Ral GTPases regulate biogenesis of cell polarity

Cell polarity is the asymmetric distribution of organelles that almost all cells use to separate individual processes and perform complex functions. Although the manner in which cells are polarized is very diverse, the processes necessary to assume polarized phenotypes are similar in many cell types. Epithelial cell polarization is of particular importance, as these cells serve form linings of organs and act as barriers distinguishing different compartments. Furthermore, loss of epithelial polarization occurs in some disease states and may result in cell invasion through underlying matrix. During initial polarization, vesicle trafficking is indispensible for assembly of structures, including apical junctional complex formation. Trafficking of new membrane and associated proteins to leading edges is also necessary for cell migration. RalA and RalB are members of the Ras superfamily of GTPases and have been implicated in several processes, including vesicle trafficking. Only 5 Ral effectors have been identified, two of which are members of the Exocyst complex, a hetero-octameric complex also involved with vesicle trafficking. I hypothesized that Ral GTPases were necessary for several aspects of cell polarization, and that they engage the Exocyst complex to mediate these processes. Initial investigation of tight junction assembly found that both RalA and RalB antagonistically affect paracellular permeability. Knockdown of RalA and RalB resulted in decreased and increased incorporation of components into assembling tight junctions, respectively. Furthermore, both RalA and RalB engaged the Exocyst in order to mediate tight junction assembly. I next examined the role of RalA-Sec5 and RalA-Exo84 interactions during tumor cell migration and invasion. Both interactions were necessary for invasion and single cell migration, although disruption of each interaction affected different aspects of migration. Furthermore, significant differences in cytoskeleton organization occurred in response to disruption of RalA-Sec5 and RalA-Exo84 interactions. Finally, I investigated the effects of RalA and RalB knockdown on growth of primary cilia and cyst formation. RalA decreased primary cilia growth and reduced average cilia length, while RalB increased cilia length. Knockdown of RalA and RalB also affected lumen formation during cystogenesis, as RalA knockdown prevented lumen formation and RalB knockdown caused formation of multiple lumens. Taken together, data presented here show that Ral engages the Exocyst to mediate distinct processes during tight junction assembly and cell migration, and implicates Ral GTPases in several different aspects of cell polarity.