Membrane differentiations at sites specialized for cell fusion

Fusion of plasma membranes between Chlamydomonas reinhardtii gametes has been studied by freeze-fracture electron microscopy of unfixed cells. The putative site of cell fusion developes during gametic differentiation and is recognized in thin sections of unmated gametes as a plaque of dense material subjacent to a sector of the anterior plasma membrane (Goodenough, U.W., and R.L. Weiss. 1975.J. Cell Biol. 67:623-637). The overlying membrane proves to be readily recognized in replicas of unmated gametes as a circular region roughly 500 nm in diameter which is relatively free of "regular" plasma membrane particles on both the P and E fracture faces. The morphology of this region is different for mating-type plus (mt+) and mt- gametes: the few particles present in the center of the mt+ region are distributed asymmetrically and restricted to the P face, while the few particles present in the center of the mt- region are distributed symmetrically in the E face. Each gamete type can be activated for cell fusion by presenting to it isolated flagella of opposite mt. The activated mt+ gamete generates large expanses of particle-cleared membrane as it forms a long fertilization tubule from the mating structure region. In the activated mt- gamete, the E face of the mating structure region is transformed into a central dome of densely clustered particles surrounded by a particle-cleared zone. When mt+ and mt- gametes are mixed together, flagellar agglutination triggeeeds to fuse with an activated mt- region. The fusion lip is seen to develop within the particle-dense central dome. We conclude that these mt- particles play an active role in membrane fusion.     

Membrane fusion

The process of membrane fusion in the case of lipid bilayers, as well as induced by influenza virus is reviewed shortly. The methods of studying fusion kinetics in pure lipid and lipid-protein systems are described. The main theories of molecular fusion machines are presented. Open questions and unsolved problems are discussed in details. In conclusion, possible ways to solve the remaining problems are suggested.     

Membrane transport: deciphering fusion

Membrane fusion events that occur in yeast have been reconstituted with a minimal set of SNARE protein components. This system has been exploited to establish the syntax underlying specificity of intracellular fusion events from yeast to mammals.     

Membrane fusion

Membrane fusion, one of the most fundamental processes in life, occurs when two separate lipid membranes merge into a single continuous bilayer. Fusion reactions share common features, but are catalyzed by diverse proteins. These proteins mediate the initial recognition of the membranes that are destined for fusion and pull the membranes close together to destabilize the lipid/water interface and to initiate mixing of the lipids. A single fusion protein may do everything or assemblies of protein complexes may be required for intracellular fusion reactions to guarantee rigorous regulation in space and time. Cellular fusion machines are adapted to fit the needs of different reactions but operate by similar principles in order to achieve merging of the bilayers.     

Progress of 1D protein structure prediction at last

Accuracy of predicting protein secondary structure and solvent accessibility from sequence information has been improved significantly by using information contained in multiple sequence alignments as input to a neural 'network system. For the Asilomar meeting, predictions for 13 proteins were generated automatically using the publicly available prediction method PHD. The results confirm the estimate of 72% three-state prediction accuracy. The fairly accurate predictions of secondary structure segments made the tool useful as a starting point for modeling of higher dimensional aspects of protein structure. © 1995 Wiley-Liss, Inc.     

Membrane permeability changes at early stages of influenza hemagglutinin-mediated fusion

While biological membrane fusion is classically defined as the leak-free merger of membranes and contents, leakage is a finding in both experimental and theoretical studies. The fusion stages, if any, that allow membrane permeation are uncharted. In this study we monitored membrane ionic permeability at early stages of fusion mediated by the fusogenic protein influenza hemagglutinin (HA). HAb2 cells, expressing HA on their plasma membrane, fused with human red blood cells, cultured liver cells PLC/PRF/5, or planar phospholipid bilayer membranes. With a probability that depended upon the target membrane, an increase of the electrical conductance of the fusing membranes (leakage) by up to several nS was generally detected. This leakage was recorded at the initial stages of fusion, when fusion pores formed. This leakage usually accompanied the "flickering" stage of the early fusion pore development. As the pore widened, the leakage reduced; concomitantly, the lipid exchange between the fusing membranes accelerated. We conclude that during fusion pore formation, HA locally and temporarily increases the permeability of fusing membranes. Subsequent rearrangement in the fusion complex leads to the resealing of the leaky membranes and enlargement of the pore.     

Membrane fusion: stalk model revisited.

Membrane fusion is believed to proceed via intermediate structures called stalks. Mathematical analysis of the stalk provided the elastic energy involved in this structure and predicted the possible evolution of the overall process, but the energies predicted by the original model were suspiciously high. This was due to an erroneous assumption, i.e., that the stalk has a figure of revolution of a circular arc. Here we abandon this assumption and calculate the correct shape of the stalk. We find that it can be made completely stress free and, hence, its energy, instead of being positive and high can become negative, thus facilitating the fusion process. Based on our new calculations, the energies of hemifusion, of complete fusion, and of the pore in a bilayer were analyzed. Implications for membrane fusion and lipid phase transitions are discussed.     

Membrane trafficking: three steps to fusion

Membrane fusion involves the action of members of the SNARE protein family as well as Sec1/Munc18 (SM) proteins, which have been found to interact with SNAREs in three distinct ways. Recent work has established that Munc18-1 directly stimulates fusion and possibly uses all three modes of SNARE interaction.     

Membrane fusion: caught in a trap

SNAREs are small coiled-coil proteins required for specific membrane fusion events in eukaryotic cells. Recent evidence points to the existence of an inhibitory class of SNAREs, i-SNAREs, which prevent incorrect fusions from occurring, adding a further layer of regulation to the process of membrane docking and fusion.     

Membrane transport: Take your fusion partners

Recent studies of how vesicles are targeted to fuse with specific membranes inside cells highlight a role for extended coiled-coil proteins in tethering partner membranes prior to formation of the 'SNARE complex' that mediates the fusion reaction. The tethering protein is recruited to membranes by a Rab family GTPase     

Membrane fusion machines of paramyxoviruses: capture of intermediates of fusion

Peptides derived from heptad repeat regions adjacent to the fusion peptide and transmembrane domains of many viral fusion proteins form stable helical bundles and inhibit fusion specifically. Paramyxovirus SV5 fusion (F) protein-mediated fusion and its inhibition by the peptides N-1 and C-1 were analyzed. The temperature dependence of fusion by F suggests that thermal energy, destabilizing proline residues and receptor binding by the hemagglutinin-neuraminidase (HN) protein collectively contribute to F activation from a metastable native state. F-mediated fusion was reversibly arrested by low temperature or membrane-incorporated lipids, and the resulting F intermediates were characterized. N-1 inhibited an earlier F intermediate than C-1. Co-expression of HN with F lowered the temperature required to attain the N-1-inhibited intermediate, consistent with HN binding to its receptor stimulating a conformational change in F. C-1 bound and inhibited an intermediate of F that could be detected until a point directly preceding membrane merger. The data are consistent with C-1 binding a pre-hairpin intermediate of F and with helical bundle formation being coupled directly to membrane fusion.     

Membrane structure and fusion-triggering conformational change of the fusion domain from influenza hemagglutinin

The N-terminal domain of the influenza hemagglutinin (HA) is the only portion of the molecule that inserts deeply into membranes of infected cells to mediate the viral and the host cell membrane fusion. This domain constitutes an autonomous folding unit in the membrane, causes hemolysis of red blood cells and catalyzes lipid exchange between juxtaposed membranes in a pH-dependent manner. Combining NMR structures determined at pHs 7.4 and 5 with EPR distance constraints, we have deduced the structures of the N-terminal domain of HA in the lipid bilayer. At both pHs, the domain is a kinked, predominantly helical amphipathic structure. At the fusogenic pH 5, however, the domain has a sharper bend, an additional 3(10)-helix and a twist, resulting in the repositioning of Glu 15 and Asp 19 relative to that at the nonfusogenic pH 7.4. Rotation of these charged residues out of the membrane plane creates a hydrophobic pocket that allows a deeper insertion of the fusion domain into the core of the lipid bilayer. Such an insertion mode could perturb lipid packing and facilitate lipid mixing between juxtaposed membranes.     

Animal navigation: longitude at last

Newly hatched sea turtles exposed to artificially generated magnetic fields with parameters characteristic of two sites 3700 km apart, differing only in longitude, can distinguish the two apparent locations and orient appropriately.     

Ion channels: open at last

Previous X-ray studies of have focused on the closed state of the potassium channel. Now the structure of a calcium-activated bacterial potassium channel has revealed the nature of the channel's open state. This provides a first view at high resolution of ion channel gating.