The membrane skeleton of acetylcholine receptor domains in rat myotubes contains antiparallel homodimers of beta-spectrin in filaments quantitatively resembling those of erythrocytes

1995 ◽  
Vol 108 (9) ◽  
pp. 3145-3154 ◽  
Author(s):  
D.W. Pumplin
Keyword(s):  
Monoclonal Antibody ◽  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Cytoplasmic Membrane ◽  
Membrane Surface ◽  
Membrane Skeleton ◽  
Immunogold Labeling ◽  
Platinum Coating ◽  
High Concentrations ◽  
Receptor Clusters

I used immunogold labeling and quick-freeze, deep-etch, rotary replication to characterize the membrane skeleton at regions with high concentrations of acetylcholine receptor domains in receptor clusters of cultured rat muscle cells. This membrane skeleton consists of a network of filaments closely applied to the cytoplasmic membrane surface. The filaments are specifically decorated by immunogold labeling with a monoclonal antibody, VIIF7, that recognizes an isoform of beta-spectrin colocalizing with acetylcholine receptors. The filaments are 32 +/- 11 nm in length and three to four filaments (average 3.1-3.3) join at each intersection to form the network. These parameters are nearly identical to those reported previously for the membrane skeleton of erythrocytes. Depending on the amount of platinum coating, filament diameters range from 9 to 11 nm in diameter, and are 1.4 nm larger on average than spectrin filaments of erythrocytes replicated at the same time. Filaments are decorated with gold particles close to one end, consistent with the location of the epitope recognized by the monoclonal antibody. Computer modeling shows that all filament intersections in the membrane skeletal network are equally capable of being labeled by the monoclonal antibody. This pattern of labeling is consistent with a network containing antiparallel homodimers of beta-spectrin.

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 Association of beta-cytoplasmic actin with high concentrations of acetylcholine receptor (AChR) in normal and anti-AChR-treated primary rat muscle cultures.

1984 ◽  
Vol 32 (9) ◽  
pp. 973-981 ◽  
Author(s):  
B W Lubit
Keyword(s):  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Morphological Changes ◽  
Muscle Cells ◽  
High Concentration ◽  
Rat Muscle ◽  
Cytoplasmic Actin ◽  
High Concentrations

Previous immunocytochemical studies in which an antibody specific for mammalian cytoplasmic actin was used showed that a high concentration of cytoplasmic actin exists at neuromuscular junctions of rat muscle fibers such that the distribution of actin corresponded exactly to that of the acetylcholine receptors. Although clusters of acetylcholine receptors also are present in noninnervated rat and chick muscle cells grown in vitro, neither the mechanism for the formation and maintenance of these clusters nor the relationship of these clusters to the high density of acetylcholine receptors at the neuromuscular junction in vivo are known. In the present study, a relationship between beta-cytoplasmic actin and acetylcholine receptors in vitro has been demonstrated immunocytochemically using an antibody specific for the beta-form of cytoplasmic actin. Networks of cytoplasmic actin-containing filaments were found in discrete regions of the myotube membrane that also contained high concentrations of acetylcholine receptors; such high concentrations of acetylcholine receptors have been described in regions of membrane-substrate contact. Moreover, when primary rat myotubes were exposed to human myasthenic serum, gross morphological changes, accompanied by an apparent rearrangement of the cytoplasmic actin-containing cytoskeleton, were produced. Although whether the distribution of cytoplasmic actin-containing structures was influenced by the organization of acetylcholine receptor or vice versa cannot be determined from these studies, these findings suggest that in primary rat muscle cells grown in vitro, acetylcholine receptors and beta-cytoplasmic actin-containing structures may be somehow connected.

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 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.

scholarly journals Actin at receptor-rich domains of isolated acetylcholine receptor clusters.

1986 ◽  
Vol 102 (4) ◽  
pp. 1447-1458 ◽  
Author(s):  
R J Bloch
Keyword(s):  
Skeletal Muscle ◽  
Monoclonal Antibody ◽  
Acetylcholine Receptor ◽  
Immunofluorescent Staining ◽  
Muscle Membrane ◽  
Rat Skeletal Muscle ◽  
Intact Cells ◽  
Adult Rat ◽  
Receptor Clusters ◽  
Low Ionic Strength

Acetylcholine receptor (AChR) clusters of cultured rat myotubes, isolated by extraction with saponin (Bloch, R. J., 1984, J. Cell Biol. 99:984-993), contain a polypeptide that co-electrophoreses with purified muscle actins. A monoclonal antibody against actin reacts in immunoblots with this polypeptide and with purified actins. In indirect immunofluorescence, the antibody stains isolated AChR clusters only at AChR domains, strips of membrane within clusters that are rich in receptor. It also stains the postsynaptic region of the neuromuscular junction of adult rat skeletal muscle. Semiquantitative immunofluorescence analyses show that labeling by antiactin of isolated analyses show that labeling by antiactin of isolated AChR clusters is specific and saturable and that it varies linearly with the amount of AChR in the cluster. Filaments of purified gizzard myosin also bind preferentially at AChR-rich regions, and this binding is inhibited by MgATP. These experiments suggest that actin is associated with AChR-rich regions of receptor clusters. Depletion of actin by extraction of isolated clusters at low ionic strength selectively releases the actin-like polypeptide from the preparation. Simultaneously, AChRs redistribute within the plane of the membrane of the isolated clusters. Similarly, brief digestion with chymotrypsin reduces immunofluorescence staining and causes AChR redistribution. Treatments that deplete AChR from clusters in intact cells also reduce immunofluorescent staining for actin in isolated muscle membrane fragments. Upon reversal of these treatments, cluster reformation occurs in regions of the membrane that also stain for actin. I conclude that actin is associated with AChR domains and that changes in this association are accompanied by changes in the organization of isolated AChR clusters.

scholarly journals Acetylcholine receptor clusters of rat myotubes have at least three domains with distinctive cytoskeletal and membranous components.

1989 ◽  
Vol 109 (2) ◽  
pp. 739-753 ◽  
Author(s):  
D W Pumplin
Keyword(s):  
Acetylcholine Receptors ◽  
Membrane Domains ◽  
Coated Pits ◽  
Microfilament Bundles ◽  
Thin Sectioning ◽  
High Concentrations ◽  
Receptor Clusters ◽  
Relationship Of ◽  
The Relationship ◽  
Coated Membrane

Cultured rat myotubes develop high concentrations of acetylcholine receptors (AChR) in specialized areas of attachment to their substrate. We examined the ultrastructure of identified AChR clusters by quick-freeze, deep-etch, rotary replication or by thin sectioning of whole myotubes fixed in the presence of saponin and tannic acid to preserve the cytoskeleton. Our findings show that AChR clusters are composed of at least three distinct domains, differing in their cytoskeletal, intramembrane, and external components. At contact domains, the myotube's ventral membrane lacked AChR and lay within 10-15 nm of the substrate; electron-dense strands connected the two. The overlying cytoplasm contained bundles of parallel microfilaments passing above and through an irregular network of globular material, resembling the relationship of microfilament bundles to focal contacts already described in fibroblasts. Coated-membrane domains lay between the microfilament bundles and were overlain by cytoplasmic plaques of a regular network of polygons having associated coated pits. These plaques closely resembled the network of polymerized clathrin described in fibroblasts and macrophages. Coated membrane also lacked AChR and adhered to the substrate by electron-dense strands, but did not anchor microfilament bundles. The cytoplasm overlying AChR domains contained a complex network composed of at least two layers. The layer closest to the membrane consisted of protrusions from the cytoplasmic surface, some connected by fine filaments less than 5 nm in diameter. An overlying layer contained larger diameter filaments, some forming an anastomotic network reminiscent of the cortical cytoskeleton of erythrocytes. Longer filaments inserting into this network appeared identical to members of nearby microfilament bundles. The morphology of AChR domains supports the idea that AChR are immobilized by a network containing actin and spectrin.

scholarly journals The distribution of acetylcholine receptor clusters and sites of transmitter release along chick ciliary ganglion neurite-myotube contacts in culture.

1987 ◽  
Vol 104 (2) ◽  
pp. 371-379 ◽  
Author(s):  
L W Role ◽  
D G Roufa ◽  
G D Fischbach
Keyword(s):  
Acetylcholine Receptor ◽  
Growth Cone ◽  
Acetylcholine Receptors ◽  
Lucifer Yellow ◽  
Extracellular Recording ◽  
Ciliary Ganglion ◽  
Nerve Muscle ◽  
Receptor Clusters ◽  
Ganglion Neurons

Acetylcholine receptors accumulate along the length of cholinergic neuron-skeletal muscle contacts in vitro. The main purpose of this study was to describe, in a quantitative way, the distribution of acetylcholine receptor clusters induced by ciliary ganglion neurons over a period of time extending from hours to weeks after contacts are established. Neurites were filled with Lucifer Yellow and receptor clusters were identified with rhodamine-bungarotoxin. A cluster located within 5 micron of a nerve process or 10 micron of the base of a growth cone was considered to be a neurite-associated receptor patch (NARP). The first synaptic potentials were evoked 20 min after growth cone-myotube contact, and, after 24 h of co-culture, greater than 60% of the nerve-muscle pairs tested were functionally connected. NARPs appear rapidly; the first clusters were detected approximately 6 h after the neurons were plated. They were composed of several small subclusters or speckles of rhodamine-bungarotoxin fluorescence. The initial accumulation of receptors may occur at the advancing tips of nerve processes because NARPs were found at greater than 80% of the growth cone-muscle contacts examined between 12 and 24 h of co-culture. Over the 3-wk period examined, the mean incidence of NARPs ranged between 1.0 and 2.6 per 100 micron of neurite-myotube contact, with the peak observed on the second day of co-culture. During the first 3 d in culture, when the neurons were multipolar, nearly all of the primary processes induced one or more clusters. With time, as the neurons become unipolar (Role and Fischbach, 1987) NARPs persisted along the remaining dominant process. Measurements made during the third day of co-culture suggest that NARPs disappear along shorter neurites before they retract. Synaptic currents were detected by focal extracellular recording at 55% of the NARPs. The fact that spontaneous or evoked responses were not recorded at 45% suggests that contacts with clusters exhibit two functional states. Two types of presynaptic specialization at identified NARPs observed by scanning electron microscopy appear to be correlated with the functional state.

scholarly journals Might prepatterned acetylcholine-receptor clusters on surface myotubes be a sign of neuromuscular-junction maturation failure?

2013 ◽  
Vol 4 ◽  
pp. 319-323
Author(s):  
Anna Fidziańska ◽  
Maria Jędrzejowska ◽  
Agnieszka Madej-Pilarczyk ◽  
Jacek Bojakowski
Keyword(s):  
Neuromuscular Junction ◽  
Acetylcholine Receptor ◽  
Receptor Clusters
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