Date of Award

Summer 8-13-2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biology

First Advisor

Wei Guo

Abstract

The exocyst, an evolutionarily conserved octameric protein complex, plays a crucial role in the tethering of post-Golgi secretory vesicles to the plasma membrane for exocytosis and cell migration. How the exocyst is targeted to sites of exocytosis and how this complex regulates cell migration are poorly understood. I have carried out experiments to characterize Exo70, a component of the exocyst complex. Firstly, I found that Exo70 directly interacts with phosphatidylinositol 4,5-bisphosphate through positively charged residues at its C-terminus, and this interaction is critical for the plasma membrane targeting of Exo70. Using the ts045 vesicular stomatitis virus glycoprotein trafficking assay, I found that the Exo70-lipid interaction is critical for the docking and fusion of post-Golgi secretory vesicles with the plasma membrane. Secondly, I demonstrate that the exocyst plays a pivotal role in tumor cell invasion. Invadopodia are actin-rich membrane protrusions formed by tumor cells that degrade the extracellular matrix for invasion. Depletion of the exocyst component Exo70 or Sec8 led to failure in invadopodia formation in MDA-MB-231 cells expressing constitutively active c-Src, whereas the overexpression of Exo70 promoted invadopodia formation. Disrupting the exocyst function by RNA interference of EXO70 or SEC8, or by expression of a dominant negative fragment of Exo70 blocked the secretion of matrix metalloproteinases. I further found that the exocyst interacts with the Arp2/3 complex in cells with high invasion potential; blocking the exocyst-Arp2/3 interaction inhibited Arp2/3-mediated actin polymerization and invadopodia formation. Finally, the exocyst also functions in directly regulating Arp2/3-mediated actin polymerization. Using pyrene actin assay and total internal reflection fluorescence microscopy, I found that Exo70 synergizes with WAVE2 to promote Arp2/3-mediated actin polymerization and branching. In addition, I have examined how the Exo70-Arp2/3 interaction is regulated in the cell downstream of growth factor signaling. Overall, these studies provide mechanistic insights to the function of the exocyst in exocytosis and cell migration.

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