Regulation Of Small Gtpases In Epithelial Cell Collective Migration

Abstract

Collective migration is a complex process in which cells migrate together with multicellular polarity while connected with each other through cell-cell junctions. Epithelial collective migration is a fundamental process in development, and occurs in many contexts, from formation of ducts and glands to tissue homeostasis and regeneration. Small GTPases, including members of the Ras and Rho subfamilies, have been reported to be involved in migration. However, the detailed mechanism of how these small GTPases regulate collective migration remains unknown. Rap1 GTPase, which belongs to the Ras subfamily, functions in cell-cell and cell-ECM connections, two important characteristics related to epithelial collective migration. I have shown that Rap1 activates Cdc42 in controlling epithelial collective migration, which may occur through a Cdc42 GEF, β-PIX, and a scaffold protein, IQGAP1. In addition to Rap1, depletion of Rho, Rac, and Cdc42 induced collective migration defects in the human bronchial epithelial cell line (16HBE cells). Since RhoGTPases are activated by guanine nucleotide factors (GEFs) and inactivated by GTPase-activating proteins (GAPs), to understand which GEFs control RhoGTPases in epithelial collective migration, I performed a small hairpin RNA screen targeting GEFs in 16HBE cells. Combining biological approaches and computational analysis, we discovered that SOS1, a Rac/Ras dual GEF, mediates Ras pathway in controlling epithelial collective migration. In addition to SOS1, we also found that ARHGEF18 controls RhoA and its downstream effector, myosin, in epithelial collective migration. Additionally, depletion of ARHGEF3, ARHGEF11, and ARHGEF28, Rho specific GEFs, also induced migration defects that were different from those induced by RhoA-depletion. Finally, depletion of several GEFs caused tight junction defects and also induced migration defects, which suggests that intact tight junctions may be required for epithelial collective migration. Overall, this study provides a potential mechanism by which Rap1 regulates epithelial collective migration and a comprehensive analysis of Rho GEFs involved in epithelial collective migration.