Investigating lipid and protein organization in cell membranes

Abstract

Cell membrane encloses the cell, separates intracellular contents from the extracellular environment, and mediates the transport of the solutes and information in and out of the cell. The core structure of the cell membrane is based on a phospholipid bilayer that forms a permeability barrier. Proteins that function as material or information carriers are inserted into the bilayer or are bound to it. In itself, the cell membrane is a self-assembled system, with both lipids and proteins distributed in a non-random manner. This organization is necessary for transport, signal transduction, cell-cell communication and identification. Lipids in cell membranes are organized laterally and transversely. In the transverse direction, `reactive¿ lipids like phosphatidyl serine (PS) are enriched in the cytoplasmic leaflet, while `non-reactive¿ lipids like phosphatidyl choline (PC) are enriched in the extracellular leaflet. This asymmetry is maintained by energy (ATP)-dependent processes. The organization in the lateral direction is less well understood. Heterogeneities in lipid composition¿sometimes termed lipid rafts¿on the order of a few tens of nanometers are thought to exist. They are also thought to be crucial for various signaling events. Model systems that mimic both the lateral and the transverse organization of lipids in cell membranes are scarce. Supported phospholipid bilayers (SLBs) are popular cell membrane models. Using SLBs on titania (TiO2), I developed a model that mimicked physiological lipid compositions as well as the asymmetric transverse organization characteristic of the cell membrane organization. While cells use ATP to sustain lipid asymmetry, in this model system, the lipid-surface adhesion energy supplies the free energy necessary to offset the entropy of mixing between the two leaflets. During developing this model, the process of SLB formation on TiO2 surfaces was investigated using a wide variety of lipid compositions and buffer conditions. Lipid diffusion and organization in these SLBs was studied. I was able to demonstrate that the electrostatic interaction between zwitterionic liposomes and negatively charged surface was repulsive, and visualize that the excess lipid leaves the surface during SLB formation in the form of elongated, tubular structures. Membrane proteins organize into clusters which are involved in cellular processes such as ion transport, signal transduction, and cell-cell adhesion. Common examples of proteins that function as dimers or oligomers include rhodopsin, OmpF porin, and F-type ATPase (ATP synthase). Oligomerization of some other proteins such as Na+, K+-ATPase, and H+, K+-ATPase, two P-type ATPases, is still under debate. Evidence supporting their oligomerization mainly comes from functional and kinetic studies. Direct evidence visualizing their organization in cell membranes is still lacking. As an example of a transmembrane protein organization, I investigated the supramolecular organization of Na+, K+-ATPase in the near-native membrane patches of the outer medulla of rabbit kidney. Using AFM, I showed that this protein is present in the near-native patches as oligomers of various orders, with tetramers (¿¿)4 being the most commonly occurring motif. This Thesis provides insights into the self-organization of lipids and proteins in cell membranes, and helps to understand the mechanisms underlying cellular behavior.