| Communication among nerve cells is the cellular basis of most, if not all, brain functions. To establish a highly precise yet plastic neuronal network, the nerve cells develop long neurites in the form of axons and dendrites. Studies in this thesis aim to characterize and define functional mechanisms of exocyst, a protein complex whose function is essential for exocytosis underlying many cell biological processes, including neurite outgrowth.;First, what is the composition and the localization of the exocyst complex inside the cell? To address this question, we generated monoclonal antibodies against individual exocyst subunits. By using these antibodies we found that multiple exocyst subunits coimmunoprecipitated, co-migrated on the density gradient and exhibited microtubule-like localization. These results suggest that the majority of exocyst subunits are likely to exist as part of an intact exocyst complex in the cell, and that the exocyst complex may associate, directly or indirectly, with microtubules.;Second, what is the functional relationship between the exocyst complex and microtubules? To address this question, we investigated the effect of exocyst subunits on microtubule dynamics. In vitro tubulin polymerization assay showed that the addition of exocyst complex or exo70 alone inhibited tubulin polymerization into microtubules. Consistently, overexpression of exo70 destabilized microtubule ends in NRK cells. These findings suggest that the exocyst complex, via the exo70 subunit, may modulate microtubule dynamics at microtubule ends.;Third, can exocyst activity influence neurite outgrowth? To our disappointment, no significant enhancement of neurite outgrowth was detected. However, we were surprised to find that exo70 overexpression enabled neurite outgrowth from hippocampal and cortical neurons to occur in the presence of Ephrin 5A and NOGO, two common neurite outgrowth inhibitors in the central nervous system. The ability of exo70 overexpression to bypass the effect of these inhibitors was due to, at least in part, its inhibition of Rho activation.;Taken together, we propose that the exocyst subunits function as a complex by coordinating the transfer of secretory vesicles from microtubules to cortical actin and the plasma membrane. |
