MOLECULAR BASIS OF SYNAPTIC VESICLE FORMATION

突触小泡形成的分子基础

• 批准号: 2774511
• 负责人: AMY W HUDSON
• 金额: $ 1.46万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2774511
• 关键词: animal genetic material tag; clathrin; complementary DNA; endocytosis; genetic library; guanosine triphosphate; laboratory rat; membrane proteins; messenger RNA; molecular cloning; nerve /myelin protein; northern blottings; nucleic acid sequence; phosphoproteins; polymerase chain reaction; protein isoforms; southern blotting; synaptic vesicles; synaptogenesis

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.

突触小泡是位于突触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 神经末梢和随之而来的钙离子内流触发突触 突触前质膜与囊泡融合与胞吐作用 发生，向突触间隙释放神经递质。之后 胞吐作用，突触囊泡膜蛋白迅速 内化并用于突触小泡的重塑 神经末梢。突触小泡重塑的分子机制 还没有被很好地理解：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，并且来自 内小体，囊泡膜发芽并掐断以改造小， 大小均匀的小泡在分泌中活跃。这份提案描述了 旨在阐明突触分子机制的实验 内小体的囊泡重塑。建议的理由是 实验是基于最近在 赞助商的实验室证明，胞质蛋白包衣的花蕾具有 动力素“环”存在于神经末梢的内小体上。 存在非水解性GTP类似物GTP-Gamma。基于这些 准备，一个体外突触囊泡萌发试验将是 它将提供一种测量突触小泡形成的方法 在各种条件下的内噬菌体。可能扮演的角色 神经元特异性蛋白Dynamin-1和p145，两个GRB-2结合蛋白， 它们会在神经末梢的作用下去磷酸化 去极化和网状蛋白相关蛋白AP-3将被检测 通过使用生化和形态分析进行了详细的分析。