Figure 1

(A) Schematic demonstrating the method to visualize newly synthesized proteins in the brain in a cell-type-specific manner. (B) The protein sequence of the catalytic core domain of the MetRS in a number of species including Danio Rerio shows strong conservation (green). Leucine 270 (bold green) was mutated to Glycine to develop cell-type-specific metabolic labeling in zebrafish. (C) Schematic of the binding pocket of the MetRS and the ribosome during translation. The wt MetRS allows the charging of Met (black) that can be incorporated during translation initiation and elongation (left). The non-canonical amino acid ANL (blue), which contains an azide group, does not fit into the binding pocket of the wt MetRS, and thus is notincorporated into nascent protein (center). The mutant MetRSL270G can charge ANL, which is then incorporated into newly synthesized proteins (in cells expressing the MetRSL270G). (D) Schematic of the UAS-CFP-MetRSL270G line transgene. Crossing the line with any Gal4-expressing line allows for the metabolic labeling of newly synthesized proteins in any accessible cell type. (E) A scheme demonstrating the use of the ELAVL3-Gal4:UAS-CFP-MetRSL270G line. Left: a zebrafish larva expressing the transgene in neurons (cyan). Following addition of ANL to the water bath, newly synthesized proteins in neurons incorporate ANL (blue). Right: a whole mount click reaction with a fluorescent alkyne reveals the newly synthesized proteins (red). (F) The effect of different ANL concentrations on swim speed after 24 hr of ANL exposure (measurement was done in the presence of ANL). 10 mM ANL, which had no significant effect on larvae swimming, was used in further experiments. N = 5 to 6 larvae for each concentration. (G) Projections of confocal images of zebrafish larval brains after click reactions demonstrating the specificity of fluorescently labeled nascent protein in the MetRSL270G larva treated with ANL, but not in controls. Scale bar = 50 μm.