Abstract Research in my laboratory has centered on the development and application of multi-disciplinary approaches to study signal transduction pathways important in normal physiology and, when de-regulated, in disease. This began with our discovery and cloning of a novel signaling partner for the epidermal growth factor receptor, the human Cdc42 protein, a small GTPase highly conserved from yeast to humans, and continued with the identification of many Cdc42 regulatory proteins and signaling targets. The conservation of Cdc42 throughout evolution accounts for the many fundamentally important roles it plays in cell biology and organism development, including the regulation of cell growth and migration, and the establishment of cellular polarity. We then discovered an unanticipated but highly significant function of Cdc42 in directing the upregulation of metabolic activities that generate the necessary building blocks for the biosynthetic processes required for a wide range of cellular functions. They also provide a mechanism to connect the various intracellular processes regulated by Cdc42 to the surrounding environment by directing the biogenesis of extracellular vesicles (EVs), which have now been implicated in mediating intercellular communication across the evolutionary spectrum from bacteria to higher organisms. Recently, we found that these metabolic activities are mediated through the assembly of large protein complexes. Understanding how these metabolic/signaling complexes assemble should hold important clues to their regulatory and catalytic mechanisms, as well as shed light on a long-standing question of how Cdc42 activates a critically important protein kinase, mTOR (mechanistic Target Of Rapamycin), as a necessary step for cap-dependent mRNA splicing, the neuronal differentiation of embryonic stem cells (ESCs), and the survival of cancer cells under stressful conditions. We have also recently discovered important roles for EVs in a number of biological contexts. Determining the biochemical and structural features of multi-protein signaling complexes that activate glutamine metabolism and mTOR, essential for the growth and survival of cells, and moving the exciting field of EVs forward, represent major research efforts for our laboratory in the next 5 years. We will address key gaps in our knowledge surrounding these broad areas of study by determining how Cdc42 directs the formation of signaling nodes with important functions in cell biology and significant implications for disease, by defining their unique biochemical characteristics and 3D structures. We also will set out to address questions representing important next steps for the development of the EV field by defining the biochemical determinants and signaling cues that dictate the loading of essential EV cargo, and by gaining insights into their structural features that enable their various biological functions. To achieve these goals, we will leverage our expertise in signal...