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

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.

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In vitro reconstitution of exocytosis from sea urchin egg plasma membrane and isolated secretory vesicles.

10.1349/ddlp.2695 ◽

1986 ◽

Author(s):

Joseph H. Crabb

Keyword(s):

Plasma Membrane ◽

Sea Urchin ◽

Secretory Vesicles ◽

Sea Urchin Egg ◽

In Vitro Reconstitution

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In vitro reconstitution of exocytosis from sea urchin egg plasma membrane and isolated cortical vesicles

Bioscience Reports ◽

10.1007/bf01362503 ◽

1987 ◽

Vol 7 (5) ◽

pp. 399-409 ◽

Cited By ~ 7

Author(s):

Joseph H. Crabb ◽

Paul A. Modern ◽

Robert C. Jackson

Keyword(s):

Plasma Membrane ◽

Human Red Blood Cells ◽

Sea Urchin Egg ◽

Cytoplasmic Face ◽

In Vitro Reconstitution ◽

Isotonic Buffer ◽

Cortical Vesicles ◽

Coated Glass Slide ◽

Egg Cortex

We have succeeded in reconstituting an exocytotically active egg cortex fraction by recombining purified cortical vesicles (CVs) with egg plasma membrane (PM). CVs were dislodged from a suspension of egg cortex by gentle homogenization in a dissociative buffer with a pH of 9.1, and purified by two rounds of differential centrifugation. Egg PM was prepared by shearing the cortical vesicles from a cortical lawn preparation with a jet of isotonic buffer. PM lawns produced by this procedure consist of an array of CV-free PM fragments attached via their extracellular surface to a polylysine coated glass slide. When a neutralized suspension of CVs was recombined with a PM lawn, CVs reassociated with the cytoplasmic face of the plasma membrane to form a reconstituted lawn (RL). RLs undergo a morphological change in response to Ca2+-containing buffers that is similar to the exocytotic release of CV contents from cortical lawns. In both reactions CV contents are vectorially transferred from the cytoplasmic to the extracytoplasmic face of the egg PM. A quantitative binding assay was developed and used to show that adherence of CVs to a heterologous PM lawn prepared from human red blood cells is minimal.

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

The Journal of Cell Biology ◽

10.1083/jcb.109.6.2801 ◽

1989 ◽

Vol 109 (6) ◽

pp. 2801-2808 ◽

Cited By ~ 37

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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Faculty Opinions recommendation of In vitro fusion between Saccharomyces cerevisiae secretory vesicles and cytoplasmic-side-out plasma membrane vesicles.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1010821.167908 ◽

2002 ◽

Author(s):

Miguel Penalva

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Membrane Vesicles ◽

Secretory Vesicles ◽

Plasma Membrane Vesicles ◽

Cytoplasmic Side

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

The Journal of Cell Biology ◽

10.1083/jcb.103.5.1829 ◽

1986 ◽

Vol 103 (5) ◽

pp. 1829-1835 ◽

Cited By ~ 13

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.

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Surface membrane vesicles from mononuclear cells stimulate erythroid stem cells to proliferate in culture

Blood ◽

10.1182/blood.v60.3.583.583 ◽

1982 ◽

Vol 60 (3) ◽

pp. 583-594 ◽

Cited By ~ 26

Author(s):

N Dainiak ◽

CM Cohen

Keyword(s):

Plasma Membrane ◽

Plasma Membranes ◽

Mononuclear Cells ◽

Surface Membrane ◽

Freeze Fracture ◽

Membrane Vesicles ◽

Cell Types ◽

Target Cells ◽

Freeze Fracture Electron Microscopy

Abstract In order to examine the contribution of cell surface materials to erythroid burst-promoting activity (BPA), we separated media conditioned by a variety of human cell types into pellets and supernatants by centrifugation. When added to serum-restricted cultures of nonadherent human marrow cells, pellets contained about half of the total stimulatory activity. Freeze-fracture electron microscopy of the pellets revealed the presence of unilamellar membrane vesicles ranging from 0.10 to 0.40 microM in diameter. The amount of BPA in culture increased with added vesicle concentration in a saturable fashion. Preparation of leukocyte conditioned medium (LCM) from 125I-wheat germ agglutinin labeled cells and studies comparing the glycoprotein composition of vesicles with that of leukocyte plasma membranes suggest that LCM-derived vesicles are of plasma membrane origin. Moreover, partially purified leukocyte plasma membrane preparations also contained BPA. While disruption of vesicles by freezing/thawing and hypotonic lysis did not alter BPA, heat, trypsin, or pronase treatment removed greater than 65% of BPA, implying that vesicle surface rather than intravesicular molecules express BPA. Results of BPA assays performed in two-layer clots indicated that proximity to target cells is required for vesicle BPA expression. We conclude that membrane vesicles spontaneously shed from cell surfaces may be important regulators of erythroid burst proliferation in vitro.

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

Infection and Immunity ◽

10.1128/iai.00326-08 ◽

2008 ◽

Vol 76 (7) ◽

pp. 2862-2871 ◽

Cited By ~ 62

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.

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Yeast actin cytoskeleton mutants accumulate a new class of Golgi-derived secretary vesicle.

Molecular Biology of the Cell ◽

10.1091/mbc.8.8.1481 ◽

1997 ◽

Vol 8 (8) ◽

pp. 1481-1499 ◽

Cited By ~ 90

Author(s):

J Mulholland ◽

A Wesp ◽

H Riezman ◽

D Botstein

Keyword(s):

Cell Wall ◽

Plasma Membrane ◽

Actin Cytoskeleton ◽

Biochemical Analysis ◽

Small Gtpase ◽

Secretory Vesicles ◽

Cytoskeleton Organization ◽

New Class ◽

Actin Cytoskeleton Organization ◽

Vectorial Transport

Many yeast actin cytoskeleton mutants accumulate large secretory vesicles and exhibit phenotypes consistent with defects in polarized growth. This, together with actin's polarized organization, has suggested a role for the actin cytoskeleton in the vectorial transport of late secretory vesicles to the plasma membrane. By using ultrastructural and biochemical analysis, we have characterized defects manifested by mutations in the SLA2 gene (also known as the END4 gene), previously found to affect both the organization of the actin cytoskeleton and endocytosis in yeast. Defects in cell wall morphology, accumulated vesicles, and protein secretion kinetics were found in sla2 mutants similar to defects found in act1 mutants. Vesicles that accumulate in the sla2 and act1 mutants are immunoreactive with antibodies directed against the small GTPase Ypt1p but not with antibodies directed against the homologous Sec4p found on classical "late" secretory vesicles. In contrast, the late-acting secretory mutants sec1-1 and sec6-4 are shown to accumulate anti-Sec4p-positive secretory vesicles as well as vesicles that are immunoreactive with antibodies directed against Ypt1p. The late sec mutant sec4-8 is also shown to accumulate Ypt1p-containing vesicles and to exhibit defects in actin cytoskeleton organization. These results indicate the existence of at least two classes of morphologically similar, late secretory vesicles (associated with Ypt1p+ and Sec4p+, respectively), one of which appears to accumulate when the actin cytoskeleton is disorganized.

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Subcellular localization and translocation of the receptor for N-formylmethionyl-leucyl-phenylalanine in human neutrophils

Biochemical Journal ◽

10.1042/bj2990473 ◽

1994 ◽

Vol 299 (2) ◽

pp. 473-479 ◽

Cited By ~ 104

Author(s):

H Sengeløv ◽

F Boulay ◽

L Kjeldsen ◽

N Borregaard

Keyword(s):

Plasma Membrane ◽

Subcellular Localization ◽

Plasma Membranes ◽

Secretory Vesicle ◽

Human Neutrophils ◽

Secretory Vesicles ◽

Beta Band ◽

Neutrophil Gelatinase Associated Lipocalin ◽

Specific Granules ◽

Inflammatory Stimuli

The subcellular localization of N-formylmethionyl-leucyl-phenylalanine (fMLP) receptors in human neutrophils was investigated. The fMLP receptor was detected with a high-affinity, photoactivatable, radioiodinated derivative of N-formyl-methionyl-leucyl-phenylalanyl-lysine (fMLFK). Neutrophils were disrupted by nitrogen cavitation and fractionated on Percoll density gradients. fMLP receptors were located in the beta-band containing gelatinase and specific granules, and in the gamma-band containing plasma membrane and secretory vesicles. Plasma membranes and secretory vesicles were separated by high-voltage free-flow electrophoresis, and secretory vesicles were demonstrated to be highly enriched in fMLP receptors. The receptors found in secretory vesicles translocated fully to the plasma membrane upon stimulation with inflammatory mediators. The receptor translocation from the beta-band indicated that the receptor present there was mainly located in gelatinase granules. A 25 kDa fMLP-binding protein was found in the beta-band. Immunoprecipitation revealed that this protein was identical with NGAL (neutrophil gelatinase-associated lipocalin), a novel protein found in specific granules. In summary, we demonstrate that the compartment in human neutrophils that is mobilized most easily and fastest, the secretory vesicle, is a major reservoir of fMLP receptors. This explains the prompt and extensive upregulation of fMLP receptors on the neutrophil surface in response to inflammatory stimuli.

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