Microvillus inclusion disease-associated MYO5B deficiency impairs endosome-to-mitochondrion iron transfer

Figures

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  Figure 1. Aberrant mitochondrial morphology in Myo5b-deficient intestinal epithelial cells. (A) Large-scale EM shows small intestine villus enterocytes of wild-type and Myo5B KO mice. Images are representative of two or three mice. Scale: 1 µm. Full-resolution zoomable dataset is available at https://www.nanotomy.org. (B) Classification of control (I) and four (II–V) aberrant mitochondrial phenotypes. (C) Quantification of mitochondrial phenotypes (see A) in the whole villus and separate lower and upper parts of the villus in wild-type and Myo5B KO mice. Statistical tests were performed as described in the Methods section. P values are indicated in the graphs. WT = wild-type, KO = knockout.

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  Figure 2. Analyses of mitochondrial morphology and mitochondrial membrane potential in control and MYO5B-deficient Caco2 cells. (A) Morphology of TOMM20-positive mitochondria and membrane potential in wild-type Caco2 cells, Caco2-M5BKO cells, Caco2-M5BKO + FL cells, and point mutants MYO5B-PL, -KA, and -LP. Magnification at the right. White arrowheads indicate swollen and fragmented mitochondria. Scale: 5 µm. (B) Quantification of mitochondria branch length. (C) Quantification of MitoRedCMXRos/TOMM20 fluorescence signal ratio. Statistical tests were performed as described in the Methods section. P values are indicated in the graphs. Experiments were carried out independently more than three times. FL = full-length, PL = Pro660Leu, KA = Lys1534Ala, LP = Leu1597Pro.

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  Figure 3. Analyses of mitochondrial respiration in control and MYO5B-deficient Caco2 cells. (A) Oxygen-consumption analyses showing State 3, state uncoupled, spare respiratory capacity, and relative proton in wild-type and Caco2/M5BKO cells. Dots in graphs represent independent experiments. (B) Transcript expression of genes involved in ETC complexes. Dots in graphs represent independent experiments. (C) Transcript expression of YY1 and PGC-1B. Dots in graphs represent independent experiments. Statistical tests were performed as described in the Methods section. P values are indicated in the graphs. Experiments were carried out independently more than three times. FL = full-length.

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  Figure 4. Analyses of mitochondrial oxidative damage in control and MYO5B-deficient Caco2 cells. (A) Detection of superoxide using MitoSOX in wild-type and Caco2/M5BKO cells. Scale: 20 µm. (B) Oxyblot showing increased level of oxidized proteins in isolated mitochondria (MW: molecular weight marker). Panel on the right shows quantification of three independent oxyblots. (C) Growth curves of wild-type and Caco2/M5BKO cells in glucose- or galactose-containing medium. Statistical tests were performed as described in the Methods section. P values are indicated in the graphs. Experiments were carried out independently more than three times. *P < 0.05, **P < 0.01, ****P < 0.0001; NS = not significant, Glu = glucose, Gal = galactose.

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  Figure 5. Co-distribution of MYO5B-positive endosomes and mitochondria in control and MYO5B-deficient Caco2 cells. (A) Immunofluorescence microscopy showing distributions of MYO5B, TfR, and mitochondria (CMXRos) in wild-type and Caco2/M5BKO cells. Scale: 5 µm. (B) Immunofluorescence microscopy showing distributions of MYO5B, Alexa488-labeled transferrin (Tf), and mitochondria (TOMM20) in wild-type and Caco2-M5BKO cells. Scale: 5 µm. (C) 3D reconstruction of TOMM20- and Alexa488-Tf-positive compartments. The purple color code indicates the nearest neighbors based on the calculated distance between the two fluorescent labels. Scale: 2 µm. (D) Quantification of (C). Statistical tests were performed as described in the Methods section. P values are indicated in the graphs. Experiments were carried out independently more than three times. TfR = transferrin receptor, Tf488 = transferrin-488.

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  Figure 6. Effect of MYO5B deficiency on mitochondrial iron in control and MYO5B-deficient Caco2 cells. (A) Staining of wild-type and Caco2/M5BKO cells with the iron-sensor RDA (green) and MitoTracker Deep Red (red). Scale: 10 µm. (B) Quantification of RDA fluorescence per cell in wild-type and Caco2/M5BKO cells, as such and corrected for MitoTracker Deep Red FM fluorescence intensity. (C) Transcript expression of genes involved in iron-related mitochondrial processes. Dots in graphs represent independent experiments. Statistical tests were performed as described in the Methods section. P values are indicated in the graphs. Experiments were carried out independently more than three times. RDA = rhodamine B 4-[(2,2’-bipyridin-4-yl) aminocarbonyl] benzyl ester, FL = full-length.

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  Figure 7. Effect of MYO5B deficiency on endosomal iron levels in control and MYO5B-deficient Caco2 cells. Staining of wild-type and Caco2/M5BKO cells, treated or not treated with the iron chelator DFO, with the endosomal iron-sensor calcein (green) and Lysotracker (red). Merged images and bright-field images are shown. Experiments were carried out independently more than three times. Scale: 10 µm. BF = bright field.