Endo-exocytic Trafficking in Regulation of Cdc42 Polarity

Leah Joy Watson: Endo-exocytic Trafficking in Regulation of Cdc42 Polarity (Under the direction of Patrick Brennwald) The precise subcellular localization of the Rho GTPase Cdc42 is essential for its spatial and temporal control of polarized growth and division. In budding yeast, the activation and clustering of Cdc42 on the cell surface designates the site of emergence for the daughter bud and it is towards this site that the actin cytoskeleton and exocytic pathways orient to promote bud formation. In turn, exocytic delivery of Cdc42 along actin cables has been suggested as a mechanism to reinforce Cdc42's own polarized localization at the bud tip. Recycling via endocytosis and GDI-dependent mechanisms are posited to contribute to Cdc42's polarized localization by offsetting lateral membrane diffusion of Cdc42 molecules from the concentrated pool. The intimate relationship between Cdc42's function in cell polarity and the maintenance of its own localization by the pathways it regulates has been extensively studied, however, the molecular mechanisms involved in the determination and maintenance of Cdc42 polarity remain unclear. Using a novel in vivo assay developed in the lab, we found that disrupting distinct stages of endocytosis severely disrupted the ability of Cdc42 to associate with secretory vesicles. This implicates the possible involvement of multiple endocytic compartments in the sorting of Cdc42 as well as the enrichment of Cdc42 iv into sites of endocytosis. We also demonstrate that GFP-tagged Cdc42 is highly defective in its ability to associate with vesicles. Although GFP-Cdc42 has been a valuable tool for understanding mechanisms involved in Cdc42 polarity, our findings demonstrate differences in the itineraries of the tagged and untagged Cdc42 proteins—which were previously assumed to be similar. We also demonstrate that the concentration of Cdc42 on vesicles is significantly lower than the concentrated pool on the cell surface, which demonstrates that vesicle delivery of Cdc42 is not, alone, sufficient to support Cdc42 polarity. This provides the first direct experimental support for a negative regulatory role for vesicle transport in Cdc42 polarity. v To my supportive parents, my grandmother and departed loved ones " …you'll always be my Joy " vi ACKNOWLEDGEMENTS