scholarly journals Dilation of the influenza hemagglutinin fusion pore revealed by the kinetics of individual cell-cell fusion events.

1996 ◽  
Vol 135 (1) ◽  
pp. 63-71 ◽  
Author(s):  
R Blumenthal ◽  
D P Sarkar ◽  
S Durell ◽  
D E Howard ◽  
S J Morris
Keyword(s):  
Membrane Potential ◽  
Cell Fusion ◽  
Individual Cell ◽  
Fluorescent Dye ◽  
Fusion Pore ◽  
Influenza Hemagglutinin ◽  
Rbc Membrane ◽  
Pore Opening ◽  
Kinetics Of ◽  
Fluorescent Lipid

We have monitored kinetics of fusion between cell pairs consisting of a single influenza hemaglutinin (HA)-expressing cell and a single erythrocyte (RBC) that had been labeled with both a fluorescent lipid (Dil) in the membrane and a fluorescent solute (calcein) in the aqueous space. Initial fusion pore opening between the RBC and HA-expressing cell produced a change in RBC membrane potential (delta psi) that was monitored by a decrease in Dil fluorescence. This event was followed by two distinct stages of fusion pore dilation: the flux of fluorescent lipid (phi L) and the flux of a large aqueous fluorescent dye (phi s). We have analyzed the kinetics of events that occur as a result of transitions between a fusion pore (FP) and a solute permissive fusion pore (FPs). Our data are consistent with a fusion pore comprising six HA trimers.

scholarly journals Membrane flux through the pore formed by a fusogenic viral envelope protein during cell fusion.

1993 ◽  
Vol 121 (3) ◽  
pp. 543-552 ◽  
Author(s):  
F W Tse ◽  
A Iwata ◽  
W Almers
Keyword(s):  
Cell Fusion ◽  
Protein A ◽  
Electrical Conductance ◽  
Viral Envelope ◽  
Fusion Pore ◽  
Viral Envelope Protein ◽  
Rbc Membrane ◽  
Membrane Flux ◽  
Quantitative Image ◽  
Fluorescent Lipid

We have investigated the mechanism of cell fusion mediated by HA, the fusogenic hemagglutinin of the Influenza viral envelope. Single erythrocytes (RBCs) were attached to fibroblasts expressing the HA on their cell surface, and fusion of the paired cells was triggered by rapid acidification. The RBC membrane was stained with fluorescent lipid, and the fusion-induced escape of lipid into the fibroblast was observed by quantitative image analysis. At the same time, the formation of an aqueous connection (i.e., the fusion pore) between the two cells was monitored electrically. Within minutes after acidification, an electrical conductance between the two cells appeared abruptly as the fusion pore opened, and then increased gradually as the pore dilated. Later, fluorescent lipid diffused into the fibroblast, approaching equilibrium over the next 5-20 min. No lipid flux was seen while the pore conductance remained 0.5 nS or less. Evidently lipid flux requires a threshold pore size. Our finding suggests that the smallest and earliest fusion pores are surrounded by a ring of protein. A fusion pore expands by breaking this ring and recruiting lipid into its circumference.

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 Cholesterol Promotes Hemifusion and Pore Widening in Membrane Fusion Induced by Influenza Hemagglutinin

2008 ◽  
Vol 131 (5) ◽  
pp. 503-513 ◽  
Author(s):  
Subrata Biswas ◽  
Shu-Rong Yin ◽  
Paul S. Blank ◽  
Joshua Zimmerberg
Keyword(s):  
Membrane Fusion ◽  
Membrane Lipid ◽  
Fusion Pore ◽  
Dye Transfer ◽  
Influenza Hemagglutinin ◽  
Human Red Blood Cells ◽  
Cholesterol Levels ◽  
Pore Opening ◽  
Two Stages ◽  
Pore Expansion

Cholesterol-specific interactions that affect membrane fusion were tested for using insect cells; cells that have naturally low cholesterol levels (<4 mol %). Sf9 cells were engineered (HAS cells) to express the hemagglutinin (HA) of the influenza virus X-31 strain. Enrichment of HAS cells with cholesterol reduced the delay between triggering and lipid dye transfer between HAS cells and human red blood cells (RBC), indicating that cholesterol facilitates membrane lipid mixing prior to fusion pore opening. Increased cholesterol also increased aqueous content transfer between HAS cells and RBC over a broad range of HA expression levels, suggesting that cholesterol also favors fusion pore expansion. This interpretation was tested using both trans-cell dye diffusion and fusion pore conductivity measurements in cholesterol-enriched cells. The results of this study support the hypothesis that host cell cholesterol acts at two stages in membrane fusion: (1) early, prior to fusion pore opening, and (2) late, during fusion pore expansion.

scholarly journals Distinct Roles for Key Karyogamy Proteins during Yeast Nuclear Fusion

2009 ◽  
Vol 20 (17) ◽  
pp. 3773-3782 ◽  
Author(s):  
Patricia Melloy ◽  
Shu Shen ◽  
Erin White ◽  
Mark D. Rose
Keyword(s):  
Fluorescence Microscopy ◽  
Nuclear Envelope ◽  
Cell Fusion ◽  
Electron Tomography ◽  
Nuclear Fusion ◽  
Live Cell ◽  
Fusion Pore ◽  
Yeast Mating ◽  
Fusion Junction ◽  
Kinetics Of

During yeast mating, cell fusion is followed by the congression and fusion of the two nuclei. Proteins required for nuclear fusion are found at the surface (Prm3p) and within the lumen (Kar2p, Kar5p, and Kar8p) of the nuclear envelope (NE). Electron tomography (ET) of zygotes revealed that mutations in these proteins block nuclear fusion with different morphologies, suggesting that they act in different steps of fusion. Specifically, prm3 zygotes were blocked before formation of membrane bridges, whereas kar2, kar5, and kar8 zygotes frequently contained them. Membrane bridges were significantly larger and occurred more frequently in kar2 and kar8, than in kar5 mutant zygotes. The kinetics of NE fusion in prm3, kar5, and kar8 mutants, measured by live-cell fluorescence microscopy, were well correlated with the size and frequency of bridges observed by ET. However the kar2 mutant was defective for transfer of NE lumenal GFP, but not diffusion within the lumen, suggesting that transfer was blocked at the NE fusion junction. These observations suggest that Prm3p acts before initiation of outer NE fusion, Kar5p may help dilation of the initial fusion pore, and Kar2p and Kar8p act after outer NE fusion, during inner NE fusion.

scholarly journals Phosphatidylserine Regulation of Ca2+-triggered Exocytosis and Fusion Pores in PC12 Cells

2009 ◽  
Vol 20 (24) ◽  
pp. 5086-5095 ◽  
Author(s):  
Zhen Zhang ◽  
Enfu Hui ◽  
Edwin R. Chapman ◽  
Meyer B. Jackson
Keyword(s):  
Pc12 Cells ◽  
Gradual Increase ◽  
Fusion Pore ◽  
Dependent Manner ◽  
Synaptotagmin I ◽  
Pore Opening ◽  
Kinetic Effects ◽  
Two Phases ◽  
Fusion Pores ◽  
Kinetics Of

The synaptic vesicle protein synaptotagmin I (Syt I) binds phosphatidylserine (PS) in a Ca2+-dependent manner. This interaction is thought to play a role in exocytosis, but its precise functions remain unclear. To determine potential roles for Syt I-PS binding, we varied the PS content in PC12 cells and liposomes and studied the effects on the kinetics of exocytosis and Syt I binding in parallel. Raising PS produced a steeply nonlinear, saturating increase in Ca2+-triggered fusion, and a graded slowing of the rate of fusion pore dilation. Ca2+-Syt I bound liposomes more tightly as PS content was raised, with a steep increase in binding at low PS, and a further gradual increase at higher PS. These two phases in the PS dependence of Ca2+-dependent Syt I binding to lipid may correspond to the two distinct and opposing kinetic effects of PS on exocytosis. PS influences exocytosis in two ways, enhancing an early step leading to fusion pore opening, and slowing a later step when fusion pores dilate. The possible relevance of these results to Ca2+-triggered Syt I binding is discussed along with other possible roles of PS.

scholarly journals Reversible Merger of Membranes at the Early Stage of Influenza Hemagglutinin-mediated Fusion

2000 ◽  
Vol 11 (7) ◽  
pp. 2359-2371 ◽  
Author(s):  
Eugenia Leikina ◽  
Leonid V. Chernomordik
Keyword(s):  
Fusion Protein ◽  
Surface Density ◽  
Early Stage ◽  
Low Ph ◽  
Fusion Pore ◽  
Complete Fusion ◽  
Physiological Temperature ◽  
Influenza Hemagglutinin ◽  
Pore Opening ◽  
Fusion Pores

Fusion mediated by influenza hemagglutinin (HA), a prototype fusion protein, is commonly detected as lipid and content mixing between fusing cells. Decreasing the surface density of fusion-competent HA inhibited these advanced fusion phenotypes and allowed us to identify an early stage of fusion at physiological temperature. Although lipid flow between membranes was restricted, the contacting membrane monolayers were apparently transiently connected, as detected by the transformation of this fusion intermediate into complete fusion after treatments known to destabilize hemifusion diaphragms. These reversible connections disappeared within 10–20 min after application of low pH, indicating that after the energy released by HA refolding dissipated, the final low pH conformation of HA did not support membrane merger. Although the dynamic character and the lack of lipid mixing at 37°C distinguish the newly identified fusion intermediate from the intermediate arrested at 4°C described previously, both intermediates apparently belong to the same family of restricted hemifusion (RH) structures. Because the formation of transient RH structures at physiological temperatures was as fast as fusion pore opening and required less HA, we hypothesize that fusion starts with the formation of multiple RH sites, only a few of which then evolve to become expanding fusion pores.

scholarly journals An Early Stage of Membrane Fusion Mediated by the Low pH Conformation of Influenza Hemagglutinin Depends upon Membrane Lipids

1997 ◽  
Vol 136 (1) ◽  
pp. 81-93 ◽  
Author(s):  
Leonid V. Chernomordik ◽  
Eugenia Leikina ◽  
Vadim Frolov ◽  
Peter Bronk ◽  
Joshua Zimmerberg
Keyword(s):  
Membrane Fusion ◽  
Lipid Bilayers ◽  
Cell Fusion ◽  
Membrane Lipids ◽  
Early Stage ◽  
Molecular Shape ◽  
Membrane Lipid ◽  
Low Ph ◽  
Fusion Pore ◽  
Influenza Hemagglutinin

While the specificity and timing of membrane fusion in diverse physiological reactions, including virus–cell fusion, is determined by proteins, fusion always involves the merger of membrane lipid bilayers. We have isolated a lipid-dependent stage of cell–cell fusion mediated by influenza hemagglutinin and triggered by cell exposure to mildly acidic pH. This stage preceded actual membrane merger and fusion pore formation but was subsequent to a low pH–induced change in hemagglutinin conformation that is required for fusion. A low pH conformation of hemagglutinin was required to achieve this lipid-dependent stage and also, downstream of it, to drive fusion to completion. The lower the pH of the medium applied to trigger fusion and, thus, the more hemagglutinin molecules activated, the less profound was the dependence of fusion on lipids. Membrane-incorporated lipids affected fusion in a manner that correlated with their dynamic molecular shape, a characteristic that determines a lipid monolayer's propensity to bend in different directions. The lipid sensitivity of this stage, i.e., inhibition of fusion by inverted cone–shaped lysophosphatidylcholine and promotion by cone-shaped oleic acid, was consistent with the stalk hypothesis of fusion, suggesting that fusion proteins begin membrane merger by promoting the formation of a bent, lipid-involving, stalk intermediate.

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.

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