Novel TIRF microscopy analyzing trafficking & signaling at the cell cortex

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My major goal is to advance knowledge about events on or near the plasma membrane. This region directly controls membrane traffic to and from the cell surface (exo- and endocytosis) and is where extracellular signals are amplified and modulated by assembly of signaling scaffolds. The introduction of total internal reflection fluorescence (TIRF) microscopy, a technique that allows unprecedented axial resolution, has revolutionized studies of dynamic processes at the cell cortex. I propose 1) to develop two highly innovative multi-angle TIRF microscopes and 2) to apply these instruments towards the elucidation of mechanisms that regulate exo- and endocytosis. These microscopes will allow the penetration depth of the light beam to be varied rapidly and avoid traditional imaging artifacts. Together with new analytical methods, they will permit high-resolution 3D imaging of a ~50- 1000 nanometer cortical region of living cells. Additionally, a highly innovative FRAP implementation will allow us to `pulse' photoactivate single vesicles and track their fate. I will use this novel instrumentation to expand our ongoing studies on exo- and endocytic traffic. A main new goal will be to elucidate mechanisms in the vesicular trafficking pathways that regulate levels of glucose transporters (Glut4) at the cell surface, a process whose dysfunction leads to type 2 diabetes. I will test the hypothesis that the exocyst complex participates in the spatial regulation of the insulin responsiveness of Glut4 vesicle exocytosis. Using photoactivatable Glut4-Dendra I will determine whether insulin signaling triggers a switch from lipid raft to clathrin-mediated endocytic pathways. To address where PI3K signaling acts, I will implement inducible dimerization technology to rapidly turn on/off PI(3,4,5)P3 at the plasma membrane. The innovative approaches of this proposal capitalize on my unique expertise in interdisciplinary research spanning instrumentation, cell biology, and quantitative biology and will fundamentally impact biology and a medically important field.