Abstract
Neurons are categorised into many subclasses, and each subclass displays different morphology, expression patterns, connectivity and function. Changes in protein synthesis are critical for neuronal function. Therefore, analysing protein expression patterns in individual neuronal subclass will elucidate molecular mechanisms for memory and other functions. In this study, we used neuronal subclass-selective proteomic analysis with cell-selective bio-orthogonal non-canonical amino acid tagging. We selected Caenorhabditis elegans as a model organism because it shows diverse neuronal functions and simple neural circuitry. We performed proteomic analysis of all neurons or AFD subclass neurons that regulate thermotaxis in C. elegans. Mutant phenylalanyl tRNA synthetase (MuPheRS) was selectively expressed in all neurons or AFD subclass neurons, and azido-phenylalanine was incorporated into proteins in cells of interest. Azide-labelled proteins were enriched and proteomic analysis was performed. We identified 4,412 and 1,834 proteins from strains producing MuPheRS in all neurons and AFD subclass neurons, respectively. F23B2.10 (RING-type domain-containing protein) was identified only in neuronal cell-enriched proteomic analysis. We expressed GFP under the control of the 5′ regulatory region of F23B2.10 and found GFP expression in neurons. We expect that more single-neuron specific proteomic data will clarify how protein composition and abundance affect characteristics of neuronal subclasses.
Highlights
Neurons are categorised into many subclasses, and each subclass displays different morphology, expression patterns, connectivity and function
Protein expression patterns of some subclasses were generated with mass spectrometry-based proteomic analysis[2,5,17]
For the first time, proteomic analyses against all neurons and AFD subclass neurons using cell-selective bio-orthogonal non-canonical amino acid tagging (BONCAT), and identify 4,412 proteins and 1,834 proteins, respectively (Figs. 3, 6)
Summary
Neurons are categorised into many subclasses, and each subclass displays different morphology, expression patterns, connectivity and function. Analysing protein expression patterns in individual neuronal subclass will elucidate molecular mechanisms for memory and other functions. Single-cell RNA-seq analysis is used to assess expression patterns in neurons of m ice[8,9], Drosophila[10] and C. elegans[11] and new neuronal subclasses are recognised. Analysis of abundance and expression patterns of proteins in target subclass neurons is needed to identify molecular mechanisms underlying neuronal functions. Protein expression patterns of some subclasses were generated with mass spectrometry-based proteomic analysis[2,5,17] In these studies, in vitro differentiation[8], laser dissection[18,19], flow cytometric s orting[20], and antibody-coupled m icrobeads[21] were used to isolate specific neuronal subclasses or all neuronal cells. Precursory Research for Embryonic Science and Technology (PREST), 7 Goban‐cho, Chiyoda‐ku, Tokyo 102‐0076, Japan. 5JST, Core Research for Evolutionary Science and Technology (CREST), 7 Goban‐cho, Chiyoda‐ku, Tokyo 102‐0076, Japan. 6Kyoto Integrated Science and Technology Bio-Analysis Center, 134 Chudoji Minamimachi, Scientific Reports | (2020) 10:13840
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