Split-Small GTPase Reassembly as a Method to Control Cellular Signaling with User-Defined Inputs

Small GTPases are critical signaling enzymes that control diverse cellular functions, such as cell migration and proliferation. However, dissecting the roles of these enzymes in cellular signaling is hindered by the lack of a plug-and-play methodology for the direct, temporal control of small GTPase activity by using user-defined inputs. Herein, we present a method that pairs split-small GTPases with user-defined chemical inducer of dimerization (CID) systems in a plug-and-play manner to directly control small GTPase signaling in living cells. The modularity of split-small GTPase systems allows for the selection of CIDs with minimal off-target effects on the pathway being studied. Our results highlight the ability to obtain consistent pathway activation with varying CID systems for direct control of MAPK signaling, filopodia formation, and cell retraction. Thus, split-small GTPase systems provide a customizable platform for the development of temporally gated systems for directly controlling cellular signaling with user-defined inputs.