Godfrey E W, Nitkin R M, Wallace B G, Rubin L L, McMahan U J
J Cell Biol. 1984 Aug;99(2):615-27. doi: 10.1083/jcb.99.2.615.
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.
肌纤维基底层鞘的突触部分有紧密结合的分子，可导致再生肌纤维上的乙酰胆碱受体（AChRs）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，接受胆碱能输入的组织经去污剂提取后的不溶性部分可能为详细表征AChR聚集分子提供丰富来源。在此，我们证明来自电鳐电器官（一种胆碱能突触浓度高的组织）的这种不溶性部分可使培养的鸡肌肉细胞上的AChRs聚集。我们已对该不溶性部分进行了部分表征，检测了肌肉细胞对其的反应，并设计了提取活性成分的方法，以期对其进行纯化并了解它们是否与神经肌肉接头处基底层中的成分相似。电器官的不溶性部分富含细胞外基质成分；它含有类似基底层鞘的结构且胶原纤维密度高。它使培养的肌管上AChR簇的数量增加了3至20倍，而对肌管的数量或大小没有显著影响。添加该部分后2至4小时首次观察到增加，24小时时达到最大值。AChR聚集效应呈剂量依赖性，至少部分是由于在添加该部分时肌肉细胞质膜中存在的AChRs横向迁移所致。活性被加热和胰蛋白酶破坏。活性成分用高离子强度或pH 5.5缓冲液从不溶性部分中提取。提取物增加了培养肌管上AChR簇的数量，而不影响表面AChRs的数量或降解速率。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。电鳐肌肉和肝脏的不溶性部分不会使培养的肌管上的AChR聚集。然而，在肌肉部分的pH 5.5提取物中检测到低水平的活性。肌肉提取物中的活性成分被针对从电器官不溶性部分提取的物质的抗血清免疫沉淀。该抗血清也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，电鳐电器官的不溶性部分富含在肌肉中也能找到的AChR聚集分子，并且其成分在抗原性上与肌纤维基底层中的成分相似。
