Single-component optogenetic tools to bidirectionally control RhoA in mechanotransduction

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ABSTRACT RhoA, a member of the Rho-family of small GTPases, centrally regulates actin organization and actomyosin contractility. RhoA dynamics coordinate actin stress fiber formation, which ultimately determines how cells generate cytoskeletal tension to transmit mechanical forces to/from neighboring cell-cell junctions and focal adhesions with the extracellular matrix (ECM). Thus, new tools to control the dynamics of RhoA signaling may enhance understanding of how cells make decisions in response to mechanical stimuli. We propose to create optogenetic tools for bi-directional (activation and inactivation) control over RhoA signaling, systematically characterize their function through a set of physiological assays in contractility and mechanotransduction, and benchmark their performance against other reported optogenetic technologies. We will use our recently discovered BcLOV4 photoreceptor that dynamically translocates via a direct light-induced protein-lipid interaction with the plasma membrane, making ii powerful for single-component optogenetic control over peripheral membrane proteins that is robust across cell types and primary cells. To demonstrate the unique capabilities of the toolbox, we will quantitatively map the cytoskeletal signaling and tensional dynamics of a known RhoA/Y AP mechanotransductive feedback loop in cytoskeletal remodeling and persistent cell motility, through simultaneous optogenetic perturbation and multi-reporter imaging. This toolbox will broadly impact cell and cytoskeletal biology by advancing control over ubiquitous RhoA signaling to probe its diverse regulatory roles in cell contractility, motility, mechanotransduction, and regeneration.