Simultaneous Imaging of Rho GTPase and RhoGEF Activation using Novel GEF Biosensors Reveals Relationships between GEF and GTPase Activation in Cell Motility

Abstract

Dbl-family RhoGEFs are broadly relevant signaling molecules that regulate cytoskeletal dynamics by integrating the effects of growth factors and adhesion molecules. The timing and localization of GEF activity precisely regulates Rho family GTPase activation to produce specific types of cellular morphodynamics. We describe a novel design approach, capable of generating biosensors for multiple GEFs, applied here to visualize activation of Vav2 and Tiam1 in living cells. A pair of fluorescent proteins was inserted in a region of the GEF with no known binding activity, such that relief of GEF autoinhibition altered FRET between the fluorescent pair. High content screening optimization increased the dynamic range of the biosensors to produce FRET ratio changes of up to 300%. These biosensors report phosphorylation of Vav2 by Src kinase and reversible Vav2 activation upon EGF stimulation; similar responses are seen with Tiam1. Using these novel biosensors we examined activation dynamics during constitutive migration of MDA-MB-231 breast cancer cells, which revealed periodic activation of GEFs at the leading edge with clearly defined relationships to Rac1 and RhoA. To extend these studies we modified our existing Rho GTPase biosensors to simultaneously visualize GEF and GTPase activities and identify spatio-temporal correlations. The combination of these biosensors allow for broad application in dissecting different GEF-GTPase pathway functions. Currently we are focusing on how multi-specific RhoGEFs such as Vav2 differentially regulate downstream targets, and whether cyclic activation patterns of Rho GTPases at the leading edge are activated in discrete regions by different RhoGEFs