has only been possible by merging the right molecular switches, several of which we pioneered, with the appropriate drug cores: and taking the resulting photopharmaceuticals through cellular to early in vivo biological testing. Our program covers diverse avenues:
● Guidelines & Reviews for cytoskeleton photopharmacology
The biologist's guide to microtubule photocontrol (Meth Mol Biol 2022)
The chemist's guide to switchable antimitotics (Molecular Photoswitches 2022, ch 36).
Swiss National Radio pop sci view on photopharmacology for drug design (@ 25:00).
● New classes of photoswitchable MT reagents to overcome azobenzene drawbacks
‣ Pioneering the GFP-orthogonal, metabolically resistant MT photoswitch SBTub (Cell Chem Biol 2021) and optimising it for in vivo photocontrol in small animals (JACS 2022)
‣ Introducing hemithioindigos for high-completion isomerisation and rational sign inversion (HOTub: ChemBioChem 2019), nanomolar potency (Beil. JOC 2020), and hetero-HTIs to match with biologists' available laser wavelengths (PHTub: Angew 2021)
‣ Photo-SAR of photoswitchable colchicinoids (Chem Sci 2024)
● Photoresponsive derivatives of potent, complex MT-binding natural products
‣ Photocaged epothilone CouEpo (Angewandte 2024)
‣ Photoswitchable epothilone STEpo (Angewandte VIP 2022)
‣ Photoswitchable taxol AzTax (Nat Commun 2020)
● High-precision cell and developmental biology: microtubule photocontrol in...
‣ showing the role of MT dynamics in neurotransmission (Comms Biol 2023), vesicle trafficking (J Neurosci 2017), and tissue morphogenesis in the fly (Nat Cell Biol 2018)
‣ redirecting cell migration in vitro (J Cell Biol 2020b) and in vivo during zebrafish neurogenesis (J Cell Biol 2020a)
‣ testing the organisation principle of early mouse embryo development (Science 2017)
● Structural Biology and Scaffold Engineering
‣ Structural biology with low-occupancy MT switches (Nat Comm 2023)
‣ The first bioactive helicene, Helistatin (JACS Au 2022)
‣ The first in vivo effective photoswitch for a cytosolic target, PST (Cell 2015)
Extended Background: Photocontrolling Microtubules for High-Precision Studies in Migration, Division, Transport, & Development
The cellular scaffolding protein tubulin is the monomeric unit that forms the complex, highly dynamic network of cellular microtubules (MTs): gigantic, noncovalent polymeric assemblies that act as roads for cellular trafficking, parking lots for resting proteins, and girders for applying or sensing forces within (and outside of) cells. MTs are continuously being directed by many proteins to undergo remodelling into rapidly changing shapes, and are modified in complex ways with biochemical flags (PTMs). These remodelling dynamics are crucial to the correct functioning of many anisotropic cellular processes, most famously, to the separation of genetic material and daughter cells during mitosis/meieosis. Many of the most powerful anticancer drugs (taxanes, vinca alkaloids, discodermolides) work by altering these microtubule dynamics so that cells can no longer divide correctly and instead go into apoptosis.
Our research has pioneered photoisomerisable microtubule inhibitors, that can be precisely controlled through reversible optical patterning. These photopharmaceutical antimitotics are allowing researchers to use light to reversibly modulate microtubule-dependent processes in eukaryotes, in 2D and 3D cell culture, as well as in vivo across model organisms from worm and fly to fish, frog, and mouse: with applications in fields from cargo transport to cell migration, cell division, and embryonic development. These results were made possible by merging the right molecular photoswitches, several of which we have pioneered, with the appropriate drug cores: and taking the resulting photopharmaceuticals through cellular to early in vivo biological testing. We are now stretching the chemical space of antimitotics and photoswitches to reach new generations of photoswitchably bioactive drugs with improved potency, in vivo compatibility, and other layers of functionality including subcellular localisation and tissue-retention.
The starting-point for this research axis was our development of photoswitchable microtubule inhibitors based around azobenzenes, for the colchicine binding site on tubulin, called PSTs. UV/blue or green illuminations switch PSTs between the inactive E isomer and the MT-inhibiting Z isomer, so they could light-specifically interrupt MT-dependent processes in cells and organisms, from mitosis to embryonic development. This cell-by-cell, reversible control had never previously been possible without genetic engineering. PSTs have since been distributed to >200 research groups for a range of cytoskeleton studies; while we have built up a reagent toolchest of photoresponsive MT inhibitors with better usability: such as compatibility with GFP/YFP for multichannel imaging studies; metabolic stability for long-term use in small animals; either rapid or slow switch-off for studies on different timescales; enhanced potency and solubility for organoid and in vivo experiments; rapid isomerisation for time-resolved structural biology; and subcellularly-precise targeting in cells. Our technical advances in these areas can also be applied to photocontrol of other protein systems, which is helping to expand the scope and applications of photopharmacology in general. In parallel though, we and others have explored the biological applications of our photoswitchable MT inhibitors as reagents to study force generation, cell migration, organoid growth, tissue morphogenesis, and embryonic development, across a range of model systems and organisms, aiming to shed more light on the many roles of MT dynamics and network structure in biology.