scholarly journals GPI-anchored influenza hemagglutinin induces hemifusion to both red blood cell and planar bilayer membranes.

1995 ◽  
Vol 131 (3) ◽  
pp. 679-691 ◽  
Author(s):  
G B Melikyan ◽  
J M White ◽  
F S Cohen
Keyword(s):  
Transmembrane Domain ◽  
Coupling Model ◽  
Planar Bilayer ◽  
Bilayer Membranes ◽  
Time Resolved ◽  
Influenza Hemagglutinin ◽  
Influenza Virus Hemagglutinin ◽  
Video Fluorescence Microscopy ◽  
Fusion Pores ◽  
Outer Monolayer

Under fusogenic conditions, fluorescent dye redistributed from the outer monolayer leaflet of red blood cells (RBCs) to cells expressing glycophosphatidylinositol-anchored influenza virus hemagglutinin (GPI-HA) without transfer of aqueous dye. This suggests that hemifusion, but not full fusion, occurred (Kemble, G. W., T. Danieli, and J. M. White. 1994. Cell. 76:383-391). We extended the evidence for hemifusion by labeling the inner monolayer leaflets of RBCs with FM4-64 and observing that these inner leaflets did not become continuous with GPI-HA-expressing cells. The region of hemifusion-separated aqueous contents, the hemifusion diaphragm, appeared to be extended and was long-lived. But when RBCs hemifused to GPI-HA-expressing cells were osmotically swollen, some diaphragms were disrupted, and spread of both inner leaflet and aqueous dyes was observed. This was characteristic of full fusion: inner leaflet and aqueous probes spread to cells expressing wild-type HA (wt-HA). By simultaneous video fluorescence microscopy and time-resolved electrical admittance measurements, we rigorously demonstrated that GPI-HA-expressing cells hemifuse to planar bilayer membranes: lipid continuity was established without formation of fusion pores. The hemifusion area became large. In contrast, for cells expressing wt-HA, before lipid dye spread, fusion pores were always observed, establishing that full fusion occurred. We present an elastic coupling model in which the ectodomain of wt-HA induces hemifusion and the transmembrane domain, absent in the GPI-HA-expressing cells, mediates full fusion.

scholarly journals Comparison of transient and successful fusion pores connecting influenza hemagglutinin expressing cells to planar membranes.

1995 ◽  
Vol 106 (5) ◽  
pp. 803-819 ◽  
Author(s):  
G B Melikyan ◽  
W D Niles ◽  
V A Ratinov ◽  
M Karhanek ◽  
J Zimmerberg ◽  
...  
Keyword(s):  
Time Course ◽  
Cell Surfaces ◽  
Bilayer Membranes ◽  
Time Resolved ◽  
Influenza Hemagglutinin ◽  
Influenza Virus Hemagglutinin ◽  
Time Courses ◽  
Fusion Pores ◽  
Planar Membranes ◽  
Pore Enlargement

Time-resolved admittance measurements were used to investigate the evolution of fusion pores formed between cells expressing influenza virus hemagglutinin (HA) and planar bilayer membranes. The majority of fusion pores opened in a stepwise fashion to semistable conductance levels of several nS. About 20% of the pores had measurable rise times to nS conductances; some of these opened to conductances of approximately 500 pS where they briefly lingered before opening further to semistable conductances. The fall times of closing were statistically similar to the rise times of opening. All fusion pores exhibited semistable values of conductance, varying from approximately 2-20 nS; they would then either close or fully open to conductances on the order of 1 microS. The majority of pores closed; approximately 10% fully opened. Once within the semistable stage, all fusion pores, even those that eventually closed, tended to grow. Statistically, however, before closing, transient fusion pores ceased to grow and reversed their conductance pattern: conductances decreased with a measurable time course until a final drop to closure. In contrast, pore enlargement to the fully open state tended to occur from the largest conductance values attained during a pore's semistable stage. This final enlargement was characterized by a stepwise increase in conductance. The density of HA on the cell surface did not strongly affect pore dynamics. But increased proteolytic treatment of cell surfaces did lead to faster growth within the semistable range. Transient pores and pores that fully opened had indistinguishable initial conductances and statistically identical time courses of early growth, suggesting they were the same upon formation. We suggest that transient and fully open pores evolved from common structures with stochastic factors determining their fate.

scholarly journals Lateral Distribution of the Transmembrane Domain of Influenza Virus Hemagglutinin Revealed by Time-resolved Fluorescence Imaging

2009 ◽  
Vol 284 (23) ◽  
pp. 15708-15716 ◽  
Author(s):  
Silvia Scolari ◽  
Stephanie Engel ◽  
Nils Krebs ◽  
Anna Pia Plazzo ◽  
Rodrigo F. M. De Almeida ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Fluorescence Imaging ◽  
Transmembrane Domain ◽  
Lateral Distribution ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Influenza Virus Hemagglutinin

scholarly journals The fusion kinetics of influenza hemagglutinin expressing cells to planar bilayer membranes is affected by HA density and host cell surface.

1995 ◽  
Vol 106 (5) ◽  
pp. 783-802 ◽  
Author(s):  
G B Melikyan ◽  
W D Niles ◽  
F S Cohen
Keyword(s):  
Cell Surface ◽  
Fusion Protein ◽  
Pore Formation ◽  
Surface Proteins ◽  
Fusion Pore ◽  
Planar Bilayer ◽  
Bilayer Membranes ◽  
Pore Evolution ◽  
Fusion Pores ◽  
Kinetics Of

Time-resolved admittance measurements were used to follow formation of individual fusion pores connecting influenza virus hemagglutinin (HA)-expressing cells to planar bilayer membranes. By measuring in-phase, out-of-phase, and dc components of currents, pore conductances were resolved with millisecond time resolution. Fusion pores developed in stages, from small pores flickering open and closed, to small successful pores that remained open until enlarging their lumens to sizes greater than those of viral nucleocapsids. The kinetics of fusion and the properties of fusion pores were studied as functions of density of the fusion protein HA. The consequences of treating cell surfaces with proteases that do not affect HA were also investigated. Fusion kinetics were described by waiting time distributions from triggering fusion, by lowering pH, to the moment of pore formation. The kinetics of pore formation became faster as the density of active HA was made greater or when cell surface proteins were extensively cleaved with proteases. In accord with this faster kinetics, the intervals between transient pore openings within the flickering stage were shorter for higher HA density and more extensive cell surface treatment. Whereas the kinetics of fusion depended on HA density, the lifetimes of open fusion pores were independent of HA density. However, the lifetimes of open pores were affected by the proteolytic treatment of the cells. Faster fusion kinetics correlated with shorter pore openings. We conclude that the density of fusion protein strongly affects the kinetics of fusion pore formation, but that once formed, pore evolution is not under control of fusion proteins but rather under the influence of mechanical forces, such as membrane bending and tension.

scholarly journals Inner but Not Outer Membrane Leaflets Control the Transition from Glycosylphosphatidylinositol-anchored Influenza Hemagglutinin-induced Hemifusion to Full Fusion

1997 ◽  
Vol 136 (5) ◽  
pp. 995-1005 ◽  
Author(s):  
Grigory B. Melikyan ◽  
Sofya A. Brener ◽  
Dong C. Ok ◽  
Fredric S. Cohen
Keyword(s):  
Outer Membrane ◽  
Negative Curvature ◽  
Pore Formation ◽  
Transmembrane Domain ◽  
Positive Curvature ◽  
Fusion Pore ◽  
Spontaneous Curvature ◽  
Wild Type ◽  
Influenza Hemagglutinin ◽  
Fusion Pores

Cells that express wild-type influenza hemagglutinin (HA) fully fuse to RBCs, while cells that express the HA-ectodomain anchored to membranes by glycosylphosphatidylinositol, rather than by a transmembrane domain, only hemifuse to RBCs. Amphipaths were inserted into inner and outer membrane leaflets to determine the contribution of each leaflet in the transition from hemifusion to fusion. When inserted into outer leaflets, amphipaths did not promote the transition, independent of whether the agent induces monolayers to bend outward (conferring positive spontaneous monolayer curvature) or inward (negative curvature). In contrast, when incorporated into inner leaflets, positive curvature agents led to full fusion. This suggests that fusion is completed when a lipidic fusion pore with net positive curvature is formed by the inner leaflets that compose a hemifusion diaphragm. Suboptimal fusion conditions were established for RBCs bound to cells expressing wild-type HA so that lipid but not aqueous dye spread was observed. While this is the same pattern of dye spread as in stable hemifusion, for this “stunted” fusion, lower concentrations of amphipaths in inner leaflets were required to promote transfer of aqueous dyes. Also, these amphipaths induced larger pores for stunted fusion than they generated within a stable hemifusion diaphragm. Therefore, spontaneous curvature of inner leaflets can affect formation and enlargement of fusion pores induced by HA. We propose that after the HA-ectodomain induces hemifusion, the transmembrane domain causes pore formation by conferring positive spontaneous curvature to leaflets of the hemifusion diaphragm.

Fusion of Large Unilamellar Liposomes Containing Hemocyanin with Planar Bilayer Membranes

1984 ◽  
Vol 39 (1-2) ◽  
pp. 147-155 ◽  
Author(s):  
Flavia Pasquali ◽  
Gianfranco Menestrina ◽  
Renzo Antolini
Keyword(s):  
Electrical Properties ◽  
Ionic Channels ◽  
Planar Bilayer ◽  
Artificial Membrane ◽  
Bilayer Membranes ◽  
Large Unilamellar Vesicles ◽  
Strong Current ◽  
Voltage Curve ◽  
Current Voltage ◽  
Current Voltage Curve

Abstract Large unilamellar vesicles were prepared by detergent removal from micelles containing phosphatidylcholine, phosphatidylethanolam ine and phosphatidylserine. Liposomes were then interacted with Megathura crenulata hemocyanin, a well studied channel former. Incubation of the resulting proteoliposomes on one side of a phosphatidylserine-containing planar bilayer under fusion conditions yielded strong current increases. Such increase is due to insertion of ionic channels from the liposomes into the planar bilayer. Studying the effects of Ba2+ on the electrical properties of the channel we could show that the protein is always inserted into a bilayer during this process, i.e. fusion of proteoliposomes with the artificial membrane occurs. The strong non linearity of the current-voltage curve of the hemocyanin pore could be used as a probe of the extent to which fusion preserves the orientation of the channel through the bilayer.

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