Fig 2: KIF5B drives mitochondrial tubulation in vitro. (A) Schematic diagram of in vitro mitochondrial tubulation. Purified mitochondria were incubated with full-length KIF5B on ice, transferred into flow chamber channels coated with polymerized microtubules and visualized in the presence of ATP. (B) Purified mitochondria were labeled with CM-DiI, incubated with KIF5B, transferred into flow chamber channels coated with polymerized microtubules, and visualized in the presence of ATP. Several controls were carried out to test the requirement for each component. Scale bar, 5 µm. (C) Time-lapse sequence of mitochondrial tubulation in the presence of ATP. Mitochondria were labeled with CM-DiI and microtubules were labeled with HyLite 647. Scale bar, 5 µm. (D) Time-lapse sequence of mitochondrial tubulation in the presence of ATP. Scale bar, 5 µm. (E) Samples from B were stained with antibody against TOM20, and observed by confocal microscopy. Scale bar, 10 µm. Red dashed box is enlarged in the lower panel. Scale bar in enlarged panel, 5 µm. (F) Samples from B were analyzed by FEISEM. Scale bar, 1 µm.

Fig 3: The relative protein levels of five nucleoside-triphosphatase activity-related proteins measured using PRM. A1: ganglionic part of male L-HSCR; A2: aganglionic part of male L-HSCR. B1: ganglionic part of male S-HSCR; B2: aganglionic part of male S-HSCR. C1: ganglionic part of female S-HSCR; C2: aganglionic part of female S-HSCR. Data represent mean ± SEM. *p = 0.05; **p = 0.01; ***p = 0.001; ****p = 0.0001. The aganglionic part was compared with the ganglionic part; the p values were calculated using an unpaired t test. A, in the A group, the ARF4 peptide QDLPNAMAISEMTDK intensity of A1 and A2 were 6.427E8 ± 3.431E7 (n = 6) and 3.328E8 ± 1.403E7 (n = 6), p < 0.0001; in the B group, the ARF4 peptide QDLPNAMAISEMTDK intensity of B1 and B2 were 7.235E8 ± 5.877E7 (n = 6) and 4.832E8 ± 4.310E7 (n = 6), p = 0.0080; in the C group, the ARF4 peptide QDLPNAMAISEMTDK intensity of C1 and C2 were 7.435E8 ± 4.452E7 (n = 6) and 3.784E8 ± 7.738E7 (n = 6), p = 0.0022. B, in the A group, sum of all detected peptide intensities in ARF4 of A1 and A2 were 1.215E10 ± 4.241E8 (n = 6) and 7.602E9 ± 1.678E8 (n = 6), p < 0.0001; in the B group, sum of all detected peptide intensities in ARF4 of B1 and B2 were 1.288E10 ± 7.956E8 (n = 6) and 1.068E10 ± 2.301E8 (n = 6), p = 0.0243; in the C group, sum of all detected peptide intensities in ARF4 of C1 and C2 were 1.442E10 ± 2.400E8 (n = 6) and 1.160E10 ± 1.000E8 (n = 5), p = 0.0086. C, in the A group, sum of all detected peptide intensities in RAB8A of A1 and A2 were 1.607E7 ± 1.070E6 (n = 6) and 6.333E6 ± 589071 (n = 6), p < 0.0001; in the B group, sum of all detected peptide intensities in RAB8A of B1 and B2 were 2.086E7 ± 3.409E6 (n = 6) and 1.199E7 ± 1.029E6 (n = 6), p = 0.0319; in the C group, sum of all detected peptide intensities in RAB8A of C1 and C2 were 1.200E7 ± 682642 (n = 6) and 8.135E6 ± 1.878E6 (n = 6), p = 0.0818. D, in the A group, sum of all detected peptide intensities in KIF5B of A1 and A2 were 7.795E9 ± 6.902E8 (n = 6) and 4.763E9 ± 3.846E8 (n = 6), p = 0.0033; in the B group, sum of all detected peptide intensities in KIF5B of B1 and B2 were 4.693E9 ± 3.017E8 (n = 6) and 3.513E9 ± 1.534E8 (n = 6), p = 0.0059; in the C group, sum of all detected peptide intensities in KIF5B of C1 and C2 were 1.693E8 ± 1.735E7 (n = 6) and 5.742E7 ± 1.075E7 (n = 5), p = 0.0006. E, in the A group, sum of all detected peptide intensities in ATP2A2 of A1 and A2 were 6.842E9 ± 1.913E8 (n = 6) and 3.972E9 ± 9.854E7 (n = 6), p < 0.0001; in the B group, sum of all detected peptide intensities in ATP2A2 of B1 and B2 were 4.810E9 ± 5.061E8 (n = 6) and 3.565E9 ± 1.181E8 (n = 6), p = 0.0376; in the C group, sum of all detected peptide intensities in ATP2A2 of C1 and C2 were 7.778E9 ± 3.011E8 (n = 6) and 6.642E9 ± 1.639E8 (n = 5), p = 0.0123. F, in the A group, sum of all detected peptide intensities in RAC2 of A1 and A2 were 2.707E9 ± 1.072E8 (n = 6) and 1.840E9 ± 9.096E7 (n = 6), p = 0.0001; in the B group, sum of all detected peptide intensities in RAC2 of B1 and B2 were 4.610E9 ± 4.172E8 (n = 6) and 3.498E9 ± 2.521E8 (n = 6), p = 0.0458; in the C group, sum of all detected peptide intensities in RAC2 of C1 and C2 were 2.443E9 ± 1.642E8 (n = 6) and 1.710E9 ± 2.632E8 (n = 6), p = 0.0397. HSCR, Hirschsprung disease; L-HSCR, long-segment HSCR; S-HSCR, short-segment HSCR.

Fig 4: A model of mitochondrial transfer in MNTs between CMs and MFsMNTs physically connect CMs and MFs. Mitochondria are transported along microtubules by the motor protein KIF5B in MNTs and can rescue H/R-induced CM apoptosis. F-actin provides the basic structure of MNTs

Fig 5: ATF4 interacted with KIF17 in the DRG tissues of mice.a–f Co-IP showing the ATF4/KIF interaction in the DRG. DRG lysates were immunoprecipitated with an ATF4 antibody and immunoblotted with a KIF17, KIF3A, KIF3B, KIF5A, KIF5B, KIFC2 or ATF4 antibody as indicated. This experiment was repeated three times. g SIM images show that the colocalization between ATF4 and KIF17 in DRG neurons. Scale bar, 5 µm. h The GST pull-down assay with two purified proteins, KIF17-GST-Flag and ATF4, showed a direct interaction between ATF4 and KIF17. This experiment was repeated three times. i Interaction between KIF17 and ATF4 mutants. The ATF4 mutants were transiently co-expressed with KIF17, and the cell lysates were immunoprecipitated with a His antibody and then immunoblotted with a His or Flag antibody as indicated. This experiment was repeated three times. j The interaction level between TRPM3 and KIF17 was examined by co-IP in scrambled and ATF4 siRNA-treated mice DRG lysates. DRG lysates were immunoprecipitated with a TRPM3 antibody and immunoblotted with a KIF17 or TRPM3 antibody as indicated. This experiment was repeated three times. t4 = 12.88, P = 0.0002. k SIM images showed the colocalization between TRPM3 and KIF17 in DRG neurons from naïve, ATF4 siRNA and ATF4-overexpresing mice. Quantification data showed the colocalization rates of KIF17 with TRPM3 (colocalized yellow spots/total KIF17 positive spots) and those of TRPM3 with KIF17 (colocalized yellow spots/total TRPM3 positive spots) in DRG neurons. n = 3 mice per group. F(2,6) = 27.17, P = 0.0224 in naïve vs. ATF4-siRNA, P = 0.0255 in naïve vs. ATF4-over in KIF17 with TRPM3. F(2,6) = 33.5, P = 0.0069 in naïve vs. ATF4-siRNA, P = 0.0375 in naïve vs. ATF4-over in TRPM3 with KIF17. Scale bar, 10 µm. j Two-tailed Independent Student’s t test. k One-way ANOVA followed by Tukey’s multiple comparisons test, *P < 0.05, **P < 0.01. The error bars indicate the SEMs.