Localization of cholinesterase at sites of high acetylcholine receptor density on embryonic amphibian muscle cells cultured without nerve

Moody-Corbett, F; Cohen, Maxime C.

doi:10.1523/jneurosci.01-06-00596.1981

Cited by 57 publications

(16 citation statements)

References 13 publications

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“…The 43-kDa protein shares the property of many postsynaptic components in that it is concentrated at sites of high AcChoR density in muscle cells cultured in the absence of neurons (20)(21)(22). The information required for assembling much of the postsynaptic structure is apparently intrinsic to the muscle cell; motor neurons, however, can trigger this process to occur at synaptic sites.…”

Section: Discussionmentioning

confidence: 99%

The subsynaptic 43-kDa protein is concentrated at developing nerve-muscle synapses in vitro.

Burden¹

1985

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

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Acetylcholine receptor (AcChoR)-rich membranes from the electric organ of electric fish are enriched in the four polypeptides of the AcChoR (subunit composition a2f3y8) (1, 2) and in nonreceptor polypeptides of 43 kDa and 270 kDa (3-6). The 43-kDa polypeptide is a peripheral membrane protein located on the cytoplasmic face of the postsynaptic membrane (7-9). Immunoelectron microscopy (9) and crosslinking studies (6) have shown that the 43-kDa protein is closely associated with the AcChoR and have raised the possibility that the 43-kDa protein can interact directly with the cytoplasmic domain(s) of the AcChoR. Antibodies against the 43-kDa protein from Torpedo electric organ react with the innervated face of the electrocyte and with synaptic sites in frozen sections of both amphibian and mammalian skeletal muscle (10, 11). Although there is no direct evidence that the crossreacting protein at neuromuscular synapses has a molecular mass of 43 kDa, the crossreacting protein is highly concentrated at synaptic sites and located on the cytoplasmic face of the postsynaptic membrane (10, 11); therefore, for convenience, this crossreacting protein will be referred to as the 43-kDa protein.The close spatial relationship of the 43-kDa protein and synaptic AcChoRs raises the possibility that the 43-kDa protein may be involved in the formation and/or maintenance of high-density clusters of AcChoRs. If the 43-kDa protein is required for AcChoRs to cluster at synapses, it should be present at developing synapses at a time when AcChoRs are being clustered. This study demonstrates that the 43-kDa protein is concentrated at synaptic sites in innervated Xenopus muscle cells in cell culture as early as synaptic AcChoR clusters are detected. Moreover, the 43-kDa protein is concentrated at AcChoR clusters that occur on noninnervated muscle cells in culture. These observations are consistent with a role for the 43-kDa protein in the formation and/or stabilization of AcChoR clusters.MATERIALS AND METHODS AcChoR-rich membranes were isolated from the electric organ of Torpedo californica, and peripheral membrane proteins were extracted at alkaline pH (6, 12). BALB/c mice were immunized with the solubilized proteins (50 pg), and their spleen cells were fused with NS-1 myeloma cells as described (6,13). Hybridoma supernatants were screened by ELISA on the immunogen, and supernatants from positive wells were further screened by immunoblotting of proteins in AcChoR-rich membranes resolved by NaDodSO4/PAGE (6,14). Hybridomas from wells whose supernatant reacted with the 43-kDa protein were cloned, and the antibodies were further assayed on immunoblots of two-dimensional gels of peripheral membrane proteins. In addition, these antibodies were assayed by indirect immunofluorescence on frozen sections of amphibian skeletal muscle (13).Myotomal muscle cells from stage 22-24 Xenopus embryos were isolated by mechanical dissociation (in a calcium-and magnesium-free solution containing 1 mM EDTA) after separating the tissue layers with the aid of col...

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Section: Discussionmentioning

confidence: 99%

The subsynaptic 43-kDa protein is concentrated at developing nerve-muscle synapses in vitro.

Burden¹

1985

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

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“…Such a mechanism has also been implicated in a study of ectopic reinnervation of adult denervated muscle in vivo (L0mo & Slater, 1980) and is supported by cross-innervation experiments which indicate that the nerve regulates the distribution of synaptic contacts along embryonic chick muscle fibres (Bennett & Pettigrew, 1974b). On the other hand, ACh receptor patches can form on cultured muscle cells in the absence of innervation or previous contact by nerve (Bekoff & Betz, 1976;Moody-Corbett & Cohen, 1981) and the site of their formation can be determined by non-neuronal factors (Peng, Cheng & Luther, 1981). Widespread ACh receptor patches also form on denervated muscle in vivo (Ko, Anderson & Cohen, 1977;Harris, 1981c;Steinbach, 1981 b), as well as on paralysed fetal muscle in vivo (Harris, 1981c).…”

mentioning

confidence: 86%

“…However, on the basis of our findings it would appear that the presence of nerve fibres is not obligatory for the expression of ACh receptor on developing muscle cells in vivo. Studies on appropriate cell cultures have likewise indicated that expression of ACh receptors and other aspects of muscle differentiation can occur in the absence of any previous contact by nerve fibres (Bekoff & Betz, 1976;Moody-Corbett & Cohen, 1981). It should be noted, however, that presumptive and developing muscle cells and spinal cord are located close to each other in young embryos.…”

Section: Acquisition Of Ach Receptorsmentioning

confidence: 99%

Developmental changes in the distribution of acetylcholine receptors in the myotomes of Xenopus laevis.

Chow¹,

Cohen²

1983

The Journal of Physiology

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SUMMARY1. The acquisition and distribution of nerve fibres and of acetylcholine (ACh) receptors were examined in the myotomes of Xenopus laevis during normal development. This muscle is well-suited for investigating temporal relationships during neuromuscular synaptogenesis because the age of the Xenopus embryo at the onset of innervation can be assessed with an accuracy of about one hour. Myotomal nerve fibres were visualized after staining them with nitroblue tetrazolium and ACh receptors were examined after exposure to x-bungarotoxin labelled with 1251 or fluorescent dye.2. Nerve fibres were seen in the myotomes of some embryos as early as stage 19(20'75 hr) and in virtually all embryos by stage 24 (26-25 hr). From the outset they were located mainly at the ends of the myotomes, but some myotomes also exhibited nerve fibres in more central regions.3. ACh receptors were already present in myotomes by stage 19 (20-75 hr) and initially had a widespread, uniform distribution. The density of extrajunctional ACh receptors increased until stage 36 (50 hr) and then declined less than 3-fold over the next 10 days of development.4. Discrete patches of high ACh receptor density began to appear at the ends of the myotomes at stage 22 (24 hr) and were seen in almost all embryos by stage 26 (29-5 hr). ACh receptor patches were also seen in central regions of some myotomes and these were usually aligned in patterns which resembled the course of nerve fibres.5. The present findings suggest that myotomal muscle cells in Xenopus embryos begin to acquire ACh receptors shortly before the arrival of nerve fibres and that discrete patches of ACh receptors begin to form at presumptive synaptic sites on the average about 3 hr after the arrival of the nerve fibres. The latter delay is considerably shorter than that in developing rat muscle.6. The temporal and spatial relationships between nerve fibres and the development of ACh receptor patches in Xenopus myotomes in vivo are consistent with findings in Xenopus cell cultures which indicate that nerve fibres can rapidly induce ACh receptor localization at sites of nerve-muscle contact.

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“…stances codistributing with clusters of AChR include basal lamina antigens [3,41], acetyl cholinesterase [33], actin [25], vinculin [4], and laminin [10], The present study demon strates additional differences in carbohydrate content between extraneous coats associated with a high density of immobile receptors and the general cell surface which contains lowdensity, mobile receptors.…”

mentioning

confidence: 83%

Distribution of Cell Surface Saccharides and Fibronectin on Cultured Chick Myotubes: Relationship to Acetylcholine Receptor Clusters

Burrage

Lentz

1982

Dev Neurosci

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This study examines the distribution of glycoconjugates on the surface of cultured chick myotubes with a battery of lectins labeled with ferritin or horseradish peroxidase. In addition, the distribution of a specific glycoprotein, fibronectin, is investigated by immunocytochemistry. Particular attention is paid to the localization of these substances in specialized patches on the cell surface previously shown to contain a high density of acetylcholine receptors as demonstrated with horseradish peroxidase labeled α-bungarotoxin. The specialized patches are found to bind a greater amount of concanavalin A, ricin agglutinin I, and soy bean agglutinin and a lesser amount of wheat germ agglutinin than the general myotube surface. Limulus lectin is distributed over the entire cell surface while other lectins do not bind to any sites. The surface patches contain a high density of acetylcholine receptors as shown by double labeling with ferritin-labeled lectins and peroxidase labeled α-bungarotoxin. Fibronectin occurs in high concentration at the surface patches and is present over other regions of the cell surface as well. These results reveal differential patterns of distribution of glycoconjugates and fibronectin over the myotube surface. These regional differences may be related to the distribution of acetylcholine receptors or to recognition and attachment by the innervating nerve.

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Localization of cholinesterase at sites of high acetylcholine receptor density on embryonic amphibian muscle cells cultured without nerve

Cited by 57 publications

References 13 publications

The subsynaptic 43-kDa protein is concentrated at developing nerve-muscle synapses in vitro.

The subsynaptic 43-kDa protein is concentrated at developing nerve-muscle synapses in vitro.

Developmental changes in the distribution of acetylcholine receptors in the myotomes of Xenopus laevis.

Distribution of Cell Surface Saccharides and Fibronectin on Cultured Chick Myotubes: Relationship to Acetylcholine Receptor Clusters

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