Investigating the spatial and temporal regulation of mammalian constitutive exocytosis

Eukaryotic cells utilize membraneous vesicles to transport or traffic proteins and macromolecules between organelles in the cell. This dissertation investigates how constitutive membrane trafficking from the Golgi to the plasma membrane is spatially controlled during the final stages of cell division known as cytokinesis. Additionally, I examine how exocytosis is regulated by the exocyst, a vesicle tethering complex that functions at the plasma membrane, which is essential for cytokinesis and many other cellular processes. I demonstrate here that during cytokinesis, both daughter cells direct post-Golgi vesicles in a symmetric fashion to the cleavage furrow region between the dividing cells. These vesicles accumulate forming a reserve membrane pool that remains distinct from other organelles in this region, and eventually undergo exocytosis. I show that despite the symmetric nature of post-Golgi vesicle trafficking into the cleavage furrow, only one of the daughter cells inherits the proteinaceous midbody structure, suggesting that cytokinesis is comprised of symmetric and asymmetric steps.;I also explore the function of the octameric exocyst protein complex in vesicle tethering at the plasma membrane in interphase cells. Tethering is the penultimate step of constitutive membrane trafficking immediately upstream of vesicle fusion. I find that depletion of any one of seven of the eight complex subunits reduces the number of tethered vesicles at the plasma membrane, and affects the duration of vesicle tethering and fusion rates. The eighth exocyst subunit, Sec8, has a unique phenotype in that depletion of this protein leads to increased exocytosis and a shorter vesicle tethering duration. I show a connection between Sec8 phosphorylation and the epidermal growth factor signaling pathway that could account for upregulation of exocytosis through encouraging complex disassembly. This finding could provide insight into regulation of constitutive membrane trafficking during cellular processes such as cytokinesis and cell migration.