Interaction of plasma membrane-associated filaments and H-2 histocompatibility antigens before and after induced patching and capping

1987 ◽  
Vol 88 (3) ◽  
pp. 313-325
Author(s):  
C.A. Feltkamp ◽  
H. Spiele ◽  
E. Roos
Keyword(s):  
Plasma Membrane ◽  
Short Distance ◽  
Coated Pits ◽  
Apparent Absence ◽  
Histocompatibility Antigens ◽  
Before And After ◽  
Prevailing Direction ◽  
Sequential Addition ◽  
Triton X ◽  
Filament Network

The interaction of H-2 antigens and plasma membrane-associated filaments was studied on dry-cleaved preparations of immunogold-labelled lymphoma cells. In prefixed cells, the plasma membrane-associated network was isotropic without any prevailing direction of the filaments, and the gold-labelled H-2 antigens were preferentially localized over or at a very short distance from membrane-associated filaments. Incubation of unfixed cells with anti-H-2 antibodies followed by fixation and incubation with anti-Ig, did not induce detectable redistribution of H-2 antigens or of the filament network. Notwithstanding this apparent absence of rearrangement of H-2 antigens and filaments, a detergent-resistant linkage to the cytoskeleton was induced. Before immune incubations, virtually all H-2 antigens were solubilized by extraction with Triton X-100, while after incubation with anti-H-2 antibodies about 50% of the H-2 antigens were linked to the Triton X-100-insoluble cytoskeleton. Sequential addition of anti-H-2 and anti-Ig antibodies to unfixed cells induced formation of patches and caps of H-2 antigens. Under these conditions, the majority of the H-2 antigens became linked to the detergent-resistant cytoskeleton. Redistribution into patches and caps was often accompanied by a local rearrangement of the isotropic network into bundles of parallel filaments immediately adjacent to the plasma membrane. Patches were seen to overly both isotropic networks and these parallel filaments. Large sheets of plasma membrane overlying parallel filaments were frequently devoid of gold-labelled H-2 antigens and coated pits, and thus most probably represented areas away from caps. This observation suggests that capping is accompanied by a rearrangement of filaments close to the membrane.

Changes Occur in Plasma Membrane Turnover when K562 Leukemia Cells are Induced to Differentiate

1982 ◽  
Vol 40 ◽  
pp. 286-287
Author(s):  
L. M. Marshall
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Intracellular Transport ◽  
Parent Cell ◽  
Membrane Turnover ◽  
Coated Pits ◽  
Surface Distribution ◽  
Specific Proteins ◽  
Erythroleukemic Cell ◽  
Specific Membrane

A human erythroleukemic cell line, metabolically blocked in a late stage of erythropoiesis, becomes capable of differentiation along the normal pathway when grown in the presence of hemin. This process is characterized by hemoglobin synthesis followed by rearrangement of the plasma membrane proteins and culminates in asymmetrical cytokinesis in the absence of nuclear division. A reticulocyte-like cell buds from the nucleus-containing parent cell after erythrocyte specific membrane proteins have been sequestered into its membrane. In this process the parent cell faces two obstacles. First, to organize its erythrocyte specific proteins at one pole of the cell for inclusion in the reticulocyte; second, to reduce or abolish membrane protein turnover since hemoglobin is virtually the only protein being synthesized at this stage. A means of achieving redistribution and cessation of turnover could involve movement of membrane proteins by a directional lipid flow. Generation of a lipid flow towards one pole and accumulation of erythrocyte-specific membrane proteins could be achieved by clathrin coated pits which are implicated in membrane endocytosis, intracellular transport and turnover. In non-differentiating cells, membrane proteins are turned over and are random in surface distribution. If, however, the erythrocyte specific proteins in differentiating cells were excluded from endocytosing coated pits, not only would their turnover cease, but they would also tend to drift towards and collect at the site of endocytosis. This hypothesis requires that different protein species are endocytosed by the coated vesicles in non-differentiating than by differentiating cells.

Caveolin-1, galectin-3 and lipid raft domains in cancer cell signalling

2015 ◽  
Vol 57 ◽  
pp. 189-201 ◽  
Author(s):  
Jay Shankar ◽  
Cecile Boscher ◽  
Ivan R. Nabi
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Cancer Cell ◽  
Cellular Response ◽  
Spatial Organization ◽  
Essential Feature ◽  
Cell Signalling ◽  
Coated Pits ◽  
Signalling Molecules ◽  
Raft Domains

Spatial organization of the plasma membrane is an essential feature of the cellular response to external stimuli. Receptor organization at the cell surface mediates transmission of extracellular stimuli to intracellular signalling molecules and effectors that impact various cellular processes including cell differentiation, metabolism, growth, migration and apoptosis. Membrane domains include morphologically distinct plasma membrane invaginations such as clathrin-coated pits and caveolae, but also less well-defined domains such as lipid rafts and the galectin lattice. In the present chapter, we will discuss interaction between caveolae, lipid rafts and the galectin lattice in the control of cancer cell signalling.

scholarly journals Calmodulin-microtubule association in cultured mammalian cells.

1984 ◽  
Vol 98 (3) ◽  
pp. 904-910 ◽  
Author(s):  
W J Deery ◽  
A R Means ◽  
B R Brinkley
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Cell System ◽  
The Other ◽  
Microtubule Assembly ◽  
Cytoplasmic Microtubule ◽  
Hypotonic Treatment ◽  
Cytoplasmic Microtubules ◽  
Triton X ◽  
Ca Sensitivity

A Triton X-100-lysed cell system has been used to identify calmodulin on the cytoskeleton of 3T3 and transformed SV3T3 cells. By indirect immunofluorescence, calmodulin was found to be associated with both the cytoplasmic microtubule complex and the centrosomes. A number of cytoplasmic microtubules more resistant to disassembly upon either cold (0-4 degrees C) or hypotonic treatment, as well as following dilution have been identified. Most of the stable microtubules appeared to be associated with the centrosome at one end and with the plasma membrane at the other end. These microtubules could be induced to depolymerize, however, by micromolar Ca++ concentrations. These data suggest that, by interacting directly with the microtubule, calmodulin may influence microtubule assembly and ensure the Ca++-sensitivity of both mitotic and cytoplasmic microtubules.

scholarly journals 209FUNCTIONAL ASSESSMENT OF WHITE RHINOCEROS CERATHOTERIUM SIMUM EPIDIDYMAL SPERMATOZOA BEFORE AND AFTER CRYOPRESERVATION

2004 ◽  
Vol 16 (2) ◽  
pp. 226 ◽  
Author(s):  
F. Martinez-Pastor ◽  
F. Olivier ◽  
T. Spies ◽  
L. Anel ◽  
P. Bartels
Keyword(s):  
Plasma Membrane ◽  
Assisted Reproduction ◽  
Phase Contrast ◽  
Egg Yolk ◽  
Phase Contrast Microscopy ◽  
White Rhinoceros ◽  
Contrast Microscopy ◽  
Before And After ◽  
Epididymal Spermatozoa

Biological Resource Banks represent a potentially valuable tool for species conservation. It is, however, necessary to understand the species-specific cryopreservation process and its consequences for spermatozoa to aid in the development of assisted reproduction as a future conservation tool. The aim of this study was to assess the in vitro functionality of white rhinoceros Cerathoterium simum epididymal spermatozoa both before and after cryopreservation. Testes from a harvested white rhino bull were removed and transported at 5°C to the laboratory within 4h. The cauda epididymis was dissected out and flushed with 2mL of Tris-citrate egg yolk extender (fraction A, Biladyl, Minitüb, Germany). A 0.1mL aliquot was removed for analysis and the balance (9mL; 2mL fraction A+7mL sperm sample) mixed with an additional 27.2mL of Tris-citrate egg yolk with glycerol (fraction B, Bidadyl). The extended sample was allowed to cool to 4°C over a 6-h period before an additional 29.2mL of cooled fraction B were added (final sperm concentration=150×106mL−1). Sperm samples were loaded into 0.25-mL straws and frozen over LN2 vapor (4cm for 20min) for later assessment. Sperm straws were thawed by placing the straws in water at 37°C for 30s. Pre-freeze and post-thaw evaluations were carried out in the same manner. Media used included: HEPES for washing (20mM HEPES, 355mM sucrose, 10mM glucose, 2.5mM KOH) and HEPES saline (197mM NaCl, instead of sucrose). An aliquot was diluted with HEPES (washing) and centrifuged for 5min at 600×g; the pellet was resuspended in HEPES saline. Sperm motility (total motility %, TM;; and progressive motility %, PM) was assessed using phase contrast microscopy (×200; 37°C). Sperm plasma membrane status was assessed using the fluorescent dye, propidium iodide (50ngmL−1 in HEPES saline;; 10min, RT). Percentage of cells with plasma membranes intact (unstained;; PMI) was recorded. Mitochondrial status was assessed with the fluorescent dye, JC-1 (7.5μM in HEPES saline;; 30min, 37°C). The % of cells with an orange-stained midpiece was recorded (active mitochondria;; MIT). Resilience to hypoosmotic shock (HOS test) was assessed by diluting a sample in 100mOsm/kg HEPES saline (1:20; 15min, RT). An aliquot was stained with PI to assess plasma membrane status (HOSPMI), and the rest was fixed with formaldehyde, and % coiled tails (positive endosmosis;; HOST) was estimated using phase contrast microscopy (×400). Evaluations of PMI, MIT and HOSPMI were performed using fluorescence microscopy (×400, 450–490nm excitation filter). The results indicated that quality was good pre-freezing (TM: 60%; PMI: 86%; MIT: 100%), except for a PM value of 15%. After thawing, although there was a drop in TM (30%), there was no decrease in PM (20%). Our in vitro functional assessment indicated a loss of quality between the pre-freeze and post-thaw evaluations, but PMI and MIT maintained their pre-thaw levels (60% and 72%, respectively). The HOS test, which indicates plasma membrane integrity, decreased from the pre-freeze level (91%) to a post-thaw value of 70%. HOSTPMI was 72% pre-freeze, and decreased to 54% post-thaw. In conclusion, epididymal spermatozoa from the white rhino may retain its functionality after cryopreservation in a commerically available cryo-extender (Bidadyl). The use of assisted reproduction techniques could someday play a role in the management and conservation of the white rhinoceros and related species.

Inhibition of clathrin-coated pit assembly by an Eps15 mutant

1999 ◽  
Vol 112 (9) ◽  
pp. 1303-1311 ◽  
Author(s):  
A. Benmerah ◽  
M. Bayrou ◽  
N. Cerf-Bensussan ◽  
A. Dautry-Varsat
Keyword(s):  
Plasma Membrane ◽  
Fusion Protein ◽  
Binding Site ◽  
Specific Marker ◽  
Coated Pits ◽  
Receptor Mediated Endocytosis ◽  
Gfp Fusion Protein ◽  
Protein Encoding ◽  
Homology Domains

Recent data have shown that Eps15, a newly identified component of clathrin-coated pits constitutively associated with the AP-2 complex, is required for receptor-mediated endocytosis. However, its precise function remains unknown. Interestingly, Eps15 contains three EH (Eps15-Homology) domains also found in proteins required for the internalization step of endocytosis in yeast. Results presented here show that EH domains are required for correct coated pit targeting of Eps15. Furthermore, when cells expressed an Eps15 mutant lacking EH domains, the plasma membrane punctate distribution of both AP-2 and clathrin was lost, implying the absence of coated pits. This was further confirmed by the fact that dynamin, a GTPase found in coated pits, was homogeneously redistributed on the plasma membrane and that endocytosis of transferrin, a specific marker of clathrin-dependent endocytosis, was strongly inhibited. Altogether, these results strongly suggest a role for Eps15 in coated pit assembly and more precisely a role for Eps15 in the docking of AP-2 onto the plasma membrane. This hypothesis is supported by the fact that a GFP fusion protein encoding the ear domain of (alpha)-adaptin, the AP-2 binding site for Eps15, was efficiently targeted to plasma membrane coated pits.

Association of the hepatic IP3 receptor with the plasma membrane: relevance to mode of action

1993 ◽  
Vol 265 (6) ◽  
pp. C1588-C1596 ◽  
Author(s):  
L. Feng ◽  
N. Kraus-Friedmann
Keyword(s):  
Plasma Membrane ◽  
Membrane Fraction ◽  
Ip3 Receptors ◽  
Ip3 Receptor ◽  
Plasma Membrane Fraction ◽  
T Lymphoma Cells ◽  
T Lymphoma ◽  
Triton X ◽  
Brain Receptor ◽  
The Brain

Studies were carried out to characterize the interaction between inositol 1,4,5-trisphosphate (IP3) receptors and the plasma membrane fraction. Extraction of the membranes with the nonionic detergents Nonidet P-40 and Triton X-100, followed by centrifugation at 100,000 g, resulted in the doubling of the IP3 receptor in the pellets, whereas no detectable binding was found in the supernatants. These data indicate that the detergents did not solubilize the receptor, that it remained associated with membrane particles, and that it is likely to be associated with the cytoskeleton. The cytoskeleton proteins actin, ankyrin, and spectrin were identified in the plasma membrane fraction. However, comparison of the amount of these proteins in different fractions of the detergent, or otherwise treated plasma membrane fractions, showed no direct correlation between the presence of any of these proteins in the plasma membrane fraction and their ability to bind [3H]IP3. This is in contrast to the brain and T-lymphoma cells in which the IP3 receptor is attached to ankyrin (L. Y. W. Bourguigon, H. Jin, N. Iida, N. R. Brandt, and S. H. Zhang. J. Biol. Chem. 268: 6477-6486, 1993; and S. K. Joseph and S. Samanta. J. Biol. Chem 268: 6477-6486, 1993). Thus the hepatic IP3 receptor, which is different from the brain receptor, might attach to the cytoskeleton by anchoring to a different protein. Because cytochalasin D treatment of livers diminishes the ability of IP3 to raise cytosolic free Ca2+ levels, the attachment of the IP3 receptor to the cytoskeleton seems to involve an association with microfilaments.

scholarly journals Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques.

1991 ◽  
Vol 115 (5) ◽  
pp. 1357-1374 ◽  
Author(s):  
L S Musil ◽  
D A Goodenough
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Gap Junction ◽  
Intracellular Transport ◽  
Surface Protein ◽  
Immunohistochemical Localization ◽  
Junctional Communication ◽  
Junction Protein ◽  
Gap Junctional ◽  
Triton X

We previously demonstrated that the gap junction protein connexin43 is translated as a 42-kD protein (connexin43-NP) that is efficiently phosphorylated to a 46,000-Mr species (connexin43-P2) in gap junctional communication-competent, but not in communication-deficient, cells. In this study, we used a combination of metabolic radiolabeling and immunoprecipitation to investigate the assembly of connexin43 into gap junctions and the relationship of this event to phosphorylation of connexin43. Examination of the detergent solubility of connexin43 in communication-competent NRK cells revealed that processing of connexin43 to the P2 form was accompanied by acquisition of resistance to solubilization in 1% Triton X-100. Immunohistochemical localization of connexin43 in Triton-extracted NRK cells demonstrated that connexin43-P2 (Triton-insoluble) was concentrated in gap junctional plaques, whereas connexin43-NP (Triton-soluble) was predominantly intracellular. Using either a 20 degrees C intracellular transport block or cell-surface protein biotinylation, we determined that connexin43 was transported to the plasma membrane in the Triton-soluble connexin43-NP form. Cell-surface biotinylated connexin43-NP was processed to Triton-insoluble connexin43-P2 at 37 degrees C. Connexin43-NP was also transported to the plasma membrane in communication defective, gap junction-deficient S180 and L929 cells but was not processed to Triton-insoluble connexin43-P2. Taken together, these results demonstrate that gap junction assembly is regulated after arrival of connexin43 at the plasma membrane and is temporally associated with acquisition of insolubility in Triton X-100 and phosphorylation to the connexin43-P2 form.

scholarly journals Initial events during phagocytosis by macrophages viewed from outside and inside the cell: membrane-particle interactions and clathrin.

1982 ◽  
Vol 94 (3) ◽  
pp. 613-623 ◽  
Author(s):  
J Aggeler ◽  
Z Werb
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Plasma Membranes ◽  
Cell Attachment ◽  
High Resolution Electron Microscopy ◽  
Peritoneal Macrophages ◽  
Coated Pits ◽  
Thin Sections ◽  
Particle Uptake ◽  
Freeze Dried

The initial events during phagocytosis of latex beads by mouse peritoneal macrophages were visualized by high-resolution electron microscopy of platinum replicas of freeze-dried cells and by conventional thin-section electron microscopy of macrophages postfixed with 1% tannic acid. On the external surface of phagocytosing macrophages, all stages of particle uptake were seen, from early attachment to complete engulfment. Wherever the plasma membrane approached the bead surface, there was a 20-nm-wide gap bridged by narrow strands of material 12.4 nm in diameter. These strands were also seen in thin sections and in replicas of critical-point-dried and freeze-fractured macrophages. When cells were broken open and the plasma membrane was viewed from the inside, many nascent phagosomes had relatively smooth cytoplasmic surfaces with few associated cytoskeletal filaments. However, up to one-half of the phagosomes that were still close to the cell surface after a short phagocytic pulse (2-5 min) had large flat or spherical areas of clathrin basketwork on their membranes, and both smooth and clathrin-coated vesicles were seen fusing with or budding off from them. Clathrin-coated pits and vesicles were also abundant elsewhere on the plasma membranes of phagocytosing and control macrophages, but large flat clathrin patches similar to those on nascent phagosomes were observed only on the attached basal plasma membrane surfaces. These resulted suggest that phagocytosis shares features not only with cell attachment and spreading but also with receptor-mediated pinocytosis.

scholarly journals Surface glycoproteins of human spermatozoa

1986 ◽  
Vol 82 (1) ◽  
pp. 11-22
Author(s):  
M. Kallajoki ◽  
I. Virtanen ◽  
J. Suominen
Keyword(s):  
Plasma Membrane ◽  
Gel Electrophoresis ◽  
Soluble Fraction ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Human Spermatozoa ◽  
Galactose Oxidase ◽  
Sperm Cells ◽  
Neuraminidase Treatment ◽  
Triton X

The surface membrane glycoprotein composition of human spermatozoa has been studied by introducing radioactive label into galactosyl (Gal) and N-acetylgalactosaminyl (GalNAc) residues by using the galactose oxidase/NaB3H4 method. Triton X-100 extracts and Triton X-100-resistant cytoskeletal residues were subjected to analysis by polyacrylamide gel electrophoresis. The distribution of the radiolabel in sperm cells was studied by light-microscopic auto-radiography. The grains were evenly distributed on the cells by the labelling methods used. The Triton X-100 treatment did not affect sperm morphology at the light-microscopic level, but in transmission electron microscopy the plasma membrane covering the acrosome was removed totally, together with most of the acrosomal membranes and acrosomal contents. Plasma membrane residues were, however, always found in the postacrosomal region. Borohydride alone without oxidative pretreatment labelled two polypeptides of molecular weights (Mr) 48,000 and 43,000 in the Triton X-100-soluble fraction. When the Gal/GalNAc residues were labelled by galactose oxidase pretreatment 120,000, 105,000, 78,000 and 68,000 Mr glycoproteins were revealed. When additional neuraminidase treatment was used to remove terminal sialic acid residues, the total labelling intensity was increased two- to fivefold and additional 36,000 and 20,000 Mr glycoproteins were revealed. The Triton X-100-resistant cytoskeletal residue contained 53–75% of the total radioactivity bound in sperm cells. When these components were analysed by polyacrylamide gel electrophoresis, all the major bands found in the Triton X-100-soluble fraction were detected and also some radioactivity was incorporated into the major bands visualized by protein staining. In the present study we describe several human sperm glycoproteins, which seem to be distributed evenly on the sperm cells. Detergent extraction, producing cytoskeletal models, appeared to leave most of the glycoproteins detectable in the extraction residues also with the apparent enrichment of a single 68,000 Mr glycoprotein.

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