scholarly journals A cell-free assay for the insertion of a viral glycoprotein into the plasma membrane.

1986 ◽  
Vol 103 (5) ◽  
pp. 1829-1835 ◽  
Author(s):  
P G Woodman ◽  
J M Edwardson
Keyword(s):  
Influenza Virus ◽  
Plasma Membrane ◽  
Trichloroacetic Acid ◽  
Plasma Membranes ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Viral Glycoprotein ◽  
Acetylneuraminic Acid ◽  
Donor And Acceptor ◽  
A Cell

A cell-free assay has been developed for the delivery of influenza virus neuraminidase to the plasma membrane. Two types of postnuclear supernatant, which acted as donor and acceptor of the enzyme, were prepared from baby hamster kidney cells. Donor preparations were obtained from cells infected with influenza virus and containing neuraminidase en route to the plasma membrane. Acceptor preparations were obtained from cells containing, bound to their plasma membranes, Semliki Forest virus with envelope glycoproteins bearing [3H]N-acetylneuraminic acid. Fusion between vesicles from these two preparations permits access of the enzyme to its substrate, which results in the release of free [3H]N-acetylneuraminic acid. This release was detected through the transfer of radioactivity from a trichloroacetic acid-insoluble to a trichloroacetic acid-soluble fraction. An ATP-dependent component of release was found, which appears to be a consequence of vesicle fusion. This component was enhanced when the donor was prepared from cells in which the enzyme had been concentrated in a compartment between the Golgi complex and the plasma membrane, which indicates that a specific exocytic fusion event has been reconstituted. The extent of fusion is greatly reduced by pre-treatment of donor and acceptor preparations with trypsin, which points to the involvement of proteins in the fusion reaction.

scholarly journals Effective in Vivo Targeting of Influenza Virus through a Cell-Penetrating/Fusion Inhibitor Tandem Peptide Anchored to the Plasma Membrane

2018 ◽  
Vol 29 (10) ◽  
pp. 3362-3376 ◽  
Author(s):  
T. N. Figueira ◽  
M. T. Augusto ◽  
K. Rybkina ◽  
D. Stelitano ◽  
M. G. Noval ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Plasma Membrane ◽  
Fusion Inhibitor ◽  
Cell Penetrating ◽  
A Cell

scholarly journals Sphingomyelin Synthase 2, but Not Sphingomyelin Synthase 1, Is Involved in HIV-1 Envelope-mediated Membrane Fusion

2014 ◽  
Vol 289 (44) ◽  
pp. 30842-30856 ◽  
Author(s):  
Yasuhiro Hayashi ◽  
Yoko Nemoto-Sasaki ◽  
Takashi Tanikawa ◽  
Saori Oka ◽  
Kiyoto Tsuchiya ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
Actin Polymerization ◽  
Plasma Membranes ◽  
Viral Envelope ◽  
Target Cells ◽  
Nonreceptor Tyrosine Kinase ◽  
Sphingomyelin Synthase ◽  
A Cell ◽  
Hiv 1

Membrane fusion between the viral envelope and plasma membranes of target cells has previously been correlated with HIV-1 infection. Lipids in the plasma membrane, including sphingomyelin, may be crucially involved in HIV-1 infection; however, the role of lipid-metabolic enzymes in membrane fusion remains unclear. In this study, we examined the roles of sphingomyelin synthase (SMS) in HIV-1 Env-mediated membrane fusion using a cell-cell fusion assay with HIV-1 mimetics and their target cells. We employed reconstituted cells as target cells that stably express Sms1 or Sms2 in Sms-deficient cells. Fusion susceptibility was ∼5-fold higher in Sms2-expressing cells (not in Sms1-expressing cells) than in Sms-deficient cells. The enhancement of fusion susceptibility observed in Sms2-expressing cells was reversed and reduced by Sms2 knockdown. We also found that catalytically nonactive Sms2 promoted membrane fusion susceptibility. Moreover, SMS2 co-localized and was constitutively associated with the HIV receptor·co-receptor complex in the plasma membrane. In addition, HIV-1 Env treatment resulted in a transient increase in nonreceptor tyrosine kinase (Pyk2) phosphorylation in Sms2-expressing and catalytically nonactive Sms2-expressing cells. We observed that F-actin polymerization in the region of membrane fusion was more prominent in Sms2-expressing cells than Sms-deficient cells. Taken together, our research provides insight into a novel function of SMS2 which is the regulation of HIV-1 Env-mediated membrane fusion via actin rearrangement.

scholarly journals In vitro reconstitution of exocytosis from plasma membrane and isolated secretory vesicles.

1985 ◽  
Vol 101 (6) ◽  
pp. 2263-2273 ◽  
Author(s):  
J H Crabb ◽  
R C Jackson
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Secretory Vesicles ◽  
Cytoplasmic Face ◽  
In Vitro Reconstitution ◽  
Transport Assay ◽  
Complex Preparation ◽  
A Cell ◽  
Vectorial Transport

We describe the reconstitution of exocytotic function through recombination of purified cortical secretory vesicles (CVs) and plasma membrane from sea urchin eggs. CVs were dislodged from a cell surface complex preparation by gentle homogenization in an isotonic dissociation buffer, and purified by differential centrifugation. CV-free plasma membrane fragments were obtained by mechanically dislodging CVs from cortical lawn (CL) preparations with a jet of CL isolation buffer. This procedure produced a "plasma membrane lawn" preparation, consisting of plasma membrane fragments attached via their vitelline layer (an extracellular glycocalyx) to a polylysine-coated microscope slide. When freshly prepared CVs were incubated with plasma membrane lawns, CVs reassociated with the cytoplasmic face of the plasma membrane, forming an exocytotically competent, reconstituted cortical lawn (RL). Exocytosis in RLs was monitored by phase-contrast microscopy, and quantitated with a sensitive microphotometric assay. Half-maximal exocytosis in RLs occurred at 18.5 microM free Ca2+; half-maximal exocytosis in control lawns occurred at 5.7 microM free Ca2+. Greater than 90% of the purified CVs that were not attached to a plasma membrane lawn remained intact when bathed in a buffer containing millimolar Ca2+. This result excluded the possibility that Ca2+-triggered CV lysis was responsible for our observations, and confirmed that the association of CVs with the plasma membrane was required for exocytosis in RLs. Evidence that the Ca2+-stimulated release of CV contents in CLs and RLs is the in vitro equivalent of exocytosis was obtained with an immunofluorescence-based vectorial transport assay, using an antiserum directed against a CV content protein: stimulation of RLs or partially CV-depleted CLs with Ca2+ resulted in fusion of the CV and plasma membranes, and the vectorial transport of CV contents from the cytoplasmic to the extracytoplasmic face of the egg plasma membrane.

A cell-free assay for delivery of a viral glycoprotein to the plasma membrane

1986 ◽  
Vol 14 (2) ◽  
pp. 293-294
Author(s):  
PHILIP G. WOODMAN ◽  
J. MICHAEL EDWARDSON
Keyword(s):  
Plasma Membrane ◽  
Viral Glycoprotein ◽  
A Cell

Direct demonstration of the endocytic function of caveolae by a cell-free assay

1999 ◽  
Vol 112 (7) ◽  
pp. 1101-1110 ◽  
Author(s):  
A. Gilbert ◽  
J.P. Paccaud ◽  
M. Foti ◽  
G. Porcheron ◽  
J. Balz ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Incubation Medium ◽  
Plasma Membranes ◽  
Cultured Cells ◽  
Significant Proportion ◽  
Membrane Domains ◽  
Direct Demonstration ◽  
Fractionation Analysis ◽  
A Cell ◽  
Endocytic Vesicles

The endocytic function of caveolae was challenged by taking advantage of a cell-free assay directly measuring the detachment of receptor-containing vesicles from isolated plasma membranes. Plasma membranes from cultured cells surface-labeled with 125I-cholera toxin (segregating in caveolae) were isolated as described previously. Following incubation of these labeled membranes in the presence of nucleotide(s) and cytosol, a significant proportion of the initially membrane-associated radioactivity was released into the incubation medium in sedimentable form (14*10(6)g). Results of biochemical, morphological, and fractionation analysis of the material containing the released radioactivity directly demonstrated that caveolae are plasma membrane domains involved in an endocytic process and resulting in the formation of caveolae-derived vesicles. In addition, these studies allowed a direct comparison of caveolae- and clathrin-coated pit-mediated endocytosis and reveal that these two processes diverge in terms of kinetics, cytosol and nucleotide requirements as well as in terms of the density and size of the endocytic vesicles formed.

scholarly journals Fusion of Semliki forest virus with the plasma membrane can be induced by low pH.

1980 ◽  
Vol 87 (1) ◽  
pp. 264-272 ◽  
Author(s):  
J White ◽  
J Kartenbeck ◽  
A Helenius
Keyword(s):  
Plasma Membrane ◽  
Semliki Forest Virus ◽  
Sendai Virus ◽  
Fusion Reaction ◽  
Low Ph ◽  
Medium Ph ◽  
Viral Membrane ◽  
Artificial Nature ◽  
Viral Nucleocapsid ◽  
Infection Pathway

When BHK-21 cells with Semliki Forest virus (SFV) bound at the plasma membrane are briefly treated with low pH medium (pH 5-6), fusion between the viral membrane and the plasma membrane occurs, releasing the viral nucleocapsid into the cytoplasm. The fusion reaction resembles that described previously for Sendai virus but with one fundamental difference; it is strictly dependent on low pH. The fusion reaction is highly efficient. Up to 86% of bound viruses fuse, and 6 X 10(6) virus spike proteins can be inserted into the plasma membrane of each cell. The process is very rapid (full activity is observed after 5 s) and it occurs over a wide temperature range and equally well with all five cell lines tested (BHK-21, HeLa B, HeLa suspension, Raji, and 3T3). Low pH-induced fusion of the virus at the plasma membrane can lead to infection of susceptible cells. The artificial nature of this infection pathway is, however, demonstrated by the facts that infection through the plasma membrane occurs only at subphysiological pH and that it is insensitive to inhibitors of the normal entry route. Nevertheless, these results indicate that low pH membrane fusion introduces the viral genome into the cytoplasm in a form suitable for replication.

scholarly journals Transcytosis via the late endocytic pathway as a cell morphogenetic mechanism

2020 ◽  
Author(s):  
R Mathew ◽  
LD Rios-Barrera ◽  
P Machado ◽  
Y Schwab ◽  
M Leptin
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Basal Membrane ◽  
Endocytic Pathway ◽  
Apical Plasma Membrane ◽  
Tracheal System ◽  
Basal Plasma ◽  
Membrane Compartments ◽  
Membrane Growth ◽  
A Cell

AbstractPlasma membranes fulfil many physiological functions. In polarized cells, different membrane compartments take on specialized roles, each being allocated correct amounts of membrane. The Drosophila tracheal system, an established tubulogenesis model, contains branched terminal cells with subcellular tubes formed by apical plasma membrane invagination. We show that apical endocytosis and late endosome-mediated trafficking determine the membrane allocation to the apical and basal membrane domains. Basal plasma membrane growth stops if endocytosis is blocked, whereas the apical membrane grows excessively. Plasma membrane is initially delivered apically, and then continuously endocytosed, together with apical and basal cargo. We describe an organelle carrying markers of late endosomes and multivesicular bodies (MVBs) that is abolished by inhibiting endocytosis, and which we suggest acts as transit station for membrane destined to be redistributed both apically and basally. This is based on the observation that disrupting MVB formation prevents growth of both compartments.

scholarly journals Dynamin at the Neck of Caveolae Mediates Their Budding to Form Transport Vesicles by GTP-driven Fission from the Plasma Membrane of Endothelium

1998 ◽  
Vol 141 (1) ◽  
pp. 101-114 ◽  
Author(s):  
Phil Oh ◽  
Deirdre P. McIntosh ◽  
Jan E. Schnitzer
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Molecular Mechanisms ◽  
Intact Cell ◽  
Subcellular Fractionation ◽  
Transport Vesicles ◽  
Cell Plasma ◽  
A Cell ◽  
Free Transport

The molecular mechanisms mediating cell surface trafficking of caveolae are unknown. Caveolae bud from plasma membranes to form free carrier vesicles through a “pinching off” or fission process requiring cytosol and driven by GTP hydrolysis (Schnitzer, J.E., P. Oh, and D.P. McIntosh. 1996. Science. 274:239–242). Here, we use several independent techniques and functional assays ranging from cell-free to intact cell systems to establish a function for dynamin in the formation of transport vesicles from the endothelial cell plasma membrane by mediating fission at the neck of caveolae. This caveolar fission requires interaction with cytosolic dynamin as well as its hydrolysis of GTP. Expression of dynamin in cytosol as well as purified recombinant dynamin alone supports GTP-induced caveolar fission in a cell-free assay whereas its removal from cytosol or the addition to the cytosol of specific antibodies for dynamin inhibits this fission. Overexpression of mutant dynamin lacking normal GTPase activity not only inhibits GTP-induced fission and budding of caveolae but also prevents caveolae-mediated internalization of cholera toxin B chain in intact and permeabilized endothelial cells. Analysis of endothelium in vivo by subcellular fractionation and immunomicroscopy shows that dynamin is concentrated on caveolae, primarily at the expected site of action, their necks. Thus, through its ability to oligomerize, dynamin appears to form a structural collar around the neck of caveolae that hydrolyzes GTP to mediate internalization via the fission of caveolae from the plasma membrane to form free transport vesicles.

scholarly journals Ectocytosis caused by sublytic autologous complement attack on human neutrophils. The sorting of endogenous plasma-membrane proteins and lipids into shed vesicles

1991 ◽  
Vol 274 (2) ◽  
pp. 381-386 ◽  
Author(s):  
J M Stein ◽  
J P Luzio
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Human Neutrophils ◽  
Cell Protection ◽  
Plasma Membrane Vesicles ◽  
Protection Mechanism ◽  
A Cell ◽  
Shed Vesicles

During sublytic complement attack on human neutrophils, plasma-membrane vesicles are shed from the cell surface as a cell-protection mechanism. By using surface-iodinated neutrophils it was found that less than 2% of surface label was recovered in shed vesicles under conditions where 40% of complement component C9 was shed. SDS/PAGE of 125I-labelled shed vesicles and plasma membranes showed differences in iodination pattern, demonstrating the sorting of membrane proteins into the shed vesicles. Analysis of 32P-labelled phospholipids after labeling of neutrophils with [32P]Pi before sublytic complement attack showed the presence of phosphatidic acid, phosphatidylcholine, phosphatidyl-ethanolamine, phosphatidylinositol and polyphosphoinositides in shed vesicles. Quantitative analysis using [3H]acetic anhydride-labelling method showed that the molar proportions of phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and sphingomyelin were the same in shed vesicles as in plasma membranes. In contrast, the molar proportions of cholesterol and diacylglycerol relative to sphingomyelin were almost twice those found in plasma membranes. The data demonstrate the existence of protein and lipid sorting mechanisms during the formation of shed vesicles when neutrophils are subject to sublytic complement attack. The term ‘ectocytosis’ is proposed to describe triggered shedding of right-side-out membrane vesicles from the surface of eukaryotic cells.

scholarly journals A specific interaction in vitro between pancreatic zymogen granules and plasma membranes: stimulation by G-protein activators but not by Ca2+.

1989 ◽  
Vol 109 (6) ◽  
pp. 2801-2808 ◽  
Author(s):  
C Y Nadin ◽  
J Rogers ◽  
S Tomlinson ◽  
J M Edwardson
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
G Protein ◽  
G Proteins ◽  
Plasma Membranes ◽  
Specific Interaction ◽  
Zymogen Granules ◽  
Fusion Event ◽  
A Cell

The molecular details of the final step in the process of regulated exocytosis, the fusion of the membrane of the secretory granule with the plasma membrane, are at present obscure. As a first step in an investigation of this membrane fusion event, we have developed a cell-free assay for the interaction between pancreatic zymogen granules and plasma membranes. We show here that plasma membranes are able to trigger the release of the granule contents, and that this effect is specific to pancreatic membranes, involves membrane fusion, requires membrane proteins, and is stimulated by activators of G-proteins but not by Ca2+. The assay is simple, reliable, and rapid, and should permit the identification of proteins that are involved in the exocytotic fusion event.

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