MICU1 and MICU2 Play an Essential Role in Mitochondrial Ca 2+ Uptake, Growth, and Infectivity of the Human Pathogen Trypanosoma cruzi

Figures

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  FIG 1. Structural domains and phylogeny of TcMCU1 and TcMICU2. (A) Domain structure of TcMICU1 and TcMICU2 highlighting the amino acid sequence of the two canonical EF-hands. (B) A phylogenetic tree was constructed using the neighbor-joining method with 33 selected MICU1 and MICU2 predicted homologs. The sequences branched into two divergent clades containing MICU1 and MICU2, which are enclosed in a green box and blue box, respectively. Bootstrap values higher than 50 are shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The scale is in units of the number of amino acid substitutions per site. Accession numbers of sequences used for panels A and B are shown in Fig. S2.

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  FIG 2. Localization of TcMICU1 and TcMICU2 and analysis of overexpression mutants. (A) TcMICU1-2×HA overexpression (MICU1-OE) was confirmed by Western blot analysis using anti-HA monoclonal antibodies, using wild-type (WT) cells as a control cell line. Tubulin was used as a loading control. (B) Immunofluorescence analysis (IFA) showed partial colocalization between TcMICU1-2×HA (HA [green]), and Mitotracker (MT [red]) in the merged image (yellow). DAPI is in blue. DIC, differential interference contrast. (C) Growth of control (epimastigotes transfected with pTREX-n empty vector [EV]) and MICU1-OE epimastigotes in LIT medium. (D) Representative traces of Ca2+ uptake by digitonin-permeabilized MICU1-OE and control epimastigotes in relative fluorescence units (RFU). The reaction was started after addition of 50 μM digitonin (DIG) in the presence of 20 μM free Ca2+, 5 mM succinate, and 0.5 μM Calcium Green-5N probe. Where indicated, 4 μM FCCP was added. Fluorescence changes were monitored using the Hitachi F-4500 spectrofluorometer. (E) Quantification of data from three experiments like that represented in panel D. (F) Western blot analysis of total protein extracts of WT and TcMICU2-2×HA…

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  FIG 3. Phenotypic changes of TcMICU1-KO and TcMICU2-KO cells in different life cycle stages. (A) Schematic representation of the strategy used to generate a TcMICU1-KO mutant by CRISPR/Cas9-induced homologous recombination. A double-stranded gDNA break was produced by Cas9 at nt +70 of the TcMICU1 ORF (1,221 bp). DNA was repaired with a blasticidin-S deaminase (Bsd) cassette containing 100-bp homologous regions from TcMICU1 5′ and 3′ untranslated regions (UTRs). (B) Primers (arrows) used to verify gene replacement by PCR. The intact locus generates a PCR product of 1,544 bp, while the disrupted locus generates a fragment of 722 bp. (C) PCR analysis showing that TcMICU1 was ablated at its genomic locus and replaced in genomic DNA of the KO cell line. Lanes: L, 1-kb plus ladder; 1, wild type; 2, TcMICU1-KO; 3, PCR negative control. (D) Southern blot analysis of wild-type (WT) and TcMICU1-KO (KO) gDNA digested with PvuII restriction enzyme. The blot was hybridized with a biotin-labeled probe corresponding to 430 bp of TcMICU1 (nt +655 to +1085). (E) Schematic representation of the strategy used to generate a TcMICU2-KO mutant by CRISPR/Cas9-induced homologous recombination. A…

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  FIG 4. Analysis of mitochondrial Ca2+ uptake and membrane potential in TcMICU1-KO and TcMICU2-KO epimastigotes. (A) Representative Ca2+ uptake traces by digitonin-permeabilized epimastigotes in relative fluorescence units (RFU). Scrambled, scrambled control cells; MICU1-KO, TcMICU1-KO cells; MICU2-KO, TcMICU2-KO cells; MCU-KO, TcMCU-KO cells; RR, 5 μM ruthenium red (RR). The reaction was started after addition of 50 μM digitonin (DIG) in the presence of 20 μM CaCl2, 5 mM succinate, and 0.5 μM Calcium Green-5N probe. Where indicated, 4 μM FCCP was added. (B) Changes in mitochondrial membrane potential (Δψm) of digitonin-permeabilized epimastigotes as detected by changes in safranin O fluorescence in scrambled, MICU1-KO, and MICU2-KO epimastigotes. Cells (5 × 107) were added to the reaction buffer (2 ml) containing 0.2% BSA, 5 mM succinate, 50 μM EGTA and 5 μM safranin O. The reaction was started with 50 μM DIG, and 250 μM ADP, 1.5 μM carboxyatractyloside (CAT), and 4 μM FCCP were added where indicated. A decrease in fluorescence after permeabilization with digitonin indicated accumulation of the dye in energized mitochondria. Addition of ADP produced a small dissipation of membrane…

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  FIG 5. Mitochondrial and autophagy changes in TcMICU1-KO and TcMICU2-KO epimastigotes. (A) Representative traces of oxygen consumption rate (OCR) by digitonin-permeabilized control (scrambled), TcMICU1-KO (MICU1-KO), and TcMICU2-KO (MICU2-KO) epimastigotes. (B) OCR of digitonin-permeabilized scrambled, MICU1-KO, and MICU2-KO cells. Bar charts show OCR after addition of 100 μM ADP (respiration stimulated by oxidative phosphorylation [OXPHOS]), 1 μg/ml oligomycin (minimal respiratory rate), and 1 μM FCCP (maximal respiratory capacity). Values are means ± SD (n = 3). *, P < 0.05, **, P < 0.01, and ***, P < 0.001, by two-way ANOVA with Dunnett’s multiple comparisons. (C) Representative Western blot of TcPDH-E1α subunit phosphorylation in TcMCU-KO, scrambled (Scr), TcMICU1-KO, and TcMICU2-KO cell lines. The antibody used detects the T. cruzi PDH-E1α subunit phosphorylated protein (expected size of 42.8 kDa). Tubulin was used as a loading control. (D) Densitometry analysis of three Western blots (as in panel C). Densitometric analysis was performed with ImageJ v1.48 software. Values are mean ± SD (n = 3). *, P < 0.05, and ***, P < 0.001, by one-way ANOVA with Dunnett’s multiple-comparison…