Endocytic regulation of epithelial polarity, proliferation and signaling in Drosophila melanogaster

How epithelial cells form distinct apical and basolateral membrane domains is a fundamental question in cell biology. This thesis focuses on the regulation of epithelial polarity in Drosophila. Specifically, how do the basic cellular trafficking pathways control apicobasal polarity? And can polarity alteration directly cause overproliferation?;To answer these questions, I take advantage of the directed-mosaic system, which generates and marks mitotic clones in a tissue-specific manner, to screen in the follicular epithelium for mutations that disrupt the highly organized epithelial architecture.;Here I report that mutations in two core components of the vesicle trafficking machinery - a syntaxin and a Rab protein - cause an expansion of the apical membrane domain of Drosophila epithelia; this polarity defect is coupled with overproliferation to form neoplastic tumors. Surprisingly, these proteins are associated not with the exocytic but rather the endocytic pathway. The syntaxin, called Avalanche (Avl), localizes to early endosomes, and loss of avl leads to the cellular accumulation of specific membrane proteins, including the Notch signaling receptor and the polarity determinant Crumbs (Crb). Protein accumulation results from a failure of endosomal entry and progression towards lysosomal degradation; these and other avl phenotypes are also seen in Rab5 null mutant cells. These findings reveal a critical and specific role for endocytic traffic in the control of both apicobasal polarity and cell proliferation.;Overexpression of Crb alone is sufficient to induce overproliferation of wild-type imaginal tissue, suggesting that polarity alterations in avl and Rab5 mutants directly contribute to tumor formation. However, eliminating Crb in imaginal discs mutant for vps45, a novel endocytic mutant that has similar phenotypes to avl and Rab5 and is likely to function in the same endocytic pathway, does not rescue vps45 phenotypes. This raises the possibility that there are other polarity regulators that function redundantly with Crb.;Despite accumulation of Notch proteins at or near the cell surface, the expression of several Notch reporters and target genes are reduced in avl mutant cells. These findings suggest that Notch signal transduction is not directly responsible for avl phenotypes. It further indicates that Avl-dependent endocytosis regulates Notch pathway activation. In Drosophila ovaries where ligands for Notch signal from the germline, Notch signaling activity is reduced in Rab5 mutant follicle cells, suggesting that endocytosis is required cell-autonomously in signal-receiving cells for Notch activation. I find reduced levels of the S3-cleaved product of Notch in avl and hypothesize that endocytosis may facilitate S3 cleavage and thus Notch activation by transporting Notch receptors to specific intracellular compartments where the active S3-cleaving enzyme gamma-secretase resides. This work on Notch trafficking and signaling provides opportunities for further in vivo studies on the role of endocytosis in intracellular signaling in Drosophila..;In summary, this study identifies endocytic trafficking as a novel mechanism in regulating epithelial polarity and proliferation and presents new insights into the regulation of Notch signaling by cellular endocytosis.