Hemifusion Diaphragm Research Articles

Hemifusomes and Interacting Proteolipid Nanodroplets Mediate Multi-Vesicular Body Formation.

The complex, pleiomorphic membrane structure of the vesicular components within the endolysosomal system has been appreciated through decades of classical electron microscopy. However, due to the heavy fixation and staining required in these approaches, in situ visualization of fragile intermediates between early endosomes, late endosomes and ultimately multivesicular bodies (MVBs), remains elusive, raising the likelihood that other structures may have also been overlooked. Here, using in situ cryo-electron tomography in four mammalian cell lines, we discover heterotypic hemifused vesicles that share an extended hemifusion diaphragm, associated with a 42nm proteolipid nanodroplet (PND). We term this previously undescribed vesicular organelle-complex, "hemifusome". Hemifusomes make up approximately 10% of the organelle pool of the endolysosomal system, but do not participate directly in transferrin-mediated endocytosis. Hemifusomes exist in compound conformations and also contain intraluminal vesicles. Based on their range of morphologies, and the consistent presence of the PND at sites of compound hemifused vesicles, we propose that hemifusomes function as platforms for vesicular biogenesis mediated by the PND. These findings offer direct in situ evidence for a long-lived hemifusion diaphragm, and a new, ESCRT-independent model for the formation of late endosomes containing intraluminal vesicles and ultimately MVBs.

Transient pores in hemifusion diaphragms

Exchange of material across two membranes, as in the case of synaptic neurotransmitter release from a vesicle, involves the formation and poration of a hemifusion diaphragm (HD). The nontrivial geometry of the HD leads to environment-dependent control, regarding the stability and dynamics of the pores required for this kind of exocytosis. This work combines particle simulations, field-based calculations, and phenomenological modeling to explore the factors influencing the stability, dynamics, and possible control mechanisms of pores in HDs. We find that pores preferentially form at the HD rim, and that their stability is sensitive to a number of factors, including the three line tensions, membrane tension, HD size, and the ability of lipids to “flip-flop” across leaflets. Along with a detailed analysis of these factors, we discuss ways that vesicles or cells may use them to open and close pores and thereby quickly and efficiently transport material.

Hemifusion diaphragm simulations suggest a molecular mechanism of IFITM3 antiviral activity

Hemifusion diaphragm simulations suggest a molecular mechanism of IFITM3 antiviral activity

The use of hemifusion to create asymmetric giant unilamellar vesicles: Insights on induced order domains.

The natural asymmetry of the lipid bilayer in biological membranes is, in part, a testament to the complexity of the structure and function of this barrier limiting and protecting cells (or organelles). These lipid bilayers consist of two lipid leaflets with different lipid compositions, resulting in unique interactions within each leaflet. These interactions, combined with interactions between the two leaflets, determine the overall behavior of the membrane. Model membranes provide the most suitable option for investigating the fundamental interactions of lipids. This report describes a comprehensive method to make asymmetric giant unilamellar vesicles (aGUVs) using the technique of hemifusion. In this method, calcium ions induce the hemifusion of giant unilamellar vesicles (GUVs) with a supported lipid bilayer (SLB), both having different lipid compositions. During hemifusion, a stalk, or a more commonly seen hemifusion diaphragm, connects the outer leaflets of GUVs and the SLB. The lateral diffusion of lipids naturally promotes the lipid exchange between the connected outer leaflets. After calcium chelation to prevent further fusion, a mechanical shear detaches aGUVs from the SLB. A fluorescence quench assay is employed to test the extent of bilayer asymmetry. A fluorescence quenching assay tests bilayer asymmetry and verifies dye and lipid migration to a GUV's outer leaflet.

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.

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.

Sterol physical properties and relationships to endo-/exocytosis.

Multiple diseases arise from sterol homeostasis disruption and are characterized at the molecular level by depletion or accumulation of sterol species. One such disease is Smith-Lemli-Opitz syndrome (SLOS) that results from genetic mutations in a particular cholesterol synthesis enzyme, which leads to depletion of cholesterol and accumulation of 7-dehydrocholesterol (7DHC). Recent in vivo work demonstrated that depleting/enriching specific sterol species modulated clathrin-mediated endocytosis (CME) that might contribute to the cellular phenotypes observed in disease states. The CME results were explained by softening mechanisms involving sterol lateral redistribution, but it remains a question as to why this occurs. Here, we take the outlook that differences in Gaussian curvature modulus and local lipid composition destabilize narrow necks in fission, reducing the membrane's apparent rigidity. Using membrane fluctuation analysis, we quantify sterol physical properties that partially define the fusion/fission energetic pathway, for example: the compressibility modulus, bending modulus, leaflet neutral surface, leaflet spontaneous curvature, bilayer thickness preference, and thickness contribution to the Gaussian modulus. Using these quantities, we make predictions on where sterol species will redistribute on strongly curved membranes. Finally, we use all-atom simulations of strongly curved fusion pore and hemifusion diaphragm geometries to quantify the sterol redistribution and compare to hypotheses. Comparing cholesterol and 7DHC physical properties, for example, the key statistically significant quantity is the thickness contribution to the Gaussian modulus, and we predicted that 7DHC would be less excluded from the fusion pore than cholesterol. All-atom molecular dynamics simulations of the fusion pore confirmed this hypothesis. The result correlates to in vivo experiments that showed reduced CME with 7DHC compared to cholesterol (p < 0.10). Other tests are ongoing.

Visualization of conformational changes and membrane remodeling leading to genome delivery by viral class-II fusion machinery

Chikungunya virus (CHIKV) is a human pathogen that delivers its genome to the host cell cytoplasm through endocytic low pH-activated membrane fusion mediated by class-II fusion proteins. Though structures of prefusion, icosahedral CHIKV are available, structural characterization of virion interaction with membranes has been limited. Here, we have used cryo-electron tomography to visualize CHIKV’s complete membrane fusion pathway, identifying key intermediary glycoprotein conformations coupled to membrane remodeling events. Using sub-tomogram averaging, we elucidate features of the low pH-exposed virion, nucleocapsid and full-length E1-glycoprotein’s post-fusion structure. Contrary to class-I fusion systems, CHIKV achieves membrane apposition by protrusion of extended E1-glycoprotein homotrimers into the target membrane. The fusion process also features a large hemifusion diaphragm that transitions to a wide pore for intact nucleocapsid delivery. Our analyses provide comprehensive ultrastructural insights into the class-II virus fusion system function and direct mechanistic characterization of the fundamental process of protein-mediated membrane fusion.

Mechanism of Membrane Fusion: Interplay of Lipid and Peptide.

Membrane fusion is an essential process for the survival of eukaryotes and the entry of enveloped viruses into host cells. A proper understanding of the mechanism of membrane fusion would provide us a handle to manipulate several biological pathways, and design efficient vaccines against emerging and re-emerging viral infections. Although fusion proteins take the central stage in catalyzing the process, role of lipid composition is also of paramount importance. Lipid composition modulates membrane organization and dynamics and impacts the lipid–protein (peptide) interaction. Moreover, the intrinsic curvature of lipids has strong impact on the formation of stalk and hemifusion diaphragm. Detection of transiently stable intermediates remains the bottleneck in the understanding of fusion mechanism. In order to circumvent this challenge, analytical methods can be employed to determine the kinetic parameters from ensemble average measurements of observables, such as lipid mixing, content mixing, and content leakage. The current review aims to present an analytical method that would aid our understanding of the fusion mechanism, provides a better insight into the role of lipid shape, and discusses the interplay of lipid and peptide in membrane fusion.Graphical

Entropic Forces, Zippering Pressure and Membrane Thinning Drive Snare-Mediated Membrane Fusion

We studied SNARE-mediated membrane fusion using ultra coarse-grained (UCG) simulations that achieved fusion of realistically sized vesicles with realistic membrane tensions. Previous simulation approaches primarily used unphysical membrane conditions to force fusion on observable timescales. Our results elucidate fundamental features of the pathway for the first time. Membrane fusion is an essential step during neurotransmitter or hormone release and trafficking. Cells use a multi-component fusion machinery whose core consists of the SNARE proteins. The mechanism of SNARE-mediated membrane fusion is not established, and the intermediate states along the fusion pathway remain controversial. Computer simulations can help uncover mechanisms, but a major obstacle is that physiological and in vitro membrane fusion timescales range from msec to sec, far beyond the range of all-atom or mildly coarse-grained (CG) approaches. Here we used UCG simulations to reveal the pathways to vesicle-vesicle and vesicle-planar membrane fusion on msec timescales, using the Cooke-Deserno force field phospholipid representation (Cooke et al., 2005). SNAREs zippered up between the two vesicles, blocking fusion, but entropic forces among the SNARE complexes and membranes then pushed the SNAREs to the periphery of the membrane-membrane contact zone where zippering forces compressed the membranes and triggered fusion along the pathway: hemifusion to hemifusion diaphragm (HD) expansion to nanopore formation in the HD. Fusion was faster with more SNAREs present, since entropic forces were greater (Mostafavi et al., 2017; McDargh et al., 2018). Transmembrane domains (TMDs) played a surprisingly vital role. With TMDs modeled on wild type SNAREs, fast fusion occurred when the TMDs were pulled deep into the membrane, causing local bilayer thinning and destabilization that triggered the fusion pathway. Modified TMDs simulating mutations failed to sufficiently penetrate the membrane and fusion was not triggered, in agreement with experiment.

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.

A Model for Myomerger Function in Myoblast Fusion

Myomerger, an integral membrane protein, has been recently established to play a crucial role in myoblast fusion by driving the transition from the hemifusion to the full fusion stage of the reaction. To accomplish this task, the ectodomains of myomerger molecules located at the proximal (outer) leaflets of the fusing plasma membranes must be able to mediate formation of a fusion pore in the hemifusion diaphragm (HD), which consist of the distal (inner) membrane leaflets. We now report that, similarly to myomerger, the phospholipid lysophosphatidylcholine (LPC) promotes hemifusion-to-fusion transition, if added to the proximal leaflets of the hemifused membranes. An apparent similarity between LPC and myomerger consists in their ability to generate a positive spontaneous curvature of lipid monolayers. This effect of LPC is known to be driven by its effective molecular shape, while myomerger could act through insertion of its N-terminal amphipathic domain into the membrane matrix. Here we propose a mechanism of indirect promotion of the fusion pore formation. The essence of the mechanism is a geometrical and mechanical coupling of the proximal and distal leaflets of the fusing membranes mediated by the HD rim. The elastic stresses generated by LPC and/or myomerger in the distal membrane leaflets are transmitted through the HD rim into the leaflets of the hemifusion diaphragm, which facilitates the fusion pore generation and growth. To substantiate the mechanism, using the theory of the membrane bending, splay and tilt elasticity, we computed the elastic stresses propagating into the HD as functions of LPC concentration in the proximal membrane leaflets, determined the effective values of HD line tension and calculated the kinetic barriers of the fusion pore formation. We predicted, quantitatively, the degree of acceleration of the fusion pore formation by relevant LPC amounts in the membrane.

Lipid flip-flop vs. lateral diffusion in the relaxation of hemifusion diaphragms

Molecular dynamics simulations of a solvent-free coarse-grained lipid model are used to characterize the mechanisms by which lipid-bilayer hemifusion diaphragm (HD) intermediates relax, across a range of global compositions of negative intrinsic curvature (NIC) lipids and neutral-curvature lipids. At low concentrations of NIC lipids, rapid fission produces a double bilayer end state through a lateral diffusion-based mechanism enabled by spontaneous rim-pore defects. At moderately higher NIC lipid concentrations, rim pores are absent and stable leaflet three-junctions persist, revealing an HD relaxation mechanism entirely reliant on lipid flip-flop, and end states that are either stable fusion pores or stable HD's. These fusogenic systems exhibit dynamics highly dependent on NIC lipid concentration via an underlying sensitivity of flip-flop rates for neutral lipids on NIC lipid concentration. This work illustrates that HD dynamics may be altered through regulation of lipid composition in the immediate three-junction region. This work further highlights the potential role of flippases in biological fusion and the importance of lipid composition on fusion dynamics.

Cholesterol suppresses membrane leakage by decreasing water penetrability.

Membrane fusion is a fundamental biological process that lies at the heart of enveloped virus infection, synaptic signaling, intracellular vesicle trafficking, gamete fertilization, and cell-cell fusion. Membrane fusion is initiated as two apposed membranes merge to a single bilayer called a hemifusion diaphragm. It is believed that the contents of the two fusing membranes are released through a fusion pore formed at the hemifusion diaphragm, and yet another possible pathway has been proposed in which an undefined pore may form outside the hemifusion diaphragm at the apposed membranes, leading to the so-called leaky fusion. Here, we performed all-atom molecular dynamics simulations to study the evolution of the hemifusion diaphragm structure with various lipid compositions. We found that the lipid cholesterol decreased water penetrability to inhibit leakage pore formation. Biochemical leakage experiments support these simulation results. This study may shed light on the underlying mechanism of the evolution pathways of the hemifusion structure, especially the understanding of content leakage during membrane fusion.

Rate of hemifusion diaphragm dissipation and ability to form three-junction bound HD determined by lipid composition.

Though the hemifusion diaphragm (HD) is widely accepted as an intermediate in bilayer membrane fusion, lipid contributions toward HD stability and dynamics are still not fully understood. In this paper, we study large, binary, protein-free HD systems at varying compositions of negative intrinsic curvature (NIC) lipids using molecular dynamics (MD) simulations of a solvent-free coarse-grained lipid model. Under MD, initially created HDs are found to relax to three major end states depending on the composition and lipid intrinsic curvature. Low compositions of NIC lipids or weak intrinsic curvature result in double-bilayer end states, and moderate compositions of moderate to strong NIC lipids result in metastable fusion pores. Importantly, high compositions of moderate NIC lipids result in a metastable HD that persists beyond μs time scales. NIC lipids stabilize the HD by filling the junction core around the HD. Sorting of NIC lipids toward the three-junction region occurs in fused-endpoint systems, but no significant sorting was seen in systems that end in a double bilayer indicating that high line tension at the triple junction drives HD dissipation faster than sorting can enrich that junction enough to lower that line tension. The appearance of three end states dependent on the NIC lipid composition highlights the necessity of NIC lipids for non-leaky fusion.

The hemifusion structure induced by influenza virus haemagglutinin is determined by physical properties of the target membranes.

SUMMARYInfluenza A virus hemagglutinin (HA) changes conformation and drives membrane fusion of viral and endosomal membrane at low pH. Membrane fusion proceeds through an intermediate called hemifusion1,2. For viral fusion the hemifusion structures are not determined3. Here, influenza virus-like particles (VLP)4 carrying wild-type (WT) HA or HA hemifusion mutant G1S5 and liposome mixtures were studied at low pH by Volta phase plate (VPP) cryo-electron tomography (cET) which improves signal-to-noise ratio close to focus. We determined two distinct hemifusion structures: a hemifusion diaphragm (HD) and a novel structure termed lipidic junction. Liposomes with lipidic junctions were ruptured with membrane edges stabilized by HA. The rupture frequency and HD diameter were not affected by G1S mutation, but decreased when the cholesterol level in the liposomes was close to physiological concentrations. We propose that HA induces merger between the viral and a target membrane by one of two independent pathways: rupture-insertion pathway leading to lipidic junction and hemifusion-stalk pathway leading to fusion pore. The latter is relevant under the conditions of influenza virus infection of cells. Cholesterol concentration functions as a pathway switch due to its negative spontaneous curvature in the target bilayer as determined by continuum analysis.

Calculating Transition Energy Barriers and Characterizing Activation States for Steps of Fusion

We use continuum mechanics to calculate an entire least energy pathway of membrane fusion, from stalk formation, to pore creation, and through fusion pore enlargement. The model assumes that each structure in the pathway is axially symmetric. The static continuum stalk structure agrees quantitatively with experimental stalk architecture. Calculations show that in a stalk, the distal monolayer is stretched and the stored stretching energy is significantly less than the tilt energy of an unstretched distal monolayer. The string method is used to determine the energy of the transition barriers that separate intermediate states and the dynamics of two bilayers as they pass through them. Hemifusion requires a small amount of energy independently of lipid composition, while direct transition from a stalk to a fusion pore without a hemifusion intermediate is highly improbable. Hemifusion diaphragm expansion is spontaneous for distal monolayers containing at least two lipid components, given sufficiently negative diaphragm spontaneous curvature. Conversely, diaphragms formed from single-component distal monolayers do not expand without the continual injection of energy. We identify a diaphragm radius, below which central pore expansion is spontaneous. For larger diaphragms, prior studies have shown that pore expansion is not axisymmetric, and here our calculations supply an upper bound for the energy of the barrier against pore formation. The major energy-requiring deformations in the steps of fusion are: widening of a hydrophobic fissure in bilayers for stalk formation, splay within the expanding hemifusion diaphragm, and fissure widening initiating pore formation in a hemifusion diaphragm.

Steroid Hormones as Transporters to Carry Exogenous Macromolecules into the Target Cell Nuclei in Vivo

Upon injection into the vascular system of rats, testosterone-bovine serum albumin conjugate (testosterone-BSA) is taken up by cells via the process of endocytosis. When it is taken up by the target cells of testosterone such as spermatogenic cells, it enters the nuclei of the cells. However, testosterone-BSA does not enter the nuclei of the non-target cells such as hepatocytes and thymocytes. Similarly, hydrocortisone-BSA conjugate enters the nuclei of its target cells such as hepatocytes and thymocytes. In the vesicular trafficking of testosterone-BSAs into the nucleoplasm, the vesicle membrane is likely to fuse with a nuclear hemifusion diaphragm. IgG coupled with hydrocortisone also enters the hormone-target cell nuclei, with its antigenicity kept intact. These results suggest that steroid hormones could act as transporters for conveying exogenous macromolecules into the target cell nuclei in vivo. Our studies provide a novel insight to the functions of steroid hormones.

Free Energy Landscape of Rim-Pore Expansion in Membrane Fusion

The productive fusion pore in membrane fusion is generally thought to be toroidally shaped. Theoretical studies and recent experiments suggest that its formation, in some scenarios, may be preceded by an initial pore formed near the rim of the extended hemifusion diaphragm (HD), a rim-pore. This rim-pore is characterized by a nontoroidal shape that changes with size. To determine this shape as well as the free energy along the pathway of rim-pore expansion, we derived a simple analytical free energy model. We argue that dilation of HD material via expansion of a rim-pore is favored over a regular, circular pore. Further, the expanding rim-pore faces a free energy barrier that linearly increases with HD size. In contrast, the tension required to expand the rim-pore decreases with HD size. Pore flickering, followed by sudden opening, occurs when the tension in the HD competes with the line energy of the rim-pore, and the rim-pore reaches its equilibrium size before reaching the critical pore size. The experimental observation of flickering and closing fusion pores (kiss-and-run) is very well explained by the observed behavior of rim-pores. Finally, the free energy landscape of rim-pore expansion/HD dilation may very well explain why some cellular fusion reactions, in their attempt to minimize energetic costs, progress via alternative formation and dilation of microscopic hemifusion intermediates.

Complexin inhibits spontaneous release and synchronizes Ca2+-triggered synaptic vesicle fusion by distinct mechanisms.

Previously we showed that fast Ca(2+)-triggered vesicle fusion with reconstituted neuronal SNAREs and synaptotagmin-1 begins from an initial hemifusion-free membrane point contact, rather than a hemifusion diaphragm, using a single vesicle-vesicle lipid/content mixing assay (Diao et al., 2012). When complexin-1 was included, a more pronounced Ca(2+)-triggered fusion burst was observed, effectively synchronizing the process. Here we show that complexin-1 also reduces spontaneous fusion in the same assay. Moreover, distinct effects of several complexin-1 truncation mutants on spontaneous and Ca(2+)-triggered fusion closely mimic those observed in neuronal cultures. The very N-terminal domain is essential for synchronization of Ca(2+)-triggered fusion, but not for suppression of spontaneous fusion, whereas the opposite is true for the C-terminal domain. By systematically varying the complexin-1 concentration, we observed differences in titration behavior for spontaneous and Ca(2+)-triggered fusion. Taken together, complexin-1 utilizes distinct mechanisms for synchronization of Ca(2+)-triggered fusion and inhibition of spontaneous fusion.