The role of Rab23 in epithelial morphogenesis, polarity and lumen formation.

Polarized epithelial sheets provide all multicellular organisms with a physical and functional barrier between the body and the external world. Epithelial cells line internal tubes and cavities as single layers surrounding a central lumen. The polarization of epithelial cells requires membrane trafficking routes that asymmetrically distribute proteins and lipids to distinct plasma membrane domains. In recent years, recycling endosomes (REs) and their associated molecules have emerged as key sorting platforms for polarized traffic in epithelial cells. Our laboratory and others have shown that RE-associated proteins, in particular members of the Rab family of small GTPases, help to regulate epithelial polarity and lumen formation. A screen for novel RE-associated proteins revealed that nearly 25% of all mammalian Rab GTPases were associated with REs. A candidate of particular interest was Rab23, which was first discovered as a negative regulator of the Hedgehog signalling pathway, and has been implicated in several human carcinomas and developmental defects. However, the trafficking functions or molecular mechanisms of Rab23 remain largely unknown. A central aim of this study was to investigate a potential role for Rab23 in epithelial cells and fibroblasts in order to elucidate its function in vertebrate development, epithelial morphogenesis and carcinogenesis. On this basis, results presented here show that GFP-Rab23 localizes on RE membranes and on the plasma membrane of epithelial Madin-Darby Canine Kidney (MDCK) cells, as well as on the Preciliary Pericentriolar Compartment (PPC), a RE-based compartment localized around the base of primary cilia in fibroblasts. The role of Rab23 in epithelial polarity and morphogenesis was examined using loss and gain-of-function approaches in MDCK monolayers and three- dimensional cyst cultures. Overexpression of GFP-Rab23 increased cell height and packing, whereas siRNA-mediated Rab23 depletion resulted in mislocalization of adherens junction proteins E-cadherin and b-catenin without affecting apical trafficking. The most profound effects of GFP-Rab23 overexpression were found in MDCK cells grown in Matrigel as cysts to emulate epithelial organs. High levels of Rab23 expression produced aberrant cysts with loss of cell polarity, absence of lumen formation and aberrant over-proliferation of cells. Furthermore, de novo lumen formation was disrupted by overexpression of wild-type and dominant-negative Rab23, as well as by shRNA-mediated Rab23 knockdown. Both overexpression and depletion of Rab23 impaired the delivery of apical marker gp135/podocalyxin to the cyst interior and the generation of an apical surface to initiate lumen formation. These findings reveal a key role for Rab23 in polarity and lumen formation, underscoring the notion that precise control of Rab23 dosage is critical for epithelial morphogenesis. Approaches taken to identify potential binding partners of Rab23, including a mass spectrometry-based proteomic assay, revealed that several members of the molecular network for lumen initiation could potentially form complexes with Rab23. Some of the relevant proteins identified in the screen that are directly implicated in epithelial biogenesis include: master polarity regulator Cdc42, RE Rabs Rab8 and Rab11, apical determinant gp135/podocalyxin and actin-binding motor protein myosin IIA. These interactions support a role for Rab23 in multiple aspects of epithelial morphogenesis such as cell polarity, cell shape, cell-cell adhesion, lumen formation and ciliogenesis. Deregulation of Rab23 in any of these cellular processes is likely to severely impact epithelial homeostasis and lead to cancer, thus we propose that Rab23 expression is subjected to precise spatiotemporal regulation and its localization, function, and molecular interactions are likely to be context-dependent throughout the polarization of cells and in tissue formation. The findings presented in this thesis significantly advance our understanding of Rab23 function, placing Rab23 in the context of epithelial homeostasis, and opening up new avenues for the study of Rab23 at the cellular and molecular levels through the analysis of Rab23 and its potential binding partners. This work establishes a basis for future investigations of Rab23 in vertebrate development and cancer, and provides new perspectives on the biological relevance of Rab GTPases and REs in epithelial morphogenesis in health and disease states.