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

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 Retrograde Transport of Golgi-localized Proteins to the ER

1998 ◽  
Vol 140 (1) ◽  
pp. 1-15 ◽  
Author(s):  
Nelson B. Cole ◽  
Jan Ellenberg ◽  
Jia Song ◽  
Diane DiEuliis ◽  
Jennifer Lippincott-Schwartz
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Fusion Proteins ◽  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Viral Glycoprotein ◽  
Lumenal Domain

The ER is uniquely enriched in chaperones and folding enzymes that facilitate folding and unfolding reactions and ensure that only correctly folded and assembled proteins leave this compartment. Here we address the extent to which proteins that leave the ER and localize to distal sites in the secretory pathway are able to return to the ER folding environment during their lifetime. Retrieval of proteins back to the ER was studied using an assay based on the capacity of the ER to retain misfolded proteins. The lumenal domain of the temperature-sensitive viral glycoprotein VSVGtsO45 was fused to Golgi or plasma membrane targeting domains. At the nonpermissive temperature, newly synthesized fusion proteins misfolded and were retained in the ER, indicating the VSVGtsO45 ectodomain was sufficient for their retention within the ER. At the permissive temperature, the fusion proteins were correctly delivered to the Golgi complex or plasma membrane, indicating the lumenal epitope of VSVGtsO45 also did not interfere with proper targeting of these molecules. Strikingly, Golgi-localized fusion proteins, but not VSVGtsO45 itself, were found to redistribute back to the ER upon a shift to the nonpermissive temperature, where they misfolded and were retained. This occurred over a time period of 15 min–2 h depending on the chimera, and did not require new protein synthesis. Significantly, recycling did not appear to be induced by misfolding of the chimeras within the Golgi complex. This suggested these proteins normally cycle between the Golgi and ER, and while passing through the ER at 40°C become misfolded and retained. The attachment of the thermosensitive VSVGtsO45 lumenal domain to proteins promises to be a useful tool for studying the molecular mechanisms and specificity of retrograde traffic to the ER.

scholarly journals Asterosap, an Egg Jelly Peptide, Elevate Intracellular Ca2+ and Activate the Motility of Spermatozoa

2013 ◽  
Vol 19 (1) ◽  
pp. 79-88 ◽  
Author(s):  
MS Islam ◽  
T Akhter ◽  
M Matsumoto
Keyword(s):  
Plasma Membrane ◽  
Cyclic Gmp ◽  
Guanylyl Cyclase ◽  
Acrosome Reaction ◽  
Nickel Chloride ◽  
Selective Inhibitor ◽  
Ion Flux ◽  
Intracellular Ca ◽  
A Cell ◽  
Egg Jelly

Components from the outer envelopes of the egg that influence the flagellar beating and acrosome reaction of spermatozoa are regulated by ion flux across the plasma membrane. Asterosap, a sperm-activating peptide from the starfish egg jelly layer, causes a transient increase in intracellular cyclic GMP (cGMP) through the activation of the asterosap receptor, a guanylyl cyclase (GC), and causes an increase in intracellular Ca2+. Here we describe the pathway of asterosap-induced Ca2+ elevation using different Ca2+ channel antagonists. Fluo-4 AM, a cell permeable Ca2+ sensitive dye was used to determine the channel caused by the asterosap-induced Ca2+ elevation in spermatozoa. Different L-type Ca2+ channel antagonists, a non specific Ca2+ channel antagonist (nickel chloride), and a store-operated Ca2+ channel (SOC) antagonist do not show any significant response on asterosap-induced Ca2+ elevation, whereas KB-R7943, a selective inhibitor against Na+/Ca2+ exchanger (NCX) inhibited effectively. We also analyzed the flagellar movement of spermatozoa in artificial seawater (ASW) containing the asterosap at 100 nM ml?1. We found that spermatozoa swam vigorously with more symmetrical flagellar movement in asterosap than in ASW and KB-R7943 significantly inhibited the flagellar movement.DOI: http://dx.doi.org/10.3329/pa.v19i1.17358 Progress. Agric. 19(1): 79 - 88, 2008 

scholarly journals Novel compound inhibitors of HIV-1NL4-3 Vpu

2021 ◽  
Author(s):  
Carolyn A Robinson ◽  
Terri D Lyddon ◽  
Hwi Min Gil ◽  
David T. Evans ◽  
Yury V Kuzmichev ◽  
...  
Keyword(s):  
Host Cell ◽  
Particle Production ◽  
Viral Glycoprotein ◽  
New Class ◽  
Host Cell Proteins ◽  
Viral Particles ◽  
Dependent Cell ◽  
A Cell ◽  
Hiv 1

HIV-1 Vpu targets the host cell proteins CD4 and BST-2/Tetherin for degradation, ultimately resulting in enhanced virus spread and host immune evasion. The discovery and characterization of small molecules that antagonize Vpu would further elucidate the contribution of Vpu to pathogenesis and lay the foundation for the study of a new class of novel HIV-1 therapeutics. To identify novel compounds that block Vpu activity, we developed a cell-based 'gain of function' assay that produces a positive signal in response to Vpu inhibition. To develop this assay, we took advantage of the viral glycoprotein, GaLV Env. In the presence of Vpu, GaLV Env is not incorporated into viral particles, resulting in non-infectious virions. Vpu inhibition restores infectious particle production. Using this assay, a high throughput screen of >650,000 compounds was performed to identify inhibitors that block the biological activity of Vpu. From this screen, we identified several positive hits but focused on two compounds from one structural family, SRI-41897 and SRI-42371. It was conceivable that the compounds inhibited the formation of infectious virions by targeting host cell proteins instead of Vpu directly, so we developed independent counter-screens for off target interactions of the compounds and found no off target interactions. Additionally, these compounds block Vpu-mediated modulation of CD4, BST-2/Tetherin and antibody dependent cell-mediated toxicity (ADCC). Unfortunately, both SRI-41897 and SRI-42371 were shown to be specific to the N-terminal region of NL4-3 Vpu and did not function against other, more clinically relevant, strains of Vpu.

scholarly journals Lysosomal Exocytosis, Exosome Release and Secretory Autophagy: The Autophagic- and Endo-Lysosomal Systems Go Extracellular

2020 ◽  
Vol 21 (7) ◽  
pp. 2576 ◽  
Author(s):  
Sandra Buratta ◽  
Brunella Tancini ◽  
Krizia Sagini ◽  
Federica Delo ◽  
Elisabetta Chiaradia ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Molecular Mechanisms ◽  
Current Knowledge ◽  
Cell Communication ◽  
Lysosomal Storage ◽  
Cellular Processes ◽  
Age Related ◽  
Extracellular Release ◽  
A Cell ◽  
Endosomal System

Beyond the consolidated role in degrading and recycling cellular waste, the autophagic- and endo-lysosomal systems play a crucial role in extracellular release pathways. Lysosomal exocytosis is a process leading to the secretion of lysosomal content upon lysosome fusion with plasma membrane and is an important mechanism of cellular clearance, necessary to maintain cell fitness. Exosomes are a class of extracellular vesicles originating from the inward budding of the membrane of late endosomes, which may not fuse with lysosomes but be released extracellularly upon exocytosis. In addition to garbage disposal tools, they are now considered a cell-to-cell communication mechanism. Autophagy is a cellular process leading to sequestration of cytosolic cargoes for their degradation within lysosomes. However, the autophagic machinery is also involved in unconventional protein secretion and autophagy-dependent secretion, which are fundamental mechanisms for toxic protein disposal, immune signalling and pathogen surveillance. These cellular processes underline the crosstalk between the autophagic and the endosomal system and indicate an intersection between degradative and secretory functions. Further, they suggest that the molecular mechanisms underlying fusion, either with lysosomes or plasma membrane, are key determinants to maintain cell homeostasis upon stressing stimuli. When they fail, the accumulation of undigested substrates leads to pathological consequences, as indicated by the involvement of autophagic and lysosomal alteration in human diseases, namely lysosomal storage disorders, age-related neurodegenerative diseases and cancer. In this paper, we reviewed the current knowledge on the functional role of extracellular release pathways involving lysosomes and the autophagic- and endo-lysosomal systems, evaluating their implication in health and disease.

scholarly journals Microgravity-Induced Cell-to-Cell Junctional Contacts Are Counteracted by Antioxidant Compounds in TCam-2 Seminoma Cells

2020 ◽  
Vol 10 (22) ◽  
pp. 8289
Author(s):  
Angela Catizone ◽  
Caterina Morabito ◽  
Marcella Cammarota ◽  
Chiara Schiraldi ◽  
Katia Corano Scheri ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Line ◽  
Simulated Microgravity ◽  
Cell Metabolism ◽  
Ultrastructural Level ◽  
Antioxidant Compounds ◽  
Signal Molecules ◽  
Beta Catenin ◽  
Autophagy Induction ◽  
A Cell

The direct impact of microgravity exposure on male germ cells, as well as on their malignant counterparts, has not been largely studied. In previous works, we reported our findings on a cell line derived from a human seminoma lesion (TCam-2 cell line) showing that acute exposure to simulated microgravity altered microtubule orientation, induced autophagy, and modified cell metabolism stimulating ROS production. Moreover, we demonstrated that the antioxidant administration prevented both TCam-2 microgravity-induced microtubule disorientation and autophagy induction. Herein, expanding previous investigations, we report that simulated microgravity exposure for 24 h induced the appearance, at an ultrastructural level, of cell-to-cell junctional contacts that were not detectable in cells grown at 1 g. In line with this result, pan-cadherin immunofluorescence analyzed by confocal microscopy, revealed the clustering of this marker at the plasma membrane level on microgravity exposed TCam-2 cells. The upregulation of cadherin was confirmed by Western blot analyses. Furthermore, we demonstrated that the microgravity-induced ROS increase was responsible for the distribution of cadherin nearby the plasma membrane, together with beta-catenin since the administration of antioxidants prevented this microgravity-dependent phenomenon. These results shed new light on the microgravity-induced modifications of the cell adhesive behavior and highlight the role of ROS as microgravity activated signal molecules.

scholarly journals Cytoskeletal-membrane interactions: a stable interaction between cell surface glycoconjugates and doublet microtubules of the photoreceptor connecting cilium.

1987 ◽  
Vol 105 (6) ◽  
pp. 2973-2987 ◽  
Author(s):  
C J Horst ◽  
D M Forestner ◽  
J C Besharse
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Molecular Mass ◽  
Lipid Bilayer ◽  
Neonatal Rat ◽  
Sds Page ◽  
A Cell ◽  
The Cross ◽  
Triton X ◽  
Cell Surface Glycoconjugates

The ciliary base is marked by a transition zone in which Y-shaped cross-linkers extend from doublet microtubules to the plasma membrane. Our goal was to investigate the hypothesis that the cross-linkers form a stable interaction between membrane or cell surface components and the underlying microtubule cytoskeleton. We have combined Triton X-100 extraction with lectin cytochemistry in the photoreceptor sensory cilium to investigate the relationship between cell surface glycoconjugates and the underlying cytoskeleton, and to identify the cell surface components involved. Wheat germ agglutinin (WGA) binds heavily to the cell surface in the region of the Y-shaped cross-linkers of the neonatal rat photoreceptor cilium. WGA binding is not removed by prior digestion with neuraminidase and succinyl-WGA also binds the proximal cilium, suggesting a predominance of N-acetylglucosamine containing glycoconjugates. Extraction of the photoreceptor plasma membrane with Triton X-100 removes the lipid bilayer, leaving the Y-shaped crosslinkers associated with the axoneme. WGA-binding sites are found at the distal ends of the crosslinkers after Triton X-100 extraction, indicating that the microtubule-membrane cross-linkers retain both a transmembrane and a cell surface component after removal of the lipid bilayer. To identify glycoconjugate components of the cross-linkers we used a subcellular fraction enriched in axonemes from adult bovine retinas. Isolated, detergent-extracted bovine axonemes show WGA binding at the distal ends of the cross-linkers similar to that seen in the neonatal rat. Proteins of the axoneme fraction were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose. WGA labeling of the nitrocellulose transblots reveals three glycoconjugates, all of molecular mass greater than 400 kD. The major WGA-binding glycoconjugate has an apparent molecular mass of approximately 600 kD and is insensitive to prior digestion with neuraminidase. This glycoconjugate may correspond to the dominant WGA-binding component seen in cytochemical experiments.

scholarly journals gp96 Is a Human Colonocyte Plasma Membrane Binding Protein for Clostridium difficile Toxin A

2008 ◽  
Vol 76 (7) ◽  
pp. 2862-2871 ◽  
Author(s):  
Xi Na ◽  
Ho Kim ◽  
Mary P. Moyer ◽  
Charalabos Pothoulakis ◽  
J. Thomas LaMont
Keyword(s):  
Plasma Membrane ◽  
Clostridium Difficile ◽  
Binding Protein ◽  
Membrane Binding ◽  
Cell Surface Receptor ◽  
Toxin A ◽  
Surface Receptor ◽  
A Cell ◽  
Plasma Membrane Binding

ABSTRACT Clostridium difficile toxin A (TxA), a key mediator of antibiotic-associated colitis, requires binding to a cell surface receptor prior to internalization. Our aim was to identify novel plasma membrane TxA binding proteins on human colonocytes. TxA was coupled with biotin and cross-linked to the surface of HT29 human colonic epithelial cells. The main colonocyte binding protein for TxA was identified as glycoprotein 96 (gp96) by coimmunoprecipitation and mass spectrum analysis. gp96 is a member of the heat shock protein family, which is expressed on human colonocyte apical membranes as well as in the cytoplasm. TxA binding to gp96 was confirmed by fluorescence immunostaining and in vitro coimmunoprecipitation. Following TxA binding, the TxA-gp96 complex was translocated from the cell membrane to the cytoplasm. Pretreatment with gp96 antibody decreased TxA binding to colonocytes and inhibited TxA-induced cell rounding. Small interfering RNA directed against gp96 reduced gp96 expression and cytotoxicity in colonocytes. TxA-induced inflammatory signaling via p38 and apoptosis as measured by activation of BAK (Bcl-2 homologous antagonist/killer) and DNA fragmentation were decreased in gp96-deficient B cells. We conclude that human colonocyte gp96 serves as a plasma membrane binding protein that enhances cellular entry of TxA, participates in cellular signaling events in the inflammatory cascade, and facilitates cytotoxicity.

scholarly journals Exocytosis of Alphaherpesvirus Virions, Light Particles, and Glycoproteins Uses Constitutive Secretory Mechanisms

mBio ◽  
2016 ◽  
Vol 7 (3) ◽  
Author(s):  
Ian B. Hogue ◽  
Julian Scherer ◽  
Lynn W. Enquist
Keyword(s):  
Plasma Membrane ◽  
Virus Particle ◽  
Immune Evasion ◽  
Host Cells ◽  
Type I ◽  
Rab Gtpases ◽  
Viral Glycoprotein ◽  
Particle Assembly ◽  
Viral Glycoproteins ◽  
Light Particles

ABSTRACTMany molecular and cell biological details of the alphaherpesvirus assembly and egress pathway remain unclear. Recently we developed a live-cell fluorescence microscopy assay of pseudorabies virus (PRV) exocytosis, based ontotalinternalreflectionfluorescence (TIRF) microscopy and a virus-encoded pH-sensitive fluorescent probe. Here, we use this assay to distinguish three classes of viral exocytosis in a nonpolarized cell type: (i) trafficking of viral glycoproteins to the plasma membrane, (ii) exocytosis of viral light particles, and (iii) exocytosis of virions. We find that viral glycoproteins traffic to the cell surface in association with constitutive secretory Rab GTPases and exhibit free diffusion into the plasma membrane after exocytosis. Similarly, both virions and light particles use these same constitutive secretory mechanisms for egress from infected cells. Furthermore, we show that viral light particles are distinct from cellular exosomes. Together, these observations shed light on viral glycoprotein trafficking steps that precede virus particle assembly and reinforce the idea that virions and light particles share a biogenesis and trafficking pathway.IMPORTANCEThe alphaherpesviruses, including the important human pathogens herpes simplex virus 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV), are among the few viruses that have evolved to exploit the mammalian nervous system. These viruses typically cause mild recurrent herpetic or zosteriform lesions but can also cause debilitating herpes encephalitis, more frequently in very young, old, immunocompromised, or nonnatural hosts. Importantly, many of the molecular and cellular mechanisms of viral assembly and egress remain unclear. This study addresses the trafficking of viral glycoproteins to the plasma membrane, exocytosis of light particles, and exocytosis of virions. Trafficking of glycoproteins affects immune evasion and pathogenesis and may precede virus particle assembly. The release of light particles may also contribute to immune evasion and pathogenesis. Finally, exocytosis of virions is important to understand, as this final step in the virus replication cycle produces infectious extracellular particles capable of spreading to the next round of host cells.

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