Optimization of clickable methionine analogues to monitor translation in living cells

(A) Frequency of methionine residues in intracellularly or extracellularly localized protein domains within the human or mouse proteins with annotated plasma membrane localization. (B) Chemical structures for methionine and its clickable analogues, AHA and HPG. (C) Schematic depicting experimental workflow. (D and E) Representative flow cytometric histograms and corresponding gMFIs of extracellular Alk-594 signal (D) or Alk-647 signal (E) after 1, 2, or 4 hours of AHA incorporation. (F and G) Representative flow cytometric histograms and corresponding gMFIs of extracellular Azd-594 signal (F) or Azd-647 signal (G) after 1, 2, or 4 hours of HPG incorporation. (H) Representative confocal images of Alk-647 signal, showing surface localization in Jurkat cells after 24 hours of Met or AHA incorporation. (I) Representative confocal images of extracellular Azd-647 signal, showing surface localization in Jurkat cells after 24 hours of Met or HPG incorporation. (J-P) Representative flow cytometric histograms of extracellular Alk-647 signal on K562 (J), MOLM-13 (K), OCI-AML3 (L), 143B (M), Calu6 (N), HCT116 (O), and H1299 (P) cell lines after 4 hours of AHA incorporation. Abbreviations: AHA = Azidohomoalanine, HPG = Homopropargylglycine, Met = Methionine, CHX = Cycloheximide, gMFI = Geometric mean fluorescence intensity Statistical significance was assessed by one-way ANOVA (D-G) followed by Tukey’s multiple comparisons test. Graphs display mean ± SD (D-G). (ns, p >0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; or ****, p ≤ 0.0001).

Validation of surface translation rate as a readout of cellular energetics

(A) Schematic depicting experimental workflow. (B) Schematic of ATP-producing pathways and corresponding metabolic inhibitors. (C) Flow cytometric histograms of extracellular Alk-647 signal on Jurkat cells incubated in AHA with varying concentrations of 2DG and Omy. (D) Correlation between changes in adenylate energy charge versus normalized Alk-647 Alkyne flow cytometric signal. (Pearson R2 = 0.8179, p = 0.0052). (E) Correlation between changes in newly synthesized ATP versus normalized Alk-647 Alkyne flow cytometric signal (Pearson R2 = 0.7840, p = 0.008). (F) Schematic depicting experimental workflow. (G-H) Seahorse glycolysis stress test (G) or MARBL-processing (H) on Jurkat cells using varying concentrations of 2DG and Omy.

Abbreviations: AHA = Azidohomoalanine, HPG = Homopropargylglycine, Met = Methionine, CHX = Cycloheximide, gMFI = geometric mean fluorescence intensity, 2DG = 2-Deoxy-D-Glucose, Omy = Oligomycin A The Pearson correlation coefficient was used to assess correlation and significance (D-E). Statistical significance was assessed by two-tailed Student’s unpaired t-test (G-H). Graphs display mean ± SD (D-E, G-H). (ns, p >0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; or ****, p ≤ 0.0001).

Development of dual-incubation MARBL workflow to measure cellular energetics in single cells

(A) Schematic depicting experimental workflow. (B-C) Representative flow cytometric contour plots of extracellular Alk-647 and Azd-594 signal on Jurkat cells (B) and corresponding cell viability after click staining (C). (D-E) Representative flow cytometric contour plots of extracellular Alk-647 and Azd-594 signal on Jurkat cells (D) and corresponding cell viability after click staining (E) in the presence of vehicle, translation (CHX = 500 µM), or metabolic (2DG = 25 mM, Omy = 2 µM) perturbation. (F) Schematic depicting experimental workflow. (G) Representative flow cytometric histograms and corresponding gMFI of extracellular Alk-647 signal with an AHA pulse followed by a Met chase. (H) Representative flow cytometric histograms and corresponding gMFI of extracellular Azd-594 signal with an HPG pulse followed by a Met chase. Abbreviations: AHA = Azidohomoalanine, HPG = Homopropargylglycine, Met = Methionine, CHX = Cycloheximide, gMFI = geometric mean fluorescence intensity, 2DG = 2-Deoxy-D-Glucose, Omy = Oligomycin A Statistical significance was assessed by one-way ANOVA (C, E, G-H) followed by Tukey’s multiple comparisons test. Graphs display mean ± SD (C, E, G-H). (ns, p >0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; or ****, p ≤ 0.0001).

Application of MARBL to primary human cells

(A) Schematic depicting experimental workflow. (B-F) Representative flow cytometric histograms of extracellular Alk-647 signal on activated CD3+ human T cells incubated with the following: Met (B), AHA + DMSO (C), AHA + CHX (D), AHA + Omy (E), or AHA + 2DG (F). (G) Quantification of CD69+ and CD69- frequency in CD3+ singlet gate (left), low AHA bin within CD3+ singlet gate (middle), and high AHA bin within CD3+ singlet gate (right). (H) Metabolic resilience indices for human CD3+ T cells stratified by CD69 activation status. Each dot depicts a different healthy human donor. Experiments (B-H) were performed in biological quadruplicate (n = 4). (I) Abbreviations: CHX = Cycloheximide, 2DG = 2-Deoxy-D-Glucose, Omy = Oligomycin A, gMFI = geometric mean fluorescence intensity, PBMC = Peripheral blood mononuclear cells Statistical significance was assessed by one-way ANOVA (H) followed by Tukey’s multiple comparisons test. Graphs display mean ± SD (G-H). (ns, p >0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; or ****, p ≤ 0.0001).

MARBL reveals energetic dependencies that stratify cell function

(A) Schematic depicting experimental workflow. (B) Representative contour plots depicting gates for cell sorting based on extracellular Azd-594 and Alk-647 signals on a 1:1 mixture of npTH17 and pTH17 cells. (C) Ratio of pTH17 cell counts relative to npTH17 cell counts in all live bin and resilient bin. (D) Fold change of IFNγ production following PMA/Ionomycin restimulation comparing resilient gate to all live bin. (E) Schematic depicting experimental workflow. (F) Representative flow cytometric histograms of MARBL-processed leukocytes enriched from B16-Ova tumors in the presence of vehicle treatment, translation inhibition (CHX = 500 µM), or metabolic perturbation (2DG = 25 mM, Omy = 2 µM). (G) Gating strategy for low and high click bins for MARBL-processed CD3+ lymphocytes with extracellular Azd-594 and Alk-647 signal. (H-I) Representative flow cytometric histograms of extracellular Azd-594 and Alk-647 signal in low (H) and high (I) click bin gates in the presence of vehicle treatment, translation inhibition (CHX = 500 µM), or metabolic perturbation (2DG = 12.5 mM and Omy = 1 µM). Abbreviations: npTH17 = non-pathogenic TH17, pTH17 = pathogenic TH17, AHA = Azidohomoalanine, Met = Methionine, CHX = Cycloheximide, 2DG = 2-Deoxy-D-Glucose, Omy = Oligomycin A Statistical significance was assessed by two-tailed Student’s unpaired t-test (C-D). Graphs display mean ± SD (C-D). (ns, p >0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; or ****, p ≤ 0.0001).

Comparison of techniques to assay bioenergetics

Supporting data for the optimization of clickable methionine analogues to monitor translation in living cells, related to Figure 1

(A) Representative flow cytometric histograms of extracellular Alk-350, Alk-405, Alk-488, Alk-594, or Alk-647 labeled Jurkat cells after 4 hours of AHA incubation. (B) Flow cytometry analysis of Jurkat cells incubated in varying concentrations of AHA or AHA + CHX (500 µM) and extracellularly clicked with Alk-647. (C) Flow cytometry analysis of Jurkat cells incubated in varying concentrations of HPG or HPG + CHX (500 µM) and extracellularly clicked with Azd-594. (D-E) Cell viability (D) and live cell count (E) in Jurkat cells cultured in Met-free media supplemented with Met and/or CMAs. (F-G) Jurkat cells extracellularly clicked with Alk-647 (F) or Azd-594 (G) after 4 hours of CMA incorporation in media supplemented with dialyzed or complete FBS. (H-I) Viability and gMFIs of Jurkat cells incubated in AHA or HPG for 4 hours with varying concentrations of CuSO4-chelating ligand BTTAA and extracellularly-clicked with Alk-647 (H) or Azd-594 (I). (J) Schematic depicting experimental workflow. Statistical significance was assessed by one-way ANOVA (B-C, H-I) or two-way ANOVA (F-G) followed by Tukey’s multiple comparisons test. Confocal images were acquired at 20X magnification with a 2X tube lens (B-C). Graphs display mean ± SD (B-I). (ns, p >0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; or ****, p ≤ 0.0001). Abbreviations: AHA = Azidohomoalanine, HPG = Homopropargylglycine, Met = Methionine, CHX = Cycloheximide, gMFI = Geometric mean fluorescence intensity, FBS = fetal bovine serum

Supporting data for validation of surface translation rate as a readout of cellular energetics, related to Figure 2

(A) Heatmap of polar metabolites after 2, 4, 6, and 8 hours of incubation in Met-free media supplemented with Met, AHA, or HPG. (B) Heatmap of methionine-derived metabolites, AHA, and HPG. (C-J) Volcano plots of metabolites differentially enriched in various media conditions after 2 (C-F) or 8 (G-J) hours of incubation. (K-M) Venn diagrams of significantly altered metabolites in various media conditions using a p-value cutoff of 0.05, comparing: HPG versus AHA after 2- or 8-hour incubations (K); HPG, AHA, or -Met compared to +Met control after a 2-hour incubation (L); and HPG, AHA, or -Met compared to + Met control after an 8-hour incubation (M). (N) Seahorse glycolysis stress test on Jurkat cells using varying concentrations of 2DG and Omy. Abbreviations: AHA = Azidohomoalanine, HPG = Homopropargylglycine, Met = Methionine, 2DG = 2-Deoxy-D-Glucose, Omy = Oligomycin A, MTA = 5’-methylthioadenosine, SAM = S-adenosylmethionine, ECAR = Extracellular acidification rate Statistical significance was calculated using a two-tailed Student’s unpaired t-test (C-M). Graphs display mean ± SD (N). (ns, p >0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; or ****, p ≤ 0.0001).

Supporting data for development of dual-incubation MARBL workflow to measure cellular energetics in single cells, related to Figure 3

(A) Schematic depicting experimental workflow. (B-C) Extracellular Alk-647 gMFI (B) and Azd-594 gMFI (C) in the presence of vehicle, translation inhibition (CHX = 500 µM), or metabolic perturbation (2DG = 25 mM, Omy = 2 µM). (D) Schematic depicting experimental workflow. (E-F) Extracellular Azd-594 gMFI (E) and Alk-647 gMFI (F) in the presence of vehicle, translation inhibition (CHX = 500 µM), or metabolic perturbation (2DG = 25 mM, Omy = 2 µM). Abbreviations: AHA = Azidohomoalanine, HPG = Homopropargylglycine, Met = Methionine, CHX = Cycloheximide, gMFI = geometric mean fluorescence intensity, 2DG = 2-Deoxy-D-Glucose, Omy = Oligomycin A Statistical significance was assessed by one-way ANOVA (B-C, E-F) followed by Tukey’s multiple comparisons test. Graphs display mean ± SD (B-C, E-F). (ns, p >0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; or ****, p ≤ 0.0001).

Supporting data for application of MARBL to primary human cells, related to Figure 4

(A) Informatic workflow for calculating metabolic resilience index. (B-C): Transformation and filtering of raw click signal on dually HPG and AHA labeled Jurkat cells treated with vehicle (B) or metabolic inhibitors (2DG = 25 mM, Omy = 2 µM). (D-E) Individual cells colored by metabolic resilience index for cells treated with vehicle (D) or metabolic inhibitors (2DG = 25 mM, Omy = 2 µM) (E). (F) Violin plots of metabolic resilience index of Jurkat cells. (G) Cell viability of PBMCs after dual-labeling and extracellular clicking. (H) Violin plots of metabolic resilience indices of human CD3+ T cells stratified by CD69+ activation status. Experiments (G-H) were performed in biological quadruplicate with four healthy donors (n = 4). Abbreviations: AHA = Azidohomoalanine, Met = Methionine, CHX = Cycloheximide, 2DG = 2-Deoxy-D-Glucose, Omy = Oligomycin A, ETO = Etomoxir, PBMC = Peripheral blood mononuclear cells Statistical significance was assessed by one-way ANOVA (G-H) followed by Tukey’s multiple comparisons test. Graphs display mean ± SD (G). (ns, p >0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; or ****, p ≤ 0.0001).

Supporting data for MARBL reveals energetic dependencies that stratify cell function, related to Figure 5

(A-B) Frequency of Tbet+ (A) and RORγt+ (B) cells in murine naïve, non-pathogenic, or pathogenic CD4+ TH17 cells. (C) Complete gating scheme for mixed npTH17 and pTH17 sorting experiment. (D) Viability as measured during cell sorting of MARBL-processed npTH17 and pTH17 cell mixtures. (E) Frequency of dually MARBL-processed npTH17 or pTH17 cells in resilient bin. (F) Viability of MARBL-processed npTH17 and pTH17 cell mixtures following PMA/Ionomycin restimulation. Abbreviations: npTH17 = non-pathogenic TH17, pTH17 = pathogenic TH17, AHA = Azidohomoalanine, Met = Methionine, CHX = Cycloheximide, 2DG = 2-Deoxy-D-Glucose, Omy = Oligomycin A, FSC = Forward scatter, SSC = Side scatter, NIR = Near-IR viability dye Statistical significance was assessed by two-tailed Student’s unpaired t-test (A-B, D) or by one-way ANOVA (E-F) followed by Tukey’s multiple comparisons test. Graphs display mean ± SD (A-B, D-F). (ns, p >0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; or ****, p ≤ 0.0001).