(263a) Composition and Sorting of Polarized Cell Plasma Membrane Compartments

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 lipids and proteins into apical and basolateral compartments between which molecular exchange is restricted. Decades ago, the apical membrane was found to be enriched in saturated lipids, glycolipids, and cholesterol. Mechanistic hypotheses to explain the biogenesis and unique composition of the apical membrane 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 plasma membrane domains in epithelial cells have been resolved. Particularly, the lipid profile of the basolateral membrane remains unstudied, leaving doubts about the differentiation and lipid separation of the apical and basolateral membrane. The general determinants of protein sorting are poorly understood, with past studies focusing on either specific proteins or trafficking machinery. Importantly, the apical membrane hosts an extensive extracellular glycocalyx consisting of glycolipids, glycoproteins, and polysaccharides. The role of these glycocalyx molecules on protein sorting has not been revealed, despite a major fraction of apical proteins being glycosylated. We use advanced lipidomics, specially designed lipid-anchored probes, 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 membrane will be enriched in highly saturated lipids and glycolipids relative to the basolateral membrane, with apical membrane biophysical properties reflecting a raft-enriched environment. Further, we systematically evaluate the transmembrane protein structural determinants of apical versus basolateral sorting including transmembrane domain features, protein raft affinity, and glycosylation using bioengineered raft and non-raft protein probes. We hypothesize that these protein features cooperatively direct protein sorting to the apical membrane.