Molecular mechanisms of membrane fusion: Steps during phospholipid and exocytotic membrane fusion

1987 ◽  
Vol 7 (4) ◽  
pp. 251-268 ◽  
Author(s):  
Joshua Zimmerberg
Keyword(s):  
Membrane Fusion ◽  
Pore Formation ◽  
Molecular Mechanisms ◽  
Fusion Pore ◽  
Pore Widening

Exocytosis is considered as four separate steps: adhesion, fusion/pore formation, pore widening, and content discharge. Experiments on both synthetic and natural membranes are presented to show each of these steps. Major differences are seen in the two fusing systems. These differences are discussed in terms of molecular mechanisms of fusion.

Mechanics of membrane fusion/pore formation

2015 ◽  
Vol 185 ◽  
pp. 109-128 ◽  
Author(s):  
Marc Fuhrmans ◽  
Giovanni Marelli ◽  
Yuliya G. Smirnova ◽  
Marcus Müller
Keyword(s):  
Membrane Fusion ◽  
Pore Formation ◽  
Fusion Pore

scholarly journals Restricted movement of lipid and aqueous dyes through pores formed by influenza hemagglutinin during cell fusion.

1994 ◽  
Vol 127 (6) ◽  
pp. 1885-1894 ◽  
Author(s):  
J Zimmerberg ◽  
R Blumenthal ◽  
D P Sarkar ◽  
M Curran ◽  
S J Morris
Keyword(s):  
Cell Membrane ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Pore Formation ◽  
Fusion Pore ◽  
Restricted Movement ◽  
Influenza Hemagglutinin ◽  
Simultaneous Measurements ◽  
Cell Membrane Capacitance ◽  
Total Fusion

The fusion of cells by influenza hemagglutinin (HA) is the best characterized example of protein-mediated membrane fusion. In simultaneous measurements of pairs of assays for fusion, we determined the order of detectable events during fusion. Fusion pore formation in HA-triggered cell-cell fusion was first detected by changes in cell membrane capacitance, next by a flux of fluorescent lipid, and finally by flux of aqueous fluorescent dye. Fusion pore conductance increased by small steps. A retardation of lipid and aqueous dyes occurred during fusion pore fluctuations. The flux of aqueous dye depended on the size of the molecule. The lack of movement of aqueous dyes while total fusion pore conductance increased suggests that initial HA-triggered fusion events are characterized by the opening of multiple small pores: the formation of a "sieve".

scholarly journals Multifaceted Sequence-Dependent and -Independent Roles for Reovirus FAST Protein Cytoplasmic Tails in Fusion Pore Formation and Syncytiogenesis

2009 ◽  
Vol 83 (23) ◽  
pp. 12185-12195 ◽  
Author(s):  
Christopher Barry ◽  
Roy Duncan
Keyword(s):  
Membrane Fusion ◽  
Fusion Proteins ◽  
Pore Formation ◽  
Syncytium Formation ◽  
Fusion Pore ◽  
Protein Fusion ◽  
Sequence Dependent ◽  
Cytoplasmic Tails ◽  
Fast Proteins ◽  
Pore Expansion

ABSTRACT Fusogenic reoviruses utilize the FAST proteins, a novel family of nonstructural viral membrane fusion proteins, to induce cell-cell fusion and syncytium formation. Unlike the paradigmatic enveloped virus fusion proteins, the FAST proteins position the majority of their mass within and internal to the membrane in which they reside, resulting in extended C-terminal cytoplasmic tails (CTs). Using tail truncations, we demonstrate that the last 8 residues of the 36-residue CT of the avian reovirus p10 FAST protein and the last 20 residues of the 68-residue CT of the reptilian reovirus p14 FAST protein enhance, but are not required for, pore expansion and syncytium formation. Further truncations indicate that the membrane-distal 12 residues of the p10 and 47 residues of the p14 CTs are essential for pore formation and that a residual tail of 21 to 24 residues that includes a conserved, membrane-proximal polybasic region present in all FAST proteins is insufficient to maintain FAST protein fusion activity. Unexpectedly, a reextension of the tail-truncated, nonfusogenic p10 and p14 constructs with scrambled versions of the deleted sequences restored pore formation and syncytiogenesis, while reextensions with heterologous sequences partially restored pore formation but failed to rescue syncytiogenesis. The membrane-distal regions of the FAST protein CTs therefore exert multiple effects on the membrane fusion reaction, serving in both sequence-dependent and sequence-independent manners as positive effectors of pore formation, pore expansion, and syncytiogenesis.

scholarly journals The block to membrane fusion differs with the site of ligand insertion in modified retroviral envelope proteins

2008 ◽  
Vol 89 (4) ◽  
pp. 1049-1058 ◽  
Author(s):  
Byoung Y. Ryu ◽  
Tatiana Zavorotinskaya ◽  
Bernadette Trentin ◽  
Lorraine M. Albritton
Keyword(s):  
Membrane Fusion ◽  
Pore Formation ◽  
Leukemia Virus ◽  
Point Mutations ◽  
Retroviral Vectors ◽  
Membrane Lipid ◽  
Fusion Pore ◽  
Subsequent Step ◽  
Cell Type Specific ◽  
Proline Rich Region

Efforts to achieve cell type-specific transduction of retroviral vectors for gene therapy have centred on modification of the envelope protein (Env). Typically, addition of a ligand to Env gives binding to the new or target receptor, but little or no infection, and affects the subunit association of the modified Env. We previously discovered two point mutations that increase targeted infection by over 1000-fold when added to an Env modified by N-terminal insertion of the receptor-binding domain from amphotropic murine leukemia virus Env. Here, we asked whether these mutations would similarly increase transduction by Env modified with a clinically relevant ligand, human interleukin-13 (IL-13L). Addition of the point mutations stabilized the weak subunit association observed in some IL-13L-modified Env proteins, but infection via the target IL-13 receptor still did not occur. Fluorescence-based cell–cell fusion assays and studies with a membrane-curving agent revealed that defects in membrane fusion differed with the site of ligand insertion. When IL-13 was inserted into the N terminus of Env, membrane fusion was blocked prior to membrane-lipid mixing, regardless of whether flanking flexible linkers were added. Unexpectedly, insertion of IL-13 in the proline-rich region showed evidence of initiation of fusion and fusion-peptide exposure, but fusion was blocked at a subsequent step prior to fusion-pore formation. Thus, the site of ligand insertion influenced initiation of membrane fusion and its progression. These observations suggest that a novel site for ligand insertion must be identified before clinically useful targeted transduction will be achieved.

scholarly journals Single-virus content mixing assay reveals cholesterol-enhanced influenza membrane fusion efficiency

2021 ◽  
Author(s):  
Katherine N Liu ◽  
Steven G. Boxer
Keyword(s):  
Membrane Fusion ◽  
Influenza A ◽  
Pore Formation ◽  
Fluorescent Labeling ◽  
Fusion Pore ◽  
Viral Fusion ◽  
Enveloped Viruses ◽  
Virus Assay ◽  
Virus Content ◽  
Target Membrane

In order to infect a cell, enveloped viruses must first undergo membrane fusion, which proceeds through a hemifusion intermediate, followed by the formation of a fusion pore through which the viral genome is transferred to a target cell. Single-virus fusion studies to elucidate the dynamics of content mixing typically require extensive fluorescent labeling of viral contents. The labeling process must be optimized depending on the virus identity and strain and can potentially be perturbative to viral fusion behavior. Here, we introduce a single-virus assay where content-labeled vesicles are bound to unlabeled influenza A virus (IAV) to eliminate the problematic step of content-labeling virions. We use fluorescence microscopy to observe individual, pH-triggered content mixing and content loss events between IAV and target vesicles of varying cholesterol compositions. We show that target membrane cholesterol increases the efficiency of IAV content mixing and decreases the fraction of content mixing events that result in content loss. These results are consistent with previous findings that cholesterol stabilizes pore formation in IAV entry and limits leakage following pore formation. We also show that content loss due to hemagglutinin fusion peptide engagement with the target membrane is independent of composition. This approach is a promising strategy for studying the single-virus content mixing kinetics of other enveloped viruses.

scholarly journals HIV gp41 Trans-Membrane Domain Promotes both Stalk and Fusion Pore Formation in Poly(Ethylene-) Glycol Mediated Membrane Fusion

2012 ◽  
Vol 102 (3) ◽  
pp. 499a-500a
Author(s):  
Hirak Chakraborty ◽  
Sekaran Moses Dennison ◽  
David G. Klapper ◽  
Barry R. Lentz
Keyword(s):  
Ethylene Glycol ◽  
Membrane Fusion ◽  
Pore Formation ◽  
Fusion Pore ◽  
Membrane Domain ◽  
Poly Ethylene Glycol ◽  
Poly Ethylene ◽  
Hiv Gp41

scholarly journals Insulin-stimulated Plasma Membrane Fusion of Glut4 Glucose Transporter-containing Vesicles Is Regulated by Phospholipase D1

2005 ◽  
Vol 16 (6) ◽  
pp. 2614-2623 ◽  
Author(s):  
Ping Huang ◽  
Yelena M. Altshuller ◽  
June Chunqiu Hou ◽  
Jeffrey E. Pessin ◽  
Michael A. Frohman
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
Glucose Uptake ◽  
Phospholipase D ◽  
Pore Formation ◽  
Glucose Transporter ◽  
Fusion Pore ◽  
Cell Periphery ◽  
Intracellular Membrane ◽  
Phospholipase D1

Insulin stimulates glucose uptake in fat and muscle by mobilizing Glut4 glucose transporters from intracellular membrane storage sites to the plasma membrane. This process requires the trafficking of Glut4-containing vesicles toward the cell periphery, docking at exocytic sites, and plasma membrane fusion. We show here that phospholipase D (PLD) production of the lipid phosphatidic acid (PA) is a key event in the fusion process. PLD1 is found on Glut4-containing vesicles, is activated by insulin signaling, and traffics with Glut4 to exocytic sites. Increasing PLD1 activity facilitates glucose uptake, whereas decreasing PLD1 activity is inhibitory. Diminished PA production does not substantially hinder trafficking of the vesicles or their docking at the plasma membrane, but it does impede fusion-mediated extracellular exposure of the transporter. The fusion block caused by RNA interference-mediated PLD1 deficiency is rescued by exogenous provision of a lipid that promotes fusion pore formation and expansion, suggesting that the step regulated by PA is late in the process of vesicle fusion.

scholarly journals The Pathway of Membrane Fusion Catalyzed by Influenza Hemagglutinin: Restriction of Lipids, Hemifusion, and Lipidic Fusion Pore Formation

1998 ◽  
Vol 140 (6) ◽  
pp. 1369-1382 ◽  
Author(s):  
Leonid V. Chernomordik ◽  
Vadim A. Frolov ◽  
Eugenia Leikina ◽  
Peter Bronk ◽  
Joshua Zimmerberg
Keyword(s):  
Membrane Fusion ◽  
Surface Density ◽  
Pore Formation ◽  
Membrane Lipid ◽  
Low Ph ◽  
Fusion Pore ◽  
Present Evidence ◽  
Membrane Lipid Composition ◽  
Influenza Hemagglutinin ◽  
Pore Opening

The mechanism of bilayer unification in biological fusion is unclear. We reversibly arrested hemagglutinin (HA)-mediated cell–cell fusion right before fusion pore opening. A low-pH conformation of HA was required to form this intermediate and to ensure fusion beyond it. We present evidence indicating that outer monolayers of the fusing membranes were merged and continuous in this intermediate, but HA restricted lipid mixing. Depending on the surface density of HA and the membrane lipid composition, this restricted hemifusion intermediate either transformed into a fusion pore or expanded into an unrestricted hemifusion, without pores but with unrestricted lipid mixing. Our results suggest that restriction of lipid flux by a ring of activated HA is necessary for successful fusion, during which a lipidic fusion pore develops in a local and transient hemifusion diaphragm.

scholarly journals Influence of Acylation Sites of Influenza B Virus Hemagglutinin on Fusion Pore Formation and Dilation

2004 ◽  
Vol 78 (21) ◽  
pp. 11536-11543 ◽  
Author(s):  
Makoto Ujike ◽  
Katsuhisa Nakajima ◽  
Eri Nobusawa
Keyword(s):  
Amino Acid ◽  
Membrane Fusion ◽  
Pore Formation ◽  
Syncytium Formation ◽  
Cysteine Residue ◽  
Fusion Pore ◽  
Influenza B Virus ◽  
Influenza B ◽  
Dye Transfer ◽  
B Virus

ABSTRACT The cytoplasmic tail (CT) of hemagglutinin (HA) of influenza B virus (BHA) contains at positions 578 and 581 two highly conserved cysteine residues (Cys578 and Cys581) that are modified with palmitic acid (PA) through a thioester linkage. To investigate the role of PA in the fusion activity of BHA, site-specific mutagenesis was performed with influenza B virus B/Kanagawa/73 HA cDNA. All of the HA mutants were expressed on Cos cells by an expression vector. The membrane fusion ability of the HA mutants at a low pH was quantitatively examined with lipid (octadecyl rhodamine B chloride) and aqueous (calcein) dye transfer assays and with the syncytium formation assay. Two deacylation mutants lacking a CT or carrying serine residues substituting for Cys578 and Cys581 promoted full fusion. However, one of the single-acylation-site mutants, C6, in which Cys581 is replaced with serine, promoted hemifusion but not pore formation. In contrast, four other single-acylation-site mutants that have a sole cysteine residue in the CT at position 575, 577, 579, or 581 promoted full fusion. The impaired pore-forming ability of C6 was improved by amino acid substitution between residues 578 and 582 or by deletion of the carboxy-terminal leucine at position 582. Syncytium-forming ability, however, was not adequately restored by these mutations. These facts indicated that the acylation was not significant in membrane fusion by BHA but that pore formation and pore dilation were appreciably affected by the particular amino acid sequence of the CT and the existence of a single acylation site in CT residue 578.

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