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SNARE proteins constitute the core of the exocytotic membrane fusion machinery. Fusion occurs when vesicle-associated and target membrane-associated SNAREs zipper into trans-SNARE complexes ('SNAREpins'), but the number required is controversial and the mechanism of cooperative fusion is poorly understood. We developed a highly coarse-grained molecular dynamics simulation to access the long fusion timescales, which revealed a two-stage process. First, zippering energy was dissipated and cooperative entropic forces assembled the SNAREpins into a ring; second, entropic forces expanded the ring, pressing membranes together and catalyzing fusion. We predict that any number of SNAREs fuses membranes, but fusion is faster with more SNAREs.

SummaryUnderstanding how functional lipid domains in live cell membranes are generated has posed a challenge. Here, we show that transbilayer interactions are necessary for the generation of cholesterol-dependent nanoclusters of GPI-anchored proteins mediated by membrane-adjacent dynamic actin filaments. We find that long saturated acyl-chains are required for forming GPI-anchor nanoclusters. Simultaneously, at the inner leaflet, long acyl-chain-containing phosphatidylserine (PS) is necessary for transbilayer coupling. All-atom molecular dynamics simulations of asymmetric multicomponent-membrane bilayers in a mixed phase provide evidence that immobilization of long saturated acyl-chain lipids at either leaflet stabilizes cholesterol-dependent transbilayer interactions forming local domains with characteristics similar to a liquid-ordered (lo) phase. This is verified by experiments wherein immobilization of long acyl-chain lipids at one leaflet effects transbilayer interactions of corresponding lipids at the opposite leaflet. This suggests a general mechanism for the generation and stabilization of nanoscale cholesterol-dependent and actin-mediated lipid clusters in live cell membranes.

Proteins embedded in the plasma membrane mediate interactions with the cell environment and play decisive roles in many signaling events. For cell-cell recognition molecules, it is highly likely that their structures and behavior have been optimized in ways that overcome the limitations of membrane tethering. In particular, the ligand binding regions of these proteins likely need to be maximally exposed. Here we show by means of atomistic simulations of membrane-bound CD2, a small cell adhesion receptor expressed by human T-cells and natural killer cells, that the presentation of its ectodomain is highly dependent on membrane lipids and receptor glycosylation acting in apparent unison. Detailed analysis shows that the underlying mechanism is based on electrostatic interactions complemented by steric interactions between glycans in the protein and the membrane surface. The findings are significant for understanding the factors that render membrane receptors accessible for binding and signaling. publishedVersion

The molecular mechanism of ethanol and its effects on neurological function is far from clear. In this study, we investigate the effects of ethanol on various structural and dynamical properties of mixed bilayers consisting of different ratios of dipalmitoylphosphatidylcholine (DPPC), sphingomyelin (SM) and cholesterol that are typical constituents of neural cell membranes (Calderon et al., 1995) using molecular dynamics (MD) simulations. The bilayer properties such as thickness, hydrophobic chain order, and diffusive motion of individual lipids as well collective properties like lateral pressure profiles are affected by the presence of ethanol molecules. The simulations show that the percentage of cholesterol present in the bilayers significantly affects the depth of penetration of ethanol molecules. In particular, presence of very high concentration of cholesterol molecules enhances the rigidity of the bilayer and renders them resistant to the penetration of the ethanol molecules, consistent with experiments. Ethanol molecules compete with cholesterol molecules for hydrogen bonding and disrupt cholesterol-lipid interactions, especially those between SM and cholesterol. Ethanol molecules also affect the lateral pressure profiles in the bilayer systems. These results may have implications in understanding the general anesthetic mechanism and role played by cholesterol on partitioning of such anesthetic/alcohol molecules into cell membranes.

Membrane proteins that couple to cortical actin show temperature-independent diffusion. The loss of this coupling and perturbation of cortical actomyosin dynamics render the diffusion temperature dependent. The findings suggest that active fluctuations arising from dynamic actin filaments at the cortex can drive diffusion on the cell membrane., Molecular diffusion at the surface of living cells is believed to be predominantly driven by thermal kicks. However, there is growing evidence that certain cell surface molecules are driven by the fluctuating dynamics of cortical cytoskeleton. Using fluorescence correlation spectroscopy, we measure the diffusion coefficient of a variety of cell surface molecules over a temperature range of 24–37°C. Exogenously incorporated fluorescent lipids with short acyl chains exhibit the expected increase of diffusion coefficient over this temperature range. In contrast, we find that GPI-anchored proteins exhibit temperature-independent diffusion over this range and revert to temperature-dependent diffusion on cell membrane blebs, in cells depleted of cholesterol, and upon acute perturbation of actin dynamics and myosin activity. A model transmembrane protein with a cytosolic actin-binding domain also exhibits the temperature-independent behavior, directly implicating the role of cortical actin. We show that diffusion of GPI-anchored proteins also becomes temperature dependent when the filamentous dynamic actin nucleator formin is inhibited. However, changes in cortical actin mesh size or perturbation of branched actin nucleator Arp2/3 do not affect this behavior. Thus cell surface diffusion of GPI-anchored proteins and transmembrane proteins that associate with actin is driven by active fluctuations of dynamic cortical actin filaments in addition to thermal fluctuations, consistent with expectations from an “active actin-membrane composite” cell surface.

The spatial distribution and remodeling dynamics of nanoscale clusters of outer-leaflet GPI-anchor proteins (GPI-APs), glycolipids and inner-leaflet Ras proteins are regulated by the activity of the underlying cortical acto-myosin machinery. Recently, we proposed a theoretical framework based on active hydrodynamics of short polar filaments interacting with myosin motors to explain the nanoclustering of the GPI-APs. This framework involves the coupling of cell-surface molecules to these dynamic filaments of actin at the inner leaflet, this in turn leads to their transient clustering. We predicted and experimentally confirmed that transmembrane proteins with cytoplasmic domains capable of directly binding filamentous actin (TM-ABD), would be organized both at nanoscale (