Abstract
Rho GTPases play crucial roles in cell motility by regulation of actin cytoskeleton dynamics. For example, Cdc42 induces the formation of filopodia, and RhoA is involved in the assembly of actin stress fibers and cell contraction. While individual roles of RhoA and Cdc42 are well studied, cellular responses to combinations of RhoA and Cdc42 activity are poorly understood. Previous methods for investigating functions of multiple proteins, such as dominant-negative mutants or siRNA, may cause secondary effects, leading to non-specific pertubations throughout the cell. Here, we co-expressed single-chain photoswitchable activators of RhoA and Cdc42, psRhoGEF and psCdc42GEF, to control ratios of RhoA and Cdc42 activity with high spatiotemporal resolutions. Utilizing this method, we discover that simultaneous activation of RhoA and Cdc42 induces strong actin fibers at the edge of the cell, with a narrow range of ratios of RhoA/Cdc42 activation promoting the response. These cortical actin fibers are mediated by a Cdc42 effector MRCK, a RhoA effector ROCK, and mDia1, a convergent target of RhoA and Cdc42. Therefore, single-chain optobiochemical systems can dissect precise functions of combinations of multiple signaling molecules, which will be critical to understanding complex and dynamic cellular processes.
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