The ability of epithelial cells to polarize with distinct apical and basolateral domains is integral to their function, with the apical membrane being morphologically structured by actin-containing microvilli. Recent work has shown that the microvillar component EPI64 regulates the presence of microvilli on epithelial cells, and that it binds Arf6-GTP and affects its regulation. This proposal seeks to illuminate the role of these molecules in the formation of microvilli, and the relationship between them. In the first aim, a mutant of EPI64 unable to bind Arf6-GTP will be constructed and its effect on Arf6 regulation determined, and on cell microvilli assessed by high-resolution spinning disk confocal microscopy. The potential pathways downstream of Arf6 in microvilli formation will then be examined, and a role for EPI64 in this Arf6 effect will be sought. A directed yeast 2-hybrid screen may be used as an alternative method to generate EPI64 mutants defective in binding Arf6. In aim 2, the direct association between EPI64 and Arf6, as well as the EPI64 binding partners EBP50 and ezrin (which binds through EBP50) will be examined using purified proteins to determine if their direct binary interactions are affected by neighboring interactions, with a focus on determining binding constants for the interactions. These assays will be complemented with an in vivo analysis of the same interactions with a particular emphasis on how these interactions are affected by Arf6 activity or cell polarization. These assays will also be exploited to determine the contribution of EBP50 to EPI64's role in microvilli formation. The final aim will seek to identify new binding partners for EPI64 that may contribute to its role on cell morphology, with special emphasis on interactions regulated by Arf6 activity levels or cell polarization. These assays use co-immunoprecipitation followed by overlay assays to identify directly-binding partners, which will then be identified by mass-spec. Alternatively, affinity chromatography utilizing immobilized EPI64 on placental extracts may be used to identify new binding-partners. Relevance: This study will provide insight into the functioning of polarized epithelial cells. This type of cell is prevalent in multicellular organisms and forms the lining of internal structures, such as the intestine or kidney. Misregulation of the functioning of these cells is found in disease states such as cancer.