Optogenetic control of cellular forces and mechanotransduction

Mechanical forces are key regulators of cell and tissue physiology. The basic molecular mechanism of fiber contraction by the sliding of actin filament upon myosin leading to conformational change has been known for decades. The regulation of force generation at the level of the cell, however, is still far from elucidated. Indeed, the magnitude of cell traction forces on the underlying extracellular matrix in culture is almost impossible to predict or experimentally control. The considerable variability in measurements of cell-traction forces indicates that they may not be the optimal readout to properly characterize cell contractile state and that a significant part of the contractile energy is not transferred to cell anchorage but instead is involved in actin network dynamics. Here we discuss the experimental, numerical, and biological parameters that may be responsible for the variability in traction force production. We argue that limiting these sources of variability and investigating the dissipation of mechanical work that occurs with structural rearrangements and the disengagement of force transmission is key for further understanding of cell mechanics.

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“…, 2015; Oakes et al. , 2017; Valon et al. , 2017), and these should be of great help in this direction.…”

Section: Discussionmentioning

confidence: 99%

Dissipation of contractile forces: the missing piece in cell mechanics

Kurzawa

Vianay

Senger

et al. 2017

MBoC

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“…Current Protocols in Cell Biology minute timescales, but RhoGEF dissociation was on the order of 20 min (Valon et al, 2017). In this case, actin accumulated and dissipated with similar kinetics as the RhoGEF.…”

Section: Of 19mentioning

confidence: 94%

“…The DH domain of LARG is a potent RhoA-specific activator and exhibits the highest catalytic activity reported for its GEF family (Jaiswal et al, 2011). Other groups have used the DHPH domain of the Drosophila-specific RhoGEF2 (Izquierdo et al, 2018), the DHPH domain of LARG (Meshik et al, 2019;O'Neill et al, 2018), or the DHPH domain of ARHGEF11 (Valon et al, 2017). Although others have included the PH domain in their recruitable GEF complexes, we recommend engineering dimerization constructs that utilize only the GEF's catalytic DH domain to reduce basal GEF activity.…”

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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“…TFM. TFM gels were prepared similarly to Valon et al 47 . Briefly, the polymerization mixture was placed in glass-bottom dish and covered with 18 mm round coverslip to polymerize for 1 hour.…”

Section: M1 Macrophagesmentioning

confidence: 99%

RHOA mediated mechanical force generation through Dectin-1

Choraghe

Kołodziej

Buser

et al. 2019

Preprint

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Dectin-1 is an innate immune pattern recognition receptor which recognizes β-glucan on the Candida albicans (C. albicans) cell wall. Recognition of β-glucan by immune cells leads to phagocytosis, oxidative burst, cytokine and chemokine production. We looked for specific mechanisms that coordinate phagocytosis downstream of Dectin-1 leading to actin reorganization and internalization of fungus. We found that stimulation of Dectin-1 by soluble b-glucan leads to mechanical force generation and areal contraction in Dectin-1 transfected HEK-293 cells and M1 macrophages. With inhibitor studies, we found this force generation is a SYK-independent, SFK (SRC Family Kinase)-dependent process mediated through the RHOA-ROCK-MLC pathway. We confirmed activation of RHOA downstream of Dectin-1 using G-LISA and stress fiber formation. Through phagocytosis assays, we found direct evidence for importance of RHOA-ROCK-MLC pathway in process of phagocytosis of C. albicans. In conclusion, we found evidence for RHOA-ROCK-MLC mediated mechanical force generation downstream of Dectin-1 for C. albicans phagocytosis.

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Optogenetic control of cellular forces and mechanotransduction

Cited by 200 publications

References 42 publications

Dissipation of contractile forces: the missing piece in cell mechanics

Dissipation of contractile forces: the missing piece in cell mechanics

Optogenetic Control of RhoA to Probe Subcellular Mechanochemical Circuitry

RHOA mediated mechanical force generation through Dectin-1

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