Date of Award
Doctor of Philosophy (PhD)
The exocyst is a conserved hetero-octameric protein complex responsible for tethering secretory vesicles to the plasma membrane prior to vesicle fusion. The exocyst associates with various factors on both the vesicular and plasma membranes throughout the process of tethering, including small GTPases and the SNARE proteins themselves. As such, these interactions influence not only where the exocyst tethers the vesicle on the plasma membrane, but also how the vesicle ultimately fuses. This theme of conferring spatial control to exocytosis has been central to decades of exocyst research, which over time has implicated the exocyst in myriad cellular processes. Consequently, genetic depletion of the exocyst is often cytotoxic or lethal, and exocyst dysfunction has been linked to various human diseases and cancers. While it is clear that proper exocyst function is essential for cellular life from yeast to mammal, it remains poorly understood how the exocyst regulates or otherwise participates in these processes. The main obstacle to such understanding is methodological in nature: conventional experimental manipulations that impair exocyst function (e.g., genetic mutation, RNAi-mediated interference) require a long time to take hold, leading to chronic or indirect effects that obscure what the exocyst actually does. In this dissertation, we surmount this obstacle primarily through two technical approaches. First, we use sensitive, two-color total internal reflection microscopy (TIRFM) to image exocyst-mediated vesicle fusion, in living cells and in real time. Second, we employ complementary acute manipulations of exocyst function— namely, plasma membrane recruitment through an optogenetic heterodimerization system, or blocking such recruitment with the small-molecule inhibitor Endosidin2 (Es2). Both manipulations take effect within minutes rather than hours, potentially revealing which processes most directly rely on the exocyst. Based on such approaches, the work presented in this thesis uncovers a direct, novel role of the exocyst in the regulation of receptor tyrosine kinase (RTK)-dependent signaling, namely the EGFR/PI3K/AKT pathway. Specifically, our imaging experiments in HeLa cells show that full fusion of exocyst-associated vesicles is spatiotemporally correlated with PIP3 production, indicating localized PI3K activation near the site of fusion. Optogenetic recruitment of the exocyst triggers PIP3 production on the plasma membrane, whereas pharmacological inhibition with Es2 impairs both basal levels and EGF-stimulated generation of PIP3. Then, through various biochemical experiments in cancer epithelial cell lines (e.g., A431 and MCF7), we show that these acute manipulations similarly affect the activation of the signaling kinase AKT, which is a downstream effector of PIP3. Further experiments show that exocyst function is required to activate AKT upon stimulation with other growth factors, while others confirm that Es2 does not disrupt total levels or activation of upstream EGFR. Together, these findings place the exocyst at a signaling node closely downstream of various RTKs. Finally, preliminary experiments that feature dual treatments with Es2 and established EGFR tyrosine kinase inhibitors (TKIs) suggest possible drug synergy and reversal of acquired TKI resistance. As such, pharmacologically targeting the exocyst to enhance the efficacy of other cancer treatments may be of clinical interest.
Anneken, Alexander Michael, "Regulation of PI3K/AKT Signaling by the Exocyst Complex" (2022). Yale Graduate School of Arts and Sciences Dissertations. 553.