Mechanistic Studies Of Membrane Remodeling Induced By Membrane Asymmetry
lipid number asymmetry
Bilayer lipid membranes are fundamental to the function of eukaryotic cells. A discernible characteristic of eukaryotic plasma membranes is the presence of asymmetry between the leaflets of the bilayer membrane. The generation and maintenance of this asymmetry is achieved by a specialized set of transmembrane proteins that consume metabolic energy. Membrane asymmetry affects a multitude of bilayer properties and critical cellular processes, several of which involve membrane remodeling. In this dissertation, I mechanistically study membrane remodeling induced by membrane asymmetry through in vitro experimental studies with biomimetic lipid bilayers. First, we investigated the mechanical consequences of a signaling lipid, PI(4,5)P2, in giant unilamellar vesicles (GUVs). By using a fluorescent marker, we found that PI(4,5)P2 lipids desorb from GUVs over time. An increase in the number of internally tubulated vesicles within minutes after dilution suggested that the desorption was asymmetric and generated membrane curvature. By means of a saturated chain homolog of PI (4,5)P2, we showed that the fast desorption of PI(4,5)P2 is facilitated by presence of an arachidonic lipid tail and is possibly due to its oxidation. Through pulling force measurements of membrane tethers, we quantified the effect of asymmetric desorption on the spontaneous membrane curvature. We found that the spontaneous curvature could be modulated by externally increasing the concentration of PI(4,5)P2 micelles. Therefore, PI(4,5)P2 could affect the formation of highly curved structures that may serve as initiators for signaling events in biological membranes. Second, to understand the generation of biological number asymmetry in vitro, we purified a bacterial ABC transporter, MsbA. This is a transmembrane protein found in the inner membrane of Gram-negative bacteria and is shown to unidirectionally translocate lipids. We sought to generate lipid number asymmetry in a bilayer by reconstituting this protein in a synthetic membrane system and observing curvature generation from its lipid translocation activity. We found that the purified and labeled MsbA preserved its secondary structure and functionality. Next, we functionally reconstituted MsbA in liposomes and subsequently into optically resolvable GUVs that allow control over membrane composition, tension, and curvature. Finally, we generated asymmetric membranes by means of two different lipid chelators — bovine serum albumin, and methyl-β-cyclodextrin. We showed that leaflet specific lipid extraction by these chelators led to increased internal tubulation. Furthermore, we quantified the extent of generated spontaneous curvature by means of tether pulling experiments.