Date of Award

2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biology

First Advisor

Wei Guo

Abstract

Dynamic shape changes of the plasma membrane are fundamental to many processes ranging from morphogenesis and cell migration to phagocytosis and viral propagation. In this study, I showed that Exo70, a component of the exocyst complex, induces tubular membrane invaginations towards the lumen of synthetic vesicles in vitro and generates actin-free protrusions on the surface of cells. Analyses using Exo70 mutants suggest that Exo70 generates negative membrane curvature through an oligomerization-based mechanism. The membrane-deformation function of Exo70 is likely to be independent of the other exocyst subunits. Exo70 thus represents a novel membrane-deforming protein for plasma membrane remodeling. Directional cell migration requires the coordination of actin assembly and membrane remodeling. Exo70 directly interacts with and activates the Arp2/3 complex, a central nucleating factor for the generation of branched actin networks for cell morphogenesis and migration. Here I found that both the stimulatory effect of Exo70 on Arp2/3 and the membrane-deformation function of Exo70 are required for lamellipodia formation and maintaining directional persistence of cell migration. Exo70 thus may couple actin dynamics and plasma membrane remodeling during cell migration. The late stage of exocytosis is regulated by the exocyst and SNARE complexes. The secretory vesicles are first tethered to the plasma membrane by the exocyst and then docked and fused to the plasma membrane by SNARE complex. Here I showed that the exocyst component Sec3, based on its sequence similarity to a SNARE-binding protein amisyn, directly binds to the plasma membrane SNARE Syntaxin4. Both Sec3 and amisyn binds to PI(4,5)P2 through basic residues on the N-terminus. Sec3 may regulate MMP and VSV-G exocytosis in cells. These results reveal a novel direct interaction between the exocyst and the plasma membrane SNARE and suggest that Sec3 may regulate the SNARE activity during exocytosis.

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