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.

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 Secretory and viral fusion may share mechanistic events with fusion between curved lipid bilayers

1998 ◽  
Vol 95 (16) ◽  
pp. 9274-9279 ◽  
Author(s):  
JinKeun Lee ◽  
Barry R. Lentz
Keyword(s):  
Membrane Fusion ◽  
Lipid Bilayers ◽  
Biological Membrane ◽  
Activation Energies ◽  
Membrane Lipid ◽  
Striking Similarity ◽  
Fusion Pore ◽  
Viral Fusion ◽  
Lipid Molecule ◽  
Free Model

Activation energies for the individual steps of secretory and viral fusion are reported to be large [Oberhauser, A. F., Monck, J. R. & Fernandez, J. M. (1992) Biophys. J. 61, 800–809; Clague, M. J., Schoch, C., Zech, L. & Blumenthal, R. (1990) Biochemistry 29, 1303–1308]. Understanding the cause for these large activation energies is crucial to defining the mechanisms of these two types of biological membrane fusion. We showed recently that the fusion of protein-free model lipid bilayers mimics the sequence of steps observed during secretory and viral fusion, suggesting that these processes may involve common lipid, rather than protein, rearrangements. To test for this possibility, we determined the activation energies for the three steps that we were able to distinguish as contributing to the fusion of protein-free model lipid bilayers. Activation energies for lipid rearrangements associated with formation of the reversible first intermediate, with conversion of this to a semi-stable second intermediate, and with irreversible fusion pore formation were 37 kcal/mol, 27 kcal/mol, and 22 kcal/mol, respectively. The first and last of these were comparable to the activation energies observed for membrane lipid exchange (42 kcal/mol) during viral fusion and for the rate of fusion pore opening during secretory granule release (23 kcal/mol). This striking similarity suggests strongly that the basic molecular processes involved in secretory and viral fusion involve a set of lipid molecule rearrangements that also are involved in model membrane fusion.

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 Genetic Control of Fusion Pore Expansion in the Epidermis ofCaenorhabditis elegans

2007 ◽  
Vol 18 (4) ◽  
pp. 1153-1166 ◽  
Author(s):  
Tamar Gattegno ◽  
Aditya Mittal ◽  
Clari Valansi ◽  
Ken C.Q. Nguyen ◽  
David H. Hall ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Cell Fusion ◽  
Fusion Pore ◽  
Membrane Domains ◽  
Cell Pair ◽  
C Elegans ◽  
Ultrastructural Studies ◽  
Different Temperatures ◽  
Multiple Cell ◽  
Extensive Information

Developmental cell fusion is found in germlines, muscles, bones, placentae, and stem cells. In Caenorhabditis elegans 300 somatic cells fuse during development. Although there is extensive information on the early intermediates of viral-induced and intracellular membrane fusion, little is known about late stages in membrane fusion. To dissect the pathway of cell fusion in C. elegans embryos, we use genetic and kinetic analyses using live-confocal and electron microscopy. We simultaneously monitor the rates of multiple cell fusions in developing embryos and find kinetically distinct stages of initiation and completion of membrane fusion in the epidermis. The stages of cell fusion are differentially blocked or retarded in eff-1 and idf-1 mutants. We generate kinetic cell fusion maps for embryos grown at different temperatures. Different sides of the same cell differ in their fusogenicity: the left and right membrane domains are fusion-incompetent, whereas the anterior and posterior membrane domains fuse with autonomous kinetics in embryos. All but one cell pair can initiate the formation of the largest syncytium. The first cell fusion does not trigger a wave of orderly fusions in either direction. Ultrastructural studies show that epidermal syncytiogenesis require eff-1 activities to initiate and expand membrane merger.

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 FUS1Regulates the Opening and Expansion of Fusion Pores between Mating Yeast

2006 ◽  
Vol 17 (5) ◽  
pp. 2439-2450 ◽  
Author(s):  
Scott Nolan ◽  
Ann E. Cowan ◽  
Dennis E. Koppel ◽  
Hui Jin ◽  
Eric Grote
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Yeast Cells ◽  
Fusion Pore ◽  
Sudden Increase ◽  
Fluorescent Markers ◽  
Fold Reduction ◽  
Fusion Pores

Mating yeast cells provide a genetically accessible system for the study of cell fusion. The dynamics of fusion pores between yeast cells were analyzed by following the exchange of fluorescent markers between fusion partners. Upon plasma membrane fusion, cytoplasmic GFP and DsRed diffuse between cells at rates proportional to the size of the fusion pore. GFP permeance measurements reveal that a typical fusion pore opens with a burst and then gradually expands. In some mating pairs, a sudden increase in GFP permeance was found, consistent with the opening of a second pore. In contrast, other fusion pores closed after permitting a limited amount of cytoplasmic exchange. Deletion of FUS1 from both mating partners caused a >10-fold reduction in the initial permeance and expansion rate of the fusion pore. Although fus1 mating pairs also have a defect in degrading the cell wall that separates mating partners before plasma membrane fusion, other cell fusion mutants with cell wall remodeling defects had more modest effects on fusion pore permeance. Karyogamy is delayed by >1 h in fus1 mating pairs, possibly as a consequence of retarded fusion pore expansion.

scholarly journals Interaction of Local Anesthetics with Biomembranes Consisting of Phospholipids and Cholesterol: Mechanistic and Clinical Implications for Anesthetic and Cardiotoxic Effects

2013 ◽  
Vol 2013 ◽  
pp. 1-18 ◽  
Author(s):  
Hironori Tsuchiya ◽  
Maki Mizogami
Keyword(s):  
Lipid Bilayers ◽  
Local Anesthetics ◽  
Cell Membranes ◽  
Membrane Lipids ◽  
Transmembrane Proteins ◽  
Membrane Lipid ◽  
Direct Inhibition ◽  
Drug Molecules ◽  
Site Of Action ◽  
Lipid Barriers

Despite a long history in medical and dental application, the molecular mechanism and precise site of action are still arguable for local anesthetics. Their effects are considered to be induced by acting on functional proteins, on membrane lipids, or on both. Local anesthetics primarily interact with sodium channels embedded in cell membranes to reduce the excitability of nerve cells and cardiomyocytes or produce a malfunction of the cardiovascular system. However, the membrane protein-interacting theory cannot explain all of the pharmacological and toxicological features of local anesthetics. The administered drug molecules must diffuse through the lipid barriers of nerve sheaths and penetrate into or across the lipid bilayers of cell membranes to reach the acting site on transmembrane proteins. Amphiphilic local anesthetics interact hydrophobically and electrostatically with lipid bilayers and modify their physicochemical property, with the direct inhibition of membrane functions, and with the resultant alteration of the membrane lipid environments surrounding transmembrane proteins and the subsequent protein conformational change, leading to the inhibition of channel functions. We review recent studies on the interaction of local anesthetics with biomembranes consisting of phospholipids and cholesterol. Understanding the membrane interactivity of local anesthetics would provide novel insights into their anesthetic and cardiotoxic effects.

Sign in / Sign up

Export Citation Format

Share Document