Regulation of cellular signaling pathways by endocytosis and protein degradation

The defined shapes and sizes of the diverse tissues that comprise multicellular organisms are intimately linked to their specialized functions. The plethora of known cellular behaviors and characteristics that contribute to tissue function and development comes in large part from any given cell's ability to receive and respond to signals from its environment. These signals are often received via transmembrane protein receptors that interpret extracellular signals and transmit them inside the cell to effect cellular changes. Cellular signaling pathways influence cellular shape, proliferative capacity, cell fate, cell movement and many other traits that are crucial for proper cellular and tissue function both during development and over the lifetime of an animal. Therefore, understanding the mechanisms by which cells regulate these signals is a central question in biology. Drosophila is an ideal model organism in which to study the regulation of cellular signaling pathways. In addition to the ease of genetic manipulation, Drosophila contains epithelial tissues comparable to those in mammals, which have defined apico-basal polarity that is essential for tissue function. Cell polarity signaling pathways in epithelial cells can be studied by the isolation of neoplastic tumor suppressor genes (TSGs) from genetic screens. Drosophila neoplastic TSGs are genes whose functions are absolutely required for proper apico-basal polarity and the regulation of epithelial cell proliferation, though the underlying mechanisms of this regulation and the links between polarity and proliferation remain unknown. In addition to studying how these genes regulate cellular polarity, the identification of certain classes of neoplastic TSGs, such as the endocytic regulators, has given us tools to investigate the regulation of other developmentally important signaling pathways. A number of cellular signaling pathways are regulated by endocytosis, which is commonly thought to play a role in the attenuation of signaling, but recently appreciated to provide a much more complex method of regulation that may in some contexts promote signaling as well. In general, the roles of both cargo internalization routes and subsequent trafficking down a degradative or recycling pathway in the regulation of cellular signaling pathways are not understood. In Chapter 2 I investigate the role of endocytosis on both trafficking and signaling regulation of the Notch/Delta pathway in Drosophila. I address the roles of specific components of the endocytic internalization machinery in the endocytosis of Notch and Delta, and define whether distinct paths of entry into the cell impact Notch signaling. In Chapter 3, I explore how endocytic trafficking regulates epithelial cell polarity and identify links between the endocytic and junctional scaffold classes of tumor suppressors. In particular, I investigate the possibilities that a common underlying mechanism of tumor suppression exists for these different classes. Another mechanism through which cells regulate signaling pathways is the ubiquitin-proteasome system. In Drosophila, one well-known example of this is the negative regulation of Hedgehog and Wingless signaling pathways via the ubiquitylation of downstream effectors by the E3 ubiquitin ligase SCFSlmb. In a screen for new neoplastic TSGs, a component of this E3 ubiquitin ligase was recovered along with many endocytic regulators. In Chapter 4, I detail my unexpected discovery that slmb regulates the epithelial cell polarity signaling pathway. I further describe my progress toward finding a presumed polarity-regulating Slmb substrate via a genetic interaction screen. In sum, my graduate work sheds new light on the ways in which basic cell biological mechanisms, including endocytosis and regulated proteolysis, regulate specific signaling pathways to control cell fate, proliferation, and polarity