The goal of the Section on Membrane Biology is to uncover the molecular mechanisms by which specific proteins first break and then reseal the lipid bilayers of the merging membranes during membrane remodeling processes such as enveloped virus entry into cells by membrane fusion and exit from the cells by membrane fission. Based on our previous work and theoretical calculations of membrane free energy, we developed the stalk-pore hypothesis of membrane fusion. The hypothesis postulates a hemifusion intermediate during membrane fusion and predicts that the transition to the hemifusion intermediate should be facilitated or inhibited depending on elastic properties of the lipids in membrane monolayers. Breaking of this hemifusion intermediate with formation of a fusion pore also involves bending of lipid monolayers and thus likewise depends on its elastic properties. Taking advantage of the wide spectrum of fusion assays available in the LCMB, we discovered that the disparate physiological and pathological fusion reactions and the fusion of phospholipid bilayer membranes converge to a common, lipid-sensitive stage in accord with the hypothesis. Furthermore, we have shown that fusion protein conformational change occurs upstream of the lipid-arrested stage and that the work accomplished by the fusion proteins is retained by the system. Subsequent relaxation of inhibitory lipid compositions regains fusion.

By altering membranes’ lipid composition to one that is nonpermissive for fusion, we have uncoupled the actual membrane rearrangement from stages preceding membrane docking and fusion triggering. We study the pathway of membrane merger as an interplay of proteins and membrane lipids. To fuse, membranes must bend, and we postulate that all biological fusion processes involve transient formation of bent lipid-involving intermediates. The hypothesis is that the energy of the intermediates depends on the elastic properties of membrane monolayers. By altering the particular geometry of fusion sites, fusion proteins may control the energy of fusion intermediates and, thus, fusion rates. To test the hypothesis, we systematically study intermediates of membrane fusion, the character of fusion protein-lipid bilayer interactions, and the mechanisms of lipid bilayer rearrangements. Our studies on the effects of the different membrane properties, the relationship between the structure and function of the proteins involved, and the physics of the membrane interaction should eventually lead us to better control over a wide array of biological fusion/fission processes.

Functional Activity of a Polypeptide Fragment of Influenza Hemagglutinin
Leikina, Chernomordik
Last year we focused on the conformational changes in the HA2 subunit of influenza hemagglutinin (HA) coupled to membrane fusion. We investigated the fusogenic activity of the polypeptide FHA2 representing 127 amino terminal residues of the ectodomain of HA2. While the conformation of FHA2 at both neutral and low pH is nearly identical to the final low pH conformation of HA2, we found that FHA2-induced lipid mixing between bound cells depends on low pH, indicating that the "spring-loaded" energy is not required for FHA2-mediated membrane merging. Although, unlike HA, FHA2 did not form an expanding fusion pore, both acidic pH and membrane concentrations of FHA2, required for lipid mixing, have been close to those required for HA-mediated fusion. Similar to what is observed for HA, FHA2-induced lipid mixing was reversibly blocked by lysophosphatidylcholine and low temperature (4�C). The same genetic modification of the fusion peptide inhibits both HA- and FHA2- fusogenic activities. The kink region of FHA2, critical for FHA2-mediated lipid mixing, was exposed in the low pH conformation of the whole HA before fusion. The close similarity between the abilities of FHA2 and HA to mediate lipid mixing is consistent with the hypothesis that hemifusion requires just a portion of the energy released in the conformational change of HA at acidic pH. In contrast, opening of an expanding fusion pore connecting two membrane compartments requires low pH-triggered refolding of multiple full-sized HA molecules. To understand more fully the energetics of a membrane pore membrane, we studied voltage-induced pores in protein-free lipid bilayers.

Properties of the Lipidic Pores
Melikov, Chernomordik
Electric fields promote pore formation in both biological and model membranes. We clamped unmodified planar bilayers at 150 to 550 mV to monitor transient single pores over long periods. We observed fast transitions between different conductance levels reflecting opening and closing of metastable lipid pores. While the mean lifetime of the pores was 3 ms (250 mV), some pores remained open for up to about 1 second. The mean amplitude of conductance fluctuations was independent of voltage and similar (about 500 pS) for bilayers of different areas (40,000 and 10 square microns), indicating the local nature of the conductive defects. The distribution of pore conductance was broad (dispersion of about 250 pS). Based on the conductance value and its dependence on ion size, we estimated the radius of the average pore to be about 1 nm. Short bursts of conductance spikes (opening and closing of pores) were often separated by periods of background conductance. Within the same burst, the conductance between spikes was indistinguishable from the background. The mean time interval between spikes in the burst was much smaller than that between adjacent bursts. The data indicate that opening and closing of lipidic pores proceed through some electrically invisible ("silent") prepores. Similar prepore defects and metastable conductive pores might be involved in remodeling cell membranes in different biologically relevant processes.