Plasma Membrane-derived Vesicles Research Articles

Regulation of MHC class I synthesis and expression by human neutrophils.

Human peripheral blood neutrophils (PMN) treated with granulocyte-macrophage CSF (GM-CSF) increase the amount of class I 42-kDa H chain and 12-kDa L chain, beta 2-microglobulin (beta 2m), that they synthesize by 2.1- and 2.6-fold, respectively. To determine whether the increase in translation was associated with an increase in levels of class I H chain transcript, RNA blot analysis was performed on PMN that had been cultured in the presence of GM-CSF. Under no conditions were there increased levels of class I H chain transcript when class I heterodimer protein synthesis was increased. In addition, there was neither an increase in the synthesis of H chain mRNA, as measured by transcription assay, nor an alteration in the degradation rates of class I H chain transcript in PMN cultured with GM-CSF. In situ hybridization demonstrated that both the percentage of PMN that expressed class I transcript and the relative amounts of transcript per cell in GM-CSF-cultured PMN were the same as those in control PMN. Although there is increased translation of class I heterodimer in PMN treated with GM-CSF, there is no increase in its expression on the plasma membrane. The maintenance of constant levels of class I on the plasma membrane is dependent on continued protein synthesis and is maintained by release of class I heterodimer and free beta 2m into the medium. Heterodimer is released in the context of plasma membrane-derived vesicles, whereas beta 2m is released as a soluble protein. Maintenance of constant levels of class I heterodimer on the plasma membrane is also regulated by constitutive internalization. Up to 30% of class I molecules bearing 125I-Fab-labeled mAb to class I are internalized over 2 h at 37 degrees C. Therefore, inducible synthesis of class I by PMN is likely a consequence of post-transcriptional regulation, whereas the continued synthesis of class I heterodimer is required for maintenance of its expression. Furthermore, there is no increase in class I expression, in spite of increased synthesis, due to the release of class I heterodimer and beta 2m and the internalization of class I heterodimer from the plasma membrane. Thus, PMN are capable of post-transcriptional regulation of protein synthesis and are able to modulate the expression of plasma membrane proteins by regulated expression, release, and internalization.

Effects of a new positive inotropic agent with a vasodilatory action, 5-methyl-6-(4-pyridyl)-2H-1,4-thiazin-3(4H)-one (ZSY-27), on calcium-movement and mechanical response in rabbit thoracic aorta.

The effects of 5-methyl-6-(4-pyridyl)-2H-1,4-thiazin-3(4H)-one (ZSY-27), a positive inotropic agent with a vasodilatory action, on a concentration-response curve of CaCl2 in the KCl-depolarized rabbit thoracic aorta and Ca-uptake by the microsomal fraction from the rabbit artery were tested. ZSY-27 (10(-5) - 10(-4) M) inhibited noncompetitively a concentration-response curve of CaCl2 in the KCl-depolarized preparation. Furthermore, ZSY-27 (10(-5) M) stimulated significantly Ca-uptake by the microsomal fraction in the presence of oxalate, as an indicator for Ca-uptake by the internal membrane, in every incubation time. However, it inhibited significantly that in the absence of oxalate, as an indicator for Ca-uptake by the plasma membrane-derived vesicle, after 6 min of incubation time. These results suggest that ZSY-27 stimulates Ca-uptake by the internal membranes and inhibits Ca-uptake by the plasma membrane-derived vesicles. Therefore, these effects of ZSY-27 are considered to be related to relaxation of vascular smooth muscle.

Plasma membrane-derived vesicles containing receptor-ligand complexes are fusogenic with early endosomes in a cell-free system.

Receptor-mediated endocytosis involves the transport of receptor-ligand complexes from the cell surface to an intracellular endocytic compartment. This study shows that plasma membrane-derived vesicles containing receptor-bound ligands (e.g. aggregated anti-dinitrophenol (DNP) IgG bound to Fc receptors) fuse with early endosomes containing DNP-beta-glucuronidase in a cell-free system. Plasma membrane vesicles were generated by homogenization of cells that had been allowed to bind ligands at 4 degrees C. Fusion between vesicles containing the two probes was assessed by (i) the formation of anti-DNP IgG-DNP-beta-glucuronidase complexes and (ii) the colocalization within closed vesicles of two different sizes of colloidal gold coated with ligands. Fusion required ATP, cytosol, and KCl. The requirements were similar to those described for endosome-endosome fusion in in vitro systems. Mild trypsinization of vesicles prior to their addition to the assay inhibited fusion. When DNP-beta-glucuronidase was chased into more mature endocytic compartments, fusion was not observed. The results indicate that cell surface regions involved in receptor-mediated endocytosis are capable of fusing to early endosomes. This fusion event may constitute the first step in the transport of ligands to an intracellular endocytic compartment.

A Monoclonal Antibody to Exfoliated Surface Vesicles That Recognizes a Membrane-Associated Erythroid Burst-Promoting Activity

A monoclonal antibody (MoAb) recognizing a membrane-associated erythroid burst-promoting factor was prepared by immunizing BALB/c mice with plasma membrane-derived vesicles exfoliated from lymphocytes under serum-free conditions. Hybrids secreting antibody reactive with lymphocyte plasma membranes were formally cloned and IgG was purified from monoclonal supernatants or from BALB/c mouse ascites fluid. Two clones (D3-E4 and D3-G9) were found to suppress burst forming unit-erythroid (BFU-E) proliferation when added directly to serum-free human marrow culture. Inhibition to a level of 100% was observed in a dose-dependent fashion over a wide range of antibody concentrations (0-200 micrograms/mL). Neither antibody altered the proliferation of colony forming unit-granulocyte macrophage (CFU-GM) or colony forming unit-granulocyte-erythroid-monocyte-megakaryocyte (CFU-GEMM) progenitor cells in human bone marrow culture. The D3-E4 clone was found to produce an IgG1 antibody which adsorbs an erythroid burst-promoting activity (BPA) from supernatants of, and octylglucoside extracts of shed vesicles present in, serum-free, lymphocyte conditioned medium (LCM), and which recognizes a vesicular protein of Mr approximate 30,000 on immunoblots of membrane proteins electrophoresed on sodium dodecyl sulfate (SDS)/polyacrylamide and transferred to nitrocellulose. In contrast, the D3-G9 clone was found to produce an IgG1 cytotoxic antibody. These antibodies will be important to the study of cell-cell and growth factor-cell interactions in vitro.

A monoclonal antibody to exfoliated surface vesicles that recognizes a membrane-associated erythroid burst-promoting activity

A monoclonal antibody (MoAb) recognizing a membrane-associated erythroid burst-promoting factor was prepared by immunizing BALB/c mice with plasma membrane-derived vesicles exfoliated from lymphocytes under serum-free conditions. Hybrids secreting antibody reactive with lymphocyte plasma membranes were formally cloned and IgG was purified from monoclonal supernatants or from BALB/c mouse ascites fluid. Two clones (D3-E4 and D3-G9) were found to suppress burst forming unit- erythroid (BFU-E) proliferation when added directly to serum-free human marrow culture. Inhibition to a level of 100% was observed in a dose- dependent fashion over a wide range of antibody concentrations (0–200 micrograms/mL). Neither antibody altered the proliferation of colony forming unit-granulocyte macrophage (CFU-GM) or colony forming unit- granulocyte-erythroid-monocyte-megakaryocyte (CFU-GEMM) progenitor cells in human bone marrow culture. The D3-E4 clone was found to produce an IgG1 antibody which adsorbs an erythroid burst-promoting activity (BPA) from supernatants of, and octylglucoside extracts of shed vesicles present in, serum-free, lymphocyte conditioned medium (LCM), and which recognizes a vesicular protein of Mr approximate 30,000 on immunoblots of membrane proteins electrophoresed on sodium dodecyl sulfate (SDS)/polyacrylamide and transferred to nitrocellulose. In contrast, the D3-G9 clone was found to produce an IgG1 cytotoxic antibody. These antibodies will be important to the study of cell-cell and growth factor-cell interactions in vitro.

Ultrastructural localization of an intracellular Toxoplasma protein that induces protection in mice

We report the ultrastructural localization of Toxoplasma antigens recognized by monoclonal antibody F3G3, which protects mice against lethal challenge. By using colloidal immunogold labeling, F3G3 failed to react with the surface of intact Toxoplasma cells, confirming previous observations that it recognizes an intracellular antigen. Immunoperoxidase labeling with F3G3 was obtained only when Toxoplasma cells were previously exposed to Triton X-100. A specific immunoperoxidase reaction was located beneath the surface membrane in the region of the pellicle and within the elaborate network of vesicles which are extruded from the surface of Toxoplasma cells during entry into host cells. Similar results were obtained with mouse polyclonal sera and a second monoclonal antibody, 6-86-1E11, both of which were produced against the F3G3 affinity-purified protein. The location of the epitope recognized by F3G3 was confirmed by purifying T. gondii-derived surface membrane vesicles, separating these proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting. Monoclonal antibody 6-86-1E11 and the analogous polyclonal sera reacted with the antigenically related proteins of 58 and 28 kilodaltons that were found both in the intact organisms and in the purified surface membrane vesicles. These results indicate that the epitope recognized by F3G3 is located beneath the Toxoplasma cell surface membrane and is contained within plasma membrane-derived vesicles.

Chapter 6 Endocytosis and Recognition Mechanisms in Dictyostelium discoideum

Publisher Summary Endocytosis is the uptake of fluid (pinocytosis) or particles (phagocytosis) by a eukaryotic cell from the extracellular environment into the cytoplasm through plasma membrane-derived vesicles. Dictyostelium discoideum is particularly well suited as an experimental system to study mechanisms of phagocytosis and cell recognition. The major experimental problem in measuring initial rates of endocytosis is to separate cells containing ingested material from the bulk of noningested material. This problem is overcome by centrifuging the cell suspension through a column of highly viscous polyethylene glycol. Extracellular fluid and small particles with a high surface/volume ratio remain on top of the column, whereas the large phagocytes are found on the bottom. Fluorescently labeled dextrans are found to be suitable fluid-phase markers to measure pinocytosis. Analysis of the Phag mutant phenotype reveals the presence of at least two membrane receptors that recognize different features of a foreign surface. A “lectin-type” receptor recognizes glucose residues and a second nonspecific receptor recognizes the relative hydrophobicity of a surface, which may be a phagocytosable particle, a solid surface, or the surface of another amoeba. Mutants defective in the nonspecific receptor can be isolated using various selection procedures, and all of the mutants independently derived display a similar phenotype.

Inhibition of Macrophage Ia Antigen Expression by Shed Plasma Membrane Vesicles From Metastatic Murine Melanoma Lines<xref ref-type="fn" rid="FN1">2</xref>

Shed plasma membrane-derived vesicles from metastatic variants of the murine B16 melanoma were examined for their ability to inhibit the induction of murine immune region-associated (Ia) antigen expression on macrophages, the initial step in the formation of an immune response. Membrane material that appears as a greater than 50 million-dalton fraction on column chromatography is found only in conditioned media from tumor cells and not in culture media from normal cells, such as murine 3T3 cells. Membrane vesicles from both metastatic variants B16-F1 (low lung colonizing) and B16-F10 (high lung colonizing) were taken up by macrophages; however, only membrane vesicles isolated from the B16-F10 cultures exhibited significant inhibitory activity for Ia induction. This inhibition appears to result from enhanced prostaglandin synthesis, since treatment with aspirin can reverse the membrane vesicle-induced inhibition. The inhibitory component(s) released into the media was demonstrated to be predominantly associated with membrane vesicles; however, the component(s) retained its activity after Triton X-100 treatment, indicating that the intact membrane vesicle was not necessary for the action of the inhibitory material. Treatments with heat (65 degrees C) and proteases (papain) indicated that the inhibitory component(s) is a heat-labile protein.

A dual action of papaverine on calcium uptake by microsomal fraction isolated from rat uterus

The effects of papaverine, cyclic AMP and MIX (3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor) on calcium uptake by a microsomal fraction from rat uterus were tested in the presence of 3 mM ATP. Papaverine potentiated calcium uptake in the presence of oxalate but inhibited it in the absence of oxalate. However, cyclic AMP and MIX did not influence calcium uptake, neither in the presence nor the absence of oxalate. These results suggest that calcium uptake by plasma membrane-derived vesicles in the absence of oxalate is inhibited by papaverine and that papaverine potentiated calcium uptake by the internal membranes in the presence of oxalate. They suggest also that the stimulatory action of papaverine involves a cyclic AMP-independent mechanism in addition to the mechanism via cyclic AMP.

POSSIBLE MECHANISMS OF STIMULATORY ACTION OF PAPAVERINE ON CALCIUM-UPTAKE BY RAT UTERINE MICROSOMAL FRACTION

Effects of papaverine and cyclic AMP on Ca-uptake by the microsomal fraction from rat uterus were studied. Papaverine (3×10-5 M) potentiated Ca-uptake by the microsomal fraction in the presence of potassium oxalate. However, cyclic AMP and MIX (3-isobutyl-1-methyl-xanthine; 1 mM), a potent phosphodiesterase inhibitor, did not influence Ca-uptake by the microsomal fraction in the presence of potassium oxalate. Cyclic AMP in concentrations of 10-8 to 10-4 M did not influence Ca-uptake by the microsomal fraction in the presence of potassium oxalate. In the absence of potassium oxalate, papaverine and Aspaminol (1,1-diphenyl-3-piperidinobutanol hydrochloride), a nonspecific smooth muscle relaxant, inhibited Ca-uptake by the microsomal fraction and cyclic AMP had no influence on this uptake. These results suggest that papaverine potentiated Ca-uptake by membranes such as sarcoplasmic reticulum, in the presence of potassium oxalate and inhibited Ca-uptake by the plasma membrane-derived vesicles in the absence of potassium oxalate. These results suggest that relaxation of smooth muscle by papaverine is related to a cyclic AMP-independent mechanism as well as to a mechanism mediated via cyclic AMP.

Possible mechanisms of stimulatory action of papaverine on calcium-uptake by rat uterine microsomal fraction.

Effects of papaverine and cyclic AMP on Ca-uptake by the microsomal fraction from rat uterus were studied. Papaverine (3 x 10(-5) M) potentiated Ca-uptake by the microsomal fraction in the presence of potassium oxalate. However, cyclic AMP and MIX (3-isobutyl-1-methylxanthine; 1 mM), a potent phosphodiesterase inhibitor, did not influence Ca-uptake by the microsomal fraction in the presence of potassium oxalate. Cyclic AMP in concentrations of 10(-8) to 10(-4) M did not influence Ca-uptake by the microsomal fraction in the presence of potassium oxalate. In the absence of potassium oxalate, papaverine and Aspaminol (1,1,-diphenyl-3-piperidinobutanol hydrochloride), a nonspecific smooth muscle relaxant, inhibited Ca-uptake by the microsomal fraction and cyclic AMP had no influence on this uptake. These results suggest that papaverine potentiated Ca-uptake by membranes such as sarcoplasmic reticulum, in the presence of potassium oxalate and inhibited Ca-uptake by the plasma membrane-derived vesicles in the absence of potassium oxalate. These results suggest that relaxation of smooth muscle by papaverine is related to a cyclic AMP-independent mechanism as well as to a mechanism mediated via cyclic AMP.

Translocation and turnover of phospholipid analogs in plasma membrane-derived vesicles from cell cultures

The time-dependent accumulation of phosphatidyldimethylethanolamine in formaldehyde-induced vesicles obtained from a somatic cell hybrid line was investigated. From a number of considerations including a two-fold enrichment of cholesterol and sphingomyelin it was concluded that these vesicles were derived from the cell plasma membrane. A progressive depletion of phosphatidylcholine, the major vesicle phospholipid, was observed in cells supplemented for various time periods with dimethylethanolamine. This depletion was accompanied by a concomitant increase in the amount of lipid analog. The time-dependent alteration of the phospholipid polar head group in intact cells was almost identical to that observed in isolated plasma membrane vesicles, suggesting a rapid equilibration of the de novo synthesized phospholipid with the cell surface compartment. From the initial velocity rate, the time required for the phosphatidylcholine pool to double was about 12 h. Agarose-linked phospholipase A 2 was used to measure the relative composition of choline- and dimethylethanolamine-phosphoglycerides in the outer surface of vesicles prepared from cells with different degrees of polar head group substitution. The gradual appearance of lysodimethylethanolamine lipid analog in vesicles treated with phospholipase A 2 suggested an asymmetric distribution of the phospholipid between the interior and the exterior part of the vesicle. This asymmetry was maximal up to about 4 h following the addition of dimethylethanolamine to the culture medium and was of a transient nature as the lipid analog accumulated on both sides of the plasma membrane. Based on these measurements a fast followed by a slow translocation component could be distinguished with apparent doubling times of 7 and 43 h for the lipid analog, respectively. As the analog becomes the predominant cellular phospholipid a significant increase in the vesicle lipid fluidity was measured.