Characterization of the molecular mechanisms by which R-Ras regulates cell adhesion

Integrin-mediated adhesion is a complex cellular event that can be broken down into specific steps: ligand engagement, spreading, polarization and focal adhesion formation. Each step is regulated by the biophysical nature of the ECM and by intracellular signal transduction pathways. R-Ras, an atypical member of the Ras subfamily of small GTPases, enhances integrin-mediated adhesion via a poorly understood mechanism. For my thesis, I have addressed molecular mechanisms by which R-Ras increases cell spreading, hoping to shed light on R-Ras biology and our basic understanding of cell-environment interactions.; Cells expressing constitutively active R-ras spread extensively upon adhesion. Using time-lapse microscopy in combination with Total Internal Fluorescence (TIRF), I demonstrated that R-Ras sustains initial membrane protrusion events that subsequently drive cell spreading. The membrane protrusions were characterized by dense, ruffling, actin structures (i.e. ruffling lamellipod) and were devoid of filopodia. Analysis of actin assembly in R-Ras cells demonstrated that the capping and severing protein gelsolin was required for the ruffling lamellipod. In contrast, the anticapping protein mena was negatively regulated, explaining the lack of filopodia during R-Ras cell spreading. Gelsolin is activated by increased cytosolic calcium concentrations and inhibited by phosphatidylinositol-4,5-bisphosphate (PIP2). Consistent with gelsolin activity in the ruffling lamellipod, I found that inhibiting phospholipase C pathways and/or depleting cytosolic calcium reduced R-Ras-mediated cell spreading. Biochemical analyses show that R-Ras directly binds and stimulates PLCepsilon. I propose a novel R-Ras effector pathway whereby R-ras stimulates phospholipase Cepsilon, resulting in downstream regulation of actin dynamics that promote integrin activation and cell spreading.; Additionally, I characterized the subcellular distribution of R-Ras and its spatio-temporal regulation in live cells using microscopic techniques and biochemical assays. With Fluorescence lifetime imaging microscopy (FLIM), I found that R-Ras associates with specific subcellular microdomains dependent on its activation state. GFP-R-Ras localizes in endosomal compartments and traffics to the plasma membrane upon activation. There, R-Ras specifically associates with cholesterol-rich membrane microdomains and plasma membrane ruffles. Importantly, the plasma membrane localization of activated R-Ras was dependent on integrin activation. Therefore, R-ras cellular activities are regulated via its membrane localization, which is dynamically regulated by integrin.