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

描述(由申请者提供)：了解神经元功能和突触的生理学和可塑性是了解滥用和成瘾药物反应的关键。已有研究表明，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来探索亚细胞分辨率的生物过程，并将其应用于单个细胞类型的选择性转录组分析和局部翻译的mRNAs分析。这项技术，我们称之为激光标签，是基于用于CLIP和SNAP表位标签的笼式底物的开发。使用单光子或双光子照明取消这些底物的标记将允许我们激活任何感兴趣的区域中的化合物，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地标记蛋白质。 波束。通过将激光标签与先前验证的方法相结合来恢复主动翻译的消息(Ribotag)，我们可以选择性地在离散细胞类型或亚细胞室中分析mRNA。在这种情况下，我们将检查树突中局部翻译的mRNAs的图谱，并测量这些群体对各种化学和遗传操作的反应变化。这些研究的总体结果不仅将加深对当地翻译及其监管方式的理解，还将为研究广泛的生物过程提供广泛的平台技术。

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.