Optogenetic control of RhoA reveals zyxin-mediated elasticity of stress fibres

Oakes, Patrick W.; Wagner, Elizabeth; Brand, Christoph A.; Probst, Dimitri; Linke, Marco; Schwarz, Ulrich S.; Glotzer, Michael; Gardel, Margaret L.

doi:10.1038/ncomms15817

Cited by 147 publications

(146 citation statements)

References 76 publications

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“…Interestingly, a recent study has demonstrated that stress fibers display elastic‐like properties on time‐scales exceeding the turnover of individual constituent components (> 60 min). This elastic‐like behavior is regulated and depends on specific stress fiber proteins such as zykin (Oakes et al , 2017). Elastic effects on surprisingly long timescales have also been observed in cell monolayers (Sunyer et al , 2016).…”

Section: Discussionmentioning

confidence: 99%

Downregulation of basal myosin‐ II is required for cell shape changes and tissue invagination

et al. 2018

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Tissue invagination drives embryo remodeling and assembly of internal organs during animal development. While the role of actomyosin‐mediated apical constriction in initiating inward folding is well established, computational models suggest relaxation of the basal surface as an additional requirement. However, the lack of genetic mutations interfering specifically with basal relaxation has made it difficult to test its requirement during invagination so far. Here we use optogenetics to quantitatively control myosin‐II levels at the basal surface of invaginating cells during Drosophila gastrulation. We show that while basal myosin‐II is lost progressively during ventral furrow formation, optogenetics allows the maintenance of pre‐invagination levels over time. Quantitative imaging demonstrates that optogenetic activation prior to tissue bending slows down cell elongation and blocks invagination. Activation after cell elongation and tissue bending has initiated inhibits cell shortening and folding of the furrow into a tube‐like structure. Collectively, these data demonstrate the requirement of myosin‐II polarization and basal relaxation throughout the entire invagination process.

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Section: Discussionmentioning

confidence: 99%

Downregulation of basal myosin‐ II is required for cell shape changes and tissue invagination

et al. 2018

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“…The most commonly used flexible linkers have stretches of Gly and Ser residues, the length and copy number of which can be optimized to separate the functional domains. For LOVpep, we have successfully used the flexible linker GGSGGSGGSPR, and for tandem PDZ we have used QSTVPRARDPPVAT (Cavanaugh et al, 2020;Oakes et al, 2017;Wagner & Glotzer, 2016). Other linkers for optogenetic tags include GSGGSGSGGT (Wang & Hahn, 2016) or GSTSGSGKPGS-GEGSTKG (Whitlow et al, 1993).…”

Section: Of 19mentioning

confidence: 99%

Optogenetic Control of RhoA to Probe Subcellular Mechanochemical Circuitry

2020

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Spatiotemporal localization of protein function is essential for physiological processes from subcellular to tissue scales. Genetic and pharmacological approaches have played instrumental roles in isolating molecular components necessary for subcellular machinery. However, these approaches have limited capabilities to reveal the nature of the spatiotemporal regulation of subcellular machineries like those of cytoskeletal organelles. With the recent advancement of optogenetic probes, the field now has a powerful tool to localize cytoskeletal stimuli in both space and time. Here, we detail the use of tunable light‐controlled interacting protein tags (TULIPs) to manipulate RhoA signaling in vivo. This is an optogenetic dimerization system that rapidly, reversibly, and efficiently directs a cytoplasmic RhoGEF to the plasma membrane for activation of RhoA using light. We first compare this probe to other available optogenetic systems and outline the engineering logic for the chosen recruitable RhoGEFs. We also describe how to generate the cell line, spatially control illumination, confirm optogenetic control of RhoA, and mechanically induce cell‐cell junction deformation in cultured tissues. Together, these protocols detail how to probe the mechanochemical circuitry downstream of RhoA signaling. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Generation of a stable cell line expressing TULIP constructs Basic Protocol 2: Preparation of collagen substrate for imaging Basic Protocol 3: Transient transfection for visualization of downstream effectors Basic Protocol 4: Calibration of spatial illumination Basic Protocol 5: Optogenetic activation of a region of interest

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“…[25][26][27] In support of these findings, FA proteins have been shown to be intrinsically mechanosensitive. Examples of this can be observed in the member of the mechanosensitive Cas family, p130Cas, which was shown to become extended and phosphorylated under tension, 28 zyxin which is recruited to strained areas along actin fibers, thereby regulating their elasticity, 29 talin, which was demonstrated to alter conformation as a function of force exertion, 30,31 and vinculin, which requires force to localize to FA compelxes. 25,27 Quantitative measurements using cytoskeleton laser nanosurgery also provided evidence of direct actin stress fiber association to the substrate, recruiting FA proteins such as zyxin to areas with highest tensed anchorage.…”

Section: The Cytoskeleton and Mechanosensitive Moleculesmentioning

confidence: 99%

Lymphocyte mechanotransduction: The regulatory role of cytoskeletal dynamics in signaling cascades and effector functions

Ben‐Shmuel¹,

Joseph²,

Sabag³

et al. 2019

Journal of Leukocyte Biology

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The process of mechanotransduction, that is, conversion of physical forces into biochemical signaling cascades, has attracted interest as a potential mechanism for regulating immune cell activation. The cytoskeleton serves a critical role in a variety of lymphocyte functions, from cellular activation, proliferation, adhesion, and migration, to creation of stable immune synapses, and execution of functions such as directed cytotoxicity. Though traditionally considered a scaffold that enables formation of signaling complexes that maintain stable immune synapses, the cytoskeleton was additionally shown to play a dynamic role in lymphocyte signaling cascades by sensing physical cues such as substrate rigidity, and transducing these mechanical features into chemical signals that ultimately influence lymphocyte effector functions. It is thus becoming clear that cytoskeletal dynamics are essential for the lymphocyte response, beyond the role of the cytoskeleton as a stationary framework. Here, we describe the transduction of extracellular forces to activate signaling pathways and effector functions mediated through the cytoskeleton in lymphocytes. We also highlight recent discoveries of cytoskeleton‐mediated mechanotransduction on intracellular signaling pathways in NK cells.

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Optogenetic control of RhoA reveals zyxin-mediated elasticity of stress fibres

Cited by 147 publications

References 76 publications

Downregulation of basal myosin‐ II is required for cell shape changes and tissue invagination

Downregulation of basal myosin‐ II is required for cell shape changes and tissue invagination

Optogenetic Control of RhoA to Probe Subcellular Mechanochemical Circuitry

Lymphocyte mechanotransduction: The regulatory role of cytoskeletal dynamics in signaling cascades and effector functions

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