MODULATION OF ENDOCYTIC AND EXOCYTIC TRAFFIC IN POLARIZED EPITHELIAL CELLS

Efficient and selective delivery and retrieval of cargo to polarized plasma membrane domains are essential for the maintenance of epithelial cell function. My studies focused on events involved in protein endocytosis from and delivery to the apical surface. Endocytosis is a multi-step process that is crucial for cells to take up nutrients and modulate receptor density. Temporal synthesis and degradation of the lipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] is critical for endocytosis to proceed. PtdIns(4,5)P2 is generated by three differentially localized phosphatidylinositol 4-phosphate 5-kinase (PIP5KI) isoforms (α, β, and γ). My studies examined the requirement for PtdIns(4,5)P2 in apical and basolateral endocytosis in polarized kidney epithelial cells by testing the effects of overexpressing individual PIP5KI isoforms. I found that PtdIns(4,5)P2 synthesis by PIP5KIβ is rate limiting for apical but not basolateral endocytosis, and that this is likely due to changes in coated pit maturation at the apical plasma membrane. The selective modulation of apical endocytosis by PtdIns(4,5)P2 has potential consequences on the maintenance of kidney cell function. Maintaining apical and basolateral membrane domain composition requires selective vesicle fusion and is mediated by Soluble N-ethylmaleimide-sensitive factor Attachment protein Receptor (SNARE) proteins. A number of pathways exist for the delivery of newly synthesized proteins to these surface domains and the post-Golgi vesicle SNAREs, or VAMPs, that mediate fusion in these pathways have not been identified. I sought to determine the role of VAMP7 in these pathways. VAMP7 expressed in polarized Madin Darby canine kidney cells colocalized primarily with LAMP2-positive compartments, and changes in VAMP7 levels modulated lysosome size, consistent with the known function of VAMP7 in lysosomal delivery. Surprisingly, VAMP7 knockdown had no effect on apical delivery of numerous cargoes tested but did decrease the length and frequency of primary cilia and disrupted cyst morphology. The effects of VAMP7 depletion on ciliogenesis and cystogenesis are not directly linked to the disruption of lysosomal function, as cilia lengths and cyst morphology were unaffected in a MDCK lysosomal storage disorder model. Together, my data suggest that VAMP7 plays an essential role in ciliogenesis and cystogenesis. This is the first study implicating an R-SNARE in ciliogenesis and cystogenesis