Investigating the roles of scribble and discs -large in epithelial polarity in Drosophila melanogaste

How cells become polarized is a fundamental and unanswered question in cell biology. The processes underlying how cells direct the asymmetric distribution of proteins and lipids to achieve and maintain this polarity have been most thoroughly studied using a simple cell type, epithelia. Epithelial tissues are also an attractive system for dissecting the progression of cancer, as the loss of polarity is coupled with the loss of growth regulation in malignant tumors. Two proteins that have been shown to be essential in Drosophila melanogaster for both epithelial structure and proliferation control are the membrane-associated scaffolding proteins Scribble (Scrib) and Discs-large (Dlg). The focus of this thesis is on understanding how Scrib and Dlg perform their roles in both polarity and growth control. What are the cellular and molecular mechanisms, and what can this reveal about the establishment and maintenance of polarity? How is epithelial polarity linked to the control of cell proliferation?;To investigate the relationship between polarity and proliferation, I performed a structure-function analysis of the multidomain scaffolding protein Scrib. Using an allelic series of mutations along with transgenic constructs, I have identified the domain requirements for polarity and proliferation control. The leucine-rich repeat (LRR) domain localizes Scrib to the plasma membrane, is both necessary and sufficient to organize a polarized epithelial monolayer, and is required for all proliferation. The PDZ domains anchor the LRR at the junctional complex, but are dispensable for overall epithelial organization. Absence of the PDZ domains leads to mild polarity defects accompanied by moderate overproliferation, but these domains alone are not sufficient to provide any Scrib function in mutant imaginal discs. This data is consistent with a model in which Scrib, via the activity of the LRR, governs proliferation primarily by regulating apical-basal polarity.;In order to identify binding partners and gain understanding as to how Scrib functions, I performed two different yeast two-hybrid screens. A pilot screen with the first two PDZ domains of Scrib as bait identified a known Scrib-binding protein, GUKH, validating the yeast two-hybrid approach. Screening cDNA libraries with the Scrib LRR domain as bait resulted in the identification of the proteins Zf30c and Ral A as Scrib partners. However, the interaction between Scrib and these two candidate partners was not confirmed using numerous secondary assays.;I also utilized biochemical approaches in order to identify associated proteins to understand how Scrib and Dlg provide their cellular functions. Using FLAG co-immunoprecipitation and analysis by mass spectrometry, Clathrin heavy chain was identified as a Dlg-associated protein. TAP pulldowns were utilized to purify Scrib-TAP and associated proteins, but these experiments must be scaled up to identify Scrib partners. In summary, this work has presented evidence that Scrib controls proliferation through the regulation of polarity and provided preliminary data that could link the two classes of neoplastic tumor suppressors in Drosophila, junctional and endocytic, with the identification of Clathrin as a Dlg-binding protein.