Neuronal subclass-selective proteomic analysis in Caenorhabditis elegans

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.

Figure 1

Experimental scheme of cell-selective BONCAT. Escherichia coli was cultured with azide-phenylalanine (Azf) to label proteins in E. coli with Azf. Next, C. elegans was cultured with the Azf-labelled E. coli to label proteins at target subclass neurons with Azf. After the extraction of total protein, azide-modified proteins in target subclass neurons were enriched using alkyne agarose. The enriched proteins were digested with trypsin and proteomic analysis was conducted with monolithic nano LC–MS/MS.

Figure 2

Confirmation of MuPheRS production and activity in the SA1 strain. (A) Confirmation of MuPheRS production in targeted cells in the SA1 strain (SAIs1[Prab-3::frs1(Thr412Gly)::fib-1/rps-16::gfp(S65C, synIVS)::unc-54 3′-UTR]). Green fluorescent protein was produced under the rab-3 promoter (All neurons). A dotted white line indicates body shape. Scale bar indicates 20 μm. (B) Confirmation of azide-phenylalanine incorporation in targeted cells. Azide-proteins were stained with dibenzocyclooctyne-PEG4-Fluor 545 (TAMRA-DBCO). Scale bar indicates 20 μm. The SA1 strain was cultured with Azf-labelled E. coli KY33 and stained with TAMRA-DBCO.

Figure 3

Comparison of the number of identified proteins. (A) The average number of identified proteins of Phe-cultured N2, Azf-cultured N2, and Azf-cultured SA1 with azide-protein enrichment. The data are shown as mean ± standard errors of the mean (N = 3). (B) Venn diagram to compare the compositions of proteins identified in worms fed on KY33 strain cultured with azide-phenylalanine. The numbers in the venn diagram indicate the number of proteins identified at least once in each sample (N2 or SA1). We performed tissue enrichment analysis with the black filled section (proteins identified only in SA1 strain cultured in the Azf-labeled KY33 strain with azide-protein enrichment procedure, Supplementary Table 3).

Figure 4

Distribution of the identified proteins in (A) isoelectric point and (B) molecular weight. The lines indicate the distribution of isoelectric point and molecular weight of the identified proteins. The black line shows the data of N2 strain cultured with the azide-labelled E. coli KY33 without azide-protein enrichment. The light grey dashed line and the dark grey line show the data of the SA1 strain cultured with the azide-labelled KY33 without or with azide-protein enrichment, respectively.

Figure 5

GFP expression under the control of the 5′ regulatory region of F23B2.10. Confocal imaging of the head ganglia of the SA3 strain (SAEx3[Pf23b2.10::GFP, Pf25b3.3::mcherry]) and N2 strain. We detected GFP fluorescence at some neurons only in the SA3 strain. Scale bar indicates 20 μm.

Figure 6

Venn diagram for comparison of proteome between the azide-enriched SA1 and SA2 strains cultured with the Azf-labelled KY33. Numbers in the Venn diagram indicate the number of identified proteins at least once in each fraction.