scholarly journals In Vitro Formation of the Endoplasmic Reticulum Occurs Independently of Microtubules by a Controlled Fusion Reaction

2000 ◽  
Vol 148 (5) ◽  
pp. 883-898 ◽  
Author(s):  
Lars Dreier ◽  
Tom A. Rapoport
Keyword(s):  
Endoplasmic Reticulum ◽  
Network Formation ◽  
Fusion Reaction ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Intermediate Stage ◽  
In Vitro System ◽  
Basic Reaction ◽  
Cytosolic Factors

We have established an in vitro system for the formation of the endoplasmic reticulum (ER). Starting from small membrane vesicles prepared from Xenopus laevis eggs, an elaborate network of membrane tubules is formed in the presence of cytosol. In the absence of cytosol, the vesicles only fuse to form large spheres. Network formation requires a ubiquitous cytosolic protein and nucleoside triphosphates, is sensitive to N-ethylmaleimide and high cytosolic Ca2+ concentrations, and proceeds via an intermediate stage in which vesicles appear to be clustered. Microtubules are not required for membrane tubule and network formation. Formation of the ER network shares significant similarities with formation of the nuclear envelope. Our results suggest that the ER network forms in a process in which cytosolic factors modify and regulate a basic reaction of membrane vesicle fusion.

Distinct egg membrane vesicles differing in binding and fusion properties contribute to sea urchin male pronuclear envelopes formed in vitro

1996 ◽  
Vol 109 (6) ◽  
pp. 1275-1283 ◽  
Author(s):  
P. Collas ◽  
D. Poccia
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Sea Urchin ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Critical Element ◽  
Marker Enzyme ◽  
Sea Urchin Egg ◽  
Nuclear Envelope Formation

We have identified three distinct membrane vesicle populations from sea urchin egg cytoplasm that cooperate in assembling the male pronuclear envelope in vitro. Membranes from sea urchin egg homogenates were separated by buoyant density into five vesicle fractions, three of which bind to demembranated sperm nuclei. Each requires a membranous element (lipophilic structure) derived from the sperm nuclear envelope at the tip and base (poles) of the nucleus in order to bind. Binding is differentially sensitive to protease, high salt and N-ethyl maleimide treatment of the membrane vesicles. MV1 binds at the poles and is required for fusion of the membrane vesicle fractions to each other and to the lipophilic structures. MV2 beta binds over the entire chromatin surface and is enriched in an endoplasmic reticulum marker enzyme. MV2 alpha binds at the nuclear poles, is enriched in a Golgi enzyme marker and is required for fusion of MV2 beta. All three fractions are required for nuclear envelope formation in vitro. The results suggest a multistep process for nuclear envelope formation involving contributions from both sperm and egg, roles for both endoplasmic reticulum and non-endoplasmic reticulum-derived vesicles, and the localization of a critical element of the fusion machinery in MV1.

scholarly journals Membrane Vesicle Release in Bacteria, Eukaryotes, and Archaea: a Conserved yet Underappreciated Aspect of Microbial Life

2012 ◽  
Vol 80 (6) ◽  
pp. 1948-1957 ◽  
Author(s):  
Brooke L. Deatherage ◽  
Brad T. Cookson
Keyword(s):  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Host Immune System ◽  
Vesicle Release ◽  
Communication Signals ◽  
Microbial Life ◽  
Functional Features

ABSTRACTInteraction of microbes with their environment depends on features of the dynamic microbial surface throughout cell growth and division. Surface modifications, whether used to acquire nutrients, defend against other microbes, or resist the pressures of a host immune system, facilitate adaptation to unique surroundings. The release of bioactive membrane vesicles (MVs) from the cell surface is conserved across microbial life, in bacteria, archaea, fungi, and parasites. MV production occurs not onlyin vitrobut alsoin vivoduring infection, underscoring the influence of these surface organelles in microbial physiology and pathogenesis through delivery of enzymes, toxins, communication signals, and antigens recognized by the innate and adaptive immune systems. Derived from a variety of organisms that span kingdoms of life and called by several names (membrane vesicles, outer membrane vesicles [OMVs], exosomes, shedding microvesicles, etc.), the conserved functions and mechanistic strategies of MV release are similar, including the use of ESCRT proteins and ESCRT protein homologues to facilitate these processes in archaea and eukaryotic microbes. Although forms of MV release by different organisms share similar visual, mechanistic, and functional features, there has been little comparison across microbial life. This underappreciated conservation of vesicle release, and the resulting functional impact throughout the tree of life, explored in this review, stresses the importance of vesicle-mediated processes throughout biology.

scholarly journals Reduced Production of Bacterial Membrane Vesicles Predicts Mortality in ST45/USA600 Methicillin-Resistant Staphylococcus aureus Bacteremia

Antibiotics ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 2 ◽  
Author(s):  
Somrita Dey ◽  
Smitha Gudipati ◽  
Christopher Giuliano ◽  
Marcus J. Zervos ◽  
Jonathan M. Monk ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Clinical Microbiology ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Bacterial Membrane ◽  
Staphylococcus Aureus Bacteremia ◽  
Immune Biomarkers ◽  
Microbial Biomarkers

Immune biomarkers can stratify mortality risk in staphylococcal bacteremia. Microbial biomarkers may provide more consistent signals during early infection. We demonstrate that in ST45/USA600 bacteremia, bacterial membrane vesicle production in vitro predicts clinical mortality (773 vs. 116 RFU, survivors vs. decedents, p < 0.0001). Using a threshold of 301 relative fluorescence units (RFU), the sensitivity and specificity of the membrane vesicles to predict mortality are 78% and 90%, respectively. This platform is facile, scalable and can be integrated into clinical microbiology lab workflows.

scholarly journals Antibiotics Stimulate Formation of Vesicles inStaphylococcus aureusin both Phage-Dependent and -Independent Fashions and via Different Routes

2018 ◽  
Vol 63 (2) ◽  
pp. e01439-18 ◽  
Author(s):  
Federica Andreoni ◽  
Masanori Toyofuku ◽  
Carmen Menzi ◽  
Ratchara Kalawong ◽  
Srikanth Mairpady Shambat ◽  
...  
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Vesicle Formation ◽  
Independent Manner ◽  
Content Type ◽  
Lactam Antibiotic ◽  
Underlying Mechanisms

ABSTRACTBacterial membrane vesicle research has so far focused mainly on Gram-negative bacteria. Only recently have Gram-positive bacteria been demonstrated to produce and release extracellular membrane vesicles (MVs) that contribute to bacterial virulence. Although treatment of bacteria with antibiotics is a well-established trigger of bacterial MV formation, the underlying mechanisms are poorly understood. In this study, we show that antibiotics can induce MVs through different routes in the important human pathogenStaphylococcus aureus. DNA-damaging agents and antibiotics inducing the SOS response triggered vesicle formation in lysogenic strains ofS. aureusbut not in their phage-devoid counterparts. The β-lactam antibiotics flucloxacillin and ceftaroline increased vesicle formation in a prophage-independent manner by weakening the peptidoglycan layer. We present evidence that the amount of DNA associated with MVs formed by phage lysis is greater than that for MVs formed by β-lactam antibiotic-induced blebbing. The purified MVs derived fromS. aureusprotected the bacteria from challenge with daptomycin, a membrane-targeting antibiotic, bothin vitroandex vivoin whole blood. In addition, the MVs protectedS. aureusfrom killing in whole blood, indicating that antibiotic-induced MVs function as a decoy and thereby contribute to the survival of the bacterium.

scholarly journals Dynamic constriction and fission of endoplasmic reticulum membranes by reticulon

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Javier Espadas ◽  
Diana Pendin ◽  
Rebeca Bocanegra ◽  
Artur Escalada ◽  
Giulia Misticoni ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Network Formation ◽  
Dynamic Balance ◽  
Ectopic Expression ◽  
Membrane Curvature ◽  
Fragmentation Mechanism ◽  
Lipid Nanotubes ◽  
Pulling Force ◽  
Organelle Morphology

AbstractThe endoplasmic reticulum (ER) is a continuous cell-wide membrane network. Network formation has been associated with proteins producing membrane curvature and fusion, such as reticulons and atlastin. Regulated network fragmentation, occurring in different physiological contexts, is less understood. Here we find that the ER has an embedded fragmentation mechanism based upon the ability of reticulon to produce fission of elongating network branches. In Drosophila, Rtnl1-facilitated fission is counterbalanced by atlastin-driven fusion, with the prevalence of Rtnl1 leading to ER fragmentation. Ectopic expression of Drosophila reticulon in COS-7 cells reveals individual fission events in dynamic ER tubules. Consistently, in vitro analyses show that reticulon produces velocity-dependent constriction of lipid nanotubes leading to stochastic fission via a hemifission mechanism. Fission occurs at elongation rates and pulling force ranges intrinsic to the ER, thus suggesting a principle whereby the dynamic balance between fusion and fission controlling organelle morphology depends on membrane motility.

scholarly journals In vitro release of physically separable factors from monocytes that exert opposing effects on erythropoiesis

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1454-1459 ◽  
Author(s):  
L Feldman ◽  
CM Cohen ◽  
N Dainiak
Keyword(s):  
Plasma Membrane ◽  
Membrane Vesicle ◽  
In Vitro Release ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Wide Range ◽  
Dose Dependent Fashion ◽  
Membrane Bound ◽  
Opposing Effects

Abstract In order to investigate the capacity of monocytes to release erythroid burst-promoting activity (BPA), we added media conditioned by homologous monocytes to both serum-free human and serum-restricted murine marrow culture. We found that soluble, membrane vesicle-free culture medium is a potent source of the growth factor. On the other hand, monocyte membranes or exfoliated plasma membrane vesicles elaborate a factor that inhibits erythroid burst formation by up to 100%. Inhibitory activity is expressed in a dose-dependent fashion over a wide range of concentrations (0.001 to 10 micrograms/mL) tested. Experiments with antilymphocyte plasma membrane IgG, which has been shown to neutralize both soluble and membrane-bound lymphocyte-derived BPA in human marrow culture, indicate that the expression of soluble BPA by monocytes is unaffected by these antibodies. Furthermore, while antimembrane IgG is capable of absorbing BPA from LCM supernatants, these antibodies are ineffective in removing BPA from MCM supernatants, suggesting that these two soluble growth factors may be antigenically distinct. Our findings indicate that while monocytes release soluble BPA, they are also a source of membrane-associated factors that exert inhibitory effects on erythropoiesis in vitro.

scholarly journals Production of Membrane Vesicles in Listeria monocytogenes Cultured with or without Sub-Inhibitory Concentrations of Antibiotics and Their Innate Immune Responses In Vitro

Genes ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 415
Author(s):  
Jung-Hwa Woo ◽  
Shukho Kim ◽  
Taewon Lee ◽  
Je-Chul Lee ◽  
Ji-Hyun Shin
Keyword(s):  
Listeria Monocytogenes ◽  
Inflammatory Responses ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Host Cells ◽  
Proteinase K ◽  
Tnf Α ◽  
Food Borne Illness ◽  
Proteinase K Treatment

Listeriosis is a food-borne illness caused by Listeria monocytogenes. Ampicillin (AMP) alone or in combination with gentamicin (GEN) is the first-line treatment option. Membrane vesicle (MV) production in L. monocytogenes under antibiotic stress conditions and pathologic roles of these MVs in hosts have not been reported yet. Thus, the aim of this study was to investigate the production of MVs in L. monocytogenes cultured with sub-minimum inhibitory concentrations (MICs) of AMP, GEN, or trimethoprim/sulfamethoxazole (SXT) and determine pathologic effects of these MVs in colon epithelial Caco-2 cells. L. monocytogenes cultured in tryptic soy broth with 1/2 MIC of AMP, GEN, or SXT produced 6.0, 2.9, or 1.5 times more MV particles, respectively, than bacteria cultured without antibiotics. MVs from L. monocytogenes cultured with AMP (MVAMP), GEN (MVGEN), or SXT (MVSXT) were more cytotoxic to Caco-2 cell than MVs obtained from cultivation without antibiotics (MVTSB). MVAMP induced more expression of tumor necrosis factor (TNF)-α gene than MVTSB, MVGEN and MVSXT, whereas MVTSB induced more expression of interleukin (IL)-1β and IL-8 genes than other MVs. Expression of pro-inflammatory cytokine genes by L. monocytogenes MVs was significantly inhibited by proteinase K treatment of MVs. In conclusion, antibiotic stress can trigger the biogenesis of MVs in L. monocytogenes and MVs produced by L. monocytogenes exposed to sub-MIC of AMP can induce strong pro-inflammatory responses by expressing TNF-α gene in host cells, which may contribute to the pathology of listeriosis.

scholarly journals In vitro release of physically separable factors from monocytes that exert opposing effects on erythropoiesis

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1454-1459
Author(s):  
L Feldman ◽  
CM Cohen ◽  
N Dainiak
Keyword(s):  
Plasma Membrane ◽  
Membrane Vesicle ◽  
In Vitro Release ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Wide Range ◽  
Dose Dependent Fashion ◽  
Membrane Bound ◽  
Opposing Effects

In order to investigate the capacity of monocytes to release erythroid burst-promoting activity (BPA), we added media conditioned by homologous monocytes to both serum-free human and serum-restricted murine marrow culture. We found that soluble, membrane vesicle-free culture medium is a potent source of the growth factor. On the other hand, monocyte membranes or exfoliated plasma membrane vesicles elaborate a factor that inhibits erythroid burst formation by up to 100%. Inhibitory activity is expressed in a dose-dependent fashion over a wide range of concentrations (0.001 to 10 micrograms/mL) tested. Experiments with antilymphocyte plasma membrane IgG, which has been shown to neutralize both soluble and membrane-bound lymphocyte-derived BPA in human marrow culture, indicate that the expression of soluble BPA by monocytes is unaffected by these antibodies. Furthermore, while antimembrane IgG is capable of absorbing BPA from LCM supernatants, these antibodies are ineffective in removing BPA from MCM supernatants, suggesting that these two soluble growth factors may be antigenically distinct. Our findings indicate that while monocytes release soluble BPA, they are also a source of membrane-associated factors that exert inhibitory effects on erythropoiesis in vitro.

scholarly journals 70-kD heat shock-related protein is one of at least two distinct cytosolic factors stimulating protein import into mitochondria.

1988 ◽  
Vol 107 (6) ◽  
pp. 2051-2057 ◽  
Author(s):  
H Murakami ◽  
D Pain ◽  
G Blobel
Keyword(s):  
Heat Shock ◽  
Matrix Protein ◽  
Protein Import ◽  
Protein S ◽  
Yeast Mitochondria ◽  
Related Protein ◽  
Free System ◽  
In Vitro System ◽  
Cytosolic Factors

We have developed an in vitro system in which the posttranslational import of Put2 (delta-pyrroline-5-carboxylate dehydrogenase), into yeast mitochondria is dependent on the addition of yeast postribosomal supernatant (PRS). When mRNA for a nuclear-encoded yeast mitochondrial matrix protein, Put2, was translated in a wheat germ cell-free system, import into posttranslationally added yeast mitochondria was negligible. However, when a yeast PRS was added, significant import was observed. The import stimulating activity of the yeast PRS was shown to consist of at least two distinct factors. One of these is the recently purified 70-kD heat shock-related protein Ssalp/Ssa2p, two proteins that are 98% homologous. The other factor is an N-ethylmaleimide-sensitive protein(s). Both factors act synergistically.

scholarly journals Immunization with outer membrane vesicles displaying conserved surface polysaccharide antigen elicits broadly antimicrobial antibodies

2018 ◽  
Vol 115 (14) ◽  
pp. E3106-E3115 ◽  
Author(s):  
Taylor C. Stevenson ◽  
Colette Cywes-Bentley ◽  
Tyler D. Moeller ◽  
Kevin B. Weyant ◽  
David Putnam ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Protective Immunity ◽  
Membrane Vesicle ◽  
Bacterial Species ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Microbial Pathogens ◽  
Polysaccharide Antigen ◽  
Protein Carriers

Many microbial pathogens produce a β-(1→6)–linked poly-N-acetyl-d-glucosamine (PNAG) surface capsule, including bacterial, fungal, and protozoan cells. Broadly protective immune responses to this single conserved polysaccharide antigen in animals are possible but only when a deacetylated poly-N-acetyl-d-glucosamine (dPNAG; <30% acetate) glycoform is administered as a conjugate to a carrier protein. Unfortunately, conventional methods for natural extraction or chemical synthesis of dPNAG and its subsequent conjugation to protein carriers can be technically demanding and expensive. Here, we describe an alternative strategy for creating broadly protective vaccine candidates that involved coordinating recombinant poly-N-acetyl-d-glucosamine (rPNAG) biosynthesis with outer membrane vesicle (OMV) formation in laboratory strains ofEscherichia coli. The glycosylated outer membrane vesicles (glycOMVs) released by these engineered bacteria were decorated with the PNAG glycopolymer and induced high titers of PNAG-specific IgG antibodies after immunization in mice. When aStaphylococcus aureusenzyme responsible for PNAG deacetylation was additionally expressed in these cells, glycOMVs were generated that elicited antibodies to both highly acetylated PNAG (∼95–100% acetate) and a chemically deacetylated dPNAG derivative (∼15% acetate). These antibodies mediated efficient in vitro killing of two distinct PNAG-positive bacterial species, namelyS. aureusandFrancisella tularensissubsp.holarctica, and mice immunized with PNAG-containing glycOMVs developed protective immunity against these unrelated pathogens. Collectively, our results reveal the potential of glycOMVs for targeting this conserved polysaccharide antigen and engendering protective immunity against the broad range of pathogens that produce surface PNAG.

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