Fig 1: The MTX-2/MIRO-1/MTX-1 complex links KHC/UNC-116 through light chain KLC-1.a Representative confocal images showing GFP::MTX-1 (single copy) and TOMM-20::mCherry in PVD cell body of wild type and mtx-2 mutant. Scale bar: 10 µm. b, c Representative confocal images of GFP::MTX-2 (b) and GFP::MTX-1 (c) in wild type, mtx-2, mtx-1, or miro-1 mutants in the anterior and posterior dendrites of PVD. Scale bar: 10 µm. d–g Quantification of mitochondria number by overexpressing GFP::MTX-2 and GFP::MTX-1 in wild type, mtx-2, mtx-1, or miro-1 mutants in anterior and posterior dendrites of PVD. Data are shown as mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons test (95% CI). p > 0.05, not significant, ****p < 0.0001. (exact p values and sample size: d PVD > gfp::mtx-2 vs. mtx-2 PVD > gfp::mtx-2, p = 0.9280; PVD > gfp::mtx-2 vs. mtx-1 PVD > gfp::mtx-2, p = 0.9917; n = 25 animals for each genotype. e PVD > gfp::mtx-2 vs. mtx-2 PVD > gfp::mtx-2, p = 0.9040; PVD > gfp::mtx-2 vs. mtx-1 PVD > gfp::mtx-2, p = 0.4083; n = 25 animals for each genotype. f PVD > gfp::mtx-1 vs. mtx-1 PVD > gfp::mtx-1, p = 0.9615; PVD > gfp::mtx-1 vs. miro-1 PVD > gfp::mtx-1, p < 0.0001; n = 25 animals for each genotype. g PVD > gfp::mtx-1 vs. mtx-1 PVD > gfp::mtx-1, p = 0.9372; PVD > gfp::mtx-1 vs. miro-1 PVD > gfp::mtx-1, p = 0.1338; n = 25 animals for each genotype). h Isolated mitochondrial lysates of HEK293T cells were immunoprecipitated with ANTI-Miro1 antibody, followed by western blot analysis with Miro1, Mtx2, Mtx1, and Tom20 antibodies. n = 3 independent experiments. In the experiment, the input was 2.5% of the total isolated mitochondrial lysates, and the IP was 25% of the total elution. n = 3 independent experiments. The cytosol fraction was included as a control. i Analytical gel-filtration analysis of the interaction between MIRO-1 and the MTX-1/MTX-2 complex. j GST pull-down assay between MIRO-1 and the MTX-1/MTX-2 complex. k Analytical gel-filtration analysis of the interaction between the MTX-1/MTX-2 complex and the TPR domain of KLC-1 (KLC-1-TPR). l GST pull-down assay of the interaction between the MTX-1/MTX-2 complex and KLC-1-TPR. m Analytical gel-filtration analysis of the formation of the MIRO-1/MTX-2/MTX-1/KLC-1-TPR complex in solution. n GST pull-down assay of the MIRO-1/MTX-2/MTX-1/KLC-1-TPR complex. n = 3 independent experiments. o A schematic diagram showing the putative molecular complex containing MTX-2, MTX-1, MIRO-1, and KLC-1 on mitochondria. Source data are provided as a Source Data file.
Fig 2: Alleviated demyelination and cognitive impairment by a high-dose treatment of D1-NBP was accompanied by decreased mitochondrial accumulation among the axons. (A–D) Representative TEM images of mitochondrial load in axons (A) and quantitative analysis of mitochondrial number per area (/μm2) (B), mitochondrial length (C) and mitochondrial diameter (D) in Sham+Placebo, Sham+L-NBP, Sham+H-NBP, BCAS+Placebo, BCAS+L-NBP, BCAS+H-NBP groups. Scale bar, 2 μm. A one-way ANOVA with Tukey's correction. For mitochondrial load per area analysis, n = 20 visual fields (4 visual fields per mouse, 5 mice per group). For mitochondrial length analysis, n = 50 (10 mitochondria per mouse, 5 mice per group). For mitochondrial diameter analysis, n = 50 (10 mitochondria per mouse, 5 mice per group). (E,F) Immunoblot (E) and quantitative analysis (F) of syntaphilin (SNPH), Miro1, and Milton in different groups. A one-way ANOVA with Tukey's correction, n = 8 mice per group. Data are represented as means ± SD (*p < 0.05; **p < 0.01; ***p < 0.001; ns, non-significant differences).
Fig 3: Impairment of mitochondrial trafficking leads to dendrite degeneration.a A schematic diagram showing the distal, proximal, and posterior dendrites of PVD. Anterior is to the left and dorsal is up. b Confocal images of PVD > GFP in wild type, miro-1, trak-1, mtx-1, and mtx-2 mutants at Day 6. Scale bar: 10 μm. c, d Quantification of the number of 2o and 4o in the distal dendrite in wild type, miro-1, trak-1, mtx-1, and mtx-2 mutants at Day 1, 2, 4, and 6. Data are shown as mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons test (95% CI). p > 0.05, not significant, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. (exact p values and sample size: c wild-type D1 vs. miro-1 D1, p = 0.9764; wild-type D1 vs. trak-1 D1, p = 0.9176; wild-type D1 vs. mtx-1 D1, p = 0.9908; wild-type D1 vs. mtx-2 D1, p = 0.3889; wild-type D2 vs. miro-1 D2, p = 0.9816; wild-type D2 vs. trak-1 D2, p = 0.7995; wild-type D2 vs. mtx-1 D2, p > 0.9999; wild-type D2 vs. mtx-2 D2, p = 0.9419; wild-type D4 vs. miro-1 D4, p = 0.0319; wild-type D4 vs. trak-1 D4, p = 0.1314; wild-type D4 vs. mtx-1 D4, p > 0.9999; wild-type D4 vs. mtx-2 D4, p = 0.0627; wild-type D6 vs. miro-1 D6, p < 0.0001; wild-type D6 vs. trak-1 D6, p < 0.0001; wild-type D6 vs. mtx-1 D6, p > 0.9999; wild-type D6 vs. mtx-2 D6, p < 0.0001; miro-1 D1 vs. miro-1 D2, p > 0.9999; miro-1 D1 vs. miro-1 D4, p = 0.7763; miro-1 D1 vs. miro-1 D6, p < 0.0001; trak-1 D1 vs. trak-1 D2, p > 0.9999; trak-1 D1 vs. trak-1 D4, p = 0.9856; trak-1 D1 vs. trak-1 D6, p < 0.0001; mtx-1 D1 vs. mtx-1 D2, p > 0.9999; mtx-1 D1 vs. mtx-1 D4, p = 0.9999; mtx-1 D1 vs. mtx-1 D6, p > 0.9999; mtx-2 D1 vs. mtx-2 D2, p > 0.9999; mtx-2 D1 vs. mtx-2 D4, p = 0.9907; mtx-2 D1 vs. mtx-2 D6, p = 0.0146. wild-type D1, n = 35; miro-1 D1, n = 19; trak-1 D1, n = 20; mtx-1 D1, n = 20; mtx-2 D1, n = 27; wild-type D2, n = 35; miro-1 D2, n = 21; trak-1 D2, n = 20;mtx-1 D2, n = 20; mtx-2 D2, n = 27; wild-type D4, n = 21; miro-1 D4, n = 18; trak-1 D4, n = 17; mtx-1 D4, n = 21; mtx-2 D4, n = 15; wild-type D6, n = 21; miro-1 D6, n = 17; trak-1 D6, n = 18; mtx-1 D6, n = 18; mtx-2 D6, n = 15 animals. d wild-type D1 vs. miro-1 D1, p = 0.9160; wild-type D1 vs. trak-1 D1, p = 0.4083; wild-type D1 vs. mtx-1 D1, p > 0.9999; wild-type D1 vs. mtx-2 D1, p = 0.9562; wild-type D2 vs. miro-1 D2, p = 0.4225; wild-type D2 vs. trak-1 D2, p = 0.0355; wild-type D2 vs. mtx-1 D2, p = 0.9994; wild-type D2 vs. mtx-2 D2, p = 0.4100; wild-type D4 vs. miro-1 D4, p < 0.0001; wild-type D4 vs. trak-1 D4, p < 0.0001; wild-type D4 vs. mtx-1 D4, p > 0.9999; wild-type D4 vs. mtx-2 D4, p < 0.0001; wild-type D6 vs. miro-1 D6, p < 0.0001; wild-type D6 vs. trak-1 D6, p < 0.0001; wild-type D6 vs. mtx-1 D6, p = 0.9972; wild-type D6 vs. mtx-2 D6, p < 0.0001; miro-1 D1 vs. miro-1 D2, p > 0.9999; miro-1 D1 vs. miro-1 D4, p = 0.0010; miro-1 D1 vs. miro-1 D6, p < 0.0001; trak-1 D1 vs. trak-1 D2, p > 0.9999; trak-1 D1 vs. trak-1 D4, p = 0.0018; trak-1 D1 vs. trak-1 D6, p < 0.0001; mtx-1 D1 vs. mtx-1 D2, p > 0.9999; mtx-1 D1 vs. mtx-1 D4, p > 0.9999; mtx-1 D1 vs. mtx-1 D6, p > 0.9999; mtx-2 D1 vs. mtx-2 D2, p > 0.9999; mtx-2 D1 vs. mtx-2 D4, p = 0.0032; mtx-2 D1 vs. mtx-2 D6, p < 0.0001. wild-type D1, n = 35; miro-1 D1, n = 30; trak-1 D1, n = 30; mtx-1 D1, n = 30;mtx-2 D1, n = 30; wild-type D2, n = 35; miro-1 D2, n = 30; trak-1 D2, n = 30; mtx-1 D2, n = 30; mtx-2 D2, n = 30; wild-type D4, n = 30; miro-1 D4, n = 30; trak-1 D4, n = 30; mtx-1 D4, n = 30; mtx-2 D4, n = 30; wild-type D6, n = 30; miro-1 D6, n = 30; trak-1 D6, n = 30; mtx-1 D6, n = 30; mtx-2 D6, n = 30 animals). Source data are provided as a Source Data file.
Fig 4: The MTX-2/MIRO-1/TRAK-1 complex mediates dynein-based mitochondria transport.a Representative confocal images showing dendritic morphology and mitochondrial distribution of PVD neuron in wild type, trak-1, dli-1 mutants, and trak-1 PVD > miro-1 animals. Magenta: PVD dendrites. Green: PVD > TOMM-20 (1-54AA)::GFP. Scale bar 10 μm. b, c Quantification of mitochondria number in the anterior (b) and posterior (c) dendrites of PVD in wild type, trak-1, dli-1 mutants, and trak-1 PVD > miro-1 animals. Data are shown as mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons test (95% CI). p > 0.05, not significant, ****p < 0.0001. (exact p values and sample size: b wild-type vs. trak-1, p < 0.0001; wild-type vs. dli-1, p < 0.0001; wild-type vs. trak-1 PVD > miro-1, p < 0.0001. wild-type, n = 72; trak-1, n = 72; dli-1, n = 35; trak-1 PVD > miro-1, n = 35 animals. c wild-type vs. trak-1, p = 0.6263; wild-type vs. dli-1, p = 0.9880; wild-type vs. trak-1 PVD> miro-1, p = 0.9015. wild-type, n = 72; trak-1, n = 60; dli-1, n = 35; trak-1 PVD> miro-1, n = 32 animals). d Representative confocal images showing mitochondria distribution of DA9 neuron in wild type, trak-1, and dli-1 mutants. Green: DA9 > TOMM-20 (1-54AA)::GFP. Scale bar: 10 μm. e, f Quantification of mitochondria number in dendrite and axon of DA9 in wild type, trak-1, and dli-1 mutants. Data are shown as mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons test (95% CI). p > 0.05, not significant, ****p < 0.0001, **p < 0.01, *p < 0.05. (exact p values and sample size: e wild-type vs. trak-1, p < 0.0001; wild-type vs. dli-1, p < 0.0001. wild-type, n = 23; trak-1, n = 28; dli-1, n = 29 animals. f wild-type vs. trak-1, p = 0.0012; wild-type vs. dli-1, p = 0.0458. wild-type, n = 15; trak-1, n = 19; dli-1, n = 19 animals). g Representative confocal images showing GFP::MTX-2 (single copy) in wild type and trak-1 mutant in the anterior and posterior dendrites of PVD. Scale bar: 10 μm. h Quantification of mitochondria number by overexpressing GFP::MTX-2 in wild type and trak-1 mutant in anterior and posterior dendrites of PVD. Data are shown as mean ± SEM. Student’s t test (95% CI). p > 0.05, not significant, ****p < 0.0001. (exact p values and sample size: anterior, PVD > gfp::mtx-2 vs. trak-1 PVD > gfp::mtx-2, p < 0.0001, n = 25 animals for each genotype. posterior, PVD > gfp::mtx-2 vs. trak-1 PVD > gfp::mtx-2, p = 0.2802, n = 25 animals for each genotype). i Representative confocal images showing single-copy transgenic GFP::MTX-1 in wild type and trak-1 mutant in anterior and posterior dendrites of PVD. Scale bar: 10 μm. j Quantification of mitochondria number of single-copy transgenic GFP::MTX-1 in wild type and trak-1 mutant in anterior and posterior dendrites of PVD. Data are shown as mean ± SEM. Student’s t test (95% CI). p > 0.05, not significant, ****p < 0.0001. (exact p values and sample size: anterior, PVD > gfp::mtx-1 vs. trak-1 PVD > gfp::mtx-1, p < 0.0001, n = 25 animals for each genotype. posterior, PVD > gfp::mtx-1 vs. trak-1 PVD > gfp::mtx-1, p = 0.0665, n = 25 animals for each genotype). k A schematic diagram showing the putative complex of MTX-2, MIRO-1, and TRAK-1 on mitochondria. Source data are provided as a Source Data file.
Fig 5: Miro1 knockdown reduces intercellular mitochondrial transfer. (a) Rhot1 knockdown efficiency was measured by quantitative real-time PCR. (b, c) Western blot confirming the efficient knockdown of Miro1. Means ± SD, n = 5. (d, e) Representative confocal images and histogram showing that CFSE+ NPCs received mitochondria from neighboring MitoTracker Red+ BMSCmiroCc and BMSCmiroLo and the mitochondrial transfer rate. Green arrowheads: NPCs; red arrowheads: BMSCs; white arrowheads: mitochondria. Scale bar, 20 μm. Means ± SD, n = 6. ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. BMSCs: bone marrow mesenchymal stem cells; BMSCmiroCc: control adenovirus-transfected BMSCs; BMSCmiroLo: BMSCs transfected with adenovirus carrying Rhot1 shRNA; NPCs: nucleus pulposus cells; rot: rotenone.
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