RESUMEN
We used video fluorescence microscopy of the vascular bed in the cremaster muscle of rat and mouse to study the transfer of plasmalemma vesicles (caveolae) across the microvessel barrier in situ. The water-soluble styryl pyridinium dye RH414, which adsorbs to and fluoresces at the membrane-water interface, was used as a marker for vesicular traffic through endothelial cells. Fluorescein isothiocyanate (FITC), similar in molecular size to the styryl pyridinium probe, was used to mark for dye transfer by the paracellular pathway. Transcellular dye flux was determined by comparing the fluorescence intensities of RH414 and FITC on either side of the vessel wall (i.e., in microvessel lumen and in muscle tissue at various distances from the microvessel wall). We observed that RH414 accumulated in the interstitium more rapidly than FITC. We next studied the role of the 60-kDa albumin-binding glycoprotein gp60, hypothesized to activate transcellular permeability, in stimulating the transcellular vesicle traffic. Introduction of anti-gp60 antibody into the microvessel to cross-link and activate gp60 markedly increased the transvascular flux of RH414. Control isotype-matched antibody had no effect on the RH414 flux. The sterol-binding agent filipin, which disassembles caveolae, inhibited the RH414 flux induced by gp60 cross-linking. The transfer of styryl pyridinium dyes in intact microvessels suggests that plasmalemmal membrane traffic across the skeletal muscle microvessel barrier is a constitutively active process. The results indicate that the gp60-dependent pathway is important in regulating endothelial permeability in situ via a transcellular mechanism.
Asunto(s)
Permeabilidad Capilar , Endotelio Vascular , Microcirculación , Animales , Bovinos , Línea Celular , Permeabilidad de la Membrana Celular , Endotelio Vascular/fisiología , Microcirculación/fisiología , Microscopía FluorescenteRESUMEN
We tested the hypothesis that the albumin-docking protein gp60, which is localized in caveolae, couples to the heterotrimeric GTP binding protein G(i), and thereby activates plasmalemmal vesicle formation and the directed migration of vesicles in endothelial cells (ECs). We used the water-soluble styryl pyridinium dye N-(3-triethylaminopropyl)-4-(p-dibutylaminostyryl) pyridinium dibromide (FM 1-43) to quantify vesicle trafficking by confocal and digital fluorescence microscopy. FM 1-43 and fluorescently labeled anti-gp60 antibody (Ab) were colocalized in endocytic vesicles within 5 min of gp60 activation. Vesicles migrated to the basolateral surface where they released FM 1-43, the fluid phase styryl probe. FM 1-43 fluorescence disappeared from the basolateral EC surface without the loss of anti-gp60 Ab fluorescence. Activation of cell-surface gp60 by cross-linking (using anti-gp60 Ab and secondary Ab) in EC grown on microporous filters increased transendothelial (125)I-albumin permeability without altering liquid permeability (hydraulic conductivity), thus, indicating the dissociation of hydraulic conductivity from the albumin permeability pathway. The findings that the sterol-binding agent, filipin, prevented gp60-activated vesicle formation and that caveolin-1 and gp60 were colocalized in vesicles suggest the caveolar origin of endocytic vesicles. Pertussis toxin pretreatment and expression of the dominant negative construct encoding an 11-amino acid G(alphai) carboxyl-terminal peptide inhibited endothelial (125)I-albumin endocytosis and vesicle formation induced by gp60 activation. Expression of dominant negative Src (dn-Src) and overexpression of wild-type caveolin-1 also prevented gp60-activated endocytosis. Caveolin-1 overexpression resulted in the sequestration of G(alphai) with the caveolin-1, whereas dn-Src inhibited G(alphai) binding to caveolin-1. Thus, vesicle formation induced by gp60 and migration of vesicles to the basolateral membrane requires the interaction of gp60 with caveolin-1, followed by the activation of the downstream G(i)-coupled Src kinase signaling pathway.
Asunto(s)
Caveolinas , Endocitosis/fisiología , Endotelio Vascular/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas de la Membrana/fisiología , Sialoglicoproteínas/metabolismo , Familia-src Quinasas/metabolismo , Animales , Bovinos , Caveolina 1 , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Permeabilidad de la Membrana Celular , Células Cultivadas , Gránulos Citoplasmáticos/fisiología , Gránulos Citoplasmáticos/ultraestructura , Endotelio Vascular/ultraestructura , Filipina/farmacología , Colorantes Fluorescentes , Humanos , Proteínas de la Membrana/genética , Microcirculación , Microscopía Confocal , Microscopía Fluorescente , Toxina del Pertussis , Circulación Pulmonar , Compuestos de Piridinio/farmacocinética , Compuestos de Amonio Cuaternario/farmacocinética , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Transfección , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
We used water-soluble styryl pyridinium dyes that fluoresce at the membrane-water interface to study vesicle traffic in endothelial cells. Cultured endothelial cells derived from bovine and human pulmonary microvessels were incubated in styryl probes, washed to remove dye from the plasmalemmal outer face, and observed by digital fluorescence microscopy. Vesicles that derived from plasmalemma by endocytosis were filled with the styryl dye. These vesicles were distributed throughout the cytosol as numerous particles of heterogeneous diameter and brightness. Vesicle formation was activated 2-fold following addition of extracellular albumin whereas a control protein, immunoglobulin G, had no effect. Dye uptake was abrogated by labeling at low temperatures and inhibitors of phosphoinositide-3-kinase (PI 3-kinase). Tyrosine kinase inhibitors (genistein and herbimycin A) prevented the albumin-induced vesicle formation. Cytochalasin B prevented vesicle redistribution indicating involvement of actin filaments in translocation of endosomes away from sites of vesicle formation. Styryl dye was lost from cells by exocytosis as evident by the disappearance of discrete fluorescent particles. N-ethylmaleimide and botulinum toxin types A and B caused cells to accumulate increased number of vesicles suggesting that exocytosis was regulated by NSF-dependent SNARE mechanism. The results suggest that phosphoinositide metabolism regulates endocytosis in endothelial cells and that extracellular albumin activates endocytosis by a mechanism involving tyrosine phosphorylation, whereas exocytosis is a distinct process regulated by the SNARE machinery. The results support the hypothesis that albumin regulates its internalization and release in vascular endothelial cells via activation of specific endocytic and exocytic pathways.
Asunto(s)
Endocitosis/fisiología , Endotelio Vascular/citología , Exocitosis/fisiología , Androstadienos/farmacología , Animales , Benzoquinonas , Toxinas Botulínicas/farmacología , Brefeldino A/farmacología , Bovinos , Membrana Celular/fisiología , Cromonas/farmacología , Citocalasina B/farmacología , Endocitosis/efectos de los fármacos , Endosomas/efectos de los fármacos , Endotelio Vascular/fisiología , Endotelio Vascular/ultraestructura , Inhibidores Enzimáticos/farmacología , Etilmaleimida/farmacología , Exocitosis/efectos de los fármacos , Colorantes Fluorescentes/farmacocinética , Genisteína/farmacología , Humanos , Lactamas Macrocíclicas , Microcirculación , Microscopía Fluorescente , Morfolinas/farmacología , Fosfatidilinositol 3-Quinasas/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/fisiología , Quinonas/farmacología , Rifabutina/análogos & derivados , Albúmina Sérica/farmacología , WortmaninaRESUMEN
We studied dynamics of cell surface expression of proteolytically activated thrombin receptor (PAR-1) in human pulmonary artery endothelial cells (HPAEC). PAR-1 activation was measured by changes in cytosolic calcium concentration ([Ca2+]i) and HPAEC retraction response (determined by real-time transendothelial monolayer electrical resistance). [Ca2+]i increase in response to thrombin was abolished by preexposure to 25 nM thrombin for >60 min, indicating PAR-1 desensitization, but preexposure to 25 nM thrombin for only 30 min or to 10 nM thrombin for up to 2 h did not desensitize PAR-1. Exposure to 10 or 25 nM thrombin decreased monolayer electrical resistance 40-60%. Cells preexposed to 10 nM thrombin, but not those preexposed to 25 nM thrombin, remained responsive to thrombin 3 h later. Loss of cell retractility was coupled to decreased cell surface PAR-1 expression as determined by immunofluorescence. Cell surface PAR-1 disappeared upon short-term (30 min) thrombin exposure but reappeared within 90 min after incubation in thrombin-free medium. Exposure to 25 nM thrombin for >60 min prevented rapid cycloheximide-insensitive PAR-1 reappearance. Cycloheximide-sensitive recovery of cell surface PAR-1 expression required 18 h. Therefore, both duration and concentration of thrombin exposure regulate the time course of recovery of HPAEC surface PAR-1 expression. The results support the hypothesis that initial recovery of PAR-1 surface expression in endothelial cells results from a rapidly mobilizable PAR-1 pool, whereas delayed recovery results from de novo PAR-1 synthesis. We conclude that thrombin itself regulates endothelial cell surface PAR-1 expression and that decreased surface expression interferes with thrombin-induced endothelial cell activation responses.
Asunto(s)
Endotelio Vascular/metabolismo , Receptores de Trombina/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Tamaño de la Célula/fisiología , Células Cultivadas , Cicloheximida/farmacología , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Impedancia Eléctrica , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Humanos , Concentración Osmolar , Inhibidores de la Síntesis de la Proteína/farmacología , Receptores de Trombina/efectos de los fármacos , Trombina/farmacología , Factores de TiempoRESUMEN
Because reactive oxygen species (ROS) can function as second messengers and regulate the activation of the transcription factor nuclear factor (NF)-kappaB, we investigated the possible role of tumor necrosis factor-alpha (TNF-alpha)-induced ROS generation in endothelial cells in signaling E-selectin gene transcription. We demonstrated that stimulation of human pulmonary artery endothelial cells with TNF-alpha (100 U/ml) resulted in ROS production using the oxidant-sensitive dye 5 (and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate bis(acetoxymethyl)ester. Pretreatment with N-acetyl-L-cysteine (NAC) or pyrrolidine dithiocarbamate (PDTC) for 0.5 h inhibited TNF-alpha-induced generation of ROS as well as activation of NF-kappaB and E-selectin mRNA and the cell surface protein expression. These findings indicate that TNF-alpha induces NF-kappaB activation and the resultant E-selectin gene expression by a pathway that involves formation of ROS and that E-selectin expression can be inhibited by the antioxidant action of NAC or PDTC. The results support the hypothesis that generation of ROS in endothelial cells induced by proinflammatory cytokines such as TNF-alpha is a critical signal mediating E-selectin expression.
Asunto(s)
Selectina E/genética , Endotelio Vascular/inmunología , FN-kappa B/metabolismo , Transcripción Genética , Factor de Necrosis Tumoral alfa/farmacología , Acetilcisteína/farmacología , Antioxidantes/farmacología , Secuencia de Bases , Sitios de Unión , Núcleo Celular/efectos de los fármacos , Núcleo Celular/fisiología , Células Cultivadas , Selectina E/biosíntesis , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Humanos , Oligodesoxirribonucleótidos/química , Oxidantes/metabolismo , Arteria Pulmonar , Pirrolidinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/farmacología , Transducción de Señal , Tiocarbamatos/farmacología , Transcripción Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Membrane fusion of a phospholipid vesicle with a planar lipid bilayer is preceded by an initial prefusion stage in which a region of the vesicle membrane adheres to the planar membrane. A resonance energy transfer (RET) imaging microscope, with measured spectral transfer functions and a pair of radiometrically calibrated video cameras, was used to determine both the area of the contact region and the distances between the membranes within this zone. Large vesicles (5-20 microns diam) were labeled with the donor fluorophore coumarin-phosphatidylethanolamine (PE), while the planar membrane was labeled with the acceptor rhodamine-PE. The donor was excited with 390 nm light, and separate images of donor and acceptor emission were formed by the microscope. Distances between the membranes at each location in the image were determined from the RET rate constant (kt) computed from the acceptor:donor emission intensity ratio. In the absence of an osmotic gradient, the vesicles stably adhered to the planar membrane, and the dyes did not migrate between membranes. The region of contact was detected as an area of planar membrane, coincident with the vesicle image, over which rhodamine fluorescence was sensitized by RET. The total area of the contact region depended biphasically on the Ca2+ concentration, but the distance between the bilayers in this zone decreased with increasing [Ca2+]. The changes in area and separation were probably related to divalent cation effects on electrostatic screening and binding to charged membranes. At each [Ca2+], the intermembrane separation varied between 1 and 6 nm within each contact region, indicating membrane undulation prior to adhesion. Intermembrane separation distances < or = 2 nm were localized to discrete sites that formed in an ordered arrangement throughout the contact region. The area of the contact region occupied by these punctate attachment sites was increased at high [Ca2+]. Membrane fusion may be initiated at these sites of closest membrane apposition.
Asunto(s)
Calcio/química , Fusión de Membrana , Membranas Artificiales , Fosfolípidos/química , Cumarinas/química , Transferencia de Energía , Polarización de Fluorescencia , Colorantes Fluorescentes , Liposomas , Imagen por Resonancia Magnética , Microscopía por Video , Conformación Molecular , Rodaminas/química , Espectrometría de Fluorescencia , Propiedades de SuperficieRESUMEN
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.
Asunto(s)
Células 3T3/metabolismo , Hemaglutininas Virales/biosíntesis , Células 3T3/citología , Células 3T3/virología , Animales , Fusión Celular/fisiología , Membrana Celular/química , Membrana Celular/fisiología , Endopeptidasas , Glicoproteínas Hemaglutininas del Virus de la Influenza , Activación del Canal Iónico/fisiología , Cinética , Membrana Dobles de Lípidos/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Porinas/metabolismo , Factores de Tiempo , Proteínas del Envoltorio Viral/biosíntesisRESUMEN
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.
Asunto(s)
Células 3T3/metabolismo , Hemaglutininas Virales/biosíntesis , Porinas/metabolismo , Células 3T3/química , Células 3T3/virología , Animales , Fusión Celular/fisiología , Electrofisiología , Glicoproteínas Hemaglutininas del Virus de la Influenza , Activación del Canal Iónico/fisiología , Membrana Dobles de Lípidos/metabolismo , Proteínas de la Membrana/fisiología , Ratones , Procesos Estocásticos , Factores de Tiempo , Proteínas del Envoltorio Viral/biosíntesisRESUMEN
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.
Asunto(s)
Hemaglutininas Virales/fisiología , Virus de la Influenza A/inmunología , Animales , Fusión Celular/fisiología , Perros , Electrofisiología , Etidio/farmacología , Fluorescencia , Glicoproteínas Hemaglutininas del Virus de la Influenza , Humanos , Membrana Dobles de Lípidos , Temperatura , TransfecciónRESUMEN
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.
Asunto(s)
Hemaglutininas Virales/fisiología , Virus de la Influenza A/inmunología , Animales , Fusión Celular/fisiología , Citoesqueleto/fisiología , Perros , Electrofisiología , Gangliósidos/fisiología , Glicoproteínas Hemaglutininas del Virus de la Influenza , Humanos , Concentración de Iones de Hidrógeno , Cinética , Membrana Dobles de LípidosRESUMEN
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.
Asunto(s)
Hemaglutininas Virales/fisiología , Fusión de Membrana/fisiología , Receptores Virales/fisiología , Sitios de Unión , Fenómenos Biofísicos , Biofisica , Glicoproteínas Hemaglutininas del Virus de la Influenza , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Cinética , Lípidos de la Membrana/química , Membranas Artificiales , Fosfolípidos/química , TermodinámicaRESUMEN
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.
Asunto(s)
Canales Iónicos/fisiología , Membrana Dobles de Lípidos , Fusión de Membrana , Fosfolípidos/química , Anfotericina B , Ergosterol , Fluoresceínas , Colorantes Fluorescentes , Formamidas , Indicadores y Reactivos , Cinética , Métodos , Modelos Biológicos , Nistatina , TermodinámicaRESUMEN
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).
Asunto(s)
Membrana Dobles de Lípidos , Fusión de Membrana , Orthomyxoviridae/fisiología , Virión/fisiología , Algoritmos , Computadores , Fluoresceínas , Colorantes Fluorescentes , Matemática , Microscopía Fluorescente/métodos , Modelos Biológicos , Factores de Tiempo , Grabación en VideoRESUMEN
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.
Asunto(s)
Virus de la Influenza A/metabolismo , Fusión de Membrana/fisiología , Lípidos de la Membrana/metabolismo , Virión/metabolismo , Concentración de Iones de Hidrógeno , Virus de la Influenza A/fisiología , Lípidos de la Membrana/fisiología , Microscopía Fluorescente/métodos , Rodaminas , Grabación en Video , Virión/fisiologíaRESUMEN
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.
Asunto(s)
Virus de la Influenza A/metabolismo , Fusión de Membrana/fisiología , Lípidos de la Membrana/metabolismo , Fosfolípidos/metabolismo , Ácidos Siálicos/fisiología , Virión/metabolismo , Colesterol/metabolismo , Gangliósidos/metabolismo , Hemaglutininas/metabolismo , Concentración de Iones de Hidrógeno , Virus de la Influenza A/fisiología , Lípidos de la Membrana/fisiología , Microscopía Fluorescente/métodos , Rodaminas , Virión/fisiologíaRESUMEN
Individual erythrocytes are visible in bright-field microscopy because their enclosed hemoglobin provides a high degree of contrast against a glass slide. Lysis of these cells is detected as the loss of contrast of individual cells caused by the leakage of cell contents. Using these optical properties of erythrocytes, we have developed a new technique to examine the time course of hemolysis induced by Sendai virus at neutral pH and by influenza virus at acidic pH. Viruses were allowed to aggregate erythrocytes at 4 degrees and the temperature was raised to allow hemolysis. Influenza-induced hemolysis at low pH, as determined by this method, occurred at a faster rate than that induced by Sendai at neutral pH. As the lysis of individual cells is detected by this method, we have discerned a cooperative "cluster" effect: Once an erythrocyte within an aggregate lyses, the likelihood of lysis of another erythrocyte in that aggregate is increased.
Asunto(s)
Hemólisis , Orthomyxoviridae/fisiología , Virus de la Parainfluenza 1 Humana/fisiología , Fusión Celular , Glicoproteínas Hemaglutininas del Virus de la Influenza , Hemaglutininas Virales/metabolismo , Humanos , Concentración de Iones de Hidrógeno , CinéticaRESUMEN
Phospholipid vesicles fuse with a planar membrane when they are osmotically swollen. Channels in the vesicle membrane are required for swelling to occur when the vesicle-containing compartment is made hyperosmotic by adding a solute (termed an osmoticant). We have studied fusion using two different channels, porin, a highly permeable channel, and nystatin, a much less permeable channel. We report that an osmoticant's ability to support fusion (defined as the magnitude of osmotic gradient necessary to obtain sustained fusion) depends on both its permeability through lipid bilayer as well as its permeability through the channel by which it enters the vesicle interior. With porin as the channel, formamide requires an osmotic gradient about ten times that required with urea, which is approximately 1/40th as permeant as formamide through bare lipid membrane. When nystatin is the channel, however, fusion rates sustained by osmotic gradients of formamide are within a factor of two of those obtained with urea. Vesicles containing a porin-impermeant solute can be induced to swell and fuse with a planar membrane when the impermeant bathing the vesicles is replaced by an isosmotic quantity of a porin-permeant solute. With this method of swelling, formamide is as effective as urea in obtaining fusion. In addition, we report that binding of vesicles to the planar membrane does not make the contact region more permeable to the osmoticant than is bare lipid bilayer. In the companion paper, we quantitatively account for the observation that the ability of a solute to promote fusion depends on its permeability properties and the method of swelling. We show that the intravesicular pressure developed drives fusion.
Asunto(s)
Membrana Dobles de Lípidos , Fusión de Membrana , Fosfolípidos , Proteínas de la Membrana Bacteriana Externa , Ósmosis , Presión Osmótica , Permeabilidad , PorinasRESUMEN
When phospholipid vesicles bound to a planar membrane are osmotically swollen, they develop a hydrostatic pressure (delta P) and fuse with the membrane. We have calculated the steady-state delta P, from the equations of irreversible thermodynamics governing water and solute flows, for two general methods of osmotic swelling. In the first method, vesicles are swollen by adding a solute to the vesicle-containing compartment to make it hyperosmotic. delta P is determined by the vesicle membrane's permeabilities to solute and water. If the vesicle membrane is devoid of open channels, then delta P is zero. When the vesicle membrane contains open channels, then delta P peaks at a channel density unique to the solute permeability properties of both the channel and the membrane. The solute enters the vesicle through the channels but leaks out through the region of vesicle-planar membrane contact. delta P is largest for channels having high permeabilities to the solute and for solutes with low membrane permeabilities in the contact region. The model predicts the following order of solutes producing pressures of decreasing magnitude: KCl greater than urea greater than formamide greater than or equal to ethylene glycol. Differences between osmoticants quantitatively depend on the solute permeability of the channel and the density of channels in the vesicle membrane. The order of effectiveness is the same as that experimentally observed for solutes promoting fusion. Therefore, delta P drives fusion. When channels with small permeabilities are used, coupling between solute and water flows within the channel has a significant effect on delta P. In the second method, an impermeant solute bathing the vesicles is isosmotically replaced by a solute which permeates the channels in the vesicle membrane. delta P resulting from this method is much less sensitive to the permeabilities of the channel and membrane to the solute. delta P approaches the theoretical limit set by the concentration of the impermeant solute.
Asunto(s)
Presión Hidrostática , Membrana Dobles de Lípidos , Fusión de Membrana , Ósmosis , Fosfolípidos , Presión , Canales Iónicos , Modelos Teóricos , Concentración Osmolar , Permeabilidad , AguaRESUMEN
We investigated the manner in which planar phospholipid membranes form when monolayers are sequentially raised. Simultaneous electrical and optical recordings showed that initially a thick film forms, and the capacitance of the film increases with the same time course as the observed thinning. The diameter of fully thinned membranes varies from membrane to membrane and a torus is readily observed. The frequency-dependent admittance of the membrane was measured using a wide-bandwidth voltage clamp whose frequency response is essentially independent of capacitative load. The membrane capacitance dominates the total admittance and the membrane dielectric is not lossy. The specific capacitance of membranes of several mixtures was measured. A schematic diagram of the formation of these membranes is presented.