Epithelial cell polarity and proliferation control in Drosophila melanogaster
The characteristic size, shape, and patterning of animal tissues and organs are regulated by the growth, division, and communication of the cells that make up those tissues. In many cases, the proper functioning of these constituent cells requires the polarized subcellular organization of proteins, lipids and cytoskeletal structures. This is especially true of apicobasally polarized epithelial cells, which make up the majority of tissues in metazoan animals, including mammals. The distinction between apical and basolateral domains in epithelial cells is essential for their roles in absorption, secretion, and protection. Furthermore, numerous diseases, including cancer, involve the loss of apicobasal polarity in epithelial tissues. Therefore, understanding the basic cell biological mechanisms controlling cell polarity is a critical step toward understanding the development and homeostasis of multicellular organisms.
The epithelia of the fruit fly Drosophila melanogaster have proven to be an invaluable model system for identifying the genes and cellular pathways controlling apicobasal polarity. Genes required for epithelial cell polarity include components of conserved polarity-regulating complexes and, surprisingly, regulators of the endocytic pathway. Moreover, genes in both classes are also required for proliferation control, and have been categorized as neoplastic tumor suppressor genes (nTSGs). However, the pathways by which nTSGs coordinate cell polarity and proliferation remain poorly characterized. Here I describe experiments which shed light on not only the molecular functions of individual regulators, but also how these regulators interact to accomplish concerted control of cell polarity and proliferation.
In the first part of this dissertation, I address how endocytosis could regulate epithelial cell polarity. The endocytic pathway has been demonstrated to control polarity not only in epithelial cells, but also in non-epithelial cells such as asymmetrically dividing neuroblasts in Drosophila and the embryo of the worm Caenorhabditis elegans. Surprisingly, polarity regulators have also recently been shown to control traffic through the endocytic pathway. I review the experimental data to support each side of this reciprocal regulation, and discuss several potential models for how the coordinated activities of endocytosis and polarity complexes together mediate cell polarity. In addition, I show how two of the first endocytic nTSGs to be identified - the endocytic syntaxin avalanche (avl) and the small GTPase Rab5 - are connected. While Rab proteins and syntaxins have well-established roles in regulating intracellular vesicle trafficking, how exactly Rab5 activity could be linked to syntaxin-mediated vesicle fusion was previously unknown. Here I provide genetic and biochemical evidence that Rab5 is molecularly coupled to Avl-mediated fusion into the endosome by Rabenosyn and Vps45, which are themselves required for endocytic polarity and proliferation control.
In the second part of this dissertation, I present experiments addressing how polarity and proliferation control could be linked, and how the known nTSGs might mediate this coregulation. In addition to controlling polarity, mutations in the endocytic and junctional nTSGs also disrupt proliferation control in the imaginal discs, though to date, the disruption in polarity has only been correlated with overproliferation; the molecular pathways linking the two phenotypes are unknown. As an approach toward discovering these pathways, I performed a forward genetic screen to identify genes that interact with the nTSG lethal giant larvae (lgl). I identified a number of regions of the genome which genetically interact with lgl, as well as several candidate genes. One such candidate is a gene required in the N-linked glycosylation pathway, which I show is mutated in the previously identified tumor suppressor tumorous imaginal discs (tid). I provide evidence that N-linked glycosylation is indeed required for epithelial growth control, and further demonstrate intriguing links between N-linked glycosylation and tumor suppression pathways.