RECEPTOR CLUSTERING ON DEVELOPING MUSCLE CELLS

发育中的肌肉细胞的受体聚集

• 批准号: 3395633
• 负责人: DANIEL AXELROD
• 金额: $ 11.7万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-07-01 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3395633
• 关键词: acetylcholine; chemical aggregate; cholinergic agents; computer graphics /printing; fluorescence; fluorescence spectrometry; laboratory rat; membrane activity; membrane channels; membrane proteins; membrane reconstitution /synthesis; mixed tissue /cell culture; molecular dynamics; myofibrils; myogenesis; neuromuscular function; nicotinic receptors; synapses; tissue /cell culture

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.

乙酰胆碱受体(AChR)在发育过程中的聚集 突触在胚胎发育过程中是一个突出的因素，但并不是很好。 在分子水平上被理解。本项目以小说的形式进行考察 荧光技术，AChR如何聚集并保持 在活体培养的大鼠肌肉细胞(肌管)和 在重组模型膜中 具体地说，我们调查几个相互关联的问题： (A)肌管上AChR自发的分子机制是什么？ 由从基础上派生的因素聚集或诱导聚集 椎板和神经？ (B)AChR在脂质双层中自我聚集的程度 环境，其中没有其他细胞的参与 蛋白质？ (C)乙酰胆碱受体聚集如何影响结合动力学 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，在什么情况下 在成年神经肌肉接头处将突触乙酰胆碱受体结合在一起， 关于涉及突触发育的遗传缺陷的性质， 突触上的AChR簇如何在某些情况下受到挑战 神经肌肉疾病及其对新功能的改造 运动系统受损后的突触。 大多数实验都是通过新的荧光来完成的 允许相对快速和灵敏地检测到 AchR聚集(由α-荧光衍生物标记) 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AChR聚集特别敏感 然后抛锚。扫描荧光相关光谱 直接测量AChR微团聚体的平均大小。 全内反射/荧光光漂白回收， 允许测量激动剂(如 作为荧光乙酰胆碱类似物)与AChR在其天然 膜，即使在许多未结合的激动剂的存在下也是如此。