Components of Torpedo electric organ and muscle that cause aggregation of acetylcholine receptors on cultured muscle cells.
Components of Torpedo electric organ and muscle that cause aggregation of acetylcholine receptors on cultured muscle cells.
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DOI：
10.1083/jcb.99.2.615
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发表时间：
1984-08
期刊：
影响因子：
--
通讯作者：
McMahan UJ
中科院分区：
文献类型：
--
作者：
Godfrey EW;Nitkin RM;Wallace BG;Rubin LL;McMahan UJ
肌纤维基底层鞘的突触部分与分子紧密结合，导致乙酰胆碱受体 (AChR) 在再生肌纤维上聚集。由于基底层和其他细胞外基质成分不溶于等渗盐水和去污剂溶液，因此接受胆碱能输入的组织的不溶性去污剂提取部分可以为详细表征提供丰富的 AChR 聚集分子来源。在这里，我们证明来自鱼雷电器官（一种具有高浓度胆碱能突触的组织）的这种不溶性部分会导致培养的小鸡肌肉细胞上的 AChR 聚集。我们对不溶性部分进行了部分表征，检查了肌肉细胞对其的反应，并设计了提取活性成分的方法，以期纯化它们并了解它们是否与神经肌肉接头处基底层中的成分相似。电器官的不溶部分富含细胞外基质成分；它含有类似于基底层鞘的结构，并具有高密度的胶原纤维。它导致培养的肌管上 AChR 簇的数量增加 3 至 20 倍，而不会显着影响肌管的数量或大小。在将级分添加到培养物中后 2-4 小时首次看到增加，并在 24 小时时达到最大。 AChR 聚集效应是剂量依赖性的，并且至少部分归因于应用该分数时肌肉细胞质膜中存在的 AChR 的横向迁移。活性被热和胰蛋白酶破坏。使用高离子强度或 pH 5.5 缓冲液从不溶性组分中提取活性成分。提取物增加了培养的肌管上 AChR 簇的数量，而不影响表面 AChR 的数量或降解率。针对溶解物质的抗血清阻断了其对AChR分布的影响并与活性成分结合。鱼雷肌肉和肝脏的不溶性部分不会导致培养的肌管上的 AChR 聚集。然而，在 pH 5.5 的肌肉部分提取物中检测到低水平的活性。通过针对从电器官不溶级分中提取的物质的抗血清，对肌肉提取物中的活性成分进行免疫沉淀。这种抗血清还与青蛙肌肉中的细胞外基质结合，包括肌纤维基底层鞘。因此，鱼雷电器官的不溶部分富含乙酰胆碱受体聚集分子，这些分子也存在于肌肉中，并且具有与肌纤维基底层中的抗原相似的成分。
The synaptic portion of a muscle fiber's basal lamina sheath has molecules tightly bound to it that cause aggregation of acetylcholine receptors (AChRs) on regenerating myofibers. Since basal lamina and other extracellular matrix constituents are insoluble in isotonic saline and detergent solutions, insoluble detergent-extracted fractions of tissues receiving cholinergic input may provide an enriched source of the AChR-aggregating molecules for detailed characterization. Here we demonstrate that such an insoluble fraction from Torpedo electric organ, a tissue with a high concentration of cholinergic synapses, causes AChRs on cultured chick muscle cells to aggregate. We have partially characterized the insoluble fraction, examined the response of muscle cells to it, and devised ways of extracting the active components with a view toward purifying them and learning whether they are similar to those in the basal lamina at the neuromuscular junction. The insoluble fraction from the electric organ was rich in extracellular matrix constituents; it contained structures resembling basal lamina sheaths and had a high density of collagen fibrils. It caused a 3- to 20-fold increase in the number of AChR clusters on cultured myotubes without significantly affecting the number or size of the myotubes. The increase was first seen 2-4 h after the fraction was added to cultures and it was maximal by 24 h. The AChR-aggregating effect was dose dependent and was due, at least in part, to lateral migration of AChRs present in the muscle cell plasma membrane at the time the fraction was applied. Activity was destroyed by heat and by trypsin. The active component(s) was extracted from the insoluble fraction with high ionic strength or pH 5.5 buffers. The extracts increased the number of AChR clusters on cultured myotubes without affecting the number or degradation rate of surface AChRs. Antiserum against the solubilized material blocked its effect on AChR distribution and bound to the active component. Insoluble fractions of Torpedo muscle and liver did not cause AChR aggregation on cultured myotubes. However a low level of activity was detected in pH 5.5 extracts from the muscle fraction. The active component(s) in the muscle extract was immunoprecipitated by the antiserum against the material extracted from the electric organ insoluble fraction. This antiserum also bound to extracellular matrix in frog muscles, including the myofiber basal lamina sheath. Thus the insoluble fraction of Torpedo electric organ is rich in AChR-aggregating molecules that are also found in muscle and has components antigenically similar to those in myofiber basal lamina.