A Photocaged Microtubule-Stabilising Epothilone Allows Spatiotemporal Control of Cytoskeletal Dynamics.

The cytoskeleton is essential for spatial and temporal organisation of a wide range of cellular and tissue-level processes, such as proliferation, signalling, cargo transport, migration, morphogenesis, and neuronal development. Cytoskeleton research aims to study these processes by imaging, or by locally manipulating, the dynamics and organisation of cytoskeletal proteins with high spatiotemporal resolution: which matches the capabilities of optical methods. To date, no photoresponsive microtubule-stabilising tool has united all the features needed for a practical high-precision reagent: a low potency and biochemically stable non-illuminated state; then an efficient, rapid, and clean photoresponse that generates a high potency illuminated state; plus good solubility at suitable working concentrations; and efficient synthetic access. We now present CouEpo, a photocaged epothilone microtubule-stabilising reagent that combines these needs. Its potency increases approximately 100-fold upon violet/blue irradiation to reach low-nanomolar values, allowing efficient photocontrol of microtubule dynamics in live cells, and even the generation of cellular asymmetries in microtubule architecture and cell dynamics. CouEpo is thus a high-performance tool compound that can support high-precision research into many microtubule-associated processes, from biophysics to transport, cell motility, and neuronal physiology.

[5]-Helistatins: Tubulin-Binding Helicenes with Antimitotic Activity.

Helicenes are high interest synthetic targets with unique conjugated helical structures that have found important technological applications. Despite this interest, helicenes have had limited impact in chemical biology. Herein, we disclose a first-in-class antimitotic helicene, helistatin 1 (HA-1), where the helicene scaffold acts as a structural mimic of colchicine, a known antimitotic drug. The synthesis proceeds via sequential Pd-catalyzed coupling reactions and a π-Lewis acid cycloisomerization mediated by PtCl2. HA-1 was found to block microtubule polymerization in both cell-free and live cell assays. Not only does this demonstrate the feasibility of using helicenes as bioactive scaffolds against protein targets, but also suggests wider potential for the use of helicenes as isosteres of biaryls or cis-stilbenes-themselves common drug and natural product scaffolds. Overall, this study further supports future opportunities for helicenes for a range of chemical biological applications.

Opto-katanin, an optogenetic tool for localized, microtubule disassembly.

Microtubules are cytoskeletal polymers that separate chromosomes during mitosis and serve as rails for intracellular transport and organelle positioning. Manipulation of microtubules is widely used in cell and developmental biology, but tools for precise subcellular spatiotemporal control of microtubules are currently lacking. Here, we describe a light-activated system for localized recruitment of the microtubule-severing enzyme katanin. This system, named opto-katanin, uses targeted illumination with blue light to induce rapid, localized, and reversible microtubule depolymerization. This tool allows precise clearing of a subcellular region of microtubules while preserving the rest of the microtubule network, demonstrating that regulation of katanin recruitment to microtubules is sufficient to control its severing activity. The tool is not toxic in the absence of blue light and can be used to disassemble both dynamic and stable microtubules in primary neurons as well as in dividing cells. We show that opto-katanin can be used to locally block vesicle transport and to clarify the dependence of organelle morphology and dynamics on microtubules. Specifically, our data indicate that microtubules are not required for the maintenance of the Golgi stacks or the tubules of the endoplasmic reticulum but are needed for the formation of new membrane tubules. Finally, we demonstrate that this tool can be applied to study the contribution of microtubules to cell mechanics by showing that microtubule bundles can exert forces constricting the nucleus.

Photocontrolling Microtubule Dynamics with Photoswitchable Chemical Reagents.

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.Here we give an introduction to using these photoswitchable microtubule inhibitors in cells. We describe the theory of small molecule photoswitching, and the unique performance features, usage requirements, and limitations that photoswitchable chemical reagents have; then we summarize the major classes of photoswitchable microtubule inhibitors that are currently available, with the properties that suit them to different applications, and troubleshooting measures for avoiding common mistakes. We outline workflows to establish cellular assays where they are used to optically control microtubule dynamics in a temporally reversible fashion with spatial specificity down to a single selected cell within a field of view. The methods in this chapter also equip the reader to tackle advanced uses of photoswitchable chemical reagents, in 3D culture and in vivo.

In Vivo Photocontrol of Microtubule Dynamics and Integrity, Migration and Mitosis, by the Potent GFP-Imaging-Compatible Photoswitchable Reagents SBTubA4P and SBTub2M.

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 Epothilone-Based Microtubule Stabilisers Allow GFP-Imaging-Compatible, Optical Control over the Microtubule Cytoskeleton.

Optical methods to modulate microtubule dynamics show promise for reaching the micron- and millisecond-scale resolution needed to decrypt the roles of the cytoskeleton in biology. However, optical microtubule stabilisers are under-developed. We introduce "STEpos" as GFP-orthogonal, light-responsive epothilone-based microtubule stabilisers. They use a novel styrylthiazole photoswitch in a design to modulate hydrogen-bonding and steric effects that control epothilone potency. STEpos photocontrol microtubule dynamics and cell division with micron- and second-scale spatiotemporal precision. They substantially improve potency, solubility, and ease-of-use compared to previous optical microtubule stabilisers, and the structure-photoswitching-activity relationship insights in this work will guide future optimisations. The STEpo reagents can contribute greatly to high-precision research in cytoskeleton biophysics, cargo transport, cell motility, cell division, development, and neuroscience.

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

We report the first cellular application of the emerging near-quantitative photoswitch pyrrole hemithioindigo, by rationally designing photopharmaceutical PHTub inhibitors of the cytoskeletal protein tubulin. PHTubs allow simultaneous visible-light imaging and photoswitching in live cells, delivering cell-precise photomodulation of microtubule dynamics, and photocontrol over cell cycle progression and cell death. This is the first acute use of a hemithioindigo photopharmaceutical for high-spatiotemporal-resolution biological control in live cells. It additionally demonstrates the utility of near-quantitative photoswitches, by enabling a dark-active design to overcome residual background activity during cellular photopatterning. This work opens up new horizons for high-precision microtubule research using PHTubs and shows the cellular applicability of pyrrole hemithioindigo as a valuable scaffold for photocontrol of a range of other biological targets.

A Robust, GFP-Orthogonal Photoswitchable Inhibitor Scaffold Extends Optical Control over the Microtubule Cytoskeleton.

Optically controlled chemical reagents, termed "photopharmaceuticals," are powerful tools for precise spatiotemporal control of proteins particularly when genetic methods, such as knockouts or optogenetics are not viable options. However, current photopharmaceutical scaffolds, such as azobenzenes are intolerant of GFP/YFP imaging and are metabolically labile, posing severe limitations for biological use. We rationally designed a photoswitchable "SBT" scaffold to overcome these problems, then derivatized it to create exceptionally metabolically robust and fully GFP/YFP-orthogonal "SBTub" photopharmaceutical tubulin inhibitors. Lead compound SBTub3 allows temporally reversible, cell-precise, and even subcellularly precise photomodulation of microtubule dynamics, organization, and microtubule-dependent processes. By overcoming the previous limitations of microtubule photopharmaceuticals, SBTubs offer powerful applications in cell biology, and their robustness and druglikeness are favorable for intracellular biological control in in vivo applications. We furthermore expect that the robustness and imaging orthogonality of the SBT scaffold will inspire other derivatizations directed at extending the photocontrol of a range of other biological targets.

Photoswitchable paclitaxel-based microtubule stabilisers allow optical control over the microtubule cytoskeleton.

Small molecule inhibitors are prime reagents for studies in microtubule cytoskeleton research, being applicable across a range of biological models and not requiring genetic engineering. However, traditional chemical inhibitors cannot be experimentally applied with spatiotemporal precision suiting the length and time scales inherent to microtubule-dependent cellular processes. We have synthesised photoswitchable paclitaxel-based microtubule stabilisers, whose binding is induced by photoisomerisation to their metastable state. Photoisomerising these reagents in living cells allows optical control over microtubule network integrity and dynamics, cell division and survival, with biological response on the timescale of seconds and spatial precision to the level of individual cells within a population. In primary neurons, they enable regulation of microtubule dynamics resolved to subcellular regions within individual neurites. These azobenzene-based microtubule stabilisers thus enable non-invasive, spatiotemporally precise modulation of the microtubule cytoskeleton in living cells, and promise new possibilities for studying intracellular transport, cell motility, and neuronal physiology.

Microtubules keep large cells in shape.

Migrating cells need to coordinate extension and retraction of their protrusions to avoid fragmenting. Kopf et al. (2020. J. Cell Biol.https://doi.org/10.1083/jcb.201907154) demonstrate that microtubules help to maintain cell coherence during amoeboid migration by controlling actomyosin contractility in retracting protrusions.

Generation and regulation of microtubule network asymmetry to drive cell polarity.

Microtubules control cell architecture by serving as a scaffold for intracellular transport, signaling, and organelle positioning. Microtubules are intrinsically polarized, and their orientation, density, and post-translational modifications both respond and contribute to cell polarity. Animal cells that can rapidly reorient their polarity axis, such as fibroblasts, immune cells, and cancer cells, contain radially organized microtubule arrays anchored at the centrosome and the Golgi apparatus, whereas stably polarized cells often acquire non-centrosomal microtubule networks attached to the cell cortex, nucleus, or other structures. Microtubule density, longevity, and post-translational modifications strongly depend on the dynamics of their plus ends. Factors controlling microtubule plus-end dynamics are often part of cortical assemblies that integrate cytoskeletal organization, cell adhesion, and secretion and are subject to microtubule-dependent feedback regulation. Finally, microtubules can mechanically contribute to cell asymmetry by promoting cell elongation, a property that might be important for cells with dense microtubule arrays growing in soft environments.

Colocation of Tpm3.1 and myosin IIa heads defines a discrete subdomain in stress fibres.

Co-polymers of tropomyosin and actin make up a major fraction of the actin cytoskeleton. Tropomyosin isoforms determine the function of an actin filament by selectively enhancing or inhibiting the association of other actin binding proteins, altering the stability of an actin filament and regulating myosin activity in an isoform-specific manner. Previous work has implicated specific roles for at least five different tropomyosin isoforms in stress fibres, as depletion of any of these five isoforms results in a loss of stress fibres. Despite this, most models of stress fibres continue to exclude tropomyosins. In this study, we investigate tropomyosin organisation in stress fibres by using super-resolution light microscopy and electron microscopy with genetically tagged, endogenous tropomyosin. We show that tropomyosin isoforms are organised in subdomains within the overall domain of stress fibres. The isoforms Tpm3.1 and 3.2 (hereafter Tpm3.1/3.2, encoded by TPM3) colocalise with non-muscle myosin IIa and IIb heads, and are in register, but do not overlap, with non-muscle myosin IIa and IIb tails. Furthermore, perturbation of Tpm3.1/3.2 results in decreased myosin IIa in stress fibres, which is consistent with a role for Tpm3.1 in maintaining myosin IIa localisation in stress fibres.

Tropomyosin concentration but not formin nucleators mDia1 and mDia3 determines the level of tropomyosin incorporation into actin filaments.

The majority of actin filaments in human cells exist as a co-polymer with tropomyosin, which determines the functionality of actin filaments in an isoform dependent manner. Tropomyosin isoforms are sorted to different actin filament populations and in yeast this process is determined by formins, however it remains unclear what process determines tropomyosin isoform sorting in mammalian cells. We have tested the roles of two major formin nucleators, mDia1 and mDia3, in the recruitment of specific tropomyosin isoforms in mammals. Despite observing poorer cell-cell attachments in mDia1 and mDia3 KD cells and an actin bundle organisation defect with mDia1 knock down; depletion of mDia1 and mDia3 individually and concurrently did not result in any significant impact on tropomyosin recruitment to actin filaments, as observed via immunofluorescence and measured via biochemical assays. Conversely, in the presence of excess Tpm3.1, the absolute amount of Tpm3.1-containing actin filaments is not fixed by actin filament nucleators but rather depends on the cell concentration of Tpm3.1. We conclude that mDia1 and mDia3 are not essential for tropomyosin recruitment and that tropomyosin incorporation into actin filaments is concentration dependent.

Co-polymers of Actin and Tropomyosin Account for a Major Fraction of the Human Actin Cytoskeleton.

Tropomyosin proteins form stable coiled-coil dimers that polymerize along the α-helical groove of actin filaments [1]. The actin cytoskeleton consists of both co-polymers of actin and tropomyosin and polymers of tropomyosin-free actin [2]. The fundamental distinction between these two types of filaments is that tropomyosin determines the functional capability of actin filaments in an isoform-dependent manner [3-9]. However, it is unknown what portion of actin filaments are associated with tropomyosin. To address this deficit, we have measured the relative distribution between these two filament populations by quantifying tropomyosin and actin levels in a variety of human cell types, including bone (U2OS); breast epithelial (MCF-10A); transformed breast epithelial (MCF-7); and primary (BJpar), immortalized (BJeH), and Ras-transformed (BJeLR) BJ fibroblasts [10]. Our measurements of tropomyosin and actin predict the saturation of the actin cytoskeleton, implying that tropomyosin binding must be inhibited in order to generate tropomyosin-free actin filaments. We find the majority of actin filaments to be associated with tropomyosin in four of the six cell lines tested and the portion of actin filaments associated with tropomyosin to decrease with transformation. We also discover that high-molecular-weight (HMW), unlike low-molecular-weight (LMW), tropomyosin isoforms are primarily co-polymerized with actin in untransformed cells. This differential partitioning of tropomyosins is not due to a lack of N-terminal acetylation of LMW tropomyosins, but it is, in part, explained by the susceptibility of soluble HMW tropomyosins to proteasomal degradation. We conclude that actin-tropomyosin co-polymers make up a major fraction of the human actin cytoskeleton.

Parallel assembly of actin and tropomyosin, but not myosin II, during de novo actin filament formation in live mice.

Many actin filaments in animal cells are co-polymers of actin and tropomyosin. In many cases, non-muscle myosin II associates with these co-polymers to establish a contractile network. However, the temporal relationship of these three proteins in the de novo assembly of actin filaments is not known. Intravital subcellular microscopy of secretory granule exocytosis allows the visualisation and quantification of the formation of an actin scaffold in real time, with the added advantage that it occurs in a living mammal under physiological conditions. We used this model system to investigate the de novo assembly of actin, tropomyosin Tpm3.1 (a short isoform of TPM3) and myosin IIA (the form of non-muscle myosin II with its heavy chain encoded by Myh9) on secretory granules in mouse salivary glands. Blocking actin polymerization with cytochalasin D revealed that Tpm3.1 assembly is dependent on actin assembly. We used time-lapse imaging to determine the timing of the appearance of the actin filament reporter LifeAct-RFP and of Tpm3.1-mNeonGreen on secretory granules in LifeAct-RFP transgenic, Tpm3.1-mNeonGreen and myosin IIA-GFP (GFP-tagged MYH9) knock-in mice. Our findings are consistent with the addition of tropomyosin to actin filaments shortly after the initiation of actin filament nucleation, followed by myosin IIA recruitment.

Coronin 1B Reorganizes the Architecture of F-Actin Networks for Contractility at Steady-State and Apoptotic Adherens Junctions.

In this study we sought to identify how contractility at adherens junctions influences apoptotic cell extrusion. We first found that the generation of effective contractility at steady-state junctions entails a process of architectural reorganization whereby filaments that are initially generated as poorly organized networks of short bundles are then converted into co-aligned perijunctional bundles. Reorganization requires coronin 1B, which is recruited to junctions by E-cadherin adhesion and is necessary to establish contractile tension at the zonula adherens. When cells undergo apoptosis within an epithelial monolayer, coronin 1B is also recruited to the junctional cortex at the apoptotic/neighbor cell interface in an E-cadherin-dependent fashion to support actin architectural reorganization, contractility, and extrusion. We propose that contractile stress transmitted from the apoptotic cell through E-cadherin adhesions elicits a mechanosensitive response in neighbor cells that is necessary for the morphogenetic event of apoptotic extrusion to occur.