Ebola Virus Glycoprotein Strongly Binds to Membranes in the Absence of Receptor Engagement.

Ebola virus (EBOV) is an enveloped virus that must fuse with the host cell membrane in order to release its genome and initiate infection. This process requires the action of the EBOV envelope glycoprotein (GP), encoded by the virus, which resides in the viral envelope and consists of a receptor binding subunit, GP1, and a membrane fusion subunit, GP2. Despite extensive research, a mechanistic understanding of the viral fusion process is incomplete. To investigate GP-membrane association, a key step in the fusion process, we used two approaches: high-throughput measurements of single-particle diffusion and single-molecule measurements with optical tweezers. Using these methods, we show that the presence of the endosomal Niemann-Pick C1 (NPC1) receptor is not required for primed GP-membrane binding. In addition, we demonstrate this binding is very strong, likely attributed to the interaction between the GP fusion loop and the membrane's hydrophobic core. Our results also align with previously reported findings, emphasizing the significance of acidic pH in the protein-membrane interaction. Beyond Ebola virus research, our approach provides a powerful toolkit for studying other protein-membrane interactions, opening new avenues for a better understanding of protein-mediated membrane fusion events.

Membrane Tension Inhibits Lipid Mixing by Increasing the Hemifusion Stalk Energy.

Fusion of biological membranes is fundamental in various physiological events. The fusion process involves several intermediate stages with energy barriers that are tightly dependent on the mechanical and physical properties of the system, one of which is membrane tension. As previously established, the late stages of fusion, including hemifusion diaphragm and pore expansions, are favored by membrane tension. However, a current understanding of how the energy barrier of earlier fusion stages is affected by membrane tension is lacking. Here, we apply a newly developed experimental approach combining micropipette-aspirated giant unilamellar vesicles and optically trapped membrane-coated beads, revealing that membrane tension inhibits lipid mixing. We show that lipid mixing is 6 times slower under a tension of 0.12 mN/m compared with tension-free membranes. Furthermore, using continuum elastic theory, we calculate the dependence of the hemifusion stalk formation energy on membrane tension and intermembrane distance and find the increase in the corresponding energy barrier to be 1.6 kBT in our setting, which can explain the increase in lipid mixing time delay. Finally, we show that tension can be a significant factor in the stalk energy if the pre-fusion intermembrane distance is on the order of several nanometers, while for membranes that are tightly docked, tension has a negligible effect.

IFITM3 blocks influenza virus entry by sorting lipids and stabilizing hemifusion.

Interferon-induced transmembrane protein 3 (IFITM3) inhibits the entry of numerous viruses through undefined molecular mechanisms. IFITM3 localizes in the endosomal-lysosomal system and specifically affects virus fusion with target cell membranes. We found that IFITM3 induces local lipid sorting, resulting in an increased concentration of lipids disfavoring viral fusion at the hemifusion site. This increases the energy barrier for fusion pore formation and the hemifusion dwell time, promoting viral degradation in lysosomes. In situ cryo-electron tomography captured IFITM3-mediated arrest of influenza A virus membrane fusion. Observation of hemifusion diaphragms between viral particles and late endosomal membranes confirmed hemifusion stabilization as a molecular mechanism of IFITM3. The presence of the influenza fusion protein hemagglutinin in post-fusion conformation close to hemifusion sites further indicated that IFITM3 does not interfere with the viral fusion machinery. Collectively, these findings show that IFITM3 induces lipid sorting to stabilize hemifusion and prevent virus entry into target cells.

The Ebola virus VP40 matrix layer undergoes endosomal disassembly essential for membrane fusion.

Ebola viruses (EBOVs) assemble into filamentous virions, whose shape and stability are determined by the matrix viral protein 40 (VP40). Virus entry into host cells occurs via membrane fusion in late endosomes; however, the mechanism of how the remarkably long virions undergo uncoating, including virion disassembly and nucleocapsid release into the cytosol, remains unknown. Here, we investigate the structural architecture of EBOVs entering host cells and discover that the VP40 matrix disassembles prior to membrane fusion. We reveal that VP40 disassembly is caused by the weakening of VP40-lipid interactions driven by low endosomal pH that equilibrates passively across the viral envelope without a dedicated ion channel. We further show that viral membrane fusion depends on VP40 matrix integrity, and its disassembly reduces the energy barrier for fusion stalk formation. Thus, pH-driven structural remodeling of the VP40 matrix acts as a molecular switch coupling viral matrix uncoating to membrane fusion during EBOV entry.

High curvature promotes fusion of lipid membranes: Predictions from continuum elastic theory.

The fusion of lipid membranes progresses through a series of hemifusion intermediates with two significant energy barriers related to the formation of stalk and fusion pore, respectively. These energy barriers determine the speed and success rate of many critical biological processes, including the fusion of highly curved membranes, for example synaptic vesicles and enveloped viruses. Here we use continuum elastic theory of lipid monolayers to determine the relationship between membrane shape and energy barriers to fusion. We find that the stalk formation energy decreases with curvature by up to 31 kBT in a 20-nm-radius vesicle compared with planar membranes and by up to 8 kBT in the fusion of highly curved, long, tubular membranes. In contrast, the fusion pore formation energy barrier shows a more complicated behavior. Immediately after stalk expansion to the hemifusion diaphragm, the fusion pore formation energy barrier is low (15-25 kBT) due to lipid stretching in the distal monolayers and increased tension in highly curved vesicles. Therefore, the opening of the fusion pore is faster. However, these stresses relax over time due to lipid flip-flop from the proximal monolayer, resulting in a larger hemifusion diaphragm and a higher fusion pore formation energy barrier, up to 35 kBT. Therefore, if the fusion pore fails to open before significant lipid flip-flop takes place, the reaction proceeds to an extended hemifusion diaphragm state, which is a dead-end configuration in the fusion process and can be used to prevent viral infections. In contrast, in the fusion of long tubular compartments, the surface tension does not accumulate due to the formation of the diaphragm, and the energy barrier for pore expansion increases with curvature by up to 11 kBT. This suggests that inhibition of polymorphic virus infection could particularly target this feature of the second barrier.

Model for ring closure in ER tubular network dynamics.

Tubular networks of the endoplasmic reticulum (ER) are dynamic structures whose steady-state conformations are maintained by a balance between the persistent generation and vanishing of the network elements. While factors producing the ER tubules and intertubular junctions have been investigated, the mechanisms behind their elimination remained unknown. Here, we addressed the ER ring closure, the process resulting in the tubule and junction removal through constriction of the network unit cells into junctional knots followed by the knot remodeling into regular junctions. We considered the ring closure to be driven by the tension existing in ER membranes. We based our consideration on the notion of Gibbs' thermodynamic tension and reviewed its relationship to other tension definitions used in the literature. We modeled, computationally, the structures of the junctional knots containing internal nanopores and analyzed their tension dependence. We analyzed the process of the pore sealing through membrane fission resulting in the formation of regular junctions. Considering the hemi-fission as the rate-limiting stage of the fission reaction, we evaluated the membrane tensions guaranteeing the spontaneous character of the pore sealing. We concluded that feasible membrane tensions explain all stages of the ER ring closure.

Myomerger promotes fusion pore by elastic coupling between proximal membrane leaflets and hemifusion diaphragm.

Myomerger is a muscle-specific membrane protein involved in formation of multinucleated muscle cells by mediating the transition from the early hemifusion stage to complete fusion. Here, we considered the physical mechanism of the Myomerger action based on the hypothesis that Myomerger shifts the spontaneous curvature of the outer membrane leaflets to more positive values. We predicted, theoretically, that Myomerger generates the outer leaflet elastic stresses, which propagate into the hemifusion diaphragm and accelerate the fusion pore formation. We showed that Myomerger ectodomain indeed generates positive spontaneous curvature of lipid monolayers. We substantiated the mechanism by experiments on myoblast fusion and influenza hemagglutinin-mediated cell fusion. In both processes, the effects of Myomerger ectodomain were strikingly similar to those of lysophosphatidylcholine known to generate a positive spontaneous curvature of lipid monolayers. The control of post-hemifusion stages by shifting the spontaneous curvature of proximal membrane monolayers may be utilized in diverse fusion processes.

Membrane Curvature and Tension Control the Formation and Collapse of Caveolar Superstructures.

Caveolae, flask-shaped pits covered by caveolin-cavin coats, are abundant features of the plasma membrane of many cells. Besides appearing as single-membrane indentations, caveolae are organized as superstructures in the form of rosette-like clusters, whose mechanism of assembly and biological functions have been elusive. Here, we propose that clustering of caveolae in mature muscle cells is driven by forces originating from the elastic energy of membrane-bending deformations and membrane tension. We substantiate this mechanism by computational modeling, which recovers the unique shapes observed for the most ubiquitous caveolar clusters. We support the agreement between the calculated and observed configurations by electron tomography of caveolar clusters. The model predicts the experimentally assessable dependence of caveolar clustering on membrane tension and on the degree of the caveolar coat assembly. We reveal a difference in conformation and, possibly, in function and formation mechanism between caveolar clusters of muscle cells and of adipocytes.

Architecture of Lipid Droplets in Endoplasmic Reticulum Is Determined by Phospholipid Intrinsic Curvature.

Lipid droplets (LDs) store fats and play critical roles in lipid and energy homeostasis. They form between the leaflets of the endoplasmic reticulum (ER) membrane and consist of a neutral lipid core wrapped in a phospholipid monolayer with proteins. Two types of ER-LD architecture are thought to exist and be essential for LD functioning. Maturing LDs either emerge from the ER into the cytoplasm, remaining attached to the ER by a narrow membrane neck, or stay embedded in the ER and are surrounded by ER membrane. Here, we identify a lipid-based mechanism that controls which of these two architectures is favored. Theoretical modeling indicated that the intrinsic molecular curvatures of ER phospholipids can determine whether LDs remain embedded in or emerge from the ER; lipids with negative intrinsic curvature such as diacylglycerol (DAG) and phosphatidylethanolamine favor LD embedding, while those with positive intrinsic curvature, like lysolipids, support LD emergence. This prediction was verified by altering the lipid composition of the ER in S. cerevisiae using mutants and the addition of exogenous lipids. We found that fat-storage-inducing transmembrane protein 2 (FIT2) homologs become enriched at sites of LD generation when biogenesis is induced. DAG accumulates at sites of LD biogenesis, and FIT2 proteins may promote LD emergence from the ER by reducing DAG levels at these sites. Altogether, our findings suggest that cells regulate LD integration in the ER by modulating ER lipid composition, particularly at sites of LD biogenesis and that FIT2 proteins may play a central role in this process.

A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division.

Mitochondrial division, essential for survival in mammals, is enhanced by an inter-organellar process involving ER tubules encircling and constricting mitochondria. The force for constriction is thought to involve actin polymerization by the ER-anchored isoform of the formin protein inverted formin 2 (INF2). Unknown is the mechanism triggering INF2-mediated actin polymerization at ER-mitochondria intersections. We show that a novel isoform of the formin-binding, actin-nucleating protein Spire, Spire1C, localizes to mitochondria and directly links mitochondria to the actin cytoskeleton and the ER. Spire1C binds INF2 and promotes actin assembly on mitochondrial surfaces. Disrupting either Spire1C actin- or formin-binding activities reduces mitochondrial constriction and division. We propose Spire1C cooperates with INF2 to regulate actin assembly at ER-mitochondrial contacts. Simulations support this model's feasibility and demonstrate polymerizing actin filaments can induce mitochondrial constriction. Thus, Spire1C is optimally positioned to serve as a molecular hub that links mitochondria to actin and the ER for regulation of mitochondrial division.