Ultra-Multiplexed Nanoscale In Situ Proteomics for Understanding Synapse Types

用于了解突触类型的超多重纳米级原位蛋白质组学

基本信息

批准号：
9108440
负责人：
Mark Bathe
金额：
$ 70.23万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-26 至 2017-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9108440
关键词：
Beds Behavior Binding Biological Assay Blinking Brain Brain region Cell Culture Techniques Cells Computer Analysis Computer software DNA DNA Probes Development Disease Distant Explosion Genetic Goals Health Image In Situ Individual Kinetics Label Laboratories Learning Maps Mental disorders Methods Microfluidics Molecular Molecular Structure Morphology Mutation Nature Neurobiology Neurons Optics Outcome Pathology Phenotype Physiological Physiology Property Proteins Proteome Proteomics Public Health Publishing Research Research Design Research Personnel Resolution Slice Software Design Spottings Synapses Technology Testing Therapeutic Training Work base brain cell brain tissue cell type combinatorial data acquisition empowered high risk imaging modality interest member nanoscale nervous system disorder neural circuit neuronal circuitry neurotechnology novel optogenetics relating to nervous system scale up synaptic function tissue fixing tool transcriptomics web site

项目摘要

DESCRIPTION (provided by applicant): Significant work is ongoing to reveal how different cell types in the brain contribute to behavior and pathology, and how they change in plasticity and disease, empowered by new genetic, optical, and physiological tools. However, the functional activity and dysregulation of neuronal circuits relies critically on the in situ molecular composition of neuronal synapses. Although it is clear that the properties of a given synapse are determined by, amongst other things, the specific types of cells that are thus connected, far less is known about the diversity of synapse types in the brain than cell types, perhaps because this is an intrinsically proteomic problem: a given neuron might make many different kinds of synapse with different targets, and thus transcriptomics (which is prevailing as a method for cell type analysis) may not suffice for synapse typing. High- throughput in situ proteomic tools are needed to characterize synapse molecular composition at the single-cell level in the context of whole brains or brain regions, and thus to connect the currently distant topics of neuronal activity and genetic aberrations associated with disease pathology. Here, we propose a high-risk, high-payoff, and as far as we know entirely novel agenda: to develop tools capable of resolving the molecular proteomic composition of synapse types, testing them in cultured neurons and intact brain tissues. To achieve this transformative goal of establishing a broadly useful tool for in situ synapse proteomics, we will build on our recent breakthrough in developing the DNA-based highly multiplexed, quantitative super-resolution imaging method DNA- PAINT (Points Accumulation for Imaging in Nanoscale Topography). DNA-PAINT exploits the transient binding of short fluorescently labeled DNA-probes for simple and easy-to-implement quantitative, highly multiplexed, super-resolution imaging with sub-10 nm resolution. In this application, we plan to develop and apply DNA-PAINT to enable quantitative, ultra-multiplexed, in situ characterization of neuronal synapse proteins for understanding synaptic types and studying cell type-specific synaptic functions. The outcome of our work will be a broadly useful in situ proteomic tool for quantification of neuronal synapse composition that can be used by diverse neurobiology laboratories to study single cell-level synapse properties in fixed tissues from whole brains or cell culture assays.

描述（由适用提供）：正在进行重要的工作，以揭示大脑中不同的细胞类型如何促进行为和病理学，以及它们如何通过新的遗传，光学和物理工具赋予能力，如何改变可塑性和疾病。然而，神经元电路的功能活性和失调依赖于神经元突触的原位分子组成。尽管很明显，给定突触的特性取决于如此连接的特定细胞的特定类型，但对大脑类型突触类型的多样性的了解少得多，也许是因为这是本质上的蛋白质组织问题：给定的神经元可能会使许多不同类型的突触与类型的转录组合进行分析，而不是分析的类型，而不是该分析的类型。需要高通量的原位蛋白质组学工具来表征整个大脑或大脑区域的单细胞水平上突触分子组成，从而连接当前不同的神经元活性和与疾病病理相关的遗传畸变的主题。在这里，我们提出了一种高风险，高付费，据我们所知，我们完全是新颖的Agerda：要开发能够解决突触类型的分子蛋白质组学组成的工具，并在培养的神经元和完整的脑组织中测试它们。为了实现为原位突触蛋白质组学建立广泛有用的工具的这种变革性目标，我们将基于我们最近在开发基于DNA的高度多重，定量的超分辨率成像方法DNA-涂料的突破性（纳米级突出中的成像点积累）。 DNA-PAINT利用短荧光标记的DNA探针的瞬时结合，以简单易用的定量，高度多重的，高分辨率的成像，分辨率低于10 nm。在此应用中，我们计划开发和应用DNA-Paint，以实现定量，超多形的原位表征神经元突触蛋白，以理解合成类型并研究细胞类型特异性突触功能。我们工作的结果将是一种可用于定量神经元突触组成的原位蛋白质组学工具，可以被多种神经生物学实验室使用，以研究来自Whole Brains或细胞培养试验的固定组织中的单细胞级突触特性。

项目成果

期刊论文数量（8）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Multiplexed neural recording along a single optical fiber via optical reflectometry.

DOI：
10.1117/1.jbo.21.5.057003
发表时间：
2016-05-01
期刊：
Journal of biomedical optics
影响因子：
3.5
作者：
Rodriques SG;Marblestone AH;Scholvin J;Dapello J;Sarkar D;Mankin M;Gao R;Wood L;Boyden ES
通讯作者：
Boyden ES

124-Color Super-resolution Imaging by Engineering DNA-PAINT Blinking Kinetics

DOI：
10.1021/acs.nanolett.9b00508
发表时间：
2019-04-01
期刊：
NANO LETTERS
影响因子：
10.8
作者：
Wade, Orsolya K.;Woehrstein, Johannes B.;Jungmann, Ralf
通讯作者：
Jungmann, Ralf

Universal Super-Resolution Multiplexing by DNA Exchange.

DOI：
10.1002/anie.201611729
发表时间：
2017-03-27
期刊：
Angewandte Chemie (International ed. in English)
影响因子：
0
作者：
Schueder F;Strauss MT;Hoerl D;Schnitzbauer J;Schlichthaerle T;Strauss S;Yin P;Harz H;Leonhardt H;Jungmann R
通讯作者：
Jungmann R

Epidemiology of intentional self-poisoning in rural Sri Lanka.