High-precision mapping of the spatial organization of synaptic-vesicle membrane proteins

突触小泡膜蛋白空间组织的高精度绘图

• 批准号: 10091526
• 负责人: Daniel T Chiu
• 金额: $ 38.88万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10091526
• 关键词: Antiepileptic Agents; Area; Behavior; Binding Sites; Biochemical; Biological Models; Brain; Buffers; Caliber; Cell membrane; Cells; Complex; Crowding; Cryoelectron Microscopy; Crystallography; Diffuse; Drug Targeting; Electron Microscopy; Eukaryotic Cell; Event; Foundations; Freedom; Functional disorder; Glutamates; Goals; Individual; Ions; Lead; Levetiracetam; Lipid Bilayers; Location; Macromolecular Complexes; Maps; Measurement; Membrane; Membrane Fusion; Membrane Proteins; Mental disorders; Methods; Modeling; Modification; Molecular; Molecular Neurobiology; Molecular Structure; Neurologic; Neurons; Neurosciences Research; Neurotransmitters; Organelles; Pathway interactions; Physiology; Play; Positioning Attribute; Presynaptic Terminals; Protein Sortings; Proteins; Proton-Translocating ATPases; Quantitative Microscopy; Recycling; Resolution; Ribosomes; Role; Rotation; Signal Transduction; Structure; Surface; Synapses; Synaptic Transmission; Synaptic Vesicles; Synaptic plasticity; Vesicle; Water; Work; base; biophysical analysis; experimental study; molecular imaging; nanoscale; neurotransmission; neurotransmitter release; neurotransmitter uptake; presynaptic; protein distribution; relating to nervous system; single molecule; synaptic function; trafficking; uptake

Project Summary/Abstract: To orchestrate neurotransmission, more than one thousand proteins are present at the presynaptic terminal and which either directly or indirectly interact with the synaptic vesicle. This high degree of convergence of presynaptic functions onto the synaptic vesicle has led to a “vesicocentric” view of neurotransmission that focuses on the synaptic vesicle as the central organelle in synaptic function. As a result, much effort in molecular neurobiology in the past decades has been spent to identify the proteins present on synaptic vesicles and how they function to control neurotransmitter release. In addition, synaptic vesicles are also used as a model trafficking organelle to understand the mechanism utilized by the eukaryotic cell to carry out membrane fusion and trafficking. Because of these central roles synaptic vesicles play in neurotransmission as well as a model system for understanding membrane trafficking in general, it is critically important to develop a detailed and quantitative understanding of the organization of the synaptic vesicle. Synaptic vesicle is the smallest organelle present in the cell. From electron-microscopy (EM) measurements, synaptic vesicle has a diameter of ~40nm. In comparison, a ribosome observed under EM has a “diameter” of 20-30nm depending on the contrast employed. In terms of size, therefore, synaptic vesicles are similar in many ways to a very large macromolecular complex, and as such, amenable to high- resolution single-molecule imaging and quantitative biophysical studies. Synaptic vesicle, however, is too large and disorganized to be studied with established structural methods, such as crystallography or cryoEM. The goal of the proposed project is to go beyond the compositional studies conducted in the past decade so as to unravel how proteins are spatially organized and interacting on the synaptic vesicle, and to construct a molecular/structural view of the synaptic vesicle. To achieve this, we have the following Aims: Aim 1: Single-Molecule Positional Mapping and Counting of Membrane Proteins on Synaptic Vesicles with Photoswitchable Pdots - this study will tell us which proteins are always located at the same positions on the vesicle, which proteins are adjacent to each other, and which are randomly distributed on the vesicle. Aim 2: Single-Molecule Orientation Mapping of Membrane Proteins on Synaptic Vesicles with Polarized Pdots - we know proteins are extremely crowded on the vesicle, but if proteins are interacting with each other or form a complex, then they are not only in close proximity but also would have the same rotational rate on the membrane or have very limited rotational freedom. This experiment will inform us which proteins are forming a complex and also whether they are in the same complex. Aim 3: Positional and Orientational Mapping of Membrane Proteins on Different Types of Synaptic Vesicle Enabled by a Nanoscale Sorter - different types of SVs might arrange the membrane proteins differently. We will use a nanoscale sorter to isolate and study different types of synaptic vesicles.

项目概要/摘要： 为了协调神经传递，在突触前存在一千多种蛋白质。 末端，并且直接或间接地与突触囊泡相互作用。这种高度 突触前功能在突触囊泡上的会聚导致了一种“囊泡中心”的观点， 神经传递，其集中于作为突触功能中的中心细胞器的突触囊泡。作为 因此，在过去的几十年里，分子神经生物学的许多努力都花在了鉴定这些蛋白质上。 以及它们如何控制神经递质的释放。此外，Synaptic 囊泡也被用作模型运输细胞器，以了解真核生物利用的机制。 细胞进行膜融合和运输。由于突触囊泡在神经元中的这些中心作用， 神经传递以及一般理解膜运输的模型系统，它是至关重要的 重要的是发展一个详细的和定量的了解组织的突触囊泡。 突触囊泡是细胞中最小的细胞器。电子显微镜（EM） 突触囊泡直径约40 nm。相比之下，在EM下观察到的核糖体 具有20- 30 nm的“直径”，这取决于所采用的对比度。因此，就大小而言，突触 囊泡在许多方面类似于非常大的大分子复合物，因此，适合于高- 分辨率单分子成像和定量生物物理研究。然而，突触囊泡也 大的和无序的，以研究与建立结构的方法，如晶体学或cryoEM。 拟议项目的目标是超越过去十年进行的成分研究 从而揭示蛋白质是如何在空间上组织和在突触囊泡上相互作用的， 突触囊泡的分子/结构视图。为了实现这一目标，我们有以下目标： 目的1：突触囊泡上膜蛋白的单分子定位和计数， 光开关Pdot-这项研究将告诉我们哪些蛋白质总是位于相同的位置上， 这些蛋白质彼此相邻，并且随机分布在囊泡上。 目的2：利用极化Pdot对突触囊泡上的膜蛋白进行单分子定位 - 我们知道蛋白质在囊泡上非常拥挤，但如果蛋白质相互作用， 形成一个复合体，那么它们不仅非常接近，而且在 膜或具有非常有限的旋转自由度。这个实验将告诉我们哪些蛋白质正在形成一个 复杂性，以及它们是否在同一个复杂性中。 目的3：不同类型突触囊泡上膜蛋白的位置和方向图 通过纳米级分选仪实现-不同类型的SV可能会以不同的方式排列膜蛋白。我们 将使用纳米级分选器分离和研究不同类型的突触囊泡。

项目成果

Error-Correction Method for High-Throughput Sizing of Nanoscale Vesicles with Single-Molecule Localization Microscopy.

使用单分子定位显微镜高通量测定纳米级囊泡的误差校正方法。

• DOI: 10.1021/acs.jpcb.2c09053
• 发表时间: 2023
• 期刊: The journal of physical chemistry. B
• 作者: Jung,Seung-Ryoung;Kim,James;Vojtech,Lucia;Vaughan,JoshuaC;Chiu,DanielT
• 通讯作者: Chiu,DanielT

Photostable Ratiometric Pdot Probe for in Vitro and in Vivo Imaging of Hypochlorous Acid.

• DOI: 10.1021/jacs.7b01545
• 发表时间: 2017-05-24
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: Wu L;Wu IC;DuFort CC;Carlson MA;Wu X;Chen L;Kuo CT;Qin Y;Yu J;Hingorani SR;Chiu DT
• 通讯作者: Chiu DT

Enhancing the Long-Term Stability of a Polymer Dot Glucose Transducer by Using an Enzymatic Cascade Reaction System.

利用酶级联反应系统增强聚合物点葡萄糖传感器的长期稳定性

• DOI: 10.1002/adhm.202001019
• 发表时间: 2021-03
• 期刊: Advanced healthcare materials
• 影响因子: 10
• 作者: Sun K;Ding Z;Zhang J;Chen H;Qin Y;Xu S;Wu C;Yu J;Chiu DT
• 通讯作者: Chiu DT

Reversible Ratiometric NADH Sensing Using Semiconducting Polymer Dots.

• DOI: 10.1002/anie.202100774
• 发表时间: 2021-05-17
• 期刊: Angewandte Chemie (International ed. in English)
• 作者: Chen H;Yu J;Men X;Zhang J;Ding Z;Jiang Y;Wu C;Chiu DT
• 通讯作者: Chiu DT

Sizing Extracellular Vesicles Using Membrane Dyes and a Single Molecule-Sensitive Flow Analyzer.

• DOI: 10.1021/acs.analchem.1c00253
• 发表时间: 2021-04-13
• 期刊: ANALYTICAL CHEMISTRY
• 影响因子: 7.4
• 作者: Andronico, Luca A.;Jiang, Yifei;Jung, Seung-Ryoung;Fujimoto, Bryant S.;Vojtech, Lucia;Chiu, Daniel T.
• 通讯作者: Chiu, Daniel T.