Fig 1.
RBP6-induced differentiation of PCF T. brucei cells in the absence of glucose.
(A) Imaging of procyclic, epimastigote, and metacyclic forms. Cells were characterized by means of morphological features such as shape, size, and the relative position of the kinetoplast to the nucleus, as well as by internalization of fluorescently labeled dextran. DNA was visualized by DAPI staining. (B) Quantification of the intracelullar distance between kDNA and nucleus. For each day, 50 cells were used for measurements. The red dots indicate cells with anterior localization of their kDNA. (C) Immunofluorescence analysis of RBP6OE cells at day 2 using anti-procyclin antibody. (D) Immunofluorescence analysis of RBP6OE cells at day 4 using anti-BARP antibody. (E) Time line for the appearance of epimastigotes and metacyclic cells upon induction of RBP6OE. (F) Western blot analysis of whole-cell lysates from RBP6OE cells. Underlying data plotted in panels B and E are provided in S1 Data. Ab, antibody; BARP, brucei alanine-rich protein; BSF, bloodstream form; k, kinetoplast (mitochondrial DNA); kDNA, kinetoplast DNA; n, nucleus; p, posterior end; PCF, procyclic form; RBP6, RNA binding protein 6.
Fig 2.
RBP6OE differentiation transcriptome and proteome.
(A) Scheme of the differentiation process and the experimental time points. The RBP6-induced cells were harvested and analyzed by RNA-Seq and label-free quantitative mass spectrometry at different time points, as indicated. The table contains an overview of the number of differentially expressed transcripts and proteins. (B) Gene expression profile of transcripts encoding surface glycoproteins GPEET (Tb927.6.510), BARP (Tb927.5.15530, Tb927.5.15550, Tb927.5.15560, Tb927.5.15600, Tb927.5.15590), and T. brucei 427 mVSG (Tb427_000106600.1, Tb427_000524500.1, Tb427_000173600.1, Tb427_000288600.1, Tb427_000304300.1, Tb427_000108300.1, Tb427_000627000.1, Tb427_000615900.1). (C) Time-course expression profiling of transcriptomic data based on K-medoids clustering. (D) Gene Ontology (GO) term analysis applied to all four transcriptomics clusters. Significant levels are depicted as follows **P < 0.01, ***P < 0.0001. (E) Volcano plots showing a comparison of protein expression levels (5,227 protein groups) between day 0 and day 2 (left panel) or day 6 (right panel) upon RBP6 induction. Log2 fold change values of averaged LFQ intensities from quadruplicate experiments are plotted against the respective −log10-transformed P values. Significantly down-regulated proteins are depicted in blue, while significantly up-regulated proteins are depicted in red. RBP6, AOX, and surface glycoproteins BARP (day 2) and metacyclic (m)VSGs (day 6) are highlighted. (F) Time-course expression profiling of proteomic data based on K-medoids clustering. (G) GO term analysis of Clusters 1, 3, and 4 showing a significant enrichment. **P < 0.01, ***P < 0.001. Underlying data plotted in panel B are provided in S1 Data. AOX, alternative oxidase; BARP, brucei alanine-rich protein; EP, procyclin rich in Glu-Pro repeats; GO, Gene Ontology; GPEET, procyclin rich in Gly-Pro-Glu-Glu-Thr repeats; k, kinetoplast; LC-MS/MS, liquid chromatography tandem mass spectrometry; LFQ, label-free quantification; mVSG, metacyclic-like variable surface glycoprotein; n, nucleus; RBP6, RNA binding protein 6.
Fig 3.
Major changes in protein abundance during RBP6 overexpression.
(A) Heatmaps showing log2 fold change of average LFQ intensities of selected proteins identified in induced samples compared to uninduced. The color key differs for each map and is always located below the heatmap. (B) Western blot analyses of whole-cell lysates from RBP6OE cells undergoing differentiation using a panel of various antibodies. Mitochondrial (mt) hsp70 serves as a loading control because its expression remains constant. AOX, alternative oxidase; AT, aminotransferase; BSF, bloodstream form; DH, dehydrogenase; FBPase, fructose 1,6-bisphosphatase; GAP DH, glyceraldehyde-3-phosphate dehydrogenase; G3P DH, glycerol-3-phosphate dehydrogenase; hsp70, heat shock protein 70; LFQ, label-free quantification; PCF, procyclic form; PDH, pyruvate dehydrogenase; PGK, phosphoglycerate kinase; PGMA, phosphoglycerate mutase; pyr-5-carb DH, pyrroline-5 carboxylate dehydrogenase; RBP6, RNA binding protein 6; SBPase, sedoheptulose 1,7-bisphosphatase; SCoAS, succinyl CoA synthetase; SDH, succinate dehydrogenase; TIM, triose-phosphate isomerase.
Fig 4.
Schematic representation of changes in selected mitochondrial and glycosomal pathways.
Enzymatic steps are represented by arrows with different thicknesses, depending on the observed abundance change of the respective protein at day 6 upon RBP6 induction. Dashed lines indicate steps that were down-regulated. Alternative dehydrogenase with unresolved orientation is in gray. The metabolic end products are highlighted by black lines. ACH, acetyl-CoA thioesterase; Aco, aconitase; AKB–CoA lyase, 2-amino-3-ketobutyrate Coenzyme A lyase; Ala TR, alanine aminotransferase; AOX, alternative oxidase; ASCT, acetate:succinate CoA-transferase; cI, complex I, NADH:ubiquinone oxidoreductase; cII, succinate dehydrogenase; cIII, complex III, ubiquinol:cytochrome c reductase; cIV, complex IV, cytochrome c oxidase; cV, FoF1-ATP synthase; CS, citrate synthase; DHAP, dihydroxyacetone phosphate; Eno, enolase; FBPase, fructose 1,6-bisphosphatase; FR, fumarate reductase; Fum, fumarase; F1,6 BP, fructose 1,6 bisphosphate; F6P, fructose-6-phosphate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Glu DH, glutamate dehydrogenase; Gly-3-P, glycerol-3-phosphate; GPI, glucose-6-phosphate isomerase; G3P DH, glycerol-3-phosphate dehydrogenase; G6P, glucose-6-phosphate; G6P DH, glucose-6-phosphate dehydrogenase; Iso DH, isocitrate dehydrogenase; IsoCit, isocitrate; KDH, α-ketoglutarate dehydrogenase; MD, malate dehydrogenase; ME, malic enzyme; OXPHOS, oxidative phosphorylation; PDH, pyruvate dehydrogenase; PEP, phosphoenolpyruvate; PEPCK, phosphoenolpyruvate carboxykinase; PG, phosphoglycerate; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; PK, pyruvate kinase; PPDK, pyruvate, phosphate dikinase; PPP, pentose phosphate pathway; Pro DH, proline dehydrogenase; pyr-5-carb, pyrroline-2-carboxylate; pyr-5-carb DH, pyrroline-5 carboxylate dehydrogenase; RBP6, RNA binding protein 6; SUBPHOS, substrate phosphorylation; SCoAS, succinyl-Coenzyme A synthetase; SucCoA, succinyl-CoA; TDH, threonine dehydrogenase; TIM, triose-phosphate isomerase.
Fig 5.
RBP6OE cells respire predominantly via AOX.
(A) The resting respiration of cells undergoing RBP6-induced differentiation was measured using the O2k-oxygraph. The ratio of complex IV–and AOX-mediated respiration was determined using KCN, a potent inhibitor of complex IV, and SHAM, a potent inhibitor of AOX. Individual values shown as dots (mean ± SD, n = 3–5), ****P < 0.0001. (B) Glycerol-3-phosphate stimulated respiration in live cells. The proportion of complex IV–and AOX-mediated respiration was determined as in (A). Individual values shown as dots (mean ± SD, n = 5–8), **P < 0.01. (C) In vitro ATP production was measured in digitonin-extracted mitochondria. The OxPhos pathway was triggered by the addition of ADP and glycerol-3-P. Treatment with 1 mM KCN serves as a control. Individual values shown as dots (mean ± SD, n = 3–4). Underlying data plotted in panels (A), (B), and (C) are provided in S1 Data. AOX, alternative oxidase; Gly-3-P, glycerol-3-phosphate; KCN, potassium cyanide; OxPhos, oxidative phosphorylation; RBP6, RNA binding protein 6; SHAM, salicylhydroxamic acid.
Fig 6.
RBP6OE-induced changes in levels and activities of ETC complexes and FoF1-ATP synthase.
In-gel activity staining and western blot analysis of respiratory complexes II, III, and IV and FoF1-ATP synthase (complex V, cV). Mitochondrial preparations were solubilized using dodecyl maltoside and the same amount of the protein samples was separated on NativePAGE 3%–12% Bis-Tris protein gels followed by in-gel activity staining specific for individual complexes (left panels) or by western blot analysis using specific antibodies (middle panels). Mitochondrial lysates were also evaluated by SDS-PAGE and western blot analysis for individual subunits of complexes II, III, IV, and V (right panels). ETC, electron transport chain; RBP6, RNA binding protein 6.
Fig 7.
Mitochondrial membrane potential (Δψm) is increased during RBP6OE.
(A) The Δψm of RBP6OE cells post induction was measured by flow cytometry using TMRE. A protonophore FCCP serves as a control for membrane depolarization (mean ± SD, n = 6–10) *P < 0.05, ***P < 0.001. (B) The proportion of Δψm that is generated by complex IV was established by treating the cells with KCN (0.5 mM) in the presence of TMRE for 30 minutes before the analysis. The graph shows a proportion of KCN-sensitive Δψm to the total Δψm measured in each individual sample (mean ± SD, n = 5). (C) Western blot analysis of FoF1-ATPase inhibitory factor TbIF1 during RBP6OE. Mitochondrial (mt) hsp70 serves as a loading control. (D) The in situ dissipation of the Δψm in response to chemical inhibition of complex IV by 1 mM NaCN was measured using safranine O dye in RBP6OE uninduced (UNIND) cells and cells induced for 4 and 6 days. The reaction was initiated with digitonin; OM, oligomycin (2.5 μg/mL), and FCCP (5 μM) were added when indicated (mean ± SD, n = 3). (E) The Δψm of RBP6OE cells that were treated (red columns) or not with oligomycin (2.5 μg/mL). Individual values shown as dots (mean ± SD, n = 3–6). Underlying data plotted in panels A, B, D, and E are provided in S1 Data. BSF, bloodstream cell; FCCP, carbonyl cyanide-4-phenylhydrazone; KCN, potassium cyanide; PCF, procyclic cell; RBP6, RNA binding protein 6; TbIF1, T. brucei inhibitory peptide 1; TMRE, tetramethyl rhodamine ethyl ester.
Fig 8.
Changes in cellular respiration and mitochondrial ATP production in RBP6OE cells.
(A, B) Oxygen consumption rates in the presence of 5 mM proline (A) or 5 mM succinate (B) in live or digitonin-permeabilized cells, respectively. Respiration via AOX was monitored in the presence of KCN (0.5 mM). Individual values shown as dots (means ± SD, n = 3–5). ****P < 0.0001. (C, D) The in vitro ATP production by oxidative or substrate phosphorylation (OXPHOS, SUBPHOS) measured in digitonin-extracted mitochondria from uninduced and RBP6-induced cells. The phosphorylation pathways are triggered by the addition of ADP and by succinate (C) or α-ketoglutarate (a-KG, D). Malonate (mal.) and KCN, specific inhibitors of succinate dehydrogenase and complex IV are used to inhibit ATP production by OXPHOS. The levels of ATP production in mitochondria isolated from uninduced RBP6OE cells are established as the reference and set to 100% (means ± SD, n = 2–4). (E) Cellular ATP content in RBP6OE cells. (means ± SD, n = 6, ****P < 0.0001). (F) Relative ADP/ATP ratios of RBP6OE cells. The ADP/ATP ratio in uninduced RBP6OE cells (between 1.75 and 6.01) is established as a reference and set to 1. In T. brucei, ADP/ATP ratio reaches unusually high levels, as also reported elsewhere [44]. The measured values are shown in S1 Data (means ± SD, n = 6–10, **P < 0.01). Underlying data plotted in panels A, B, C, D, E, and F are provided in S1 Data. AOX, alternative oxidase; KCN, potassium cyanide; OXPHOS, oxidative phosphorylation; RBP6, RNA binding protein 6; SHAM, salicylhydroxamic acid; SUBPHOS, substrate phosphorylation.
Fig 9.
Metabolomics profiling of RBP6OE cells.
(A) Volcano plot showing the full metabolome (698 metabolites) analyzed at day 0 and day 2 upon RBP6 induction. Log2 fold change values of the average of mean peak area from quadruplicate experiments are plotted against the respective −log10 transformed P values. Few key metabolites are highlighted. (B) Volcano plot showing the full metabolome analyzed at day 2 (gray) and 8 (blue) upon RBP6 induction compared to day 0. Log2 fold change values of the average of mean peak area from quadruplicate experiments are plotted against the respective −log10 transformed P values. (D, E, F) Heatmaps showing log2 fold change of average of mean peak area of selected metabolites identified in induced samples compared to uninduced (day 0). The color key differs for each map and is always located below the heatmap. Heatmaps were generated with GraphPad prism 8.2.0. RBP6, RNA binding protein 6; TCA, tricarboxylic acid.
Fig 10.
Elevated ROS generated during the differentiation of T. brucei are crucial for driving forward RBP6-induced differentiation.
(A) Metabolites glutathione and glutathione disulfide as detected by LC-MS analyses. The size of the bars represents the total abundance of the metabolite (mean ± SD, n = 4, ***P < 0.001). (B) Mitochondrial and cellular ROS detection reagents (MitoSox and H2DCFDA, respectively) were quantified by FACS (means ± SD, n = 5, ***P < 0.001, ****P < 0.0001). (C) Representative growth curves of RBP6OE and RBP6OE_catalase cells induced by tetracycline. Total number of experiments n = 5. The inset shows subcellular localization of v5-tagged catalase in RBP6OE_catalase cells induced for 48 hours. Immunoblots were labeled with anti-v5, anti-adenosine phosphoribosyl transferase (APRT), and anti-mt hsp70 antibodies to visualize catalase, cytosolic APRT, and mitochondrial localized hsp70, respectively. (D) Time line for the appearance of epimastigotes and metacyclic cells upon induction of RBP6OE and RBP6OE_catalase. Total number of experiments n = 3. (E) Western blot analysis of whole-cell lysates from RBP6OE and RBP6OE_catalase cells using available antibodies. (F) FACS analyses of RBP6OE and RBP6OE_catalase cells treated with 5 mM bathophenanthroline disulphonic acid (BPS) and labeled with polyclonal anti-BARP and anti-procyclin antibodies. (G) The Δψm of RBP6OE_catalase cells post induction measured by flow cytometry using TMRE (means ± SD, n = 4), *P < 0.05. (H) Mitochondrial ROS detection reagent MitoSox in RBP6OE_catalase cells quantified by FACS (means ± SD, n = 5), *P < 0.05, ***P < 0.001. (I) Cellular ROS detection reagent H2DCFDA in RBP6OE_catalase cells quantified by FACS (means ± SD, n = 5). Underlying data plotted in panels A, B, C, D, F, G, H, and I are provided in S1 Data. AOX, alternative oxidase; APRT, adenine phosphoribosyl transferase; BARP, brucei alanine-rich protein; CYT, cytosol; hsp70, heat shock protein 70; H2DCFDA, 2′,7′-dichlorofluorescin diacetate; LC-MS, liquid chromatography–mass spectrometry; ns, statistically not significant; ORG, organellar fraction; RBP6, RNA binding protein 6; ROS, reactive oxygen species; SCoAS, succinyl Co-A synthetase; TMRE, tetramethyl rhodamine ethyl ester; WCL, whole-cell lysate.