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.
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