RECEPTOR CLUSTERING ON DEVELOPING MUSCLE CELLS

发育期乙酰胆碱受体（AChR）的聚集 突触在胚胎发育过程中是一个突出的，但它不是很好， 在分子水平上理解。 这个项目通过小说 荧光技术，乙酰胆碱受体如何成为集群，并保持 聚集在活的培养的大鼠肌肉细胞（肌管）中， 在重构的模型膜中 具体来说，我们调查几个相互关联的问题： (a)肌管上的乙酰胆碱受体是通过什么样的分子机制自发产生的 由来自基部的因子聚集或诱导聚集 椎板和神经 (b)AChR在脂双层中自聚集到什么程度 环境，其中没有其他细胞的参与 蛋白质？ (c)乙酰胆碱受体的聚集如何影响 乙酰胆碱？ 这些问题的答案最终关系到 神经肌肉连接在胎儿发育过程中形成，在什么 在成年神经肌肉接头处将突触AChR保持在一起， 关于涉及突触发育的遗传缺陷的本质， 关于突触上的AChR簇如何在某些 神经肌肉疾病，以及新的功能重建 运动系统损伤后的突触。 大多数实验是通过新颖的荧光 这些技术允许相对快速和灵敏地检测 AChR聚集（如由α-乙酰胆碱受体的荧光衍生物标记的） 银环蛇毒素）及其对活细胞的影响。 极化 荧光光漂白恢复测量旋转扩散 它可能对乙酰胆碱受体的聚集特别敏感 和锚定。 扫描荧光相关光谱 直接测量AChR微聚集体的平均尺寸。 全内反射/荧光光漂白恢复， 允许测量激动剂（例如 作为荧光乙酰胆碱类似物）与其天然的乙酰胆碱受体 膜，即使在存在大量未结合的激动剂的情况下。

The clustering of acetylcholine receptors (AChR) at developing synapses is a prominent during embryogenesis, yet it is not well understood at a molecular level. This project examines, by novel fluorescence techniques, how AChR become clustered and remain clustered in both living cultured rat muscle cells (myotubes) and in reconstituted model membranes Specifically, we investigate several interrelated questions: (a) By what molecular mechanisms are AChR on myotubes spontaneously clustered or induced to cluster by factors derived from basal lamina and nerve? (b) To what extent do AChR self-aggregate in a lipid bilayer environment, where there is no involvement of other cellular proteins? (c) How does AChR clustering affect the binding kinetics of acetylcholine? The answers to these questions have an ultimate bearing on how nerve-muscle connections form during fetal development, on what holds the synaptic AChR together at adult neuromuscular junctions, on the nature of genetic defects involving synaptic development, on how AChR clusters at synapses are challenged in certain neuromuscular diseases, and on reformation of new functional synapses after injury to the motor system. Most of the experiments are performed by novel fluorescence techniques which allow relatively rapid and sensitive detection of AChR aggregation (as marked by a fluorescent derivative of alpha- bungarotoxin) and its consequences on living cells. Polarized fluorescence photobleaching recovery measures rotational diffusion which is likely to be particularly sensitive to AChR aggregation and anchoring. Scanning fluorescence correlation spectroscopy directly measures the average size of microaggregations of AChR. Total internal reflection/fluorescence photobleaching recovery, allows the measurement of binding/unbinding rates of agonists (such as fluorescent acetylcholine analogs) to AChR in its natural membrane, even in the presence of much unbound agonist.