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

Cold Spring Harbor Laboratory, Cold Spg Hbr NY

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Abstract

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.