Structural Biology of Presynaptic Scaffolds

描述(申请人提供)：脊柱结构的宏观形态变化，以及与可卡因和安非他明等滥用药物相伴的突触数量表明，突触可塑性在成瘾大脑的重新连接中发挥重要作用。在分子水平上，回路的改变是通过突触活性的变化来发出的，突触活性的变化关键依赖于内吞作用的细胞过程，因为它直接参与表面受体的清除和神经转运体囊泡/转运体的循环。这项探索性研究的目标是通过打开细胞膜和细胞质之间的动态界面来进行结构探索，从而能够从机械上理解基本的突触过程，如内吞作用。作为起点，我们的工作将集中在bar-和F-bar结构域蛋白上，这些蛋白具有诱导和稳定膜曲率的能力，使它们处于膜重塑、囊泡分裂和细胞骨架重排之间的战略交叉点。利用纯化的bar-/F-bar结构域及其相应的全长蛋白质(内亲素、两体素、Syndapin 1、FBP17和CIP4)，我们寻求获得用于电子冷冻显微镜结构测定的样品，其中这些蛋白质单独或与下游相互作用伙伴如裂变GTP酶Dynamin、细胞骨架调节因子CDC42和神经性Wiskott-Aldrich综合征蛋白(N-WASP)结合在一起。接触可卡因和安非他明等滥用药物会导致大脑某些部分发生持久的形态变化，表明突触可塑性在成瘾生物学中发挥着重要作用。在分子水平上，突触重塑的线索源于突触活动的变化，而在分子水平上，突触活动严重依赖于细胞内吞作用的过程。为了促进我们对成瘾生物学基础的复杂过程的基本理解，本研究最终旨在从分子水平揭示对内吞过程至关重要的蛋白质复合体促进突触可塑性的机制。

DESCRIPTION (provided by applicant): The macroscopic morphological changes in spine structure, and number of synapses that accompany exposure to drugs of abuse such as cocaine and amphetamines indicate that synaptic plasticity plays an important role in the re-wiring of an addicted brain. Descending to a molecular level, cues for changes in the circuitry are issued through changes in synaptic activity, which critically depends on the cellular process of endocytosis because of its direct involvement in receptor clearance from the surface and neurotramsitter vesicle/transporter recycling. The goal of this exploratory study is to enable mechanistic understanding of fundamental synaptic processes, such as endocytosis, by opening the dynamic interface between cellular membranes and the cytoplasm to structural exploration. As a starting point, our work will focus on BAR- and F-BAR-domain proteins whose ability to induce, and to stabilize membrane curvature places them at a strategic intersection between membrane remodeling, vesicle fission, and cytoskeletal re-arrangements. Using purified BAR-/F-BAR-domains and their corresponding full-length proteins (endophilin, amphiphysin, syndapin 1, FBP17 and CIP4), we seek to obtain samples for structure determination by electron cryomicroscopy, in which these proteins are bound to lipid bilayers, either alone or in complex with downstream interaction partners like the fission GTPase dynamin, the cytoskeletal regulators Cdc42 and neural Wiskott-Aldrich-Syndrome Protein (N-WASP). Exposure to drugs of abuse such as cocaine and amphetamines results in lasting morphological changes in some parts of the brain, indicating that synaptic plasticity plays an important role in the biology of addiction. At the molecular level, cues for remodeling of synapses stem from changes in synaptic activities, which at the molecular level critically depend on the cellular process of endocytosis. To advance our basic understanding of the complex processes underlying the biology of addiction, this study ultimately aims at revealing, at a molecular scale, the mechanisms through which protein complexes that are critically important for endocytotic processes contribute to synaptic plasticity.