Abstract
Cytoskeletal mechanics regulates cell morphodynamics and many physiological processes. While contractility is known to be largely RhoA-dependent, the process by which localized biochemical signals are translated into cell-level responses is poorly understood. Here we combine optogenetic control of RhoA, live-cell imaging and traction force microscopy to investigate the dynamics of actomyosin-based force generation. Local activation of RhoA not only stimulates local recruitment of actin and myosin but also increased traction forces that rapidly propagate across the cell via stress fibres and drive increased actin flow. Surprisingly, this flow reverses direction when local RhoA activation stops. We identify zyxin as a regulator of stress fibre mechanics, as stress fibres are fluid-like without flow reversal in its absence. Using a physical model, we demonstrate that stress fibres behave elastic-like, even at timescales exceeding turnover of constituent proteins. Such molecular control of actin mechanics likely plays critical roles in regulating morphodynamic events.
Highlights
Cytoskeletal mechanics regulates cell morphodynamics and many physiological processes
During stimulation by blue light, a cytosolic fusion protein, photo-recruitable guanine exchange factor (GEF), consisting of tandem PDZ domains fused to the DH domain of the RhoA-specific GEF LARG6, is recruited to the plasma membrane where it activates RhoA (Fig. 1b)
To illustrate the local recruitment of photo-recruitable GEF (prGEF), we tagged it with the fluorophore mCherry and imaged an NIH 3T3 fibroblast expressing the constructs on a glass coverslip (Fig. 1c)
Summary
Cytoskeletal mechanics regulates cell morphodynamics and many physiological processes. We demonstrate that stress fibres behave elastic-like, even at timescales exceeding turnover of constituent proteins Such molecular control of actin mechanics likely plays critical roles in regulating morphodynamic events. The current understanding is that, at timescales up to those of typical kinetic processes, the actin cytoskeleton behaves like an elastic solid Such elasticity enables rapid force transmission across the cell and reversible deformations to preserve cytoskeletal architecture. At longer timescales, it is thought that dynamic processes make the cytoskeleton behave predominately like a viscous fluid, enabling cytoskeletal flows and remodelling These dynamic processes, including exchange of proteins from the cytosol, are typically on the order of tens of seconds in structures like the cortex[24,25] and on the order of a minute in stress fibres[26]. Little is known about how small changes in activity can regulate cell contractility, actin architecture and adhesion
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