New roles of Rac-GEFs in Neutrophils and in Glucose Homeostasis

Abstract

Rac-GEFs (guanine-nucleotide exchange factors) are proteins that activate Rac GTPases, thereby enabling Rac-dependent cytoskeletal dynamics and cellular processes such as adhesion and migration. I used mice with genetically modified Rac-GEFs to identify new functional roles of these proteins in two distinct biological systems, neutrophil adhesion/migration and glucose homeostasis. In the first part of my thesis, I investigated cytoskeletal dynamics controlled by the Rac-GEF Tiam1 in neutrophil adhesion/migration. We previously found a paradoxical increase in the adhesion of Tiam1–/– neutrophils (unpublished). This was surprising, as deficiencies in other neutrophil Rac-GEFs impair adhesion. I showed deregulated neutrophil polarisation, Filamentous-actin (F-actin) polarity, F-actin dynamics and focal adhesion structures in Tiam1–/– neutrophils adhering to integrin ligands. I demonstrated increased integrin avidity in Tiam1–/– neutrophils stimulated with CXCL1, and increased migration of Tiam1–/– neutrophils under shear stress. These results contribute to our elucidation of the mechanisms underlying the paradoxical increase in the adhesion of Tiam1-deficient neutrophils. In the second part, I investigated spatiotemporal patterns of Rac activity generated during neutrophil adhesion/migration by several major neutrophil Rac-GEFs. The aim was to identify specific roles for these Rac-GEFs which all signal in response to the activation of GPCRs. I used our Rac activity FRET reporter mouse strain (RFC) (Johnsson, Dai et al. 2014), crossed to mice deficient in the Rac-GEFs P-Rex1/Vav1, DOCK2 or Tiam1. I demonstrated that Rac activity is increased in RFC Tiam1–/– neutrophils adhering to integrin ligands, which may explain the increased adhesion. In contrast, RFC DOCK2–/– and RFC P-Rex1–/– Vav1–/– neutrophils had reduced Rac activity, as expected for Rac-GEF deficient cells, confirming a unique and paradoxical role of Tiam1 in limiting Rac activity and Rac-dependent cell responses. The loss of Rac activity was global in RFC DOCK2–/– neutrophils but more localised in RFC P-Rex1–/– Vav1–/– cells. This project has identified specific roles of various Rac-GEFs in neutrophil adhesion and migration. In the third and final part, I investigated adaptor functions of P-Rex family Rac-GEFs P-Rex1 and P-Rex2 in glucose homeostasis. We previously showed that P-Rex1 and P-Rex2 deficient mice have accelerated glucose clearance during glucose challenge, along with low fasting blood glucose levels and altered insulin sensitivity (unpublished). In order to address the underlying mechanisms, I used mice with catalytically inactive P-Rex1 or P-Rex2 (GEF-dead mice) which we recently generated (unpublished). I demonstrated that the increased glucose clearance is an adaptor function of P-Rex Rac-GEFs, whereas fasting blood glucose levels and insulin sensitivity are Rac-GEF activity dependent. I showed increased plasma insulin levels in P-Rex1–/– and P-Rex2–/– mice upon glucose challenge and increased glucose-stimulated insulin secretion from P-Rex1–/– and P-Rex2–/– pancreatic islets. Use of P-Rex1 GEF-dead mice showed that these latter phenotypes were again adaptor functions, suggesting that these responses contribute to the accelerated glucose clearance in P-Rex-deficient mice. Combined, my work has provided a substantial body of data identifying unexpected novel roles for Rac-GEFs in both neutrophil biology and in glucose homeostasis, providing mechanistic insight in addition to new functions in both systems.