Formation and dispersal of acetylcholine receptor clusters in muscle cells

1986 ◽  
Vol 9 ◽  
pp. 125-129 ◽  
Author(s):  
H. Benjamin Peng ◽  
Mu-ming Poo
Keyword(s):  
Acetylcholine Receptor ◽  
Muscle Cells ◽  
Receptor Clusters

scholarly journals Development of Acetylcholine Receptor Clusters on Cultured Muscle Cells

1973 ◽  
Vol 70 (1) ◽  
pp. 270-274 ◽  
Author(s):  
A. J. Sytkowski ◽  
Z. Vogel ◽  
M. W. Nirenberg
Keyword(s):  
Acetylcholine Receptor ◽  
Muscle Cells ◽  
Receptor Clusters

scholarly journals Freeze-fracture and electrophysiological studies of newly developed acetylcholine receptors in Xenopus embryonic muscle cells.

1984 ◽  
Vol 98 (6) ◽  
pp. 2160-2173 ◽  
Author(s):  
P C Bridgman ◽  
S Nakajima ◽  
A S Greenberg ◽  
Y Nakajima
Keyword(s):  
Size Distribution ◽  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Freeze Fracture ◽  
Muscle Cells ◽  
Embryonic Muscle ◽  
Freeze Fracture Electron Microscopy ◽  
Receptor Clusters ◽  
Alpha Bungarotoxin ◽  
Solitary Form

The development of acetylcholine receptors on Xenopus embryonic muscle cells both in culture and in situ was studied using electrophysiology and freeze-fracture electron microscopy. Acetylcholine sensitivity first appeared at developmental stage 20 and gradually increased up to about stage 31. Freeze-fracture of muscle cells that were nonsensitive to acetylcholine revealed diffusely distributed small P-face intramembraneous particles. When cells acquired sensitivity to acetylcholine, a different group of diffusely distributed large P-face particles began to appear. This group of particles was analyzed by subtracting the size distribution found on nonsensitive cells from that found on sensitive cells. We call this group of particles difference particles. The sizes of difference particles were large (peak diameter 11 nm). The density of difference particles gradually increased with development. The density of small particles (less than 9 nm) did not change with development. At later stages (32-36) aggregates of large particles appeared, which probably represent acetylcholine receptor clusters. The size distribution of difference particles was close to that of the aggregated particles, suggesting that at least part of difference particles represent diffusely distributed acetylcholine receptors. Difference particles exist mostly in solitary form (occasionally double), indicating that an acetylcholine receptor can be functional in solitary form. This result also shows that diffuse acetylcholine receptors that have previously been observed with 125I-alpha-bungarotoxin autoradiography do indeed exist in solitary forms not as microaggregates.

scholarly journals Microinjection of a monoclonal antibody against a 37-kD protein (tropomyosin 2) prevents the formation of new acetylcholine receptor clusters.

1989 ◽  
Vol 109 (5) ◽  
pp. 2337-2344 ◽  
Author(s):  
G Marazzi ◽  
F Bard ◽  
M W Klymkowsky ◽  
L L Rubin
Keyword(s):  
Monoclonal Antibody ◽  
Acetylcholine Receptor ◽  
Rous Sarcoma Virus ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Muscle Cells ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Receptor Clusters

We have shown previously that chick muscle cells transformed with Rous sarcoma virus are unable to form clusters of acetylcholine receptors (AChRs) (Anthony, D. T., S. M. Schuetze, and L. L. Rubin. 1984. Proc. Natl. Acad. Sci. USA. 81:2265-2269) and are missing a 37-KD tropomyosin-like protein (TM-2) (Anthony, D. T., R. J. Jacobs-Cohen, G. Marazzi, and L. L. Rubin. 1988. J. Cell Biol. 106:1713-1721). In an attempt to clarify the role of TM-2 in the formation and/or maintenance of AChR clusters, we have microinjected a monoclonal antibody specific for TM-2 (D3-16) into normal chick muscle cells in culture. D3-16 injection blocks the formation of new clusters but does not affect the preexisting ones. In addition, TM-2 is concentrated at rat neuromuscular junctions. These data suggest that TM-2 may play an important role in promoting the formation of AChR clusters.

scholarly journals Localization of intracellular proteins at acetylcholine receptor clusters induced by electric fields in Xenopus muscle cells

1989 ◽  
Vol 94 (1) ◽  
pp. 73-83
Author(s):  
M.W. Rochlin ◽  
H.B. Peng
Keyword(s):  
Electric Field ◽  
Acetylcholine Receptor ◽  
Electric Fields ◽  
Matrix Material ◽  
Muscle Cells ◽  
Ultrastructural Level ◽  
Anchoring Proteins ◽  
Intracellular Proteins ◽  
Receptor Clusters ◽  
Electric Field Treatment

Electric fields cause acetylcholine receptor (AChR) patches to form on the cathodal sides of cultured muscle cells. These patches are stable for several hours following cessation of an electric field treatment, indicating that the receptors are anchored to the cluster sites. Furthermore, at the ultrastructural level, AChR patches induced by electric fields are marked by an accumulation of extracellular matrix material and a sarcolemmal density. Thus, these AChR patches are similar to those induced by other stimuli, including nerve, polycation-coated beads, and the tissue culture substratum. Proteins that may be involved in anchoring AChRs have been colocalized with AChR patches induced by the latter three stimuli, but not at AChR patches induced by electric fields. In this study, we demonstrate that three putative anchoring proteins, 43K (K = 10(3) Mr) protein, 58K protein and talin, are associated with field-induced AChR patches. We also show that these proteins persist at field-induced AChR patches following removal of the field, indicating that they are stabilized at the AChR patch. Our data are consistent with the possibility that these proteins contribute to the stabilization of AChRs at patches induced by the electric field. Since 43K, 58K and talin are intracellular proteins, and therefore could not undergo field-induced lateral electrophoresis, our observations support the notion that the electric field triggers the formation of an AChR-stabilizing specialization.

scholarly journals Distribution of filipin-sterol complexes on cultured muscle cells: cell-substratum contact areas associated with acetylcholine receptor clusters.

1983 ◽  
Vol 96 (2) ◽  
pp. 363-372 ◽  
Author(s):  
P C Bridgman ◽  
Y Nakajima
Keyword(s):  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Freeze Fracture ◽  
Muscle Cells ◽  
Distinct Pattern ◽  
Intramembrane Particles ◽  
Membrane Topography ◽  
Interference Contrast Microscopy ◽  
Receptor Clusters ◽  
Alpha Bungarotoxin

Specialized areas within broad, close, cell-substratum contacts seen with reflection interference contrast microscopy in cultures of Xenopus embryonic muscle cells were studied. These areas usually contained a distinct pattern of light and dark spots suggesting that the closeness of apposition between the membrane and the substratum was irregular. They coincided with areas containing acetylcholine receptor clusters identified by fluorescence labeled alpha-bungarotoxin. Freeze-fracture of the cells confirmed these observations. The membrane in these areas was highly convoluted and contained aggregates of large P-face intramembrane particles (probably representing acetylcholine receptors). If cells were fixed and then treated with the sterol-specific antibiotic filipin before fracturing, the pattern of filipin-sterol complex distribution closely followed the pattern of cell-substratum contact. Filipin-sterol complexes were in low density in the regions where the membrane contained clustered intramembrane particles. These membrane regions were away from the substratum (bright white areas in reflection interference contrast; depressions of the P-face in freeze-fracture). Filipin-sterol complexes were also in reduced density where the membrane was very close to the substratum (dark areas in reflection interference contrast; bulges of the P-face in freeze-fracture). These areas were not associated with clustered acetylcholine receptors (aggregated particles). This result suggests that filipin treatment causes little or no artefact in either acetylcholine receptor distribution or membrane topography of fixed cells and that the distribution of filipin-sterol complexes may closely parallel the microheterogeneity of membranes that exist in living cells.

Concentration of pp125 focal adhesion kinase (FAK) at the myotendinous junction

1994 ◽  
Vol 107 (6) ◽  
pp. 1485-1497
Author(s):  
L.P. Baker ◽  
D.F. Daggett ◽  
H.B. Peng
Keyword(s):  
Neuromuscular Junction ◽  
Focal Adhesion Kinase ◽  
Acetylcholine Receptor ◽  
Focal Adhesion ◽  
Cultured Cells ◽  
Muscle Cells ◽  
Relative Molecular Mass ◽  
Myotendinous Junction ◽  
Receptor Clusters

Focal adhesion kinase is a recently characterized tyrosine kinase that is concentrated at focal contacts in cultured cells. It is thought to play an important role in the regulation of the integrin-based signal transduction mechanism involved in the assembly of this membrane specialization. In this study, we examined the immunocytochemical distribution of focal adhesion kinase in Xenopus skeletal muscle and its role in the formation of two sarcolemmal specializations, the myotendinous junction and the neuromuscular junction, using a monoclonal antibody (2A7) against this protein. Immunoprecipitation of Xenopus embryonic tissues with this antibody demonstrated a single band at a relative molecular mass of 116 kDa. A distinct concentration of immunolabeling for focal adhesion kinase was observed at the myotendinous junction of muscle fibers in vivo. At this site, the labeling for this protein is correlated with an accumulation of phosphotyrosine immunolabeling. Focal adhesion kinase was not concentrated at the neuromuscular junction in muscle cells either in vivo or in vitro. However, it was localized at spontaneously formed acetylcholine receptor clusters in cultured Xenopus myotomal muscle cells, although its distribution was not exactly congruent with that of the receptors. In these cells, the accumulation focal adhesion kinase was induced by polystyrene microbeads. In addition, beads also induce the formation of acetylcholine receptor clusters and myotendinous junction-like specializations. By following the appearance of the focal adhesion kinase relative to the formation of these sarcolemmal specializations at bead-muscle contacts in cultured muscle cells, we conclude that the accumulation of this protein was in pace with the development of the myotendinous junction, but occurred well after the clustering of acetylcholine receptors. These results suggest that focal adhesion kinase may be involved in the development and/or maintenance of the myotendinous junction through an integrin-based signaling system. Although it can accumulate at acetylcholine receptor clusters formed in culture, it does not appear to be involved in the development of the neuromuscular junction.

scholarly journals Association of the postsynaptic 43K protein with newly formed acetylcholine receptor clusters in cultured muscle cells.

1985 ◽  
Vol 100 (5) ◽  
pp. 1698-1705 ◽  
Author(s):  
H B Peng ◽  
S C Froehner
Keyword(s):  
Acetylcholine Receptor ◽  
Binding Sites ◽  
Electric Organ ◽  
Muscle Cells ◽  
Postsynaptic Membrane ◽  
Latex Beads ◽  
Peripheral Membrane Protein ◽  
Double Label ◽  
Torpedo Electric Organ ◽  
Receptor Clusters

The postsynaptic membrane from Torpedo electric organ contains, in addition to the acetylcholine receptor (AChR), a major peripheral membrane protein of approximately 43,000 mol wt (43K protein). Previous studies have shown that this protein is closely associated with AChR and may be involved in anchoring receptors to the postsynaptic membrane. In this study, binding sites for monoclonal antibodies (mabs) to the 43K protein have been compared to the distribution of AChR in Xenopus laevis muscle cells in culture. In double label immunofluorescence experiments, clusters of AChR that occur spontaneously on these cells were stained with anti-43K mabs. Newly formed receptor clusters induced with positive polypeptide-coated latex beads were also stained with anti-43K mabs as early as 12 h after the application of the beads. Exact correspondence in the distribution of the anti-43K protein binding sites and the AChR was found in both types of clusters. These results suggest that the 43K protein becomes associated with AChR clusters during a period of active postsynaptic membrane differentiation. Thus, this protein may participate in the clustering process.

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