Fig. 4: The glutamine response of cilia is mediated by the enzyme ASNS.
From: Primary cilia sense glutamine availability and respond via asparagine synthetase
a, Box-and-whisker plots of the levels of intracellular glutamine and asparagine in MEFCtrl and MEFIft88 cells in HBSS ± l-glutamine (Q) assessed by LC–MS targeted metabolomics as in Fig. 3a, n = 5 biological replicates. b, Western blot for ASNS and pS6RPS235/236 of total cell lysates from mIMCD3 transiently knocked down for Asns (siAsns) compared with control (Scr) after 24 h in either 0% FBS or HBSS ± Q (4 mM). c, Average cilia length values from six independent experiments (in each counting >50 cilia per condition) in siAsns mIMCD3 compared with Scr after 24 h culture in either DMEM/F12 + 0% FBS or HBSS ± Q. d, Quantification of cilia length of one representative experiment in mIMCD3 after 24 h in either 0% FBS ± Asnase (5 U ml−1), HBSS ± Q, or Q and Asnase, or asparagine (N) (0.1 mM). e, Left: analysis of OCR measurement of one representative experiment in siAsns mIMCD3 compared with Scr after 4 h culture in HBSS (Scr, blue; siAsns, red) followed by acute injection (+) of Q (Scr, green; siAsns, purple), in basal condition and after sequential addition of oligomycin (O), FCCP and antimycin A/rotenone (A/R). Right: quantification of acute response as in left. f, Scheme of 15N2-glutamine usage by ASNS. g, Levels of intracellular glutamine m + 2 and asparagine m + 1 release assessed by 15N2-glutamine labelled LC–MS targeted metabolomics in MEFCtrl (CT) and MEFIft88 (KO) at 4 and 24 h. n = 5 biological replicates. h, Top: scheme of cilia enrichment by subcellular fractionation. Bottom: representative western blot for TSC1, IFT88, ASNS and S6RP of fraction 1 (Fr1: cytoplasmic), fraction 2 (Fr2: organelles), fraction 3 (Fr3: cilia) and total extract (TE) from mIMCD3 as in top. i, Fluorescence live imaging of eGFP-ASNS transiently transfected mIMCD3 cells in 0% FBS. eGFP-ASNS (green, arrows), nuclei (Hoechst, blue). Scale bar, 10 µm. Insets show magnification. Scale bar, 5 µm. j, Representative IF images of eGFP-ASNS transiently transfected mIMCD3 cells in 0% FBS. eGFP-ASNS (green), cilia (ARL13B, red), nuclei (DAPI, blue). Scale bar, 10 µm. Arrows indicate eGFP-ASNS at the base of cilia. Insets are magnifications (merged and single channels). Scale bar, 5 µm. k, Representative IF images of mNeon-ASNS stable mIMCD3 lines in HBSS (8 h). mNeon-ASNS (green), centrosomes (γ-tubulin, red), cilia (ARL13B, white), nuclei (DAPI, blue). Scale bar, 10 µm. Arrows indicate co-localization with centrosomes. Insets are magnifications (merged and single channels). Scale bar, 5 µm. Box-and-whisker plots show median and minimum to maximum; data in dot and bar plots are mean ± standard deviation. Average plot is mean ± standard error of the mean. Statistical analysis: one-way ANOVA, followed by Tukey’s (a, d, e, g) or Bonferroni’s (c) multiple comparisons test; NS, not significant, ****P < 0.0001. n reported in brackets. Additional replicate experiments in d, e and h are shown in Supplementary Fig. 7.
