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 HIV-1 Envelope Proteins Complete Their Folding into Six-helix Bundles Immediately after Fusion Pore Formation

2003 ◽  
Vol 14 (3) ◽  
pp. 926-938 ◽  
Author(s):  
Ruben M. Markosyan ◽  
Fredric S. Cohen ◽  
Grigory B. Melikyan
Keyword(s):  
Fusion Proteins ◽  
Pore Formation ◽  
Temperature Jump ◽  
Fusion Pore ◽  
Target Cells ◽  
Pore Growth ◽  
Essential Function ◽  
Intermediate Value ◽  
Fusion Pores ◽  
Hiv 1

Fusion proteins of many viruses, including HIV-1 envelope protein (Env), fold into six-helix bundle structures. Fusion between individual Env-expressing cells and target cells was studied by fluorescence microscopy, and a temperature jump technique, to determine whether folding of Env into a bundle is complete by the time fusion pores have formed. Lowering temperature to 4°C immediately after a pore opened halted pore growth, which quickly resumed when temperature was raised again. HIV gp41-derived peptides that inhibit bundle formation (C34 or N36) caused the cold-arrested pore to quickly and irreversibly close, demonstrating that bundle formation is not complete by the time a pore has formed. In contrast, lowering the temperature to an intermediate value also halted pore growth, but the pore was not closed by the bundle-inhibiting peptides, and it enlarged when temperature was again elevated. This latter result shows that bundle formation is definitely required for the fusion process, but surprisingly, some (if not all) bundle formation occurs after a pore has formed. It is concluded that an essential function of the bundle is to stabilize the pore against collapse and ensure its growth.

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 Fusion Pore Formation Observed During SNARE-Mediated Vesicle Fusion with Pore-Spanning Membranes

2020 ◽  
Author(s):  
P. Mühlenbrock ◽  
K. Herwig ◽  
L. Vuong ◽  
I. Mey ◽  
C. Steinem
Keyword(s):  
Fluorescence Microscopy ◽  
Pore Formation ◽  
Three Dimensional ◽  
Rare Event ◽  
Vesicle Fusion ◽  
Fusion Process ◽  
Fusion Pore ◽  
Dual Color ◽  
Fusion Pores

ABSTRACTPlanar pore-spanning membranes (PSMs) have been shown to be a versatile tool to resolve docking and elementary steps of the fusion process with single large unilamellar vesicles (LUVs). However, in previous studies, we monitored only lipid mixing and did not gather information about the formation of fusion pores. To address this important step of the fusion process, we entrapped sulforhodamine B at self-quenching concentrations into LUVs containing the v-SNARE synaptobrevin 2, which were docked and fused with lipid-labeled PSMs containing the t-SNARE acceptor complex ΔN49 prepared on porous silicon substrates. By dual color spinning disc fluorescence microcopy with a time resolution of 20 ms, we could unambiguously distinguish between bursting vesicles and fusion pore formation. Owing to the aqueous compartment underneath the PSMs, vesicle bursting turned out to be an extremely rare event (< 0.01 %). From the time-resolved dual color fluorescence time traces, we were able to identify different fusion pathways including remaining three-dimensional postfusion structures with released content and flickering fusion pores. Our results on fusion pore formation and lipid diffusion from the PSM into the fusing vesicle let us conclude that the content release, i.e., fusion pore formation follows the merger of the two lipid membranes by only about 40 ms.STATEMENT OF SIGNIFICANCEDespite great efforts to develop in vitro fusion assays to understand the process of neuronal fusion, there is still a huge demand to provide single vesicle fusion assays that simultaneously report on all intermediate states including three-dimensional postfusion structures and fusion pore formation including flickering pores without the underlying artifact of vesicle bursting. Here, we show that pore-spanning membranes (PSMs) are ideal candidates to fulfill these demands. Owing to their planarity and the second aqueous compartments, they are readily accessible by fluorescence microscopy and provide sufficient space so that vesicle bursting becomes negligible. Dual color fluorescence microscopy allows distinguishing between different fusion intermediates and fusion pathways such as “kiss and run” fusion as well as flickering fusion pores.

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.

scholarly journals A bone sialoprotein-binding protein from Staphylococcus aureus: a member of the staphylococcal Sdr family

2000 ◽  
Vol 345 (3) ◽  
pp. 611-619 ◽  
Author(s):  
Hui-shan TUNG ◽  
Bengt GUSS ◽  
Ulf HELLMAN ◽  
Lena PERSSON ◽  
Kristofer RUBIN ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Surface ◽  
Fusion Protein ◽  
Binding Protein ◽  
Joint Infection ◽  
Bone Sialoprotein ◽  
Amino Acid Sequences ◽  
Surface Proteins ◽  
Aureus Strain ◽  
Chromosomal Dna

Staphylococcus aureus bacteria, isolated from bone and joint infections, specifically interact with bone sialoprotein (BSP), a glycoprotein of bone and dentine extracellular matrix, via a cell-surface protein of Mr 97000 [Yacoub, Lindahl, Rubin, Wendel, Heinegård and Rydén, (1994) Eur. J. Biochem. 222, 919-925]. Amino acid sequences of seven trypsin fragments from the 97000-Mr BSP-binding protein were determined. A gene encoding a protein encompassing all seven peptide sequences was identified from chromosomal DNA isolated from S. aureus strain O24. This gene encodes a protein with 1171 amino acids, called BSP-binding protein (Bbp), which displays similarity to recently described proteins of the Sdr family from S. aureus. SdrC, SdrD and SdrE encode putative cell-surface proteins with no described ligand specificity. Bbp also shows similarity to a fibrinogen-binding protein from S. epidermidis called Fbe. A serine-aspartic acid repeat sequence was found close to the cell-wall-anchoring Leu-Pro-Xaa-Thr-Gly sequence in the C-terminal end of the protein. Escherichia coli cells were transformed with an expression vector containing a major part of the bbp gene fused to the gene for glutathione S-transferase. The affinity-purified fusion protein bound radiolabelled native BSP, and inhibited the binding of radiolabelled BSP to staphylococcal cells. Serum from patients suffering from bone and joint infection contained antibodies that reacted with the fusion protein of the BSP-binding protein, indicating that the protein is expressed during an infection and is immunogenic. The S. aureus Bbp protein may be important in the localization of bacteria to bone tissue, and thus might be of relevance in the pathogenicity of osteomyelitis.

scholarly journals Molecular mechanism of fusion pore formation driven by the neuronal SNARE complex

2018 ◽  
Vol 115 (50) ◽  
pp. 12751-12756 ◽  
Author(s):  
Satyan Sharma ◽  
Manfred Lindau
Keyword(s):  
Pore Formation ◽  
Structural Model ◽  
Snare Complex ◽  
Coarse Grained ◽  
Fusion Pore ◽  
Protein Mutants ◽  
Experimental Values ◽  
Dynamics Simulations ◽  
External Forces ◽  
Fusion Pores

Release of neurotransmitters from synaptic vesicles begins with a narrow fusion pore, the structure of which remains unresolved. To obtain a structural model of the fusion pore, we performed coarse-grained molecular dynamics simulations of fusion between a nanodisc and a planar bilayer bridged by four partially unzipped SNARE complexes. The simulations revealed that zipping of SNARE complexes pulls the polar C-terminal residues of the synaptobrevin 2 and syntaxin 1A transmembrane domains to form a hydrophilic core between the two distal leaflets, inducing fusion pore formation. The estimated conductances of these fusion pores are in good agreement with experimental values. Two SNARE protein mutants inhibiting fusion experimentally produced no fusion pore formation. In simulations in which the nanodisc was replaced by a 40-nm vesicle, an extended hemifusion diaphragm formed but a fusion pore did not, indicating that restricted SNARE mobility is required for rapid fusion pore formation. Accordingly, rapid fusion pore formation also occurred in the 40-nm vesicle system when SNARE mobility was restricted by external forces. Removal of the restriction is required for fusion pore expansion.

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.

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