-
ACS Chemical Biology Jul 2022Paramyxoviruses are enveloped viruses harboring a negative-sense RNA genome that must enter the host's cells to replicate. In the case of the parainfluenza virus, the...
Paramyxoviruses are enveloped viruses harboring a negative-sense RNA genome that must enter the host's cells to replicate. In the case of the parainfluenza virus, the cell entry process starts with the recognition and attachment to target receptors, followed by proteolytic cleavage of the fusion glycoprotein (F) protein, exposing the fusion peptide (FP) region. The FP is responsible for binding to the target membrane, and it is believed to play a crucial role in the fusion process, but the mechanism by which the parainfluenza FP (PIFP) promotes membrane fusion is still unclear. To elucidate this matter, we performed biophysical experimentation of the PIFP in membranes, together with coarse grain (CG) and atomistic (AA) molecular dynamics (MD) simulations. The simulation results led to the pinpointing of the most important PIFP amino acid residues for membrane fusion and show that, at high concentrations, the peptide induces the formation of a water-permeable porelike structure. This structure promotes lipid head intrusion and lipid tail protrusion, which facilitates membrane fusion. Biophysical experimental results validate these findings, showing that, depending on the peptide/lipid ratio, the PIFP can promote fusion and/or membrane leakage. Our work furthers the understanding of the PIFP-induced membrane fusion process, which might help foster development in the field of viral entry inhibition.
Topics: Humans; Lipids; Membrane Fusion; Paramyxoviridae Infections; Peptides; Viral Fusion Proteins
PubMed: 35500279
DOI: 10.1021/acschembio.2c00208 -
Proceedings of the National Academy of... May 2023Among the current five Variants of Concern, infections caused by SARS-CoV-2 B.1.617.2 (Delta) variant are often associated with the greatest severity. Despite recent...
Among the current five Variants of Concern, infections caused by SARS-CoV-2 B.1.617.2 (Delta) variant are often associated with the greatest severity. Despite recent advances on the molecular basis of elevated pathogenicity using recombinant proteins, the architecture of intact Delta virions remains veiled. Moreover, pieces of molecular evidence for the detailed mechanism of S-mediated membrane fusion are missing. Here, we showed the pleomorphic nature of Delta virions from electron beam inactivated samples and reported the in situ structure and distribution of S on the authentic Delta variant. We also captured the virus-virus fusion events, which provided pieces of structural evidence for Delta's attenuated dependency on cellular factors for fusion activation, and proposed a model of S-mediated membrane fusion. Besides, site-specific glycan analysis revealed increased oligomannose-type glycosylation of native Delta S than that of the WT S. Together, these results disclose distinctive factors of Delta being the most virulent SARS-CoV-2 variant.
Topics: Humans; SARS-CoV-2; COVID-19; Membrane Fusion; Glycosylation; Spike Glycoprotein, Coronavirus
PubMed: 37094167
DOI: 10.1073/pnas.2213332120 -
Molecular Biology of the Cell Mar 2023The conserved catalysts of intracellular membrane fusion are Rab-family GTPases, effector complexes that bind Rabs for membrane tethering, SNARE proteins of the R, Qa,...
The conserved catalysts of intracellular membrane fusion are Rab-family GTPases, effector complexes that bind Rabs for membrane tethering, SNARE proteins of the R, Qa, Qb, and Qc families, and SNARE chaperones of the SM, Sec17/SNAP, and Sec18/NSF families. Yeast vacuole fusion is regulated by phosphatidylinositol-3-phosphate (PI3P). PI3P binds directly to the vacuolar Qc-SNARE and to HOPS, the vacuolar tethering/SM complex. We now report several distinct functions of PI3P in fusion. PI3P binds the N-terminal PX domain of the Qc-SNARE to enhance its engagement for fusion. Even when Qc has been preassembled with the Qa- and Qb-SNAREs, PI3P still promotes -SNARE assembly and fusion between these 3Q proteoliposomes and those with R-SNARE, whether with the natural HOPS tether or with a synthetic tether. With HOPS, efficient -SNARE complex formation needs PI3P on the 3Q-SNARE proteoliposomes, in to the Qc. PI3P is also needed for HOPS to confer resistance to Sec17/Sec18. With a synthetic tether, fusion is supported by PI3P on either fusion partner membrane, but this fusion is blocked by Sec17/Sec18. PI3P thus supports multiple stages of fusion: the engagement of the Qc-SNARE, -SNARE complex formation with preassembled Q-SNAREs, HOPS protection of SNARE complexes from Sec17/Sec18, and fusion per se after tethering and Q-SNARE assembly.
Topics: Humans; Adenosine Triphosphatases; Membrane Fusion; Qc-SNARE Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; SNARE Proteins; Vacuoles; Vesicular Transport Proteins; Phosphatidylinositol Phosphates
PubMed: 36735517
DOI: 10.1091/mbc.E22-10-0486 -
Traffic (Copenhagen, Denmark) May 2016There is a fundamental gap in our understanding of how a eukaryotic cell apportions the limited space within its cell membrane. Upon infection, a cell competes with... (Review)
Review
There is a fundamental gap in our understanding of how a eukaryotic cell apportions the limited space within its cell membrane. Upon infection, a cell competes with intracellular pathogens for control of this same precious resource. The struggle between pathogen and host provides us with an opportunity to uncover the mechanisms regulating subcellular space by understanding how pathogens modulate vesicular traffic and membrane fusion events to create a specialized compartment for replication. By comparing several important intracellular pathogens, we review the molecular mechanisms and trafficking pathways that drive two space allocation strategies, the formation of tight and spacious pathogen-containing vacuoles. Additionally, we discuss the potential advantages of each pathogenic lifestyle, the broader implications these lifestyles might have for cellular biology and outline exciting opportunities for future investigation.
Topics: Host-Pathogen Interactions; Membrane Fusion; Vacuoles
PubMed: 26842840
DOI: 10.1111/tra.12382 -
Cell Calcium Jul 2022Exocytosis operates through two distinct modes. Full-fusion leads to rapid expulsion of the entire content of a vesicle; kiss-and-run leads to slow and partial...
Exocytosis operates through two distinct modes. Full-fusion leads to rapid expulsion of the entire content of a vesicle; kiss-and-run leads to slow and partial expulsion. These two modes have important biological consequences for endocrine regulation and synaptic transmission. Amperometry recordings of catecholamine release from chromaffin cells reveal single-vesicle fusion events corresponding to both of these modes, but classification is often difficult. This study introduces a new method of analyzing amperometry data to improve this classification. The ratio of the average amplitude to the peak amplitude differs between full-fusion and kiss-and-run, and the probability distribution of this ratio is well fitted by a double-Gaussian. Kiss-and-run events identified by this method have fusion pores with kinetic properties different from pores associated with full-fusion. They have slower transition rates and lifetime distributions indicative of irreversible transitions. The total-charge of an amperometric spike is expected to scale with vesicle volume during a full-fusion event. The cube root of this quantity should therefore scale with diameter, but the distribution of this quantity differs from the distribution of vesicle diameter seen in the electron microscope. Fusion pore lifetimes associated with full-fusion depend on vesicle size, and this makes the choice of mode size dependent. The fusion pore thus bifurcates after opening, and vesicle size influences this choice. The secretory vesicle protein synaptophysin influences the size dependence of fusion pore lifetime and the choice of release mode. Incorporating vesicle size into an analysis of release mode reconciled the kinetics of fusion pores, as well as the distributions of vesicle diameter and catecholamine content. Thus, the initial fusion pore emerges as a critical focus in endocrine regulation. By modulating the size-dependence of the mode of exocytosis, changes in the molecular makeup of the exocytotic apparatus can impact the shape and size of an amperometric event, and the speed and composition of secretion.
Topics: Catecholamines; Chromaffin Cells; Exocytosis; Membrane Fusion; Secretory Vesicles
PubMed: 35636152
DOI: 10.1016/j.ceca.2022.102606 -
Cellular and Molecular Life Sciences :... Feb 2018The mitochondrial network constantly changes and remodels its shape to face the cellular energy demand. In human cells, mitochondrial fusion is regulated by the large,... (Review)
Review
The mitochondrial network constantly changes and remodels its shape to face the cellular energy demand. In human cells, mitochondrial fusion is regulated by the large, evolutionarily conserved GTPases Mfn1 and Mfn2, which are embedded in the mitochondrial outer membrane, and by OPA1, embedded in the mitochondrial inner membrane. In contrast, the soluble dynamin-related GTPase Drp1 is recruited from the cytosol to mitochondria and is key to mitochondrial fission. A number of new players have been recently involved in Drp1-dependent mitochondrial fission, ranging from large cellular structures such as the ER and the cytoskeleton to the surprising involvement of the endocytic dynamin 2 in the terminal abscission step. Here we review the recent findings that have expanded the mechanistic model for the mitochondrial fission process in human cells and highlight open questions.
Topics: Animals; Humans; Membrane Fusion; Mitochondrial Dynamics; Mitochondrial Membranes; Mitochondrial Proteins
PubMed: 28779209
DOI: 10.1007/s00018-017-2603-0 -
The Journal of Chemical Physics Nov 2017Despite catalyzed by fusion proteins of quite different molecular architectures, intracellular, viral, and cell-to-cell fusions are found to have the essential common...
Despite catalyzed by fusion proteins of quite different molecular architectures, intracellular, viral, and cell-to-cell fusions are found to have the essential common features and the nearly same nature of transition states. The similarity inspires us to find a more general catalysis mechanism for membrane fusion that minimally depends on the specific structures of fusion proteins. In this work, we built a minimal model for membrane fusion, and by using dissipative particle dynamics simulations, we propose a mechanism that the pulling force generated by fusion proteins initiates the fusion process and the membrane tension regulates the subsequent fusion stages. The model shows different features compared to previous computer simulation studies: the pulling force catalyzes membrane fusion through lipid head overcrowding in the contacting region, leading to an increase in the head-head repulsion and/or the unfavorable head-tail contacts from opposing membranes, both of which destabilize the contacting leaflets and thus promote membrane fusion or vesicle rupture. Our simulations produce a variety of shapes and intermediates, closely resembling cases seen experimentally. Our work strongly supports the view that the tight pulling mechanism is a conserved feature of fusion protein-mediated fusion and that the membrane tension plays an essential role in fusion.
Topics: Lipids; Membrane Fusion; Molecular Dynamics Simulation; Proteins; Surface Tension
PubMed: 29166098
DOI: 10.1063/1.4997393 -
Talanta May 2021Membrane fusion is fundamental to biological activity of cells, so disclosingits relevant mechanism is very important for understanding various cell functions. Although...
Membrane fusion is fundamental to biological activity of cells, so disclosingits relevant mechanism is very important for understanding various cell functions. Although artificial model systems have been developed to uncover the mechanism of membrane fusion, key factors determining the mode of membrane fusion remain unclear. Based on the construction of different types of liposome vesicles, we used a dynamic fluorescence imaging method to investigate the effect of membrane protein distribution density on membrane fusion. Time-resolved imaging revealed that protein-free pure phospholipid vesicles themselves occurred full membrane fusion. Moreover, we prepared proteoliposomes with increasing protein-to-lipid ratio to better reflect the characteristic of membrane structure in vivo. Our data showed that pure phospholipid vesicles no longer fused with the proteoliposomes that in a higher protein proportion, indicating dense membrane proteins may hinder membrane fusion. A further comparative analysis of the interactions of pure phospholipid vesicles with the cell membrane / giant plasma membrane vesicles (GPMVs) / protein-free giant unilamellar vesicles (GUVs) confirmed the inhibitory effect of dense membrane proteins on membrane fusion. Our work demonstrates the membrane protein density influences the mode of membrane fusion and lays a foundation for constructing quasi-native membrane fusion models in vitro.
Topics: Cell Membrane; Membrane Fusion; Membrane Proteins; Optical Imaging; Unilamellar Liposomes
PubMed: 33676648
DOI: 10.1016/j.talanta.2021.122091 -
Langmuir : the ACS Journal of Surfaces... Feb 2022Membrane active compounds are able to induce various types of membrane perturbations. Natural or biomimetic candidates for antimicrobial treatment or drug delivery...
Membrane active compounds are able to induce various types of membrane perturbations. Natural or biomimetic candidates for antimicrobial treatment or drug delivery scenarios are mostly designed and tested for their ability to induce membrane permeabilization, also termed leakage. Furthermore, the interaction of these usually cationic amphiphiles with negatively charged vesicles often causes colloidal instability leading to vesicle aggregation or/and vesicle fusion. We show the interplay of these modes of membrane perturbation in mixed phosphatidyl glycerol (PG)/phosphatidyl ethanolamine (PE) by the statistical copolymer MM:CO comprising, both, charged and hydrophobic subunits. MM:CO is a representative of partially hydrophobic, highly active, but less selective antimicrobial polycations. Cryo-electron microscopy indicates vesicle fusion rather than vesicle aggregation upon the addition of MM:CO to negatively charged PG/PE (1:1) vesicles. In a combination of fluorescence-based leakage and fusion assays, there is support for membrane permeabilization and pronounced vesicle fusion activity as distinct effects. To this end, membrane fusion and aggregation were prevented by including lipids with polyethylene glycol attached to their head groups (PEG-lipids). The leakage activity of MM:CO is very similar in the absence and presence of PEG-lipids. Vesicle aggregation and fusion however are largely suppressed. This strongly suggests that MM:CO induces leakage by asymmetric packing stress because of hydrophobically driven interactions which could lead to leakage. As a further membrane perturbation effect, MM:CO causes lipid clustering in model vesicles. We address potential artifacts and misinterpretations of experiments characterizing leakage and fusion. Additional to the leakage activity, the pronounced fusogenic activity of the polymer and potentially of many other similar compounds likely has implications for antimicrobial activity and beyond.
Topics: Anti-Infective Agents; Cluster Analysis; Cryoelectron Microscopy; Membrane Fusion; Polyelectrolytes; Static Electricity
PubMed: 35148117
DOI: 10.1021/acs.langmuir.1c03445 -
Molecular Biology of the Cell Mar 2017Recent studies suggest revisions to the SNARE paradigm of membrane fusion. Membrane tethers and/or SNAREs recruit proteins of the Sec 1/Munc18 family to catalyze SNARE... (Review)
Review
Recent studies suggest revisions to the SNARE paradigm of membrane fusion. Membrane tethers and/or SNAREs recruit proteins of the Sec 1/Munc18 family to catalyze SNARE assembly into -complexes. SNARE-domain zippering draws the bilayers into immediate apposition and provides a platform to position fusion triggers such as Sec 17/α-SNAP and/or synaptotagmin, which insert their apolar "wedge" domains into the bilayers, initiating the lipid rearrangements of fusion.
Topics: Animals; Humans; Lipids; Membrane Fusion; Membrane Proteins; Munc18 Proteins; Protein Binding; SNARE Proteins; Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins
PubMed: 28292915
DOI: 10.1091/mbc.E16-07-0517