Optical regulation of endogenous RhoA reveals selection of cellular responses by signal amplitude

How protein signaling networks respond to different input strengths is an important but poorly understood problem in cell biology. For example, RhoA can promote focal adhesion (FA) growth or disassembly, but how RhoA activity mediates these opposite outcomes is not clear. Here, we develop a photoswitchable RhoA guanine nucleotide exchange factor (GEF), psRhoGEF, to precisely control endogenous RhoA activity. Using this optical tool, we discover that peak FA disassembly selectively occurs upon activation of RhoA to submaximal levels. We also find that Src activation at FAs selectively occurs upon submaximal RhoA activation, identifying Src as an amplitude-dependent RhoA effector. Finally, a pharmacological Src inhibitor reverses the direction of the FA response to RhoA activation from disassembly to growth, demonstrating that Src functions to suppress FA growth upon RhoA activation. Thus, rheostatic control of RhoA activation by psRhoGEF reveals that cells can use signal amplitude to produce multiple responses to a single biochemical signal. [Display omitted] •A photoswitchable RhoA GEF enables rheostatic control of endogenous RhoA activity•Submaximal levels of RhoA activity preferentially induce focal adhesion disassembly•Src functions as an amplitude-dependent RhoA effector for focal adhesion disassembly•Amplitude modulation of a single signal can produce distinct cell shape outcomes Ju et al. develop photoswitchable RhoA GEF that enables rheostatic control of endogenous RhoA activity. Fine modulation of RhoA signal amplitude reveals that submaximal levels of RhoA activity preferentially induce focal adhesion disassembly through selective activation of Src kinase, suggesting that amplitude of a single signal can produce distinct cell outcomes.