Localization of actin, beta-spectrin, 43 × 10(3) Mr and 58 × 10(3) Mr proteins to receptor-enriched domains of newly formed acetylcholine receptor aggregates in isolated myotube membranes

1990 ◽  
Vol 97 (4) ◽  
pp. 615-626
Author(s):  
M.P. Daniels
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Acetylcholine Receptor ◽  
Plasma Membranes ◽  
Subsequent Treatment ◽  
Brain Extract ◽  
Early Stages ◽  
Cytoplasmic Face ◽  
Alpha Bungarotoxin ◽  
Immunofluorescence Labeling

I have examined the possible involvement of specific cytoskeletal and peripheral membrane proteins in the early stages of acetylcholine receptor (AChR) aggregation in rat myotubes in culture by immunofluorescence localization of these proteins on the cytoplasmic face of isolated plasma membranes. A culture procedure utilizing selective replating of myoblasts and subsequent treatment with cytosine arabinoside was devised to obtain large, multipolar myotubes with extensive upper surfaces that are free of fibroblasts. These cultures were exposed for 4–6 h to embryonic pig brain extract (EBX) to induce AChR aggregate formation on the upper cell surface, and the AChRs were labeled with TRITC-conjugated alpha-bungarotoxin. Large sheets of plasma membranes from the upper cell surface were isolated by adhesion to a coverslip coated with a polypeptide adhesive (Cell-Tak) that was pressed on top of the culture. The membranes were labeled by indirect immunofluorescence with monoclonal antibodies against the 43 × 10(3) Mr and 58 × 10(3) Mr proteins, originally identified in the AChR-enriched membranes of Torpedo electroplaques, and with monoclonal antibodies against isoforms of actin and beta-spectrin. The labeling patterns showed that all four of these proteins are concentrated in the punctate AChR-enriched domains within the aggregates, suggesting that they may be involved in the early stages of AChR aggregation. Immunofluorescence labeling with monoclonal antibodies against vinculin and clathrin, and with an antiserum to talin, showed that these proteins are also associated with AChR aggregates; however, their labeling patterns did not correspond closely to the AChR-enriched domains. Furthermore, vinculin and talin dissociated from most of the membrane during isolation. The concentration of beta-spectrin and actin isoforms on the cytoplasmic fact of the AChR-enriched domains is consistent with the formation, early in the aggregation process, of a membrane-cytoskeleton association similar to that of erythrocytes.

scholarly journals Glycosaminoglycans on fibroblasts accelerate thrombin inhibition by protease nexin-1

1987 ◽  
Vol 245 (2) ◽  
pp. 543-550 ◽  
Author(s):  
D H Farrell ◽  
D D Cunningham
Keyword(s):  
Complex Formation ◽  
Cell Surface ◽  
Plasma Membranes ◽  
Chondroitin Sulphate ◽  
Human Fibroblasts ◽  
Heparan Sulphate ◽  
Order Rate Constant ◽  
Subsequent Treatment ◽  
Protease Nexin ◽  
Inhibition Of Thrombin

Protease nexin-1 (PN-1) is a proteinase inhibitor that is secreted by human fibroblasts in culture. PN-1 inhibits certain regulatory serine proteinases by forming a covalent complex with the catalytic-site serine residue; the complex then binds to the cell surface and is internalized and degraded. The fibroblast surface was recently shown to accelerate the rate of complex-formation between PN-1 and thrombin. The present paper demonstrates that the accelerative activity is primarily due to cell-surface heparan sulphate, with a much smaller contribution from chondroitin sulphate. This conclusion is supported by the effects of purified glycosaminoglycans on the second-order rate constant for the inhibition of thrombin by PN-1. Also, treatment of 35SO4(2-)-labelled cells with heparitin sulphate lyase or chondroitin sulphate ABC lyase demonstrated two discrete pools of 35S-labelled glycosaminoglycans; subsequent treatment of plasma membranes with these glycosidases showed that heparitin sulphate lyase treatment abolished about 80% of the accelerative activity and chondroitin sulphate ABC lyase removed the remaining 20%. These results show that two components are responsible for the acceleration of PN-1-thrombin complex-formation by human fibroblasts. Although dermatan sulphate is also present on fibroblasts, it did not accelerate the inhibition of thrombin by PN-1.

scholarly journals Interaction of the cytoskeletal framework with acetylcholine receptor on th surface of embryonic muscle cells in culture.

1982 ◽  
Vol 92 (1) ◽  
pp. 231-236 ◽  
Author(s):  
J Prives ◽  
A B Fulton ◽  
S Penman ◽  
M P Daniels ◽  
C N Christian
Keyword(s):  
Cell Surface ◽  
Internal Structure ◽  
Acetylcholine Receptor ◽  
Muscle Cells ◽  
Cells In Culture ◽  
Detergent Extraction ◽  
Embryonic Muscle ◽  
Alpha Bungarotoxin ◽  
Triton X

To monitor the interaction of cell surface acetylcholine (AcCho) receptors with the cytoskeleton, cultured muscle cells were labeled with radioactive or fluorescent alpha-bungarotoxin and extracted with Triton X-100, using conditions that preserve internal structure. A significant population of the AcCho receptors is retained on the skeletal framework remaining after detergent extraction. The skeleton organization responsible for restricting AcCho receptors to a patched region may also result in their retention after detergent extraction.

scholarly journals Fibroblasts transfected with Torpedo acetylcholine receptor beta-, gamma-, and delta-subunit cDNAs express functional receptors when infected with a retroviral alpha recombinant.

1989 ◽  
Vol 108 (6) ◽  
pp. 2277-2290 ◽  
Author(s):  
T Claudio ◽  
H L Paulson ◽  
W N Green ◽  
A F Ross ◽  
D S Hartman ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Acetylcholine Receptor ◽  
Protein Complex ◽  
Half Life ◽  
Fibroblast Cell ◽  
Beta Subunit ◽  
Alpha Beta ◽  
Alpha Bungarotoxin ◽  
Delta Subunit

Torpedo californica acetylcholine receptor (AChR) alpha-, beta-, gamma-, and delta-subunit cDNAs were each stably introduced into muscle and/or fibroblast cell lines using recombinant retroviral vectors and viral infection, or using SV-40 vectors and DNA-mediated cotransfection. The expressed proteins were characterized in terms of their molecular mass, antigenicity, posttranslational processing, cell surface expression, stability in fibroblasts, stability in differentiated and undifferentiated muscle cells, and ability (of alpha) to bind alpha-bungarotoxin (BuTx). We demonstrated that the alpha, beta, gamma, and delta polypeptides acquired one, one, two, and three units of oligosaccharide, respectively. If all four subunits were expressed in the same cell, fully functional cell surface AChRs were produced which had a Kd for BuTx of 7.8 X 10(-11) M. In contrast, subunits expressed individually were not detected on the surface of fibroblasts and the Kd for BuTx binding to individual alpha polypeptides was only approximately 4 X 10(-7) M. The half-lives of the alpha, gamma, and delta subunits at 37 degrees C were all found to be quite short (approximately 43 min), while the half-life of the beta subunit was found to be even shorter (approximately 12 min). The unique half-life of the beta subunit suggests that it might perform a key regulatory role in the process of AChR subunit assembly. One stable fibroblast cell line was established by transfection that expressed beta, gamma, and delta subunits simultaneously. When this cell line was infected with a retroviral alpha recombinant, fully functional cell surface AChRs were produced. The successful expression of this pentameric protein complex combining transfection and infection techniques demonstrates one strategy for stably introducing the genes of a heterologous multisubunit protein complex into cells.

scholarly journals Regulation of acetylcholine receptor synthesis at the level of translation in rat primary muscle cells.

1989 ◽  
Vol 108 (5) ◽  
pp. 1817-1822 ◽  
Author(s):  
O Horovitz ◽  
V Spitsberg ◽  
M M Salpeter
Keyword(s):  
Ascorbic Acid ◽  
Cell Surface ◽  
Acetylcholine Receptor ◽  
Muscle Cells ◽  
Alpha Subunit ◽  
Brain Extract ◽  
Primary Cells ◽  
Active Components ◽  
Subunit Mrna ◽  
Threefold Increase

Previous studies have shown that rat primary muscle cells do not respond to crude rat brain extract or one of its active components, ascorbic acid, with a significant increase in surface acetylcholine receptor (AChR) number. We report here that, although little or no response is seen on the cell surface, rat primary muscle cells do respond to both crude brain extract and to ascorbic acid with an approximately threefold increase in AChR alpha-subunit mRNA. The response of the mRNA is similar to that seen in the cloned L5 cells. However, while in L5 cells the increase in alpha-subunit mRNA is further translated into increased levels of alpha-subunit protein, there is no such increase in alpha-subunit synthesis in the primary cells. This study thus shows a regulation of surface AChR synthesis in rat primary cells at the level of alpha-subunit translation. This level of regulation is different from that involving subunit transcription or subunit assembly reported by others.

scholarly journals Role of the cytoskeleton in the formation, stabilization, and removal of acetylcholine receptor clusters in cultured muscle cells.

1984 ◽  
Vol 99 (1) ◽  
pp. 148-154 ◽  
Author(s):  
J A Connolly
Keyword(s):  
Cell Surface ◽  
Cytochalasin D ◽  
Brain Extract ◽  
The Stability ◽  
Receptor Clusters ◽  
Alpha Bungarotoxin ◽  
Large Clusters ◽  
The Brain ◽  
Ach Receptors

We have examined the effects of microtubule- and microfilament-disrupting drugs on the stability, formation, and removal of acetylcholine (ACh) receptors and ACh receptor clusters on the surface of aneurally cultured chick embryonic myotubes. (a) In muscle cell cultures, cytochalasin D (0.2 microgram/ml) or B (2.0 micrograms/ml) causes the dispersal of 50-60% of the existing clusters over a 24-h period (visualized with rhodamine-conjugated alpha-bungarotoxin); Colcemid (0.5 micrograms/ml) has no affect on these clusters. The total number of cell surface ACh receptors does not decline during this period (measured by [125I]alpha-bungarotoxin binding) in the presence of either drug. (b) When cells are treated with biotinylated alpha-bungarotoxin and fluorescent avidin, ACh receptors are cross-linked and rapidly internalized (Axelrod, D., 1980, Proc. Natl. Acad. Sci. USA., 77: 4823-4827). Within 6 h, I have found that 0-15% of the existing large clusters remain. Cytochalasin D or B had no effect on this removal of clusters; however, Colcemid completely prevented the removal of clusters from the cell surface. (c) Addition of chick brain extract to chick myotubes causes an increase in the synthesis and clustering of ACh receptors (Jessell et al., 1979, Proc. Natl. Acad. Sci. USA. 76: 5397-5401). Cytochalasin D caused a slight increase in the number of receptors synthesized in the presence of brain extract whereas Colcemid had no effect on the synthesis and insertion of new receptors into the plasma membrane induced by the brain extract. However, both drugs prevented the increase in the number of receptor clusters. These results are consistent with the hypothesis that receptor clusters are stabilized by actin-containing filaments, but that the movement of receptors in the plane of the membrane requires Colcemid-sensitive microtubules.

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