The aim of these studies is to elucidate structural and dynamic mechanisms of molecular machinescomposed of phosphoinositide/protein complexes as they function in modulating membrane processes. Wefocus on insulin-mediated GLUT4 glucose transporter translocation to the plasma membrane of adipocytesas our model system, a seminal problem in biology that merges the fields of cell signaling and membranetrafficking. Application of unique TIRF technology already developed within this Program Project, and newtechnology to be developed in Project 4, positions us to answer previously intractable questions in this field.We propose to define trajectories and dynamics of GLUT4-containing vesicles within about 200nm of theplasma membrane (TIRF zone), using novel methods that enable localization of particles at resolutions of50nm or less. New data obtained since the last submission shows we can now identify and quantifydocking and fusion events during insulin-mediated exocytosis of these vesicles in the TIRF zone.Thus, we can now address the central hypothesis that insulin regulates the kinetics of GLUT4-containing vesicle docking/fusion processes through modulating vesicles associated with either of 2specific functional phosphoinositide-based protein complexes. We hypothesize that one subset ofGLUT4-containing exocytic vesicles rapidly recycle and fuse through a mechanism that depends uponPI(3)P/Rabenosyn-5/EHD1/EHBP1/Rab5/Rab4 complexes. We hypothesize that a second subset of GLUT4-containing exocytic vesicles more slowly recycle and fuse through a mechanism that depends uponPI(3,4,5)P3/FIP2/EHD1/EHBP1/Rab11 complexes. All our efforts are now directed to testing these focusedhypotheses related to the nature of GLUT4-containing exocytic vesicles that proceed to docking and fusionwith the plasma membrane. We propose to define the 3D paths that GLUT4-containing vesicles traverseduring recycling and exocytosis relative to other cargo(EGFR and transferrin receptor with Project 2). Usinghigh resolution TIRF microscopy with exofacially labeled myc-GLUT4-EGFP, we propose to track trajectoriesof insulin-sensitive, GLUT4-containing vesicles to test whether GLLJT4 in Rab5-positive early endosomesrapidly recycles to fuse with the plasma membrane in a 'short circuit' pathway. The combination of uniqueimaging tools with biochemical approaches provides a powerful way to dissect out the functions ofphosphoinositide-based complexes containing EHD1 and Rab proteins in GLUT4 dynamics. We shall alsotest whether complexes of PI(3,4,5)P3/Akt2/substrates function in docking/fusion of GLUT4-containingvesicles and regulate specific steps in these pathways in the TIRF zone. With Project 3, we will also definethe membrane topographies and structures of molecular complexes that act on GLUT4-containing vesicles inthe TIRF zone, and elucidate the mechanisms involved in their actions.
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