UC San Diego

Beyond compartmentalization and protection, cellular membranes are highly dynamic and specialized structures that perform molecular transport and serve as platforms for protein localization and modulation of function. The lipid composition of membranes defines their identity, material properties, interacting partners, and cellular functions. While bilayer lipids establish the dominant fluid lamellar topology, nonbilayer lipids destabilize uniform lipid packing and introduce elastic curvature stress that enables morphological changes, membrane fission and fusion, and protein interaction. Cells expend considerable energy to establish specific lipid compositions for specialized membrane compartments, and a growing body of literature has provided evidence for tight homeostatic regulation of lipid composition and biophysical properties. Through combinations of headgroups, linkages, and acyl chain chemistry, eukaryotic cells produce more than 2000 species of lipids, many of whose functional roles remain unknown. Thus, the central goal of my thesis has been to identify nonbilayer lipids in cells, characterize their biophysical contributions to membrane function, and identify lipidomic signatures of pathways by which cells regulate intrinsic curvature.In Chapter 1, I introduce fundamental concepts in lipid biophysics, focusing on lipid intrinsic curvature and nonbilayer lipids, and provide a review of modern methods for studying the biophysical properties of membranes and lipids. I highlight the diversity of nonbilayer lipids in organelle and cell types, the roles of nonbilayer lipids in membrane fission and fusion, how nonbilayer lipids modulate membrane protein localization and activity, and open questions in the field pertaining to active regulation of nonbilayer lipid composition in cell membranes.In Chapter 2, I identify nonbilayer lipids as an evolutionary adaptation of deep-sea ctenophores to extreme hydrostatic pressure environments. SAXS analysis of polar lipid extracts from shallow and deep sea ctenophores indicated that lipid adaptations preserve access between lamellar and nonlamellar membrane topologies under compression. Using lipidomic profiling, I find that these organisms selectively enrich plasmalogens, a nonbilayer lipid class. Characterization of plasmalogens using small angle X-ray scattering (SAXS) showed that plasmalogens have high negative spontaneous curvature, and the heterologous expression of plasmalogen biosynthesis in Escherichia coli improved their pressure tolerance.In Chapter 3, I extend these observations to model eukaryotic organisms and show that cells acclimate their lipidomes to curvature stress imposed by hydrostatic pressure incubation by increasing production of negative curvature lipids. A human cancer cell line showed increases in ether-linked lipids under pressure, while budding yeast increased production of phosphatidylinositol, which I characterized to bear negative spontaneous curvature.In Chapter 4, I utilize hydrostatic pressure as an experimental variable to interrogate calcium-induced fusion of large unilamellar vesicles across a range of nonbilayer-to-bilayer lipid ratios. Kinetic analysis reveals that lipid intrinsic curvature features an exponential relationship with the rate of hemifusion stalk formation, demonstrating the biophysical link between lipid composition and membrane fusion energetics.In summary, this dissertation presents work demonstrating that the balance between bilayer and nonbilayer lipids in biological membranes is important to cellular fitness. I present biophysical and lipidomic evidence that lipid intrinsic curvature is actively regulated by eukaryotic cells in ways similar to how deep-sea marine life has adapted to survive under extreme hydrostatic pressure. Hydrostatic pressure is established as an experimental tool to induce lipid curvature stress in both cells and synthetic membranes. These findings will motivate subsequent work linking the biophysical properties of nonbilayer lipids to mechanisms governing specialized membrane behavior, its regulation, and pathologies arising from their disruption.