Shadiack A M, Nitkin R M
Department of Biological Sciences, Rutgers University, Newark, New Jersey 07102.
J Neurobiol. 1991 Sep;22(6):617-28. doi: 10.1002/neu.480220607.
Agrin induces discrete high-density patches of acetylcholine receptors (AChRs) and other synaptic components on cultured myotubes in a manner that resembles synaptic differentiation. Furthermore, agrin-like molecules are present at developing neuromuscular junctions in vivo. This provides us with a unique opportunity to manipulate AChR patching in order to examine the role of cytoskeletal components. Cultured chick myotubes were fixed and labeled to visualize the distributions of actin, alpha-actinin, filamin, tropomyosin, and vinculin. Overnight exposure to agrin caused a small amount of alpha-actinin, filamin, and vinculin to reorganize into discrete clusters. Double-labeling studies revealed that 78% of the AChR clusters were associated with detectable concentrations of filamin, 70% with alpha-actinin, and 58% with vinculin. Filamin even showed congruence to AChRs within clustered regions. By contrast, actin (visualized with fluorescein-phalloidin) and tropomyosin did not show specific associations with agrin-induced AChR clusters. The accumulation of cytoskeletal components at AChRs clusters raised the possibility that cytoskeletal rearrangements direct AChR clustering. However, a time course of agrin-induced clustering that focused on filamin revealed that most of the early AChR clusters (3-6 h) were not associated with detectable amounts of cytoskeletal material. The accumulation of cytoskeletal material at later times (12-18 h) may imply a role in maintenance and stabilization, but it appears unlikely that these cytoskeletal elements initiate AChR clustering on myotubes.
聚集蛋白以类似于突触分化的方式，在培养的肌管上诱导乙酰胆碱受体（AChR）及其他突触成分形成离散的高密度斑块。此外，聚集蛋白样分子在体内发育中的神经肌肉接头处也有存在。这为我们提供了一个独特的机会来操控AChR斑块形成，以研究细胞骨架成分的作用。将培养的鸡肌管固定并标记，以观察肌动蛋白、α-辅肌动蛋白、细丝蛋白、原肌球蛋白和纽蛋白的分布。过夜暴露于聚集蛋白会使少量的α-辅肌动蛋白、细丝蛋白和纽蛋白重新组织成离散的簇。双重标记研究显示，78%的AChR簇与可检测浓度的细丝蛋白相关，70%与α-辅肌动蛋白相关，58%与纽蛋白相关。在聚集区域内，细丝蛋白甚至与AChRs显示出一致性。相比之下，肌动蛋白（用荧光素-鬼笔环肽可视化）和原肌球蛋白并未显示出与聚集蛋白诱导的AChR簇有特异性关联。细胞骨架成分在AChRs簇处的积累增加了细胞骨架重排指导AChR簇集的可能性。然而，一项聚焦于细丝蛋白的聚集蛋白诱导簇集的时间进程研究表明，大多数早期AChR簇（3 - 6小时）与可检测量的细胞骨架物质无关。细胞骨架物质在后期（12 - 18小时）的积累可能意味着其在维持和稳定方面的作用，但这些细胞骨架元件似乎不太可能启动肌管上的AChR簇集。
