Cholesterol modulates the bilayer structure of biological membranes in multiple ways. It changes the fluidity, thickness, compressibility, water penetration and intrinsic curvature of lipid bilayers. In multi-component lipid mixtures, cholesterol induces phase separations, partitions selectively between different coexisting lipid phases, and causes integral membrane proteins to respond by changing conformation or redistribution in the membrane. But, which of these often overlapping properties are important for membrane fusion? – Here we review a range of recent experiments that elucidate the multiple roles that cholesterol plays in SNARE-mediated and viral envelope glycoprotein-mediated membrane fusion.

SNAREs mediate membrane fusion in intracellular vesicle traffic and neuronal exocytosis. Reconstitution of membrane fusion in vitro proved that SNAREs constitute the minimal fusion machinery. However, the slow fusion rates observed in these systems are incompatible with those required in neurotransmission. Here we present a single vesicle fusion assay that records individual SNARE-mediated fusion events with millisecond time resolution. Docking and fusion of reconstituted synaptobrevin vesicles to target SNARE complex-containing planar membranes are distinguished by total internal reflection fluorescence microscopy as separate events. Docking and fusion are SNAP-25-dependent, require no Ca 2؉ , and are efficient at room temperature. Analysis of the stochastic data with sequential and parallel multi-particle activation models reveals six to nine fast-activating steps. Of all the tested models, the kinetic model consisting of eight parallel reaction rates statistically fits the data best. This might be interpreted by fusion sites consisting of eight SNARE complexes that each activate in a single ratelimiting step in 8 ms.Neurotransmitter release in synaptic transmission by fusion of synaptic vesicles with the presynaptic membrane is tightly regulated and is probably the fastest membrane fusion event in mammalian cells. Synaptic vesicles are primed and docked to the plasma membrane but do not fuse until triggered by an influx of Ca 2ϩ from opened Ca 2ϩ channels. After electrical stimulation, neurotransmitter release is observed in less than 1 ms (1-3). The neuronal fusion and disassembly machinery is composed of the soluble N-ethylmaleimide-sensitive factor, N-ethylmaleimide-sensitive factor attachment proteins (SNAPs), 2 and the SNAP receptors (SNAREs) syntaxin1a (Syx1a), SNAP-25, and synaptobrevin2 (Syb). Proteins such as the Ca 2ϩ sensor synaptotagmin, complexin, Sec1/Munc18 homologs, Munc13, and synaptophysin are involved in regulating the fusion process, and Rab GTPases function as upstream tethering factors (4 -6). SNARE proteins, which assemble during fusion with equimolar stoichiometry into a parallel four-helix coiled-coil structure with their C termini oriented toward their respective membranes (7-9), play the most essential role in this machinery (10 -12). Energy released from a proposed N 3 C folding process pulls the two membranes together, deforms them, and eventually fuses them in a process that is mechanistically still poorly understood. SNARE-mediated fusion between target (t)-SNARE (Syx1a and SNAP-25) and vesicle (v)-SNARE (Syb) liposomes has been reconstituted in vitro (12). This and many subsequent similar studies were initially criticized because the reaction was very slow (minutes to hours). Adding a C-terminal fragment (residues 49 -96) of Syb to the Syx1a/SNAP-25 heterodimer, resulting in a ternary acceptor-SNARE complex, increased the rate of fusion with Syb liposomes by more than an order of magnitude, presumably by preventing the formation of a nonproductive 2:1 Syx1a⅐SNAP-25 complex (13)...

Cell membranes have complex lipid compositions, including an asymmetric distribution of phospholipids between the opposing leaflets of the bilayer. Although it has been demonstrated that the lipid composition of the outer leaflet of the plasma membrane is sufficient for the formation of raft-like liquid-ordered (l(o)) phase domains, the influence that such domains may have on the lipids and proteins of the inner leaflet remains unknown. We used tethered polymer supports and a combined Langmuir-Blodgett/vesicle fusion (LB/VF) technique to build asymmetric planar bilayers that mimic plasma membrane asymmetry in many ways. We show that directly supported LB monolayers containing cholesterol-rich l(o) phases are inherently unstable when exposed to water or vesicle suspensions. However, tethering the LB monolayer to the solid support with the lipid-anchored polymer 1,2-dimyristoyl phophatidylethanolamine-N-[poly(ethylene glycol)-triethoxysilane] significantly improves stability and allows for the formation of complex planar-supported bilayers that retain >90% asymmetry for 1-2 h. We developed a single molecule tracking (SPT) system for the study of lipid diffusion in asymmetric bilayers with coexisting liquid phases. SPT allowed us to study in detail the diffusion of individual lipids inside, outside, or directly opposed to l(o) phase domains. We show here that l(o) phase domains in one monolayer of an asymmetric bilayer do not induce the formation of domains in the opposite leaflet when this leaflet is composed of palmitoyl-oleoyl phosphatidylcholine and cholesterol but do induce domains when this leaflet is composed of porcine brain phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and cholesterol. The diffusion of lipids is similar in l(o) and liquid-disordered phase domains and is not affected by transbilayer coupling, indicating that lateral and transverse lipid interactions that give rise to the domain structure are weak in the biological lipid mixtures that were employed in this work.

Lipid rafts in plasma membranes have emerged as possible platforms for entry of HIV and other viruses into cells. However, how lipid phase heterogeneity contributes to viral entry is little known due to the fine-grained and still poorly understood complexity of biological membranes. We used model systems mimicking HIV envelopes and T-cell membranes and showed that raft-like (Lo phase) lipid domains are necessary and sufficient for efficient membrane targeting and fusion. Interestingly, membrane binding and fusion was low in homogeneous Ld and Lo phase membranes, indicating that lipid phase heterogeneity is essential. The HIV fusion peptide preferentially targeted to Lo/Ld boundary regions and promoted full fusion at the interface between ordered and disordered lipids. Ld phase vesicles proceeded only to hemifusion. Thus, we propose that the edges, but not the areas of raft-like ordered lipid domains are vital for HIV entry and membrane fusion.

Pannexin 1 (PANX1) subunits form oligomeric plasma membrane channels that mediate nucleotide release for purinergic signalling, which is involved in diverse physiological processes such as apoptosis, inflammation, blood pressure regulation, and cancer progression and metastasis. Here we explore the mechanistic basis for PANX1 activation by using wild type and engineered concatemeric channels. We find that PANX1 activation involves sequential stepwise sojourns through multiple discrete open states, each with unique channel gating and conductance properties that reflect contributions of the individual subunits of the hexamer. Progressive PANX1 channel opening is directly linked to permeation of ions and large molecules (ATP and fluorescent dyes) and occurs during both irreversible (caspase cleavage-mediated) and reversible (α1 adrenoceptor-mediated) forms of channel activation. This unique, quantized activation process enables fine tuning of PANX1 channel activity and may be a generalized regulatory mechanism for other related multimeric channels.