Project Summary Epithelial cell polarization is an essential biological process, serving many physiological roles including tissue morphogenesis and wound healing. A defining feature of polarization is the separation of cell plasma membrane (PM) lipids and proteins into apical and basolateral compartments between which molecular exchange is restricted. Decades ago, the apical PM was found to be enriched in saturated lipids, glycolipids, and cholesterol. Mechanistic hypotheses to explain the biogenesis and unique composition of the apical PM include self- assembling membrane domains (i.e., lipid rafts), specific protein sorting motifs, and post-translational modifications mediating protein sorting. However, neither the detailed composition nor the mechanisms of protein and lipid sorting between PM domains in epithelial cells have been resolved. The lipid profile of the basolateral PM remains unresolved, leaving doubts about the differentiation and lipid separation of the apical and basolateral PM. The general determinants of protein sorting are poorly understood, with past studies focusing on either specific proteins or trafficking machinery. Importantly, the apical PM hosts an extensive extracellular glycocalyx consisting of glycolipids, glycoproteins, and polysaccharides, which can be crosslinked by sugar binding proteins called lectins natively present in the extracellular space. The role of these glycocalyx molecules on protein sorting has not been revealed, despite a major fraction of apical proteins being glycosylated. Finally, the apical PM of polarized epithelia takes on highly outward-curved membrane shapes such as cilia or microvilli, but must also be simultaneously capable of forming inward membrane invaginations for cell signaling and uptake from the extracellular space. The driving forces for the formation of these highly-curved membrane structures remain undetermined. We will investigate these knowledge gaps in membrane and epithelial biology. In Aim 1, we will use advanced lipidomics and imaging techniques to characterize the changes in lipid organization, membrane composition, and membrane properties during the cellular polarization process. We hypothesize that the apical PM will be enriched in highly saturated lipids and glycolipids relative to the basolateral PM, with apical PM biophysical properties reflecting a raft-enriched environment. In Aim 2, we will systematically evaluate the transmembrane protein structural determinants of apical versus basolateral sorting including transmembrane domain features, protein raft affinity, and glycosylation. We hypothesize that these protein features cooperatively direct protein sorting to the apical PM. In Aim 3, we will explore the role of the apical PM glycocalyx in driving membrane bending. We hypothesize that lectin-mediated interactions control the curvature and organization of membranes. Successful execution of these aims will address several largely open questions in membrane biolog...