Clusters of intramembranous particles on cultured myotubes at sites that are highly sensitive to acetylcholine.

Yee, Amy S.; Fischbach, G D; Karnovsky, Morris J.

doi:10.1073/pnas.75.6.3004

Cited by 45 publications

(30 citation statements)

References 28 publications

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“…Freeze-fracture studies have shown that a single cluster is made up of numerous small aggregates of intramembrane particles representing AcChoRs (5)(6)(7). In response to innervation, a redistribution of the AcChoRs occurs that involves the disruption of preexisting clusters and the appearance of new clusters at the sites of neuromuscular contacts (8,9).…”

mentioning

confidence: 99%

Membrane lipid heterogeneity associated with acetylcholine receptor particle aggregates in Xenopus embryonic muscle cells.

Bridgman¹,

Nakajima²

1981

Proc. Natl. Acad. Sci. U.S.A.

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Filipin, digitonin, and saponin react with membrane cholesterol to produce unique membrane alterations (sterolspecific complexes) that are easily discernible in freeze-fracture replicas. We have treated both noninnervated and innervated Xenopus embryonic muscle cells in culture with these agents. Freeze-fracture of these treated muscle cells showed that most areas of the muscle plasma membrane contain sterol-specific complexes (19-to 40-nm protuberances and dimples with filipin, a scalloped appearance with digitonin, or an irregular, rough appearance with saponin). However, these complexes were virtually absent from membrane areas of junctional and nonjunctional aggregates of acetylcholine receptor particles. This result suggests that the membrane matrix of these aggregates is low in cholesterol and that this membrane lipid heterogeneity may be linked to the mechanisms involved in their formation and stabilization on muscle cells in culture.Embryonic skeletal muscle cells grown in culture show discrete areas of high acetylcholine receptor (AcChoR) density (1-4), which are commonly referred to as nonjunctional AcChoR clusters or hot spots. Freeze-fracture studies have shown that a single cluster is made up of numerous small aggregates of intramembrane particles representing AcChoRs (5-7). In response to innervation, a redistribution of the AcChoRs occurs that involves the disruption of preexisting clusters and the appearance of new clusters at the sites of neuromuscular contacts (8,9). The causes of the formation and stabilization of these clusters are not known, although there is some circumstantial evidence that cytoskeletal elements may be involved (4, 10-13).There have been a number of studies of the role of cholesterol in membrane fluidity and the interaction between cholesterol and membrane proteins (14-17). The possibility that differences in the lipid composition and fluidity of the plasma membrane may play a role in the formation and maintenance of AcChoR aggregates and the subsequent formation of AcChoR clusters has not been investigated. We have taken advantage of the recent development of cytochemical agents that can be used to specifically detect the presence of cholesterol in freezefractured membranes (18)(19)(20) to test whether heterogeneity of membrane lipid distribution exists. We have used three such agents-the polyene antibiotic filipin, the glycosylated sterol digitonin, and saponin. All three bind specifically to cholesterol in cell membranes (16,(21)(22)(23)(24)(25) and produce distinctive membrane structural alterations, "sterol-specific complexes," in freeze-fracture replicas (18)(19)(20) (Calbiochem). Fixation in the presence of these agents was carried out at room temperature for 15-30 min. Controls were fixed in fixative without the sterol-specific agents, with and without 1.0% dimethyl sulfoxide, for 15-30 min at room temperature and subsequently handled identically to the treated cultures. Cultures were washed several times with buffer and then prepared for freeze-fracture ...

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mentioning

confidence: 99%

Membrane lipid heterogeneity associated with acetylcholine receptor particle aggregates in Xenopus embryonic muscle cells.

Bridgman¹,

Nakajima²

1981

Proc. Natl. Acad. Sci. U.S.A.

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“…Finally, the particle aggregates closely resemble the aggregates of intramembrane particles seen in freeze-fracture replicas of Xenopus AChR clusters [Peng et al, 1980;Bridgman and Nakajima, 19831. A similar organization of intramembrane particles is evident at AChR clusters in chick myotubes [Yee et al, 1978;Cohen and Pumplin, 19791. On the other hand, our results do not indicate that all of the particles within these receptor aggregates represent AChR.…”

Section: Discussionmentioning

confidence: 76%

“…Scale bars: (A) 2 pm [applies also to (B-D)]; (E) 0.25 pm; (F) 0.5 pm; (G) 0.1 pm; (H) 0.05 pm. studies [Yee et al, 1978;Cohen and Pumplin, 19791. In contrast, AChR in the postsynaptic membrane of electrocytes form a continuous plaque [Heuser and Salpeter, 1979;Bridgman et al, 19871, whereas AChR clusters in cultured rat myotubes consist of individual receptors, separated from each other in the sarcolemma [Pumplin and Bloch, 1983;Pumplin and Strong, 19891.…”

Section: Discussionmentioning

confidence: 99%

Clustered acetylcholine receptors have two levels of organization in Xenopus muscle cells

Luther

Samuelsson

Bloch

et al. 1994

Cell Motil. Cytoskeleton

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We studied the organization of acetylcholine receptor (AChR) clusters by shearing cultured Xenopus muscle cells with a stream of buffer, and preparing rotary replicas of the exposed cytoplasmic surface of the sarcolemma. AChR clusters contained numerous particles that protruded from the sarcolemma and formed an irregular array composed of discrete aggregates. AChR were located within these particle aggregates, as shown by comparison of the replicas to labeling by fluorescent alpha-bungarotoxin, and by immunogold cytochemistry with antibodies specific for the receptor. The aggregates were cross-linked by a dense network of 7 nm filaments that replicated with the banded pattern characteristic of actin microfilaments. The organization of receptors into the small aggregates was independent of the organization of these aggregates into clusters, as alkaline extraction removed the microfilament network and disrupted the irregular array of particle aggregates, but did not disperse individual receptors from the aggregates. We conclude that two levels of interactions organize AChR clusters in Xenopus muscle cells: short-range interactions that assemble individual AChR into small aggregates, and long-range interactions, perhaps mediated by actin microfilaments, that anchor the aggregates into larger clusters.

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“…The numerous intramembrane particle aggregates (arrowheads) seen at the presynaptic P face (P-F) represent gap junctions. The distal part of the lateral dendrite w a s excised from 50-pm brain stem slices after fixation and then investigated with the double replica freeze-fracture method (Yee et al, 1978). synapses, we conducted freeze-fracture, thin section and immunocytochemical electron microscopic studies of the LMCE synapses and the SVB synapses on the distal part of the lateral dendrite.…”

Section: Introductionmentioning

confidence: 99%

Synapses on the mauthner cell of the goldfish: Thin section, freeze‐fracture, and immunocytochemical studies

Nakajima¹,

Tuttle²,

Masuko³

et al. 1987

J. Elec. Microsc. Tech.

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Large myelinated club ending and small-vesicle bouton synapses on the distal part of the lateral dendrite of the goldfish Mauthner cell were investigated with thin section, freeze-fracture, and immunocytochemical electron microscopic methods. Large myelinated club endings form mixed synapses, having both gap junctions and chemical synaptic junctions. The correlation of the number of gap junction particles (connexons) and the data from electrophysiological studies of single large myelinated club ending synapses suggest that only a small fraction of gap junction channels are open at any given time during electrical synaptic transmission. The chemical synaptic junctions at the large myelinated club ending synapse have large, round synaptic vesicles, indicating that they are excitatory. This result is in agreement with electrophysiological data demonstrating the excitatory nature of this chemical synapse. Freeze-fracture of these excitatory chemical synaptic junctions reveals the presence of the intramembrane particle aggregates in the postsynaptic E face.Small-vesicle boutons form chemical synaptic junctions with small, flat or oval synaptic vesicles. These structural data, in combination with previous electrophysiological studies, suggest that the small-vesicle bouton synapses are inhibitory. In support of this theory, the cytoplasmic side of the postsynaptic membrane of some of these synapses show positive immunocytochemical reaction to monoclonal antibodies against the rat glycine receptor. Freezefracture data reveal intramembrane particle aggregates in the postsynaptic P face of some small-vesicle bouton synapses which could possibly represent glycine receptor aggregates.

show abstract

Clusters of intramembranous particles on cultured myotubes at sites that are highly sensitive to acetylcholine.

Cited by 45 publications

References 28 publications

Membrane lipid heterogeneity associated with acetylcholine receptor particle aggregates in Xenopus embryonic muscle cells.

Membrane lipid heterogeneity associated with acetylcholine receptor particle aggregates in Xenopus embryonic muscle cells.

Clustered acetylcholine receptors have two levels of organization in Xenopus muscle cells

Synapses on the mauthner cell of the goldfish: Thin section, freeze‐fracture, and immunocytochemical studies

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