A light-regulated protein tagging method to study local translation in neurons

研究神经元局部翻译的光调节蛋白标记方法

• 批准号: 8623127
• 负责人: Graham Ellis-Davies
• 金额: $ 22.1万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8623127
• 关键词: Affinity; Binding; Biochemical Process; Biochemistry; Biological Process; Biology; Biotin; Brain; Cataloging; Catalogs; Cells; Chemistry; Chimeric Proteins; Complex; Coupling; Cues; Dendrites; Development; Drug Addiction; Drug abuse; Enzymes; Epitopes; Gene Expression Profile; Genes; Goals; Individual; Knowledge; Lasers; Learning; Light; Lighting; Link; Long-Term Depression; Long-Term Potentiation; Maps; Measurement; Measures; Memory; Messenger RNA; Methodology; Methods; Molecular Biology; Mus; Neurons; Neurosciences; Neurotransmitters; O(6)-Methylguanine-DNA Methyltransferase; O(6)-benzylguanine; Optics; Outcome Study; Physiology; Population; Process; Proteins; RNA; Reaction; Recovery; Research Personnel; Resolution; Ribosomal Proteins; S-nitro-N-acetylpenicillamine; Signal Transduction; Signaling Molecule; Stimulus; Synapses; Technology; Testing; Translating; Translations; addiction; base; cell type; chemical genetics; drug of abuse; fluorophore; genetic manipulation; innovation; interest; mutant; photolysis; public health relevance; response; signal processing; single molecule; synaptic function; two-photon

DESCRIPTION (provided by applicant): Understanding neuronal function and synaptic physiology and plasticity is key to understanding the response to drugs of abuse and addiction. It is well demonstrated that local protein translation impacts both short- and long-term responses of synapses, including long-term potentiation (LTP) and long-term depression (LTD). Here, we propose to develop an innovative method for probing biological processes at subcellular resolution and to apply it to selective transcriptome profiling of individual cell type and to profiling of locally translated mRNAs. This technology, which we term Laser Tag, is based on the development of caged substrates for the CLIP and SNAP epitope tags. Uncaging of these substrates using single or two-photon illumination will allow us to activate the compounds in any region of interest, either intracellularly or extracellularly, and enable selectiv tagging of proteins with a high degree of spatial resolution that is designated solely by the light beam. By combining Laser Tag with a previously validated methodology to recover actively translating messages (Ribotag), we can profile mRNAs selectively in discrete cell types or subcellular compartments. In this case, we will examine profiles of locally translated mRNAs in dendrites and measure changes in these populations in response to a variety of chemical and genetic manipulations. The overall outcome of these studies will not only be a deeper understanding of local translation and how it is regulated but also a broad platform technology that can be used to investigate a wide range of biological processes.

描述（由申请人提供）：了解神经元功能、突触生理学和可塑性是了解对滥用和成瘾药物的反应的关键。事实证明，局部蛋白质翻译会影响突触的短期和长期反应，包括长期增强（LTP）和长期抑制（LTD）。在这里，我们建议开发一种创新方法，以亚细胞分辨率探测生物过程，并将其应用于个体细胞类型的选择性转录组分析和局部翻译的 mRNA 分析。这项技术，我们称之为激光标签，是基于用于 CLIP 和 SNAP 表位标签的笼状基质的开发。使用单光子或双光子照明释放这些底物将使我们能够激活任何感兴趣区域的化合物，无论是细胞内还是细胞外，并能够以仅由光指定的高空间分辨率选择性标记蛋白质 光束。通过将激光标签与先前验证的方法相结合以恢复主动翻译信息 (Ribotag)，我们可以选择性地分析离散细胞类型或亚细胞区室中的 mRNA。在这种情况下，我们将检查树突中局部翻译的 mRNA 的概况，并测量这些群体响应各种化学和遗传操作的变化。这些研究的总体成果不仅是对本地翻译及其监管方式的更深入了解，而且是可用于研究广泛生物过程的广泛平台技术。