Fig. 5: THRONCAT allows in vivo analysis of protein synthesis in Drosophila melanogaster.
From: THRONCAT: metabolic labeling of newly synthesized proteins using a bioorthogonal threonine analog
a Schematic overview of THRONCAT workflow in Drosophila larvae. CNS, central nervous system; TAMRA-N3, 5-carboxytetramethylrhodamine-azide. b Representative images of in vivo THRONCAT in Drosophila larval CNS selectively expressing membrane-tethered GFP in motor neurons (OK371-GAL4 > UAS-mCD8::GFP). Larvae were exposed to medium containing either 0 mM or 4 mM βES, followed by conjugation to TAMRA-N3. Merged images of the green (GFP) and red (TAMRA) channels are shown. Scale bar: 20 μm. c, d Relative fluorescent signal intensity in motor neurons (c) and muscle (d) of larvae exposed to 0 mM or 4 mM βES for 48 h and subjected to TAMRA-N3 conjugation. Signals are normalized to that of 4 mM βES, which is set to 100%. c n = 6 and d n = 3 (0 mM) or 6 (4 mM) larvae per concentration; ***P = 0.0022 (c), *P = 0.0238 (d) by two-tailed Mann–Whitney test. Error bars represent SEM. e Relative fluorescent signal intensity in larvae exposed to 0, 0.04, 0.4, or 4 mM βES for 48 h, after conjugation to TAMRA-N3. Signals are normalized to that of 4 mM βES, which is set to 100%. n = 8 (0 mM), 9 (0.04 mM), 8 (0.4 mM) or 6 (4 mM) larvae per concentration; ***P = 0.0019 (0 vs. 0.4 mM), ***P < 0.0001 (0 vs. 4 mM), ***P = 0.0048 (0.04 vs 4 mM) by Kruskall–Wallis test. Error bars represent SEM. f Relative fluorescent signal intensity after conjugation to TAMRA-N3 in larvae exposed to 4 mM βES or 4 mM HPG for 2 h, 4 h, 8 h, 16 h, or 48 h. n = 6–10 larvae per treatment group and time point, For βES from low to high concentrations, n = 8, 9, 9, 10, or 10. For HPG from low to high concentrations, n = 6, 9, 8, 9 or 9; *P = 0.0395 (8 h), **P = 0.005 (16 h); ***P < 0.0005 (48 h) by unpaired two-tailed t test (2 h, 16 h, 48 h) or two-tailed Mann–Whitney test (4 h, 8 h) per time point, with Bonferroni correction for multiple testing. See Supplementary Table 1 for exact p-values. Error bars represent SEM. g Relative fluorescent signal intensity after conjugation to TAMRA-N3 in larvae exposed to 4 mM βES versus OK371-GAL4 > UAS-MetRSL262G larvae conjugated with TAMRA-alkyne after exposure to 4 mM ANL for 0 h, 4 h, 8 h, 16 h, or 48 h. n = 6–10 larvae per treatment group and time point, For βES from low to high concentrations, n = 8, 8, 9, 9, or 6. For ANL from low to high concentrations, n = 8, 7, 10, 9 or 10; ***P = 0.0015 (4 h), ***P < 0.0005 (8 h), ***P < 0.0005 (16 h), ***P < 0.001 (48 h) by two-tailed Mann–Whitney test per time point, with Bonferroni correction for multiple testing. Error bars represent SEM. h Representative images of THRONCAT in Drosophila larvae selectively expressing membrane-tethered GFP in motor neurons (OK371-GAL4 > UAS-mCD8::GFP), with or without co-expression of G240R mutant human glycyl-tRNA synthetase (UAS-hGlyRS_G240R). Larvae were exposed to 4 mM βES for 48 h, followed by conjugation to TAMRA-N3. The red (TAMRA) channel alone and merged images of the green (GFP) and red channels are shown. Scale bar: 20 μm. i Relative translation rate in motor neurons (OK371-GAL4) as determined by THRONCAT in larvae expressing hGlyRS_G240R versus driver-only control. Signals are normalized to that of control, which is set to 100%. n = 14 larvae per genotype; ***P < 0.0001 by unpaired two-tailed t test. Error bars represent SEM. Source data are provided as a Source Data file.
