scholarly journals Membrane proteins of the vacuolar system. III. Further studies on the composition and recycling of endocytic vacuole membrane in cultured macrophages

1983 ◽  
Vol 96 (1) ◽  
pp. 29-36 ◽  
Author(s):  
WA Muller ◽  
RM Steinman ◽  
ZA Cohn
Keyword(s):  
Cell Surface ◽  
Plasma Membranes ◽  
Adjacent Area ◽  
Cell Surface Antigens ◽  
Membrane Flow ◽  
Two Dimensional Gel Electrophoresis ◽  
Electron Microscope Autoradiography ◽  
Vacuole Membrane ◽  
Lysosomal Ph ◽  
Immune Precipitation

In previous publications (Muller, W.A., R.M. Steinman, Z.A. Cohn. 1980, J.Cell Biol. 86:292-314), we found that the membrane of macrophage phagolysosomes could be selectively radioiodinated in living cells, The technique required phagocytosis of lactoperoxidase covalently coupled to latex spheres (LPO-latex), followed by iodination on ice with Na(125)I and hydrogen peroxide. In this paper, we use the LPO-latex system to further analyze the composition and recycling of phagocytic vacuole membrane. Three approaches were employed to examine the polypeptide composition of the phagolysosome (PL) and plasma membranes (PM). (a) The efficiency of intracellular iodination was increased by increasing lysosomal pH with chloroquine. By one-dimensional SDS PAGE, the heavily labeled chloroquine-treated PL exhibited the same labeled polypeptides as PM iodinated extracellularly with LPO-latex. (b) Iodinated PL and PM were compared by two-dimensional gel electrophoresis. No differences in the isoelectric point and molecular weight of the major iodinated species were detected. (c) Quantitative immune precipitation was performed with five specific antibodies directed against cell surface antigens. Four antibodies precipitated similar relative amounts of labeled antigen on the cell surface and endocytic vacuole. One antibody, secreted by hybridoma 2.6, detected a 21-kdalton polypeptide that was enriched sevenfold in PL membrane. This enrichment was cell surface-derived, since the amount of labeled 2.6 was increased sevenfold when iodinated PM was driven into the cell during latex uptake. Therefore, intracellular iodination primarily detects PL proteins that are identical to their PM counterparts. Additional studies employed electron microscope autoradiography to monitor the centrifugal flow of radiolabeled polypeptides from PL to PM. Cells were iodinated intralysosomally and returned to culture for only 5-10 min at 37 degrees C. Most of the cell-associated label then redistributed to the cell surface or its adjacent area. Significant movement out of the lysosome compartment occurred even at 2 degrees C and 22 degrees C. Extensive and rapid membrane flow through the secondary lysosome presumably contributes to the great similarity between PM and PL membrane polypeptides.

scholarly journals Large-scale co-aggregation of fluorescent lipid probes with cell surface proteins.

1994 ◽  
Vol 125 (4) ◽  
pp. 795-802 ◽  
Author(s):  
J L Thomas ◽  
D Holowka ◽  
B Baird ◽  
W W Webb
Keyword(s):  
Cell Surface ◽  
Large Scale ◽  
Plasma Membranes ◽  
Immunoglobulin E ◽  
Large Fraction ◽  
Surface Antigens ◽  
Specific Cell ◽  
Cell Surface Antigens ◽  
Receptor Complexes ◽  
Ige Receptors

Large scale aggregation of fluorescein-labeled immunoglobulin E (IgE) receptor complexes on the surface of RBL cells results in the co-aggregation of a large fraction of the lipophilic fluorescent probe 3,3'-dihexadecylindocarbocyanine (diI) that labels the plasma membranes much more uniformly in the absence of receptor aggregation. Most of the diI molecules that are localized in patches of aggregated receptors have lost their lateral mobility as determined by fluorescence photobleaching recovery. The diI outside of patches is mobile, and its mobility is similar to that in control cells without receptor aggregates. It is unlikely that the co-aggregation of diI with IgE receptors is due to specific interactions between these components, as two other lipophilic probes of different structures are also observed to redistribute with aggregated IgE receptors, and aggregation of two other cell surface antigens also results in the coredistribution of diI at the RBL cell surface. Quantitative analysis of CCD images of labeled cells reveals some differences in the spatial distributions of co-aggregated diI and IgE receptors. The results indicate that cross-linking of specific cell surface antigens causes a substantial change in the organization of the plasma membrane by redistributing pre-existing membrane domains or causing their formation.

scholarly journals Production of a monoclonal antibody against cell-surface glycoprotein of guinea pig adrenocortical cells.

1993 ◽  
Vol 41 (2) ◽  
pp. 235-243 ◽  
Author(s):  
Y Kameda ◽  
C Hirota
Keyword(s):  
Monoclonal Antibody ◽  
Sialic Acid ◽  
Cell Surface ◽  
Guinea Pig ◽  
Plasma Membranes ◽  
Surface Glycoprotein ◽  
Zona Fasciculata ◽  
Cell Surface Antigens ◽  
Adrenocortical Cells ◽  
Cell Surface Glycoprotein

A monoclonal antibody (MAb) that reacted with the cell-surface antigens of adrenocortical cells was generated against cell suspensions from guinea pig adrenal glands. Cell-surface membranes of the adrenocortical cells in all zones, i.e., zona glomerulosa, zona fasciculata, and zona reticularis, were labeled with the antibody. Adrenal medulla remained unlabeled. Immunoelectron microscopy showed that entire plasma membranes, i.e., plasma membranes between adjacent cells and free cell-surface membranes, including sinusoidal microvilli, were immunoreactive to the antibody. Immunoblot analysis demonstrated that the antibody bound to two prominent bands at molecular weights of approximately 62,000 and 110,000. Two bands were stained with lectin-digoxigenin conjugates. The 110 KD band reacted with Datura stramonium (DSA) and Maackia amurensis (MAA) agglutinins, indicating the presence of N-acetyl-glucosamine and sialic acid-linked alpha (2-3) to galactose; the 62 KD band reacted with SNA, indicating the presence of sialic acid-linked alpha (2-6) to galactose. In adrenocortical cells, the reaction pattern of Sambucus nigra (SNA) agglutinin was similar to that of the (MAb), whereas reaction patterns of DSA and MAA were different. Both neuraminidase digestion and prior absorption of the antibody with N-acetyl-neuraminic acid completely prevented the immunolabeling of adrenocortical cells. These results indicate that the MAb mainly recognizes the 2-6 sialylated cell-surface antigen of adrenocortical cells.

Feeding in Ciliated Protozoa

1974 ◽  
Vol 15 (2) ◽  
pp. 379-401
Author(s):  
JOHN. A. KLOETZEL
Keyword(s):  
Cell Surface ◽  
Buccal Cavity ◽  
Surface Membrane ◽  
Food Vacuole ◽  
Food Ingestion ◽  
Membrane Flow ◽  
Vacuole Membrane ◽  
Vacuole Formation ◽  
Food Vacuoles ◽  
Lamellar Material

The ciliate Euplotes is able to expend a very large amount of membrane in the formation of food vacuoles. Calculations based on the rate of ingestion of the food organism Tetrahymena indicate that an amount of food vacuole membrane equivalent to approximately 50-150% of the total Euplotes cell surface area can be produced within 5-10 min. An aggregation of osmiophilic, membrane-limited ‘pharyngeal disks’ is found packed in the cytoplasm just beneath the cell surface membrane in the region of the cell mouth and cytopharynx. These disks, which can be seen also in living cells, have average dimensions of 2 µm diameter by 100 nm thickness, and contain tightly packed layers of a thin lamellar material. Electron micrographs have revealed the apparent fusion of the limiting membrane of disks with the cell's plasma membrane at the base of the gullet. The lamellar disk contents are thereby released to the exterior medium in the buccal cavity, where they form a loosely packed layer over the surface membrane. It is postulated that the pharyngeal disks represent a repository of preformed membrane for use in food vacuole formation. The disk contents may also play a role in food ingestion, although this is not well defined at present. The myeloid content of old food vacuoles is very similar to that of nearby disks in the cytoplasm, suggesting that the disks may form by pinching from shrinking food vacuoles during the digestive cycle. Thus a cycle of membrane flow is envisaged, with the pharyngeal disks (1) coalescing with the surface membrane during food vacuole formation, (2) reforming by pinching from these food vacuoles during digestion, and (3) migrating back to the oral region to serve as a membrane store for subsequent food vacuole formation.

Isolation of plasma membranes from purified mouse spermatogenic cells

1980 ◽  
Vol 43 (1) ◽  
pp. 279-299
Author(s):  
C.F. Millette ◽  
D.A. O'Brien ◽  
C.T. Moulding
Keyword(s):  
Cell Surface ◽  
Germ Cells ◽  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Lectin Binding ◽  
Round Spermatid ◽  
Ultrastructural Observation ◽  
Spermatogenic Cells ◽  
Cell Surface Antigens ◽  
Total Recovery

Plasma membranes have been prepared from purified pachytene spermatocytes, round spermatids and residual bodies of the adult mouse testis using procedures modified from other authors'. Isolated membranes have been examined using electron microscopy, lectin binding and enzymic assays. Ultrastructural observation reveals smooth unit-membrane vesicles from 0.4-1.7 micrometer diameter. No contamination by nuclei, mitochondria or lysosomes is detected microscopically. Radiolabelled lectin-binding experiments [125I-RCAI, 125I-green pea lectin] indicate that cell surface label cofractionates with material identified morphologically as plasma membrane. Estimates of total recovery of membrane, based upn the lectin data, average 33%. Biochemical analysis of subcellular markers reveal that no detectable DNA and only 1.2% of the total cellular RNA cofractionate with membranes. A variety of enzyme assays suggests little contamination by cytosol enzymes, Golgi material or mitochondria. Assays of 5′-nucleotidase (E.C. 3.1.3.5) indicate that this enzyme is not a major component of developing mouse spermatogenic cell membranes. Instead, Sertoli cells represent the most important source of this enzyme in the adult seminiferous tubule. Polyacrylamide gel analysis of membranes isolated from purified germ cells reveals significant differences in the protein compositions of pachytene spermatocyte and round spermatid membranes. The preparation of highly purified plasma membranes from homogeneous populations of spermatogenic cells should facilitate the biochemical characterization of cell surface antigens specific to developing male germ cells.

A pre-embedding, protein a-gold immunolabelling technique for cytocentrifuged cell monolayers for lm and tem

1986 ◽  
Vol 44 ◽  
pp. 220-221
Author(s):  
K. Chien ◽  
I.P. Shintaku ◽  
A.F. Sassoon ◽  
R.L. Van de Velde ◽  
R. Heusser
Keyword(s):  
Cell Surface ◽  
Staining Method ◽  
Protein A ◽  
Surface Antigens ◽  
Cell Suspensions ◽  
Cellular Morphology ◽  
Cellular Phenotype ◽  
Cell Surface Antigens ◽  
Cell Monolayers ◽  
Diagnostic Histopathology

Identification of cellular phenotype by cell surface antigens in conjunction with ultrastructural analysis of cellular morphology can be a useful tool in the study of biologic processes as well as in diagnostic histopathology. In this abstract, we describe a simple pre-embedding, protein A-gold staining method which is designed for cell suspensions combining the handling convenience of slide-mounted cell monolayers and the ability to evaluate specimen staining specificity prior to EM embedding.

Quantification of gold labeled cell surface antigens by SEM: Current progress and remaining problems

1990 ◽  
Vol 48 (3) ◽  
pp. 34-35
Author(s):  
Etienne de Harven ◽  
Davide Soligo ◽  
Roy McGroarty ◽  
Hilary Christensen ◽  
Richard Leung ◽  
...  
Keyword(s):  
Cell Surface ◽  
Immunogold Labeling ◽  
Target Antigen ◽  
Facs Analysis ◽  
Becton Dickinson ◽  
Cell Surface Antigens ◽  
Human T Lymphocyte ◽  
Backscattered Electron ◽  
Imaging Mode ◽  
Electron Imaging

Taking advantage of the high elemental contrast of particles of colloidal gold observed in the backscattered electron imaging(BEI) mode of the SEM (1,2), the human T lymphocyte was chosen as a model system to study the potential value of immunogold labeling for the quantification of cell surface expressed molecules. The CD3 antigen which is expressed on all human T lymphocytes and is readily identified by the LEU-4 murine monoclonal antibody (Becton Dickinson, Mountain View, CA) followed by a gold conjugated goat anti-mouse Ig polyclonal antibody was chosen as a model target antigen. When quantified by non-EM methods, using radio-iodinated probes or FACS analysis, approximately 30,000 to 50,000 copies of this antigen per cell are enumerated.The following observations were made while attempting to quantify the same molecule by SEM after specific immunogold labeling:Imaging in the SE vs BE mode: The numbers of gold markers counted in the secondary electron (SE) imaging mode are considerably lower than those counted on the same cells in the backscattered electron (BE) imaging mode.

scholarly journals EFFECT OF 12-O-TETRADECANOYLPHORBOL-13-ACETATE ON EXPRESSION OF CELL SURFACE ANTIGENS IN TWO SUBCLONES OF HUMAN LEUKEMIA K562 CELLS

1993 ◽  
Vol 16 (10) ◽  
pp. 1054-1056
Author(s):  
Dai SASAKI ◽  
Satoshi KOSUNAGO ◽  
Takeshi MIKAMI ◽  
Tatsuji MATSUMOTO ◽  
Masuko SUZUKI
Keyword(s):  
Cell Surface ◽  
K562 Cells ◽  
Surface Antigens ◽  
Human Leukemia ◽  
Cell Surface Antigens ◽  
Human Leukemia K562 Cells
Sign in / Sign up

Export Citation Format

Share Document