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.
Abstract
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.
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.
Microtubules keep large cells in shape Meiring, Joyce C M; Akhmanova, Anna
The Journal of cell biology,
06/2020, Letnik:
219, Številka:
6
Journal Article
Recenzirano
Odprti dostop
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.
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.
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•Actomyosin network reorganization is necessary for effective junctional contractility•Coronin 1B recruits to E-cadherin contacts, where it regulates F-actin architecture•Actomyosin network reorganization supports apoptotic contractility•E-cadherin recruits coronin 1B for effective apoptotic contractility
Michael et al. show that coronin 1B regulates F-actin network architecture reorganization at cell-cell junctions. This not only maintains junctional tension in epithelia at steady state but is also required for apoptotic cell extrusion, suggesting that extrusion requires a mechanosensitive response in neighboring cells elicited by contractile stress in the apoptotic cell.
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.
Pyrrole hemithioindigos can be near‐quantitatively photoisomerised by the laser wavelengths available on confocal microscopes. The first pyrrole hemithioindigo‐based photopharmaceuticals, PHTubs, which are photoswitchable tubulin inhibitors, have been developed. PHTubs optically inhibit microtubule dynamics in live cells with single‐cell, second‐scale spatiotemporal precision, with both lit‐active and dark‐active compounds demonstrated in action.
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.
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•Blue-light-induced katanin targeting triggers fast, localized microtubule clearance•Opto-katanin-mediated microtubule disassembly is rapidly reversible•Different microtubule anchors can be used for katanin targeting and severing•Opto-katanin can be employed to locally block transport and probe cell mechanics
Meiring et al. engineer an optogenetic tool for fast, reversible, and highly localized disassembly of microtubules and maintenance of microtubule-free areas in cells by controlling the recruitment of katanin. This tool was used to study microtubule-dependent ER and cargo dynamics and to show that microtubule bundles can exert forces on the nucleus.
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.
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•SBTub3 photocontrols microtubule dynamics, organization, and dependent processes•Microtubule photocontrol is cell and sub-cellularly precise and temporally reversible•SBT photocontrol is orthogonal to GFP/YFP imaging and SBTs are metabolically stable•The SBT scaffold is promising for photopharmaceuticals for other protein targets
Photocontrollable reagents have unique potential as high spatiotemporal precision modulators of biological systems. Here, Gao et al. demonstrate a GFP-orthogonal and metabolically stable photoswitch that allows optical control over microtubule dynamics and architecture with subcellular resolution. The photoswitch scaffold also offers new possibilities for photopharmaceutical design against other targets.
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.
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.
A photoswitchable epothilone‐based microtubule stabiliser, STEpo, is introduced for high‐precision cytoskeleton photocontrol in live cells. STEpo features a novel styrylthiazole photoswitch that is isosteric to azobenzene, but is compatible with GFP imaging, and can photomodulate hydrogen‐bonding as well as sterics. STEpo photocontrols microtubule dynamics and cell division with micron‐ and second‐scale resolution, and has mid‐nanomolar potency.
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