Extended Data Fig. 5: Characterization of dhgd-1, hphd-1, and immt-1 mutants, expression of MICOS components, and 3-HP biding with IMMT-1.
From: A feedback loop engaging propionate catabolism intermediates controls mitochondrial morphology
a, Representative TEM images of mitochondria in the hypodermis and muscle cells in N2, dhgd-1(tm6671) and hphd-1(ok3580) animals grown on E. coli OP50. Bars, 2 μm. Quantifications of abnormal mitochondria are shown on the right. ≥6 mitochondria in each cell type in animals of each genotype were examined, the exact number of mitochondria is provided as source data. n = 3 independent biological samples. b, Graphic depiction of the MICOS complex. SAM, sorting and assembly machinery. c, Diagram of the immt-1 gene and the encoded protein. Filled boxes and thin lines represent exons and introns, respectively. The yq160 point mutation is indicated with a red line. In the protein, blue, red, green and purple boxes indicate the transmembrane domain (TM), the coiled-coil domain, the lipid binding sites (LBS), and the mitofilin domain, respectively. d, Representative images of mitochondria in the hypodermis of immt-1(yq160) mutants. Mitochondria are labeled with Mito-GFP and TOMM-20::mCh. Bars, 5 µm. e, Representative TEM images of mitochondria in hypodermal and muscle cells in immt-1(yq160) mutants grown on E. coli OP50. Red and yellow arrows indicate vacuoles and inner membrane stacks, respectively. Bars, 2 μm. Quantifications of hypodermal (top) and muscle (bottom) mitochondria containing only vacuoles (Vacuole only), only inner membrane stacks (Stack only), or both (Vacuole + Stack) are shown on the right. The indicated numbers of mitochondria were analyzed in n = 8 independent immt-1(yq160) animals. f, Relative mRNA levels of immt-1, immt-2, chch-3 (Mic19) and F54A3.5 (Mic10) in N2, hphd-1(ok3580) and dhgd-1(tm6671) animals. 300 animals were examined for each genotype. n = 3 independent experiments. g, Immunoblotting of IMMT-1 in N2, immt-1(yq160), hphd-1(ok3580) and dhgd-1(tm6671) animals. Quantification of IMMT-1 protein levels from two independent experiments with similar results is shown at the bottom. h, Left: Graphic depiction of the full-length and truncated IMMT-1 (top) and the corresponding His6-tagged proteins (indicated with *) used for MST assays. Right: Binding curves of 3-HP with the different His6-tagged IMMT-1 proteins shown in the left panel. Data (mean) are from two independent experiments. For statistical analyses, P values were determined using one-way ANOVA (a and g) and two-way ANOVA (f). Individual data points (mean ± s.e.m. and exact P value) are shown. ****P < 0.0001; ns, P > 0.05. Source numerical data and unprocessed gels and blots are available in source data.
