Elucidating the functional significance of DOCK/ELMO Multimerization in Membrane Binding and Activation

Abstract

The ability of immune cells to polarizes, migrate, and clear host pathogens requires signal transduction at the cellular plasma membrane. Following chemokine receptor activation, lipid kinases rapidly generate secondary messengers, such as phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) at the plasma membrane. This recruits protein machinery required for downstream signaling events. The generation of PI(3,4,5)P3 is regulated in part by small GTPases. In the context neutrophils, Rac1 GTPase serves a critical role controlling actin network assemble to drive cell polarity and migration. Functioning upstream of Rac1, guanine nucleotide exchange factors (GEFs) control the spatiotemporal dynamics of Rac1 activation. Thus, understanding the molecular mechanisms of GEFs will give insight is important for deciphering cells polarize and migrate in complex environments. In immune cells, the Dedicator of cytokinesis (DOCK) and Engulfment and cell motility (ELMO) protein complex functions a GEF that controls Rac1 activation. Biochemical studies have shown DOCK/ELMO forms an autoinhibited quaternary complex composed for two heterodimers (i.e. (DOCK/ELMO)2). Biochemistry studies in solution have shown that models suggest the small GTPase RhoG can relieve DOCK/ELMO autoinhibition. However, the mechanisms controlling DOCK/ELMO activation on a lipid membrane have not been investigated. Furthermore, the physiological significance of multimerization in controlling DOCK/ELMO membrane and activity on membranes remains unclear. For my thesis, I used Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize monomeric and dimeric DOCK/ELMO complexes recruitment to supported lipid bilayers with single molecule resolution. Using this technique, I showed that the dimerization of DOCK/ELMO and RhoG affects membrane binding. RhoG also increases the GEF activity of dDOCK2/ELMO1. Mutation that preliminarily suggest that multimerization of DOCK2/ELMO1 reduces the catalytic activity. Overall, this work illustrates how DOCK/ELMO cofactors and dimerization influence activity along a lipid membrane.