A Modular Approach for the Optogenetic Control of Small GTPase Activity in Living Cells

Small GTPases are critical regulators of cellular processes, such as cell migration, and comprise a family of over 167 proteins in the human genome. Despite the essential role these proteins play in maintaining cell homeostasis, there is no generalizable method for directly controlling the activity of these signaling enzymes with subcellular precision. Herein, we report the design and implementation of a modular, optogenetic platform for the spatial control of small GTPase activity within living cells. By combining split-small GTPases with improved Light-Inducible Dimerization (iLID) domains, we obtain spLIT-small GTPase constructs that enable spatially precise control of cytoskeletal dynamics such as filopodia formation (spLIT-Cdc42), membrane retraction (spLIT-RhoA), and directed cell migration (spLIT-Rac1). These results establish spLIT-small GTPases as a modular optogenetic platform for spatially controlled activation of small GTPase signaling within living cells and demonstrate the ability to probe spatiotemporal aspects of cell signaling using this approach.