Proper membrane physiology requires maintenance of biophysical properties, which must be buffered from external perturbations. While homeostatic adaptation of membrane fluidity to temperature variation is a ubiquitous feature of ectothermic organisms, such responsive membrane adaptation to external inputs has not been directly observed in mammals. Here, we report that challenging mammalian membranes by dietary lipids leads to robust lipidomic remodeling to preserve membrane physical properties. Specifically, exogenous polyunsaturated fatty acids are rapidly incorporated into membrane lipids, inducing a reduction in membrane packing. These effects are rapidly compensated both in culture and in vivo by lipidome-wide remodeling, most notably upregulation of saturated lipids and cholesterol, resulting in recovery of membrane packing and permeability. Abrogation of this response results in cytotoxicity when membrane homeostasis is challenged by dietary lipids. These results reveal an essential mammalian mechanism for membrane homeostasis wherein lipidome remodeling in response to dietary lipid inputs preserves functional membrane phenotypes.

Extracellular vesicles (EVs) including exosomes and microvesicles play critical roles in intercellular communication by exchanging proteins, lipids, and genetic materials between cells. However, the origin and biogenesis of these vesicles remain unclear. It has been noted that almost all EVs present the anionic membrane lipid phosphatidylserine (PS) on their outer leaflet, in opposition to their cells of origin, which almost exclusively retain PS in the cytoplasmic leaflet. The "scrambling” of PS from the inner to the outer leaflet of the bilayer is facilitated by the lipid channel Ano6. This process of lipid scrambling has been implicated in a variety of physiological contexts, including apoptotic cell removal, bone mineralization, viral infection and blood coagulation. However, the biophysical role of PS externalization in the formation of extracellular vesicles is still a mystery. Using micrometer-sized giant plasma membrane vesicles (GPMVs) that are produced by membrane blebbing from mouse B-cells (BaF3), we studied the role of PS externalization in membrane vesiculation. We first found that a scramblase (i.e. Ano6) knockout BaF3 cell line was deficient in producing large vesicles compared with the wild type cell line, indicating that PS externalization is necessary for membrane budding. Importantly, through fluorescence lifetime imaging microscopy (FLIM) using a reporter of lipid packing (Di4), we found that PS exposure lead to a decrease of plasma membrane packing, potentially make the membrane softer for bending and thus facilitating membrane budding. To confirm this assumption, we artificially decreased the lipid order on the plasma membrane by increasing the incubation temperature or treating the cells with methyl-β-cyclodextrin (MβCD). Under all conditions which reduced membrane stiffness, large vesicles were efficiently produced from the PS externalization-deficient Ano6 knockout cell line, confirming the crucial role of membrane stiffness in cell vesiculation.

Compositional asymmetry between the two leaflets of a membrane bilayer is a fundamental feature of eukaryotic plasma membranes. Maintaining lipid asymmetry is energetically demanding, requiring active transport of phospholipids from one leaflet to the other. Phospholipid asymmetry has been described largely in the context of apoptosis, where translocation of the anionic lipid phosphatidylserine from the inner to the outer leaflet of the plasma membrane is a marker for macrophage engulfment. However, it has recently become evident that lipid asymmetry can also be reversible, and regulates many physiological processes, including immune cell signalling and cell-cell fusion. Owing to their distinct compositions, the two leaflets of the plasma membrane are believed to have distinct physical properties. Besides the commonly noted surface charge, the hypothesized differences in sphingolipid content and acyl chain composition suggest that other physical properties (lipid packing, lateral diffusion, permeability) would also be distinct between the two leaflets. Here, we test this hypothesis directly by using a combination of fluorescence lifetime imaging of order sensitive probes with microinjection and selective quenching. Our results show that in live cell plasma membranes, inner leaflet order is slightly, but significantly, lower compared to the outer leaflet. The magnitude of the difference is smaller than expected from compositional asymmetry, suggesting that strong coupling between leaflets in asymmetric membranes prevents high lipid order differentials. We also find that a loss of membrane asymmetry during phospholipid scrambling or apoptosis drastically reduces the order of the plasma membrane and eliminates the order differences between leaflets. Finally, we observe that these biophysical differences are important for cell physiology, as inhibition of lipid scrambling suppresses the functional activation of immune cells. These findings document the biophysical asymmetry of the plasma membrane and suggest an important role for this asymmetry in cell function.