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

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，局部蛋白质翻译影响突触的短期和长期反应，包括长时程增强（LTP）和长时程抑制（LTD）。在这里，我们建议开发一种创新的方法，用于探测在亚细胞分辨率的生物过程，并将其应用于选择性转录组分析的个别细胞类型和分析本地翻译的mRNA。这项技术，我们称之为激光标签，是基于CLIP和SNAP表位标签的笼状底物的开发。使用单光子或双光子照射来解开这些底物的束缚将允许我们激活任何感兴趣区域中的化合物，无论是细胞内还是细胞外，并且能够以仅由光指定的高度空间分辨率选择性地标记蛋白质。 梁通过将激光标签与先前验证的恢复主动翻译信息的方法（Ribotag）相结合，我们可以在离散细胞类型或亚细胞区室中选择性地分析mRNA。在这种情况下，我们将检查局部翻译的mRNA在树突和测量这些人口的变化，以应对各种化学和遗传操作的配置文件。这些研究的总体成果不仅将是对本地翻译及其调控方式的更深入了解，而且还将是一个广泛的平台技术，可用于研究广泛的生物过程。

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.