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: 8534507
• 负责人: Graham Ellis-Davies
• 金额: $ 24.32万
• 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/8534507
• 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; computerized data processing; drug of abuse; fluorophore; genetic manipulation; innovation; interest; mutant; photolysis; public health relevance; response; 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）：了解神经元功能和突触生理学和可塑性是了解药物滥用和成瘾反应的关键。研究表明，局部蛋白翻译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，我们建议开发一种在亚细胞分辨率下探测生物过程的创新方法，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mrna的分析。这项技术，我们称之为激光标签，是基于为CLIP和SNAP表位标签开发的笼状底物。使用单光子或双光子照明来解开这些底物，将使我们能够激活细胞内或细胞外任何感兴趣区域的化合物，并能够以高度的空间分辨率选择性地标记仅由光指定的蛋白质