Quantitative Subcellular Control of Cdc42, Rac1 and RhoA GTPases using the Cry2/CIBN Optogenetic Dimerizer

Abstract

Cell polarization is established and maintained through complex mechanisms involving signaling networks regulated in space and time at the sub-cellular level. How a cell coordinates multiple local signaling modules to create a global polarization is still unclear. Recently developed optogenetic methods have been recognized as promising tools to dissect these intracellular signaling networks by allowing perturbations to be spatially and temporally controlled. First, we characterized the biophysical properties of the Cry2/CIBN light gated dimerization system in order to establish the quantitative relationship between patterns of light stimulation and corresponding gradients of induced signaling activity. We analyzed the processes involved in the recruitment and localization of Cry2 including the 3D cytoplasmic diffusion of Cry2, the 2D diffusion of Cry2/CIBN complexes at the cell membrane and the disassociation kinetics of this complex. From these experiments we determined that it is possible to induce subcellular gradients of recruited proteins of any chosen profile up to a spatial resolution of 5µm and a temporal one of ca. 3 minutes. Second, we applied our quantitative optogenetic method to the regulation of Cdc42, Rac1 and RhoA, the three canonical RhoGPTases involved in cell polarity and migration. We quantified the effect of the local activation of Cdc42 using cell displacement and cell shape changes as reporters of cell polarization and migration. We qualitatively characterized the effects of local activation of RhoA and Rac1 on different cellular effectors including actin filaments and focal adhesion complexes. Altogether, our quantitative optogenetic method provides a new step for the optogenetic dissection of subcellular signaling networks by allowing the simultaneous measurement of the perturbation and the cell response in a straightforward and reproducible way.