One hallmark of neuromuscular synapse formation is accumulation of acetylcholine receptors (AChRs). Our studies of early synapse formation reveal other cell surface remodeling changes: neuronal growth cone filopodial insertion into coated regions of the postsynaptic plasma membrane and appearance of coated pits and vesicles, some of which show alpha-bungarotoxin (alpha-BTX) and mAb35 (a monoclonal antibody which binds to the AChR alpha-subunit) binding and contain acetylcholinesterase (AChE) histochemistry. In this investigation we will study key features of postsynaptic membrane remodeling and AChR expression, particularly at early stages of development. We will determine how coated pits and vesicles contribute to postsynaptic membrane remodeling and AChR expression in early stages of synapse formation. For this purpose we have made monoclonal antibodies to vesicular membrane proteins, and will use these antibodies to determine the contribution of coated pits and vesicles to postsynaptic membrane remodeling. Because the growth cone is likely to influence specific interactions between the neuron and the postsynaptic cell, we have designed studies to investigate membrane organization and coated vesicle distribution as defined by the relationship of the growth cone to the postsynaptic cell. Recently, using in situ hybridization, we have shown that nuclei in a multinucleated myotube show striking differences in the AChR mRNA expression. To relate the nuclei that actively express AChR alpha-subunit mRNA to AChR clusters and coated vesicles, we will use in situ hybridization and immunocytochemistry to detect mRNA and proteins (AChR, vesicle and pit proteins) in the same cell. Our work will employ double label experiments with monoclonal antibodies to vesicular proteins and in situ hybridization in primary co-cultures of muscle and ciliary and/or spinal neurons (with dorsal root ganglia as a non-cholinergic control). In addition to traditional cultures we will use microcultures and laser microbeam cell surgery to determine how the motor neuron regulates the nuclear activity, AChR synthesis, and postsynaptic membrane remodeling at a single cell level. These studies will elucidate whether there are specific plasma membrane domains where AChRs cluster, which coated pits and vesicles contain AChR, which serve as sources of new AChR rich plasma membrane, which share common determinants with plasma membrane, whether these synaptic proteins are synthesized near the site of function, and how their expression is regulated.
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