Fusion of human erythrocytes induced by Sendai virus: freeze-fracture aspects

1980 ◽  
Vol 43 (1) ◽  
pp. 419-432
Author(s):  
P.P. da Silva ◽  
K. Shimizu ◽  
C. Parkison
Keyword(s):  
Sendai Virus ◽  
Freeze Fracture ◽  
Fracture Morphology ◽  
Human Erythrocytes ◽  
Erythrocyte Membranes ◽  
Radial Expansion ◽  
Viral Membrane ◽  
Membrane Bound ◽  
Viral Envelopes ◽  
Membrane Components

Analysis of the freeze-fracture morphology of human erythrocytes during Sendai virus-induced fusion indicates that it occurs in several spots, with the formation of membrane-bound cytoplasmic connexions. Radial expansion of these connexions causes the formation of a polymorphic network of membrane tubules with a lumen continuous with the external space. Our results are best explained assuming that the viral envelopes do not necessarily participate directly in inter-membrane fusion but, instead, indirectly through conditioning and modification of the erythrocyte membranes by viral membrane components.

scholarly journals Human erythrocytes adhering to schistosomula of Schistosoma mansoni lyse and fail to transfer membrane components to the parasite.

1985 ◽  
Vol 101 (1) ◽  
pp. 158-166 ◽  
Author(s):  
J P Caulfield ◽  
C M Cianci
Keyword(s):  
Schistosoma Mansoni ◽  
Erythrocyte Membrane ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Human Erythrocytes ◽  
Transmission Microscopy ◽  
Carbocyanine Dyes ◽  
Host Membrane ◽  
Membrane Components ◽  
20 Nm

We studied the adherence of human erythrocytes to larvae of the intravascular parasite Schistosoma mansoni by transmission microscopy, freeze fracture, and fluorescence techniques. In addition, we used the adherent cells to investigate the problem of host antigen acquisition. Schistosomula were cultured for from 24 to 48 h after transformation in order to clear the remnants of the cercarial glycocalyx. In some cases, the worms were preincubated with wheat germ agglutinin to promote adherence of the erythrocytes. The results were similar with and without the lectin except that more cells attached to the lectin-coated parasites. Erythrocytes adhered within a few hours and, unlike neutrophils, did not fuse with the parasite. A layer of 10-20-nm electron dense material separated the outer leaflets of the tegumental and plasma membranes. In addition, many deformed and lysed cells were seen on the parasite surface. The ability of the worm to acquire erythrocyte membrane constituents was tested with carbocyanine dyes, fluorescein covalently conjugated to glycophorin, monoclonal antibodies against B and H blood group glycolipids, and rabbit alpha-human erythrocyte IgG. In summary, glycophorin, erythrocyte proteins, and glycolipids were not transferred to the parasite membrane within 48 h. Carbocyanine dyes were rapidly transferred to the parasite with or without lectin preincubation. Thus, the dye in the worm membrane came from both adherent and nonadherent cells. These studies suggest that, in the absence of membrane fusion, the parasite may acquire some lipid molecules similar in structure to host membrane glycolipids by simple transfer through the medium but that B and H glycolipids and erythrocyte membrane proteins are not transferred from adhering cells to the worm.

Studies of membrane fusion. V. Fusion of erythrocytes with non-haemolytic Sendai virus

1979 ◽  
Vol 36 (1) ◽  
pp. 85-96
Author(s):  
S. Knutton
Keyword(s):  
Membrane Fusion ◽  
Cell Fusion ◽  
Erythrocyte Membrane ◽  
Sendai Virus ◽  
Lateral Diffusion ◽  
Viral Envelope ◽  
Cell Swelling ◽  
Viral Membrane ◽  
Cytoplasmic Bridges ◽  
Viral Envelopes

The fusion of human erythrocytes with non-haemolytic ‘1-day’ Sendai virus has been studied by electron microscopy. The mechanism of viral envelope-cell fusion is the same as that described previously for haemolytic ‘3-day’ Sendai virus except that fusion is frequently arrested at an initial stage when 2 segments of smooth linear viral membrane fuse and become incorporated into the erythrocyte membrane. After longer periods of incubation at 37 degrees C, in addition to many partly fused virus particles, long (up to 4 micrometer) lengths of smooth linear viral membrane are seen within the erythrocyte membrane which arise by linear aggregation of shorter (approximately 0.25 micrometer long) segments of smooth linear membrane derived from individual fused viral envelopes. Cell-Cell fusion, as a result of the fusion of a viral envelope with 2 adjacent erythrocytes also occurs but, in the absence of cell swelling, fusion is arrested at this stage with cells joined by one (or more) small cytoplasmic bridges. Typical fused cells are produced if such cells are swollen with hypotonic buffer. These observations provide further evidence that membrane fusion and cell swelling are distinct events in cell fusion and that cell swelling is the driving force both for completing the incorporation of the viral envelope into the cell membrane and for expanding cells connected by small cytoplasmic bridges to form spherical fused cells. Little lateral diffusion of viral envelope components occurs in the absence of cell swelling; in fact, some aggregation of components occurs. Comparison with previous studies using haemolytic ‘3-day’ Sendai virus suggests that virally induced cell swelling perturbs membrane structure so as to allow the rapid lateral diffusion of integrated viral envelope components.

scholarly journals Guanine nucleotide-dependent translocation of RhoA from cytosol to high affinity membrane binding sites in human erythrocytes

1998 ◽  
Vol 330 (3) ◽  
pp. 1391-1398 ◽  
Author(s):  
A. Andrey BOUKHAROV ◽  
M. Carl COHEN
Keyword(s):  
Binding Sites ◽  
Human Erythrocytes ◽  
Affinity Membrane ◽  
Protease Digestion ◽  
Peripheral Proteins ◽  
Cellular Signal ◽  
Number Of Binding Sites ◽  
Membrane Bound ◽  
Membrane Components ◽  
Membrane Binding Sites

The translocation of the small GTP-binding protein Rho from the cytosolic to membrane-bound form is an early step in many cellular signal-transduction events, but little is known regarding the mechanism of Rho association with the plasma membrane. We have used membranes from human erythrocytes to uncover a novel class of integral membrane components involved in the Rho-membrane association. Membranes of human erythrocytes contain several proteins of the Ras superfamily. Using specific antibodies and C3 exoenzyme of Clostridium botulinum we have identified one of them as RhoA. This protein was detected in both cytosol and membrane fractions of hypotonically lysed erythrocytes. We found that cytosolic Rho bound specifically to the cytoplasmic surface of the erythrocyte membrane and that the translocation of Rho to the membrane was absolutely dependent on the prior incubation of the cytosol with guanosine 5ʹ-[γ-thio]triphosphate (1-50 μM) at low Mg2+ concentration. Rho binding sites could not be extracted from the membrane using conditions that extracted all other peripheral proteins and were unaffected by heat treatment and protease digestion. Rho binding was saturable, with a Kd in the range 1-5.0 nM, and the number of binding sites was estimated to be approx. (1-2)×103 sites per cell. This is the first report of Rho binding to integral membrane components. The identity of these components may reveal novel aspects of the mechanism by which Rho exerts its multiple biochemical effects.

scholarly journals Rearrangements of integral membrane components during in vitro aging of sheep erythrocyte membranes

1977 ◽  
Vol 74 (2) ◽  
pp. 389-398 ◽  
Author(s):  
HU Lutz ◽  
AJ Lomant ◽  
P McMillan ◽  
E Wehrli
Keyword(s):  
Membrane Proteins ◽  
Particle Density ◽  
Sheep Erythrocyte ◽  
Freeze Fracture ◽  
Erythrocyte Membranes ◽  
Intramembranous Particles ◽  
In Vitro Aging ◽  
Membrane Components ◽  
Fracture Electron

In vitro aged sheep erythrocytes and sheep erythrocyte ghosts spontaneously release vesicles that consist of long protrusions affixed to flattened headlike structures. The intramembranous particles seen on the protoplasmic face of freeze fracture electron micrographs of vesicle protrusions are arranged in paired particle rows. On the equivalent fracture face of headlike structures, the particle density is low; if particles are present, they are clustered along the rim of the flattened headlike structure and at the junction with the protrusion. The released vesicles are depleted of the intramembranous particles seen on the exoplasmic face of ghost but retain almost exclusively particles of the protoplasmic face. Correspondingly, the exoplasmic face of ghosts that have released vesicles reveals a 28 percent higher density of intramembranous particles than that of fresh ghosts. Purified vesicles are depleted of spectrin but retain integral membrane proteins, with one of an apparent mol wt of 160,000 accounting for nearly 50 percent of the total protein (Lutz, H.U.,R. Barber, and R.F. McGuire. 1976. J. Biol. Chem. 251:3500-3510). When vesicles are modified with the cleavable cross-linking reagent [(35)S]dithiobis (succinimidyl propionate)at 0 degrees C, the 160,000 mol wt protein is rapidly converted to disulfide-linked dimers and higher oligomers. Exposure of intact ghosts to the reagent in the same way fails to yield equivalent polymers. A comparison of the morphological and biochemical aspects of ghosts and vesicles suggest that a marked rearrangement of membrane proteins accompanies the supramolecular redistribution of intramembranous particles during spontaneous vesiculation. The results also suggest that the paired particles of the protoplasmic face of vesicle protrusions are arranged in paired helices and contain the 160,000 mol wt protein as dimers.

Movement of Ca(2+)-ATPase molecules within the sarcoplasmic/endoplasmic reticulum in skeletal muscle

1996 ◽  
Vol 109 (10) ◽  
pp. 2529-2537
Author(s):  
Z. Kaprielian ◽  
S.W. Robinson ◽  
D.M. Fambrough ◽  
P.D. Kessler
Keyword(s):  
Skeletal Muscle ◽  
Endoplasmic Reticulum ◽  
Sarcoplasmic Reticulum ◽  
Cell Fusion ◽  
Sendai Virus ◽  
Mouse Cells ◽  
Membrane Bound ◽  
Species Specific ◽  
Membrane Components ◽  
P Type

The endoplasmic reticulum undergoes rapid, microscopic changes in its structure, including extension and anastomosis of tubular elements. Such dynamism is expected to manifest itself also as rapid intermixing of membrane components, at least within subdomains of the endoplasmic reticulum. Here we present evidence of a similar dynamism in the sarcoplasmic reticulum of developing skeletal muscle. The sarcoplasmic reticulum is sometimes considered a specialized type of endoplasmic reticulum, but it appears to be a rather static set of membrane-bound elements, repetitively arranged to enwrap each sarcomere of each myofibril. Both endoplasmic reticulum and sarcoplasmic reticulum contain P-type Ca(2+)-ATPases that transport calcium from the cytosol into their lumen. In the experiments reported here, chicken and mouse cells were fused by polyethylene glycol, natural myogenic cell fusion, or Sendai virus. The redistribution of Ca(2+)-ATPase molecules between chick and mouse endoplasmic reticulum/sarcoplasmic reticulum was followed by immunofluorescence microscopy in which species-specific monoclonal antibodies to chick and mouse Ca(2+)-ATPases were used. Redistribution was time- and temperature-dependent but independent of protein synthesis as well as the method of cell fusion. Intermixing occurred on a time scale of tens of minutes at 37 degrees C. These results verify the dynamic nature of the sarcoplasmic reticulum and illustrate an aspect of the special relationship between endoplasmic reticulum and sarcoplasmic reticulum.

Effect of Integral Proteins on Frozen Membrane Fracture

1980 ◽  
Vol 38 ◽  
pp. 756-759 ◽  
Author(s):  
S. Kirchanski ◽  
D. Branton
Keyword(s):  
Lipid Bilayers ◽  
Freeze Fracture ◽  
Covalent Bond ◽  
Erythrocyte Membranes ◽  
Glycophorin A ◽  
Experimental Protocol ◽  
Transmembrane Glycoprotein ◽  
Bond Breakage ◽  
Human Erythrocyte Membranes ◽  
Integral Proteins

We have investigated the effect of integral membrane proteins upon the fracturing of frozen lipid bilayers. This investigation has been part of an effort to develop freeze fracture labeling techniques and to assess the possible breakage of covalent protein bonds during the freeze fracture process. We have developed an experimental protocol utilizing lectin affinity columns which should detect small amounts of covalent bond breakage during the fracture of liposomes containing purified (1) glycophorin (a transmembrane glycoprotein of human erythrocyte membranes). To fracture liposomes in bulk, frozen liposomes are ground repeatedly under liquid nitrogen. Failure to detect any significant covalent bond breakage (contrary to (2)) led us to question the effectiveness of our grinding procedure in fracturing and splitting lipid bilayers.

An improved approach to freeze-fracture morphology of monolayer cell cultures

1998 ◽  
Vol 82 (1) ◽  
pp. 97-103 ◽  
Author(s):  
Elisabeth Bugnard ◽  
Patrick Sors ◽  
Alain Bloc ◽  
Françoise Loctin ◽  
Yves Dunant
Keyword(s):  
Cell Cultures ◽  
Freeze Fracture ◽  
Fracture Morphology ◽  
Monolayer Cell

scholarly journals Isolation and characterization of a membrane protein from rat erythrocytes which inhibits lysis by the membrane attack complex of rat complement

1992 ◽  
Vol 284 (1) ◽  
pp. 169-176 ◽  
Author(s):  
T R Hughes ◽  
S J Piddlesden ◽  
J D Williams ◽  
R A Harrison ◽  
B P Morgan
Keyword(s):  
Affinity Purification ◽  
Membrane Attack Complex ◽  
Erythrocyte Membranes ◽  
Dependent Manner ◽  
Inhibitory Protein ◽  
Rat Erythrocytes ◽  
Butanol Extract ◽  
Isolation And Characterization ◽  
Membrane Bound

The membrane attack complex (MAC) of complement in humans is regulated by several membrane-bound proteins; however, no such proteins have so far been described in other species. Here we report the isolation and characterization of a rat erythrocyte membrane glycoprotein of molecular mass 21 kDa which inserts into cell membranes and is a potent inhibitor of the rat MAC. This protein, here called rat inhibitory protein (RIP), was first partially purified by column chromatography from a butanol extract of rat erythrocyte membranes. Monoclonal antibodies (Mabs) were raised against RIP and used for its affinity purification. Affinity-purified RIP was shown to inhibit in a dose-dependent manner the cobra venom factor (CVF)-mediated ‘reactive’ lysis of guinea pig erythrocytes by rat complement. Conversely, the anti-RIP MAbs 6D1 and TH9 were shown to markedly enhance the CVF-mediated lysis of rat erythrocytes by rat complement. RIP acted late in the assembly of the MAC (at or after the C5b-8 stage) and was releasable from the membranes of rat erythrocytes by phosphatidylinositol-specific phospholipase C. These features, together with its size, deglycosylation pattern and N-terminal amino acid sequence, lead us to conclude that RIP is the rat homologue of the human MAC-inhibitory protein CD59 antigen.

scholarly journals Sendai virus recruits cellular villin to remodel actin cytoskeleton during fusion with hepatocytes

2017 ◽  
Vol 28 (26) ◽  
pp. 3801-3814 ◽  
Author(s):  
Sunandini Chandra ◽  
Raju Kalaivani ◽  
Manoj Kumar ◽  
Narayanaswamy Srinivasan ◽  
Debi P. Sarkar
Keyword(s):  
Membrane Fusion ◽  
Viral Entry ◽  
Sendai Virus ◽  
Viral Envelope ◽  
Bioactive Molecules ◽  
Host Cells ◽  
Actin Dynamics ◽  
Envelope Glycoproteins ◽  
Animal Cells ◽  
Viral Envelopes

Reconstituted Sendai viral envelopes (virosomes) are well recognized for their promising potential in membrane fusion–mediated delivery of bioactive molecules to liver cells. Despite the known function of viral envelope glycoproteins in catalyzing fusion with cellular membrane, the role of host cell proteins remains elusive. Here, we used two-dimensional differential in-gel electrophoresis to analyze hepatic cells in early response to virosome-induced membrane fusion. Quantitative mass spectrometry together with biochemical analysis revealed that villin, an actin-modifying protein, is differentially up-regulated and phosphorylated at threonine 206—an early molecular event during membrane fusion. We found that villin influences actin dynamics and that this influence, in turn, promotes membrane mixing through active participation of Sendai viral envelope glycoproteins. Modulation of villin in host cells also resulted in a discernible effect on the entry and egress of progeny Sendai virus. Taken together, these results suggest a novel mechanism of regulated viral entry in animal cells mediated by host factor villin.

Sign in / Sign up

Export Citation Format

Share Document