Molecular Mechanisms of Rapid Synaptic Vesicle Endocytosis

快速突触小泡内吞作用的分子机制

基本信息

  • 批准号:
    9299552
  • 负责人:
  • 金额:
    $ 27.91万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2017
  • 资助国家:
    美国
  • 起止时间:
    2017-03-01 至 2019-02-28
  • 项目状态:
    已结题

项目摘要

PROJECT SUMMARY Endocytosis sustains synaptic transmission. The continuous retrieval of fused synaptic vesicle membrane remnants from the presynaptic plasma membrane via the coupled, compensatory events of endocytosis replenishes the synaptic vesicle pool for sustained neurotransmitter release. Defects in synaptic vesicle endocytosis underlie Epilepsy, Down’s syndrome, Alzheimer’s, Parkinson’s and Huntington’s diseases. The molecular mechanisms that accomplish rapid synaptic vesicle endocytosis at the pre-synaptic plasma membrane are poorly understood, as are the roles of the molecules involved in effecting rapid vesicle scission. The long- term goal of this proposal is to address such issues. Dynamin 1, endophilin A1 and synaptojanin 1 are three interacting protein partners essential for rapid synaptic vesicle endocytosis. However, very little is known about the coupling or coordination of their molecular mechanisms, either in space or in time, during this membrane remodeling event. Several unknown or unresolved fundamental issues essential for understanding the roles, mechanisms, and regulation of these molecules will be addressed by the experiments proposed in this application. These include 1) the cooperative mechanisms underlying the formation of a dynamin 1-endophilin A1 copolymer around the endocytic vesicle neck, and 2) the role of the polyphosphoinositol lipid phosphatase, synaptojanin 1, in the regulation of endophilin A1- and dynamin 1-membrane interactions during membrane fission. These experiments will test the central, paradigm-shifting hypothesis that dynamin 1 and synaptojanin 1 act as regulators and/or catalysts of endophilin A1-effected membrane fission. We will use a vast array of innovative fluorescence spectroscopic and microscopic approaches, in combination with various biochemical, biophysical and cellular assays, to address these issues. These include the use of: (i) environmentally sensitive fluorescence probes for measuring protein-membrane insertion, (ii) membrane-restricted quenchers for measuring protein membrane insertion-depth, (iii) Förster resonance energy transfer (FRET) probes for determining protein polymerization and copolymerization, (iv) stopped-flow kinetics to determine the rates of protein-membrane binding, -membrane insertion, and -self-assembly, and (v) fluorescence imaging on model Giant Unilamellar Vesicles (GUVs) to visualize membrane remodeling and fission. Successful outcomes of this research will provide (i) a fundamentally improved understanding of the cooperative molecular mechanisms underlying rapid synaptic vesicle scission, and (ii) a molecular foundation for the design of drugs and therapeutics that can beneficially modulate synaptic vesicle endocytosis under various disease states.
项目摘要 内吞作用维持突触传递。融合的突触囊泡膜的连续恢复 突触前质膜的残余物通过内吞作用的偶联、补偿事件 使突触囊泡池保持神经递质的持续释放。突触囊泡缺陷 内吞作用是癫痫、唐氏综合症、阿尔茨海默氏症、帕金森氏症和亨廷顿氏病的基础。的 在突触前质膜实现快速突触囊泡内吞作用的分子机制 人们对这些分子的作用知之甚少,同样的,这些分子参与了囊泡的快速分裂。很长的- 本提案的长期目标就是解决这些问题。发动蛋白1、内啡肽A1和突触连接蛋白1是三种 相互作用的蛋白质伴侣的快速突触囊泡内吞作用必不可少。然而,人们对 在这种膜过程中,它们的分子机制在空间或时间上的耦合或协调 重塑事件几个未知或未解决的基本问题对于理解这些角色至关重要, 机制和调节这些分子将解决的实验中提出的, 应用程序.这些包括:1)发动蛋白1-内啡肽形成的基础合作机制 A1共聚物在内吞囊泡颈部周围,和2)聚磷酸肌醇脂质磷酸酶的作用, synaptojanin 1,在调节内亲素A1和发动蛋白1-膜相互作用的膜 裂变这些实验将检验发动蛋白1和突触蛋白1 作为内亲素A1影响的膜分裂的调节剂和/或催化剂。我们将使用大量的 创新的荧光光谱和显微镜方法,结合各种生物化学, 生物物理和细胞分析,以解决这些问题。这些措施包括使用: 用于测量蛋白质-膜插入的荧光探针,(ii)用于测量蛋白质-膜插入的膜限制性猝灭剂, 测量蛋白质膜插入深度,(iii)Förster共振能量转移(FRET)探针, 确定蛋白质聚合和共聚,(iv)停流动力学,以确定 蛋白质-膜结合、-膜插入和-自组装,以及(v)模型上的荧光成像 巨大单层囊泡(GUV),用于可视化膜重塑和分裂。成功的结果 研究将提供(i)从根本上改善对合作分子机制的理解 潜在的快速突触囊泡切断,和(ii)药物和治疗设计的分子基础 其可以在各种疾病状态下有益地调节突触囊泡内吞作用。

项目成果

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Rajesh Ramachandran其他文献

Rajesh Ramachandran的其他文献

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{{ truncateString('Rajesh Ramachandran', 18)}}的其他基金

Conformational Dynamics of the Dynamin PH domain in Synaptic Vesicle Endocytosis
突触小泡胞吞作用中 Dynamin PH 结构域的构象动力学
  • 批准号:
    10057144
  • 财政年份:
    2020
  • 资助金额:
    $ 27.91万
  • 项目类别:
Molecular Mechanisms of Dynamin-related Protein 1-Mediated Mitochondrial Fission
动力相关蛋白1介导的线粒体分裂的分子机制
  • 批准号:
    10251912
  • 财政年份:
    2017
  • 资助金额:
    $ 27.91万
  • 项目类别:
Molecular Mechanisms of Dynamin-related Protein 1-Mediated Mitochondrial Fission
动力相关蛋白1介导的线粒体分裂的分子机制
  • 批准号:
    9895369
  • 财政年份:
    2017
  • 资助金额:
    $ 27.91万
  • 项目类别:
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