Membrane fusion by the influenza hemagglutinin: the fusion pore.
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Animals↗
Biomedical subjects
F S Cohen
Publications and source records attributed to F S Cohen.
Membrane mechanics can account for fusion pore dilation in stages.
Once formed, fusion pores rapidly enlarge to semi-stable conductance values. The membranes lining the fusion pore are continuous bilayer structures, so variations of conductance in time reflect bending and stretching of membranes. We therefore modeled the evolution of fusion pores using the theory of the mechanics of deforming homogeneous membranes. We calculated the changes in length and width of theoretical fusion pores according to standard dynamical equations of motion. Theoretical fusion pores quickly achieve semi-stable dimensions, which correspond to energy minima located in a canyon between energy barriers. The height of the barrier preventing pore expansion diminishes along the dimensions of length and width. The bottom of the canyon slopes gently downward along increasing length. As a consequence, theoretical fusion pores slowly lengthen and widen as the dimensions migrate along the bottom of the canyon, until the barrier vanishes and the pore rapidly enlarges. The dynamics of growth is sensitive to tension, spontaneous curvature, bending elasticity, and mobilities. This sensitivity can account for the quantitative differences in pore evolution observed in two experimental systems: HA-expressing cells fusing to planar bilayer membranes and beige mouse mast cell degranulation. We conclude that the mechanics of membranes could cause the phenomenon of stagewise growth of fusion pores.
Animals↗
GPI-anchored influenza hemagglutinin induces hemifusion to both red blood cell and planar bilayer membranes.
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.
Animals↗
The fusion kinetics of influenza hemagglutinin expressing cells to planar bilayer membranes is affected by HA density and host cell surface.
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.
3T3 Cells↗
Comparison of transient and successful fusion pores connecting influenza hemagglutinin expressing cells to planar membranes.
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.
3T3 Cells↗
Structure-function of the channel-forming colicins.
The channel-forming colicins are plasmid-encoded bacteriocins that kill E. coli and related cells and whose mode of action is of interest in related problems of protein import and toxicology. Colicins parasitize metabolite receptors in the outer membrane and translocate across the periplasm with the aid of the Tol or Ton protein systems. X-ray structure data for the channel domain and colicin are available. Residues have been identified that affect the channel ion selectivity and particular helices implicated in channel structure and in conformational changes required for binding or insertion of the channel into the membrane. Unique aspects of the colicin channel system are the involvement of protein import in the gating process, the existence of multiple open and closed states, and the existence and action of an immunity protein that involves specific intramembrane helix-helix interactions with transmembrane helices of the colicin channel-forming domains.
Amino Acid Sequence↗
Exocytotic fusion pores exhibit semi-stable states.
Rapid-freezing/freeze-fracture electron microscopy and whole-cell capacitance techniques were used to study degranulation in peritoneal mast cells of the rat and the mutant beige mouse. These studies allowed us to create a time-resolved picture for fusion pore formation. After stimulation, a dimple in the plasma membrane formed a small contact area with the secretory granule membrane. Within this zone of apposition no ordered proteinaceous specializations were seen. Electrophysiological technique measured a small fusion pore which widened rapidly to 1 nS. Thereafter, the fusion pore remained at semi-stable conductances between 1 and 20 nS for a wide range of times, between 10 and 15,000 msec. These conductances correspond to pore diameters 25-36 nm. Ultrastructural data confirmed small pores of hourglass morphology, composed of biological membrane coplanar with both the plasma and granular membranes. Later, the fusion pore rapidly increased in conductance, consistent with the observed morphology of omega-figures. The hallmarks of channel-like behavior, instantaneous jumps in pore conductance between defined levels, and sharp peaks in histograms of conductance dwell-time, were not seen. Since the morphology of small pores shows contiguous fracture planes, the electrical data represent pores that contain lipid. These combined morphological and electrophysiological data are consistent with a lipid/protein complex mediating both the initial and later stages of membrane fusion.
Animals↗
Reconstituting channels into planar membranes: a conceptual framework and methods for fusing vesicles to planar bilayer phospholipid membranes.
Protocols to reconstitute channels into planar bilayers via fusion methods have now been developed. The greater the intravesicular pressures generated, the greater is the fusion. These pressures can be calculated exactly for any experimental configuration. For some of the configurations, adding nystatin channels to the vesicle membrane will greatly aid fusion. The configurations of the 1990 Method (Figs. 4 and 5) are optimal for fusing vesicles that are reconstituted with ion-selective channels to planar membranes. Greater binding, and ultimately greater fusion, is achieved by ejecting vesicles directly at the membrane rather than by simply adding material to the cis compartment.
Amphotericin B↗
Single event recording shows that docking onto receptor alters the kinetics of membrane fusion mediated by influenza hemagglutinin.
The initial steps of membrane fusion, receptor binding and membrane destabilization, are mediated by the envelope glycoprotein hemagglutinin of influenza virus. Interaction between these functions was determined from the time course of individual virion fusions to a planar membrane with and without receptor. With receptor, fusion was described by a Poisson process. In the absence of receptor, the time course was more complicated and could not be described with exponential rate constants. The conversion of a non-Markovian process into a simple Markov chain is direct evidence that receptor binding fundamentally alters the route of fusion.
Binding Sites↗
Influenza hemagglutinin-mediated fusion pores connecting cells to planar membranes: flickering to final expansion.
We have studied the fusion between voltage-clamped planar lipid bilayers and influenza virus infected MDCK cells, adhered to one side of the bilayer, using measurements of electrical admittance and fluorescence. The changes in currents in-phase and 90 degrees out-of-phase with respect to the applied sinusoidal voltage were used to monitor the addition of the cell membrane capacitance to that of the lipid bilayer through a fusion pore connecting the two membranes. When ethidium bromide was included in the solution of the cell-free side of the bilayer, increases in cell fluorescence accompanied tee admittance changes, independently confirming that these changes were due to formation of a fusion pore. Fusion required acidic pH on the cell-containing side and depended on temperature. For fusion to occur, the influenza hemagglutinin (HA) had to be cleaved into HA1 and HA2 subunits. The incorporation of gangliosides into the planar bilayers greatly augmented fusion. Fusion pores developed in four distinct stages after acidification: (a) a pre-pore, electrically quiescent stage; (b) a flickering stage, with 1-2 nS pores opening and closing repetitively; (c) an irreversibly opened stage, in which pore conductances varied between 2 and 100 nS and exhibited diverse kinetics; (d) a fully opened stage, initiated by an instantaneous, time-resolution limited, increase in conductance leveling at approximately 500 nS. The expansion of pores by stages has also been shown to occur during exocytosis in mast cells and fusion of HA-expressing cells and erythrocytes. We conclude that essential features of fusion pores are produced with proteins in just one of the two fusing membranes.
Animals↗
Influenza virus hemagglutinin-induced cell-planar bilayer fusion: quantitative dissection of fusion pore kinetics into stages.
Cells expressing the influenza virus hemagglutinin (HA) fuse to planar bilayer membranes under acidic conditions. After an electrically quiescent perfusion stage (Q), a fusion pore forms that enlarges in three subsequent stages. A repetitively flickering pore stage (R) develops into a securely open stage (S) that exhibits conductances ranging from a few to tens of nS. The pore then expands to a terminal stage (T) with a large conductance on the order of one microSiemens. We have studied how virus strain, HA receptors in the target bilayer membrane, and cytoskeleton affect the time a fusion pore remains in each stage. These intervals are referred to as waiting times. In the quiescent stage, waiting times were very sensitive to the virus strain and presence of gangliosides (HA receptors) in the bilayer. When bilayers contained gangliosides, the waiting times in the Q stage for cells infected with the PR/8/34 strain of virus were exponentially distributed, whereas waiting times for cells infected with the Japan/305/57 strain were not so distributed. Without gangliosides, the waiting time distribution for PR/8/34 infected cells was complex. The waiting times in the R and S stages of pore growth were exponentially distributed under all tested conditions. Within the R stage, we analyzed the kinetics of the flickering pore by fitting the open and closed time distributions with a sum of two exponentials. Neither the open and closed time distributions nor the flickering pore conductance distributions were appreciably affected by virus strain or gangliosides. Colchicine and cytochalasin B increased the flicker rates, without affecting the waiting time in the R stage. We conclude that variations in amino acid sequences of the HAs and the presence of gangliosides as receptors within the target membrane critically affect the kinetics of fusion pore formation, but do not affect subsequent stages.
Animals↗
Computer detection of the rapid diffusion of fluorescent membrane fusion markers in images observed with video microscopy.
We have developed an algorithm for automated detection of the dynamic pattern characterizing flashes of fluorescence in video images of membrane fusion. The algorithm detects the spatially localized, transient increases and decreases in brightness that result from the dequenching of fluorescent dye in phospholipid vesicles or lipid-enveloped virions fusing with a planar membrane. The flash is identified in video images by its nonzero time derivative and the symmetry of its spatial profile. Differentiation is implemented by forward and backward subtractions of video frames. The algorithm groups spatially connected pixels brighter than a user-specified threshold into distinct objects in forward- and backward-differentiated images. Objects are classified as either flashes or noise particles by comparing the symmetries of matched forward and backward difference profiles and then by tracking each profile in successive difference images. The number of flashes identified depends on the brightness threshold, the size of the convolution kernel used to filter the image, and the time difference between the subtracted video frames. When these parameters are changed so that the algorithm identifies an increasing percentage of the flashes recognized by eye, an increasing number of noise objects are mistakenly identified as flashes. These mistaken flashes can be eliminated by a human observer. The algorithm considerably shortens the time needed to analyze video data. Tested extensively with phospholipid vesicle and virion fusion with planar membranes, our implementation of the algorithm accurately determined the rate of fusion of influenza virions labeled with the lipophilic dye octadecylrhodamine (R18).
Algorithms↗
Dynamic properties of the colicin E1 ion channel.
The mechanism of channel formation and action of channel-forming colicins is a paradigm for the study of dynamic aspects of membrane-protein interactions. The following experimental results concerning interaction of the colicin E1 channel domain with target membranes, in vitro and in vivo, are discussed: (1) the nature of the translocation-competent state of the channel-forming domain; (2) unfolding of the colicin channel peptide during in vitro binding and anchoring of the channel to liposome membranes at acidic pH; (3) reversal of channel peptide binding to liposomes by an alkaline-directed pH shift; (4) voltage-driven translocation and gating of the ion channel, discussed in the context of a four-helix model for a monomeric channel; (5) rescue of colicin-treated cells by high levels of external K+; (6) trypsin rescue of cells depolarized by the colicin ion channel; and (7) interaction of the channel domain with its immunity protein.
Amino Acid Sequence↗
Fusion of influenza virions with a planar lipid membrane detected by video fluorescence microscopy.
The fusion of individual influenza virions with a planar phospholipid membrane was detected by fluorescence video microscopy. Virion envelopes were loaded with the lipophilic fluorescent marker octadecylrhodamine B (R18) to a density at which the fluorescence of the probe was self-quenched. Labeled virions were ejected toward the planar membrane from a micropipette in a custom-built video fluorescence microscope. Once a virion fused with the planar membrane, the marker was free to diffuse, and its fluorescence became dequenched, producing a flash of light. This flash was detected as a transient spot of light which increased and then diminished in brightness. The diffusion constants calculated from the brightness profiles for the flashes are consistent with fusion of virus to the membrane with consequent free diffusion of probe within the planar membrane. Under conditions known to be fusigenic for influenza virus (low pH and 37 degrees C), flashes appeared at a high rate and the planar membrane quickly became fluorescent. To further establish that these flashes were due to fusion, we showed that red blood cells, which normally do not attach to planar membranes, were able to bind to membranes that had been exposed to virus under fusigenic conditions. The amount of binding correlated with the amount of flashing. This indicates that flashes signaled the reconstitution of the hemagglutinin glycoprotein (HA) of influenza virus, a well-known erythrocyte receptor, into the planar membrane, as would be expected in a fusion process. The flash rate on ganglioside-containing asolectin membranes increased as the pH was lowered. This is also consistent with the known fusion behavior of influenza virus with cell membranes and with phospholipid vesicles. We conclude that the flashes result from the fusion of individual virions to the planar membrane.
Hydrogen-Ion Concentration↗
The role of N-acetylneuraminic (sialic) acid in the pH dependence of influenza virion fusion with planar phospholipid membranes.
It is known that fusion of influenza virus to host cell membranes is strongly promoted by acidic pH. We have determined conditions required to obtain pH-dependent fusion of influenza virus to planar bilayer membranes. The rate of viral fusion was determined from the flash rate of R18-labeled virions delivered to the surface of the planar membrane by pressure-ejection from a pipette. For a bilayer formed only of phospholipids and cholesterol, the fusion rate was independent of pH and unaffected by the phospholipid composition. When the gangliosides GD1a + GT1b were included in the planar membrane, however, the fusion rate varied steeply with pH. The rate at pH 7.4 in the presence of the gangliosides was about an order of magnitude less than in their absence. At pH less than approximately 5.5, the rate was about an order of magnitude greater in the presence of gangliosides than in their absence. The fusion rate with planar membranes containing globoside, a ceramide-backboned glycolipid, was also independent of pH, indicating that the pH dependence required sialic acid on the carbohydrate moiety of the glycolipid. The gangliosides GM1a and GM3, both of which possess sialic acid, produced the same pH-dependent fusion rate as seen with GD1a + GT1b, indicating that the presence, but not the location, of terminal sialic acids is critical. Incubating virus with soluble sialyllactose blocked fusion to both ganglioside-free and ganglioside-containing planar membranes. These results show that the pH dependence of influenza virion fusion arises from the interaction of the sialic acid receptor with the influenza hemagglutinin. A model for sialic acid-hemagglutinin interactions accounting for pH-dependent fusion is presented.
Cholesterol↗
Video microscopy studies of vesicle-planar membrane adhesion and fusion.
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Calcium↗
A lipid/protein complex hypothesis for exocytotic fusion pore formation.
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Cell Membrane↗
Membrane topography of ColE1 gene products: the hydrophobic anchor of the colicin E1 channel is a helical hairpin.
The paucity of crystallographic data on the structure of intrinsic membrane proteins necessitates the development of additional techniques to probe their structures. The colicin E1 ion channel domain contains one prominent hydrophobic region near its COOH terminus that has been proposed to be an anchor for the assembly of the channel. Saturation site-directed mutagenesis of the hydrophobic anchor region of the colicin E1 ion channel was used to probe whether it spanned the bilayer once or twice. A nonpolar amino acid was replaced by a charged residue in 29 mutations made at 26 positions in the channel domain. Substitution of the charged amino acid at all positions except those in the center of the hydrophobic region and the periphery of the hydrophobic region caused a large decrease in the cytotoxicity of the purified mutant colicin E1 protein. This result implies that the hydrophobic domain spans the membrane bilayer twice in a helical hairpin loop, with the center of this domain residing in an aqueous or polar phase. The lengths of the trans-membrane helices appear to be approximately 18 and 16 residues. The absence of significant changes in ion selectivity in five of nine mutants indicated that these mutations did not cause a large change in the channel structure. The ion selectivity changes in four mutants and those previously documented for the flanking Lys residues imply that the hydrophobic hairpin is part of the channel lumen. Water may "abhor" the hydrophobic side of the channel, explaining the small effects of residue charge changes on ion selectivity.
Amino Acid Sequence↗