MOLECULAR BASIS OF SYNAPTIC VESICLE FORMATION

突触囊泡是位于突触膜上的高度特化的细胞器。 含有化学神经递质的神经末梢。去极化 和随之而来的钙离子内流触发突触 囊泡与突触前质膜融合和胞吐作用 发生，释放神经递质进入突触间隙。后 胞吐作用，突触囊泡膜蛋白迅速 内化并用于突触囊泡的重组， 神经末梢突触小泡再形成的分子机制 并没有得到很好的理解：令人信服的证据表明，突触 囊泡膜蛋白存在于早期的内体中， 在核内体中，囊泡膜出芽并被夹断以改革小的， 大小一致的囊泡，分泌活跃。该提案描述了 旨在阐明突触的分子机制的实验 囊泡从内体重新形成。建议的理由 实验是基于最近的初步数据产生的， 申办方实验室证明，具有网格蛋白包被的芽 发动蛋白“项圈”存在于神经末梢的内体上， 存在不可水解的GTP类似物GTP-γ S。基于这些 制备，将进行体外突触囊泡出芽测定。 它将提供一种测量突触囊泡形成的方法， 在各种条件下的内体。的可能作用 神经元特异性蛋白dynamin-1和p145，两种Grb-2结合蛋白， 其在神经末梢的刺激下发生去磷酸化 去极化，网格蛋白相关蛋白AP-3，将被检查 通过使用生物化学和形态学分析详细地描述。

Synaptic vesicles are highly specialized organelles located at synaptic nerve terminals which contain chemical neurotransmitters. Depolarization of the nerve terminal and consequent calcium ion influx triggers synaptic vesicle fusion with the presynaptic plasma membrane and exocytosis occurs, releasing neurotransmitters into the synaptic cleft. After exocytosis, the synaptic vesicle membrane proteins are rapidly internalized and used in the reformation of synaptic vesicles at the nerve terminal. The molecular mechanism of synaptic vesicle reformation is not well understood: compelling evidence suggests that synaptic vesicle membrane proteins are present in early endosomes, and from endosomes, the vesicle membranes bud and pinch off to reform the small, uniformly-sized vesicles active in secretion. This proposal describes experiments designed to elucidate the molecular mechanism of synaptic vesicle reformation from endosomes. The rationale underlying the proposed experiments is based upon recent preliminary data generated in the sponsor's laboratory demonstrating that clathrin coated buds possessing dynamin "collars" are present on endosomes in the nerve terminal in the presence of the non-hydrolyzable GTP analogue, GTP-gammaS. Based on these preparations, an in vitro synaptic vesicle budding assay will be developed which will provide a measure of synaptic vesicle formation from endosomes under a variety of conditions. The possible roles of the neuron-specific proteins dynamin-1 and p145, two Grb-2 binding-proteins, which undergo dephosphorylation in response to nerve terminal depolarization, and of clathrin associated protein AP-3, will be examined in detail through the use of biochemical and morphological assays.