Optogenetic control of small GTPases reveals RhoA-mediated intracellular calcium signaling

Abstract

Rho/Ras family small GTPases are known to regulate numerous cellular processes, including cytoskeletal reorganization, cell proliferation, and cell differentiation. These processes are also controlled by Ca2+, and consequently, crosstalk between these signals is considered likely. However, systematic quantitative evaluation is not yet reported. Thus, we developed optogenetic tools to control the activity of small GTPases (RhoA, Rac1, Cdc42, Ras, Rap, and Ral) using an improved light-inducible dimer system (iLID). Using these optogenetic tools, we investigated calcium mobilization immediately after small GTPase activation. Unexpectedly, we found that only RhoA activation induced a transient intracellular calcium elevation in RPE1 and HeLa cells. Transients were also observed in MDCK and HEK293T cells by RhoA activation, but interestingly, molecular mechanisms were identified to be different among cell types. In RPE1 and HeLa cells, RhoA directly activated phospholipase C (PLC)ε at the plasma membrane, which in turn induced Ca2+ release from the endoplasmic reticulum (ER). The RhoA-PLCε axis induced calcium-dependent NFAT nuclear translocation, suggesting it does activate intracellular calcium signaling.