( D , E ) Interactions of the WT or five full - length TmMR mutants with φx174RFII were examined in the absence ( D ) or presence of AMP - PNP / Mg2 + ( E ) . Reaction conditions are same as those in Fig 3B .

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(D, E) Interactions of the WT or five full-length TmMR mutants with φx174RFII were examined in the absence (D) or presence of AMP-PNP/Mg2+ (E). Reaction conditions are same as those in Fig 3B.


( F ) Nuclease activities of free MjMre11 ( lane 3 , 4 ) , nucleotide unbound - ( 5 , 6 ) and - bound MjMRcd complex ( 7 , 8 ) towards a hairpin DNA . Each protein sample was incubated with a hairpin DNA ( 10 nM ) in a 1 : 10 or 1 : 20 molar ratio ( protein : DNA ) for 30 min on 55 ° C . The error bars for the quantified values of uncleaved substrates on the right panel are calculated from the standard deviation from three repetitions of each experiment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Nuclease activities of free MjMre11 (lane 3, 4), nucleotide unbound- (5, 6) and -bound MjMRcd complex (7, 8) towards a hairpin DNA. Each protein sample was incubated with a hairpin DNA (10 nM) in a 1:10 or 1:20 molar ratio (protein: DNA) for 30 min on 55°C. The error bars for the quantified values of uncleaved substrates on the right panel are calculated from the standard deviation from three repetitions of each experiment.


( G ) Nuclease activities of free TmMre11 alone ( lane 3 , 4 ) , nucleotide unbound - ( lane 5 , 6 ) and - bound ( lane 7 , 8 ) full - length TmMR complex towards a hairpin DNA .

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(G) Nuclease activities of free TmMre11 alone (lane 3, 4), nucleotide unbound- (lane 5, 6) and -bound (lane 7, 8) full-length TmMR complex towards a hairpin DNA.


( A ) Superposition of the ATPγS - MjMR - DNA complex ( green ) and apo - MjMR ( 3AV0 , magenta ) structures . Alignment was done using their NBDs ( 1 . 3 Å rmsd in the positions of 311 Cα atoms ) . The coiled - coils of the DNA - bound Rad50 molecules are shifted ( arrow ) to more parallel orientation . The entire structures can be superimposed with a 1 . 9 Å rmsd in 692 Cα positions .

O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Superposition of the ATPγS-MjMR-DNA complex (green) and apo-MjMR (3AV0, magenta) structures. Alignment was done using their NBDs (1.3 Å rmsd in the positions of 311 Cα atoms). The coiled-coils of the DNA-bound Rad50 molecules are shifted (arrow) to more parallel orientation. The entire structures can be superimposed with a 1.9 Å rmsd in 692 Cα positions.


( B , C ) Comparison of the local structures between the apo ( B ) and the DNA - bound MjMR complex ( C ) . Upon DNA binding , helix α6 translates to DNA backbone , resulting in the tighter packing of Leu155 and Leu156 against a hydrophobic pocket formed by lobe I and II . Arrows indicate the direction of the helix movement by DNA binding . See Fig EV5 for a close - up view .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O

(B, C) Comparison of the local structures between the apo (B) and the DNA-bound MjMR complex (C). Upon DNA binding, helix α6 translates to DNA backbone, resulting in the tighter packing of Leu155 and Leu156 against a hydrophobic pocket formed by lobe I and II. Arrows indicate the direction of the helix movement by DNA binding. See Fig EV5 for a close-up view.


( A ) Stereo view of the superposition of the ADP - MjRad50 ( 3AUX ) complex ( black ) onto the ATPγS - MjMR - DNA complex ( green and blue ) by aligning their lobe II domains . The α1 - α2 loop that could collide with parts of the DNA molecule is indicated . ( B ) Aligning the lobe II domains of two structures in Fig 5A shows that rotation of the lobe I opens the gate for Mre11 , which could allow the access of the unwound DNA . Color schemes are same as those in Fig 5A .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O

(A) Stereo view of the superposition of the ADP-MjRad50 (3AUX) complex (black) onto the ATPγS-MjMR-DNA complex (green and blue) by aligning their lobe II domains. The α1-α2 loop that could collide with parts of the DNA molecule is indicated. (B) Aligning the lobe II domains of two structures in Fig 5A shows that rotation of the lobe I opens the gate for Mre11, which could allow the access of the unwound DNA. Color schemes are same as those in Fig 5A.


( C ) DNA - unwinding activities of TmMR . Lane 2 , no protein ; lanes 3 and 4 , no ATP ; lanes 5 and 6 , with ATPγS ; lanes 7 and 8 , with ATP . The DNA : protein ratio was 1 : 2 for lanes 3 , 5 , and 7 , and 1 : 5 for lanes 4 , 6 , and 8 . Plotted data represent the mean of three experiments , with error bars indicating one standard deviation . A graph of the quantified data is shown in the lower panel .

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(C) DNA-unwinding activities of TmMR. Lane 2, no protein; lanes 3 and 4, no ATP; lanes 5 and 6, with ATPγS; lanes 7 and 8, with ATP. The DNA: protein ratio was 1:2 for lanes 3, 5, and 7, and 1:5 for lanes 4, 6, and 8. Plotted data represent the mean of three experiments, with error bars indicating one standard deviation. A graph of the quantified data is shown in the lower panel.


( D ) Comparison of the ATP - dependent DNA unwinding activities of WT and mutant TmMR proteins . Lane 2 , no protein ; lane 3 and 4 , WT TmMR ; lane 5 and 6 , Rad50 ( Δ54 - 56 ) mutant ; lane 7 and 8 , R87E mutant ; lane 9 and 10 , K95E mutant ; lane 11 and 12 , R94E / K95E mutant ; lane 13 and 14 , K115E mutant . For each TmMR , reaction contains 1 : 2 or 1 : 5 ratio of DNA : protein .

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(D) Comparison of the ATP-dependent DNA unwinding activities of WT and mutant TmMR proteins. Lane 2, no protein; lane 3 and 4, WT TmMR; lane 5 and 6, Rad50 (Δ54-56) mutant; lane 7 and 8, R87E mutant; lane 9 and 10, K95E mutant; lane 11 and 12, R94E/K95E mutant; lane 13 and 14, K115E mutant. For each TmMR, reaction contains 1:2 or 1:5 ratio of DNA: protein.


( A ) Endonuclease activities of the TmMR WT and mutant proteins towards a duplex DNA in the absence ( left panel ) or presence of ATP ( right panel ) . Major products are marked with dots . The 5 ' - 32P - label is indicated by a star . See Appendix Fig S4A and S4B for the quantified values .

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(A) Endonuclease activities of the TmMR WT and mutant proteins towards a duplex DNA in the absence (left panel) or presence of ATP (right panel). Major products are marked with dots. The 5'-32P-label is indicated by a star. See Appendix Fig S4A and S4B for the quantified values.


( B ) Quantification of a bubble DNA binding by WT or mutant TmMR in the presence or absence of AMP - PNP . See Appendix Fig S3E and S3F for the original data .

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(B) Quantification of a bubble DNA binding by WT or mutant TmMR in the presence or absence of AMP-PNP. See Appendix Fig S3E and S3F for the original data.


( C ) Endonuclease activities of the WT and mutant TmMR proteins towards a bubble DNA molecule in the absence ( left panel ) or presence of ATP ( right panel ) . See Appendix Fig S4C and S4D for the quantified values .

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(C) Endonuclease activities of the WT and mutant TmMR proteins towards a bubble DNA molecule in the absence (left panel) or presence of ATP (right panel). See Appendix Fig S4C and S4D for the quantified values.


( D ) The effects of single or multiple point mutations in the DNA - contacting or neighboring residues of S . cerevisiae rad50 on the sensitivity to chemical drugs . Sensitivities of rad50 expressing the mutants towards indicated concentrations of CPT , PHL , and MMS were examined . ( E ) The effects of Rad50 mutation on recombination stimulated by inverted Alu repeats . See Appendix Table S1 for the quantified values .

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(D) The effects of single or multiple point mutations in the DNA-contacting or neighboring residues of S. cerevisiae rad50 on the sensitivity to chemical drugs. Sensitivities of rad50 expressing the mutants towards indicated concentrations of CPT, PHL, and MMS were examined. (E) The effects of Rad50 mutation on recombination stimulated by inverted Alu repeats. See Appendix Table S1 for the quantified values.


( E ) The effects of Rad50 mutation on recombination stimulated by inverted Alu repeats . See Appendix Table S1 for the quantified values .

O O O O O O O O O O O O O O O O O O O O O O O O O

(E) The effects of Rad50 mutation on recombination stimulated by inverted Alu repeats. See Appendix Table S1 for the quantified values.


( A - C ) Extra - mitochondrial puncta formation of CHCHD2T61I in MEFs . Chchd2KO cells were transfected with the CHCHD2WT - HA and CHCHD2T61I - HA plasmids . At the indicated times , cells were fixed and stained with anti - HA and anti - Tom20 antibodies , and observed by confocal microscopy . In ( A ) , representative images are shown . Magnified images of the areas within the dashed squares are shown in the insets . Representative images at lower magnification are shown in Appendix Fig . S1D . ( B , C ) Quantification of cells displaying mitochondrial CHCHD2 and extra - mitochondrial CHCHD2 puncta ( n ≥ 100 cells in each experiment ) . In ( B , C , data are shown as the mean ± SD ( n = 3 ) . Comparisons were performed using unpaired two - tailed Student t - tests and one - way ANOVA followed by the Tukey post - hoc tests . * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-C) Extra-mitochondrial puncta formation of CHCHD2T61I in MEFs. Chchd2KO cells were transfected with the CHCHD2WT-HA and CHCHD2T61I-HA plasmids. At the indicated times, cells were fixed and stained with anti-HA and anti-Tom20 antibodies, and observed by confocal microscopy. In (A), representative images are shown. Magnified images of the areas within the dashed squares are shown in the insets. Representative images at lower magnification are shown in Appendix Fig. S1D. (B, C) Quantification of cells displaying mitochondrial CHCHD2 and extra-mitochondrial CHCHD2 puncta (n ≥ 100 cells in each experiment). In (B, C, data are shown as the mean ± SD (n = 3). Comparisons were performed using unpaired two-tailed Student t-tests and one-way ANOVA followed by the Tukey post-hoc tests. **p < 0.01


( D , E ) Extra - mitochondrial puncta formation of CHCHD2T61I in Neuro2a cells . Neuro2a cells were transfected with the CHCHD2WT - HA and CHCHD2T61I - HA plasmids for 4 hr , then cultured with medium containing 2 % FBS and 10 µM retinoic acid for neuronal differentiation . At 28 hr after transfection , cells were fixed and stained with anti - HA and anti - Tom20 antibodies . In ( D ) , representative images are shown . Magnified images of the areas within the dashed squares are shown in the insets . Representative images at lower magnification are shown ( E ) Quantification of cells displaying mitochondrial CHCHD2 and extra - mitochondrial CHCHD2 puncta ( n ≥ 100 cells in each experiment ) . In E , data are shown as the mean ± SD ( n = 3 ) . Comparisons were performed using unpaired two - tailed Student t - tests and one - way ANOVA followed by the Tukey post - hoc tests . * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D, E) Extra-mitochondrial puncta formation of CHCHD2T61I in Neuro2a cells. Neuro2a cells were transfected with the CHCHD2WT-HA and CHCHD2T61I-HA plasmids for 4 hr, then cultured with medium containing 2% FBS and 10 µM retinoic acid for neuronal differentiation. At 28 hr after transfection, cells were fixed and stained with anti-HA and anti-Tom20 antibodies. In (D), representative images are shown. Magnified images of the areas within the dashed squares are shown in the insets. Representative images at lower magnification are shown (E) Quantification of cells displaying mitochondrial CHCHD2 and extra-mitochondrial CHCHD2 puncta (n ≥ 100 cells in each experiment). In E, data are shown as the mean ± SD (n = 3). Comparisons were performed using unpaired two-tailed Student t-tests and one-way ANOVA followed by the Tukey post-hoc tests. **p < 0.01


( F ) Neuro2a cells were treated as described in ( D ) , and cell lysates were fractionated into cytosol , organelles ( including mitochondria ) , nuclei , and cytoskeleton ( including insoluble matter ) . The expression of each protein was analyzed by western blotting using an anti - HA antibody . Cox4 was used as a control . ( G ) A semiquantitative analysis of protein expression in ( F ) is shown . In G ) , data are shown as the mean ± SD ( n = 3 ) . Comparisons were performed using unpaired two - tailed Student t - tests and one - way ANOVA followed by the Tukey post - hoc tests . * * p < 0 .

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(F) Neuro2a cells were treated as described in (D), and cell lysates were fractionated into cytosol, organelles (including mitochondria), nuclei, and cytoskeleton (including insoluble matter). The expression of each protein was analyzed by western blotting using an anti-HA antibody. Cox4 was used as a control. (G) A semiquantitative analysis of protein expression in (F) is shown. In G), data are shown as the mean ± SD (n = 3). Comparisons were performed using unpaired two-tailed Student t-tests and one-way ANOVA followed by the Tukey post-hoc tests. **p < 0.01


Neuro2a cells were transfected with the CHCHD2WT - HA and CHCHD2T61I - HA plasmids for 4 hr , and then cultured in medium containing 2 % FBS and 10 µM retinoic acid to induce neuronal differentiation . At 28 hr after transfection , cells were fixed and stained with an anti - HA antibody and ProteoStat protein aggregation dye ( A , B ) , anti - HA and anti - Nefl antibodies ( C , D ) , anti - HA and anti - p - Nefl473 antibodies ( E , F ) , In ( A , C , E , representative images are shown . Magnified images of the areas within the dashed squares are shown in the inset . Arrowheads indicate colocalized puncta between CHCHD2T61I - HA and aggresomes or the indicated proteins . Representative images at lower magnification are shown ( B , D , F , The amount of aggresomes ( B ) , Nefl ( D ) , p - Nefl473 ( F ) was measured as the fluorescence intensity per cell ( n = 30 cells in each experiment ) . Red bars indicate mean values . In ( B , D , F , comparisons were performed using unpaired two - tailed Student t - tests . * p < 0 . 05 . * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Neuro2a cells were transfected with the CHCHD2WT-HA and CHCHD2T61I-HA plasmids for 4 hr, and then cultured in medium containing 2% FBS and 10 µM retinoic acid to induce neuronal differentiation. At 28 hr after transfection, cells were fixed and stained with an anti-HA antibody and ProteoStat protein aggregation dye (A, B), anti-HA and anti-Nefl antibodies (C, D), anti-HA and anti-p-Nefl473 antibodies (E, F), In (A, C, E, representative images are shown. Magnified images of the areas within the dashed squares are shown in the inset. Arrowheads indicate colocalized puncta between CHCHD2T61I-HA and aggresomes or the indicated proteins. Representative images at lower magnification are shown (B, D, F, The amount of aggresomes (B), Nefl (D), p-Nefl473 (F) was measured as the fluorescence intensity per cell (n  = 30 cells in each experiment). Red bars indicate mean values. In (B, D, F, comparisons were performed using unpaired two-tailed Student t-tests. *p < 0.05. **p < 0.01


Neuro2a cells were transfected with the CHCHD2WT - HA and CHCHD2T61I - HA plasmids for 4 hr , and then cultured in medium containing 2 % FBS and 10 µM retinoic acid to induce neuronal differentiation . At 28 hr after transfection , cells were fixed and stained with anti - HA and anti - p - α - Synuclein129 ( p - α - Syn129 ) antibodies ( G , H ) . In G ) , representative images are shown . Magnified images of the areas within the dashed squares are shown in the inset . Arrowheads indicate colocalized puncta between CHCHD2T61I - HA and the indicated proteins . Representative images at lower magnification are shown H ) The amount of p - α - Syn129 ( H ) was measured as the fluorescence intensity per cell ( n = 30 cells in each experiment ) . Red bars indicate mean values . In H , comparisons were performed using unpaired two - tailed Student t - tests . * p < 0 . 05 . * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Neuro2a cells were transfected with the CHCHD2WT-HA and CHCHD2T61I-HA plasmids for 4 hr, and then cultured in medium containing 2% FBS and 10 µM retinoic acid to induce neuronal differentiation. At 28 hr after transfection, cells were fixed and stained with anti-HA and anti-p-α-Synuclein129 (p-α-Syn129) antibodies (G, H). In G), representative images are shown. Magnified images of the areas within the dashed squares are shown in the inset. Arrowheads indicate colocalized puncta between CHCHD2T61I-HA and the indicated proteins. Representative images at lower magnification are shown H) The amount of p-α-Syn129 (H) was measured as the fluorescence intensity per cell (n  = 30 cells in each experiment). Red bars indicate mean values. In H, comparisons were performed using unpaired two-tailed Student t-tests. *p < 0.05. **p < 0.01


In ( I ) , cell lysates were harvested at 28 hr , and the expression of each protein was analyzed by western blotting . ( J ) A semiquantitative analysis of protein expression in ( I ) is shown . Data are shown as the mean ± SD ( n = 3 ) . In J ) , comparisons were performed using unpaired two - tailed Student t - tests . * p < 0 . 05 . * * p < 0 .

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In (I), cell lysates were harvested at 28 hr, and the expression of each protein was analyzed by western blotting. (J) A semiquantitative analysis of protein expression in (I) is shown. Data are shown as the mean ± SD (n = 3). In J), comparisons were performed using unpaired two-tailed Student t-tests. *p < 0.05. **p < 0.01


( E , F ) Interaction between CHCHD2T61I and Csnk1e / d . Neuro2a cells were transfected with the CHCHD2WT - HA and CHCHD2T61I - HA plasmids together with pmax - GFP ( to detect transfected cells ) , and were differentiated into neuronal cells at 4 hr after transfection . At 28 hr after transfection , cells were fixed and the PLA was performed using anti - HA and anti - Csnk1e / d antibodies and Duolink PLA reagents . In ( E ) , PLA signals were clearly observed in cells expressing CHCHD2T61I - HA , but were faint and localized in the cytosol in cells expressing CHCHD2WT - HA . Representative images at lower magnification are shown In ( F ) , the number of PLA signals were counted ( n = 50 cells in each experiment ) . Red bars indicate mean values . In F ) , comparisons were performed using the unpaired two - tailed Student t - test and one - way ANOVA followed by the Tukey post - hoc test . * p < 0 . 05 . * * p < 0 . 01 . NS : not

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E, F) Interaction between CHCHD2T61I and Csnk1e/d. Neuro2a cells were transfected with the CHCHD2WT-HA and CHCHD2T61I-HA plasmids together with pmax-GFP (to detect transfected cells), and were differentiated into neuronal cells at 4 hr after transfection. At 28 hr after transfection, cells were fixed and the PLA was performed using anti-HA and anti-Csnk1e/d antibodies and Duolink PLA reagents. In (E), PLA signals were clearly observed in cells expressing CHCHD2T61I-HA, but were faint and localized in the cytosol in cells expressing CHCHD2WT-HA. Representative images at lower magnification are shown In (F), the number of PLA signals were counted (n  = 50 cells in each experiment). Red bars indicate mean values. In F), comparisons were performed using the unpaired two-tailed Student t-test and one-way ANOVA followed by the Tukey post-hoc test. *p < 0.05. **p < 0.01. NS: not significant


( D - I ) Neuro2a cells were transfected with the CHCHD2T61I - HA plasmid together with the indicated siRNAs , and were differentiated into neuronal cells after 4 hr . At 28 hr after transfection , cells were fixed and stained with anti - HA , anti - p - Nefl473 , and anti - p - α - Syn129 antibodies , or ProteoStat aggresome detection dye . Similar experiments were performed by the addition of PF - 670462 ( 10 µM ) after 4 hr of transfection instead of gene silencing . In ( D , F , H ) , representative images are shown . Magnified images of the areas within the dashed squares are shown in the insets . Arrowheads indicate colocalized puncta between CHCHD2T61I - HA and the indicated phosphorylated proteins or aggresomes . In ( E , G , I ) , the amount of p - Nefl473 ( E ) , p - α - Syn129 ( G ) , and aggresomes ( I ) was measured as the fluorescence intensity per cell ( n = 30 cells in each experiment ) . Red bars indicate mean values . In E , G , I ) , comparisons were performed using one - way ANOVA followed by the Tukey post - hoc tests or unpaired two - tailed Student t - test . * p < 0 . 05 ; * * p < 0 . 01 . NS : not

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D-I) Neuro2a cells were transfected with the CHCHD2T61I-HA plasmid together with the indicated siRNAs, and were differentiated into neuronal cells after 4 hr. At 28 hr after transfection, cells were fixed and stained with anti-HA, anti-p-Nefl473, and anti-p-α-Syn129 antibodies, or ProteoStat aggresome detection dye. Similar experiments were performed by the addition of PF-670462 (10 µM) after 4 hr of transfection instead of gene silencing. In (D, F, H), representative images are shown. Magnified images of the areas within the dashed squares are shown in the insets. Arrowheads indicate colocalized puncta between CHCHD2T61I-HA and the indicated phosphorylated proteins or aggresomes. In (E, G, I), the amount of p-Nefl473 (E), p-α-Syn129 (G), and aggresomes (I) was measured as the fluorescence intensity per cell (n = 30 cells in each experiment). Red bars indicate mean values. In E, G, I), comparisons were performed using one-way ANOVA followed by the Tukey post-hoc tests or unpaired two-tailed Student t-test. *p < 0.05; **p < 0.01. NS: not significant


Reduction in TH signals in the SNpc of knock - in mice . In ( D ) , brain cryosections were immunostained with the dopaminergic cell marker TH . Representative images of the SNpc and ventral tegmental area ( VTA ) are shown . Dashed lines indicate the SNpc and VTA regions . Bars = 200 µm . In ( E ) , TH signals in the SNpc ( average fluorescence intensity per region ) are shown as the mean ± SD ( n = 3 ) . In E ) , comparisons were performed using one - way ANOVA followed by the Tukey post - hoc tests . * p < 0 . 05 ; * * p < 0 . 01 . NS : not

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Reduction in TH signals in the SNpc of knock-in mice. In (D), brain cryosections were immunostained with the dopaminergic cell marker TH. Representative images of the SNpc and ventral tegmental area (VTA) are shown. Dashed lines indicate the SNpc and VTA regions. Bars = 200 µm. In (E), TH signals in the SNpc (average fluorescence intensity per region) are shown as the mean ± SD (n = 3). In E), comparisons were performed using one-way ANOVA followed by the Tukey post-hoc tests. *p < 0.05; **p < 0.01. NS: not significant


Reduction in TH signals in the SNpc of knock - in mice . In ( F ) , cryosections of the SNpc were immunostained with anti - Chchd2 and anti - TH antibodies . Arrowheads indicate Chchd2T61I puncta in TH - positive cells . Dashed lines indicate cell shapes . ( G - K ) Extra - mitochondrial aggresome formation by p - Nefl473 , p - α - Syn129 , and Csnk1e / d in the SNpc of knock - in mice . Cryosections of the SNpc were immunostained with anti - Chchd2 and anti - Ant1 / 2 ( G ) , anti - p - Nefl473 ( H ) , anti - p - α - Syn129 ( I ) , and anti - Csnk1e / d antibodies ( J ) , and with ProteoStat dye ( K ) . Ant1 / 2 are mitochondrial membrane proteins and arrowheads indicate extra - mitochondrial Chchd2T61I ( G ) . In ( H - K ) , arrowheads indicate the colocalization of puncta with Chchd2T61I and the indicated proteins or aggresomes . Dashed lines indicate cell shapes .

Reduction in TH signals in the SNpc of knock-in mice. In (F), cryosections of the SNpc were immunostained with anti-Chchd2 and anti-TH antibodies. Arrowheads indicate Chchd2T61I puncta in TH-positive cells. Dashed lines indicate cell shapes. (G-K) Extra-mitochondrial aggresome formation by p-Nefl473, p-α-Syn129, and Csnk1e/d in the SNpc of knock-in mice. Cryosections of the SNpc were immunostained with anti-Chchd2 and anti-Ant1/2 (G), anti-p-Nefl473 (H), anti-p-α-Syn129 (I), and anti-Csnk1e/d antibodies (J), and with ProteoStat dye (K). Ant1/2 are mitochondrial membrane proteins and arrowheads indicate extra-mitochondrial Chchd2T61I (G). In (H-K), arrowheads indicate the colocalization of puncta with Chchd2T61I and the indicated proteins or aggresomes. Dashed lines indicate cell shapes.


( L ) Isolated midbrain and diencephalon lysates were subjected to western blotting .

(L) Isolated midbrain and diencephalon lysates were subjected to western blotting.


( F - J ) The same experiments as Fig . 5G - K were performed using PF - 670462 - infused and vehicle - infused Chchd2T61I knock - in mice .

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(F-J) The same experiments as Fig. 5G-K were performed using PF-670462-infused and vehicle-infused Chchd2T61I knock-in mice.


( A ) Photos of the midbrain of a control subject ( left ) and PD patient harboring the CHCHD2T61I mutation ( right ) . The dashed lines indicate the SNpc . A portion of the midbrain of the control subject was lost when slicing .

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(A) Photos of the midbrain of a control subject (left) and PD patient harboring the CHCHD2T61I mutation (right). The dashed lines indicate the SNpc. A portion of the midbrain of the control subject was lost when slicing.


( B - G ) Colocalization of extra - mitochondrial CHCHD2T61I with CSNK1E / D , p - NEFL472 , and p - α - SYN129 in the brain of a PD patient harboring the CHCHD2T61I mutation . Each brain section was deparaffinized and immunostained with anti - CHCHD2 and anti - ANT1 / 2 ( B ) , anti - CSNK1E / D ( C ) , anti - p - NEFL472 ( D , E ) , or anti - p - α - SYN129 antibodies ( F , G ) . Arrowheads indicate extra - mitochondrial CHCHD2T61I puncta ( B ) , and puncta showing colocalization of CHCHD2T61I with CSNK1E / D ( C ) , p - NEFL472 ( D ) , and p - α - SYN129 ( F ) . Arrows indicate weaker colocalization between CHCHD2T61I and the indicated proteins in the control brain ( C , D , F ) . Dashed lines indicate cell shapes . In ( E , G ) , the amount of p - NEFL472 ( E ) and p - α - SYN129 ( G ) was measured by the average fluorescence intensity per cell ( n = 30 cells in each experiment ) . Red bars indicate mean values . Comparisons were performed using the unpaired two - tailed Student t - tests . * * p < 0 .

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(B-G) Colocalization of extra-mitochondrial CHCHD2T61I with CSNK1E/D, p-NEFL472, and p-α-SYN129 in the brain of a PD patient harboring the CHCHD2T61I mutation. Each brain section was deparaffinized and immunostained with anti-CHCHD2 and anti-ANT1/2 (B), anti-CSNK1E/D (C), anti-p-NEFL472 (D, E), or anti-p-α-SYN129 antibodies (F, G). Arrowheads indicate extra-mitochondrial CHCHD2T61I puncta (B), and puncta showing colocalization of CHCHD2T61I with CSNK1E/D (C), p-NEFL472 (D), and p-α-SYN129 (F). Arrows indicate weaker colocalization between CHCHD2T61I and the indicated proteins in the control brain (C, D, F). Dashed lines indicate cell shapes. In (E, G), the amount of p-NEFL472 (E) and p-α-SYN129 (G) was measured by the average fluorescence intensity per cell (n  = 30 cells in each experiment). Red bars indicate mean values. Comparisons were performed using the unpaired two-tailed Student t-tests. **p < 0.01


iPSCs were prepared from a healthy control and a PD patient harboring the CHCHD2T61I mutation . An isogenic control iPSC line was generated by the correction of the gene mutation Then , these iPSC cells were differentiated into dopaminergic ( DA ) neurons , and analyzed by immunofluorescence using anti - CHCHD2 ( A - E ) , anti - ANT1 / 2 ( A ) , anti - CSNK1E / D ( B ) , anti - p - NEFL472 ( C ) , and anti - p - α - SYN129 antibodies ( D ) Arrowheads indicate extra - mitochondrial CHCHD2T61I ( A ) , puncta showing colocalization of CHCHD2T61I with CSNK1E / D ( B ) , p - NEFL472 ( C , , and p - α - SYN129 ( D , Dashed lines indicate cell shapes .

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iPSCs were prepared from a healthy control and a PD patient harboring the CHCHD2T61I mutation. An isogenic control iPSC line was generated by the correction of the gene mutation Then, these iPSC cells were differentiated into dopaminergic (DA) neurons, and analyzed by immunofluorescence using anti-CHCHD2 (A-E), anti-ANT1/2 (A), anti-CSNK1E/D (B), anti-p-NEFL472 (C), and anti-p-α-SYN129 antibodies (D) Arrowheads indicate extra-mitochondrial CHCHD2T61I (A), puncta showing colocalization of CHCHD2T61I with CSNK1E/D (B), p-NEFL472 (C, , and p-α-SYN129 (D, Dashed lines indicate cell shapes.


CHCHD2T61I iPSC - derived DA neurons were treated with PF - 670462 ( 10 µM ) for 20 hr and analyzed by immunofluorescence using anti - CHCHD2 and anti - p - α - SYN129 antibodies ( G ) , and ProteoStat aggresome dye ( H ) . Arrowheads indicate puncta showing colocalization of CHCHD2T61I with p - α - SYN129 G ) or aggresomes H ) . Dashed lines indicate cell shapes .

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CHCHD2T61I iPSC-derived DA neurons were treated with PF-670462 (10 µM) for 20 hr and analyzed by immunofluorescence using anti-CHCHD2 and anti-p-α-SYN129 antibodies (G), and ProteoStat aggresome dye (H). Arrowheads indicate puncta showing colocalization of CHCHD2T61I with p-α-SYN129 G) or aggresomes H). Dashed lines indicate cell shapes.


A . MDCK cells were cultured on cell culture inserts for 4 days . On the last day , the culture medium was replaced with EV - depleted medium . EVs released from the apical and basolateral sides of MDCK cells were purified by PEG precipitation . Cell lysates and EV proteins in PEG pellets were analyzed by immunoblotting with the antibodies indicated . Note that the PEG pellets did not contain mitochondrial protein TOMM20 , suggesting that the PEG pellets were not contaminated by intracellular organelles .

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A. MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. EVs released from the apical and basolateral sides of MDCK cells were purified by PEG precipitation. Cell lysates and EV proteins in PEG pellets were analyzed by immunoblotting with the antibodies indicated. Note that the PEG pellets did not contain mitochondrial protein TOMM20, suggesting that the PEG pellets were not contaminated by intracellular organelles.


B . PEG pellets were subjected to OptiPrep flotation analysis .

B. PEG pellets were subjected to OptiPrep flotation analysis.


C . MDCK cells stably expressing human CD63 were cultured sEVs were isolated from the pre - cleared medium by direct immunoaffinity capture using anti - CD63 antibody .

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C. MDCK cells stably expressing human CD63 were cultured sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD63 antibody.


D . sEVs were eluted from the beads with a glycine buffer and analyzed by nanoparticle tracking analysis ( NTA ) . Representative NTA traces were shown . E . Quantification of the NTA data obtained in five independent experiments .

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D. sEVs were eluted from the beads with a glycine buffer and analyzed by nanoparticle tracking analysis (NTA). Representative NTA traces were shown. E. Quantification of the NTA data obtained in five independent experiments.


F . MDCK cells were cultured sEVs were isolated from the pre - cleared medium by direct immunoaffinity capture using anti - CD9 antibody .

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F. MDCK cells were cultured sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody.


G . sEVs were eluted from the beads with a glycine buffer and analyzed by NTA . Representative NTA traces were shown . H . Quantification of the NTA data obtained in five independent experiments .

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G. sEVs were eluted from the beads with a glycine buffer and analyzed by NTA. Representative NTA traces were shown. H. Quantification of the NTA data obtained in five independent experiments.


A . MDCK cells were cultured on cell culture inserts for 4 days . On the last day , the culture medium was replaced with serum - free medium . sEVs released from the apical and basolateral sides of MDCK cells were purified by ultracentrifugation . Apical and basolateral sEVs ( P100 ) were analyzed by silver staining .

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A. MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the culture medium was replaced with serum-free medium. sEVs released from the apical and basolateral sides of MDCK cells were purified by ultracentrifugation. Apical and basolateral sEVs (P100) were analyzed by silver staining.


C . MDCK cells were cultured on cell culture inserts for 4 days . On the last day , the culture medium was replaced with EV - depleted medium . sEVs released from the apical and basolateral sides of MDCK cells were purified by PEG precipitation . Cell lysates and sEV proteins in PEG pellets were analyzed by immunoblotting with the antibodies indicated . GPRC5C was detected only in the apical sEV

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C. MDCK cells were cultured on cell culture inserts for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. sEVs released from the apical and basolateral sides of MDCK cells were purified by PEG precipitation. Cell lysates and sEV proteins in PEG pellets were analyzed by immunoblotting with the antibodies indicated. GPRC5C was detected only in the apical sEV samples


D . PEG pellets were subjected to OptiPrep flotation analysis . GPRC5C was detected only in the apical sEV

D. PEG pellets were subjected to OptiPrep flotation analysis. GPRC5C was detected only in the apical sEV samples


E . MDCK cells were cultured sEVs were isolated from the pre - cleared medium by direct immunoaffinity capture using anti - CD9 antibody . GPRC5C was detected only in the apical sEV

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E. MDCK cells were cultured sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. GPRC5C was detected only in the apical sEV samples


F . MDCK cells stably expressing human CD63 were cultured sEVs were isolated from the pre - cleared medium by direct immunoaffinity capture using anti - CD63 antibody .

F. MDCK cells stably expressing human CD63 were cultured sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD63 antibody.


A . MDCK cells were transfected with siControl or the siRNAs indicated , and the cells were transferred to cell culture inserts and cultured for 4 days . On the last day , the culture medium was replaced with EV - depleted medium . sEVs were isolated from the pre - cleared medium by direct immunoaffinity capture using anti - CD9 antibody . Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated .

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A. MDCK cells were transfected with siControl or the siRNAs indicated, and the cells were transferred to cell culture inserts and cultured for 4 days. On the last day, the culture medium was replaced with EV-depleted medium. sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated.


B . The intensity of the bands was measured in three independent experiments .

B. The intensity of the bands was measured in three independent experiments.


C . sEVs were eluted from the beads with a glycine buffer and analyzed by NTA . Representative NTA traces were shown .

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C. sEVs were eluted from the beads with a glycine buffer and analyzed by NTA. Representative NTA traces were shown.


D . Quantification of the NTA data obtained in five independent experiments .

D. Quantification of the NTA data obtained in five independent experiments.


A . MDCK cells were transfected with siControl or siALIX , and the cells were transferred to cell culture inserts and cultured for 4 days . On the last day , the culture medium was replaced with EV - depleted medium with or without 10 nM GW4869 . sEVs were isolated from the pre - cleared medium by direct immunoaffinity capture using anti - CD9 antibody . Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated .

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A. MDCK cells were transfected with siControl or siALIX, and the cells were transferred to cell culture inserts and cultured for 4 days. On the last day, the culture medium was replaced with EV-depleted medium with or without 10 nM GW4869. sEVs were isolated from the pre-cleared medium by direct immunoaffinity capture using anti-CD9 antibody. Cell lysates and sEV samples were analyzed by immunoblotting with the antibodies indicated.


( A ) Hep3B hepatocytes were treated with nontargeting control siRNA ( siNT ) or siRNA targeting Dyn2 ( siDyn2 ) , followed by re - expression of either vector alone , or GFP - tagged versions of Dyn2 , loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0 . 1 % FBS . LDs were visualized by immunofluorescence using Oil Red O staining . Cell boundaries are outlined and those cells re - expressing GFP , GFP - wtDyn2 , or GFP - - K44A are denoted with asterisks . Bars , 20 µM . ( B ) Representative blot showing the efficiency of the Dyn2 knockdown in these cells . ( C and D ) Quantitation of the average LD number and area ( in pixels2 ) per cell from three independent experiments . The data are represented as mean ± SE . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 . NS , not significant .

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(A) Hep3B hepatocytes were treated with nontargeting control siRNA (siNT) or siRNA targeting Dyn2 (siDyn2), followed by re-expression of either vector alone, or GFP-tagged versions of Dyn2, loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0.1% FBS. LDs were visualized by immunofluorescence using Oil Red O staining. Cell boundaries are outlined and those cells re-expressing GFP, GFP-wtDyn2, or GFP--K44A are denoted with asterisks. Bars, 20 µM. (B) Representative blot showing the efficiency of the Dyn2 knockdown in these cells. (C and D) Quantitation of the average LD number and area (in pixels2) per cell from three independent experiments. The data are represented as mean ± SE. *, P ≤ 0.05; **, P ≤ 0.01. NS, not significant.


HuH - 7 ( A ) and Hep3B ( B ) hepatocytes were loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0 . 1 % FBS in the presence of Dyn2 inhibitors or DMSO as indicated . Representative images of inhibitor - treated and control cells ( stained with Oil Red O ) are shown in A and B together with the quantitation of the average LD area per cell from three independent experiments . Pharmacological inhibitors used were : Dynasore ( inhibits Dyn2 GTPase activity ) , MiTMAB ( targets PH domain and interferes with membrane binding ) , Dynole 34 - 2 ( allosteric GTPase inhibitor ) , and Dynole 31 - 2 ( negative control for Dynole 34 - 2 ) . Bars , 20 µM .

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HuH-7 (A) and Hep3B (B) hepatocytes were loaded with 150 µM oleate overnight and starved for 48 h in medium containing 0.1% FBS in the presence of Dyn2 inhibitors or DMSO as indicated. Representative images of inhibitor-treated and control cells (stained with Oil Red O) are shown in A and B together with the quantitation of the average LD area per cell from three independent experiments. Pharmacological inhibitors used were: Dynasore (inhibits Dyn2 GTPase activity), MiTMAB (targets PH domain and interferes with membrane binding), Dynole 34-2 (allosteric GTPase inhibitor), and Dynole 31-2 (negative control for Dynole 34-2). Bars, 20 µM.


( C ) Representative images from control and Dyn2 knockout MEFs after an overnight lipid loading with 400 µM Dyn2 for 17 h . Knockout of Dyn2 was induced by treatment with 2 µM 4 - hydroxy - tamoxifen for 7 d and was confirmed by immunostaining of endogenous Dyn2 ( top row ) and by immunoblot ( D ) . Bars , 20 µM . ( E and F ) Average LD number ( E ) and area ( F ) per cell from five independent experiments . All data are represented as mean ± SE . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 .

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(C) Representative images from control and Dyn2 knockout MEFs after an overnight lipid loading with 400 µM Dyn2 for 17 h. Knockout of Dyn2 was induced by treatment with 2 µM 4-hydroxy-tamoxifen for 7 d and was confirmed by immunostaining of endogenous Dyn2 (top row) and by immunoblot (D). Bars, 20 µM. (E and F) Average LD number (E) and area (F) per cell from five independent experiments. All data are represented as mean ± SE. *, P ≤ 0.05; **, P ≤ 0.01.


( G ) Whole - cell lysates and LD fractions isolated from HuH - 7 hepatocytes under resting or starved ( 2 h HBSS starvation ) conditions .

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(G) Whole-cell lysates and LD fractions isolated from HuH-7 hepatocytes under resting or starved (2 h HBSS starvation) conditions.


( H ) Primary hepatocyte expressing Dyn2 - GFP , showing an absence of colocalization with the LD surface ( stained with Oil Red O ) . Bar , 20 µM . Inset shows magnification of boxed region ( bar , 2 µM ) .

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(H) Primary hepatocyte expressing Dyn2-GFP, showing an absence of colocalization with the LD surface (stained with Oil Red O). Bar, 20 µM. Inset shows magnification of boxed region (bar, 2 µM).


Hep3B cells treated with either a nontargeting control siRNA ( A and B , siNT ) or an siRNA targeting human Dyn2 ( C and D , siDyn2 ) were fixed and co - stained with antibodies specific for LAMP1 ( red ) and LC3 ( green ) . After Dyn2 knockdown , a juxtanuclear aggregation and enlargement of the LAMP1 - positive compartment is observed ( C and D , arrows ) . Increased labeling of LC3 is also detectable after knockdown of Dyn2 .

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Hep3B cells treated with either a nontargeting control siRNA (A and B, siNT) or an siRNA targeting human Dyn2 (C and D, siDyn2) were fixed and co-stained with antibodies specific for LAMP1 (red) and LC3 (green). After Dyn2 knockdown, a juxtanuclear aggregation and enlargement of the LAMP1-positive compartment is observed (C and D, arrows). Increased labeling of LC3 is also detectable after knockdown of Dyn2.


( E ) Western blotting of Hep3B lysates after a 3 - d treatment with either the control or Dyn2 - targeted siRNA and further treatment with or without 50 µM leupeptin . Densitometry - based analysis of six independent experiments is shown at the bottom of the figure .

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(E) Western blotting of Hep3B lysates after a 3-d treatment with either the control or Dyn2-targeted siRNA and further treatment with or without 50 µM leupeptin. Densitometry-based analysis of six independent experiments is shown at the bottom of the figure.


( F ) Western blotting of Hep3B lysates after treatment for 2 h with DMSO or 80 µM Dynasore , in the presence or absence of 50 µM leupeptin . Quantitation of LC3 - II levels relative to control are shown below the blots . The data are represented as mean ± SE ; * , P ≤ 0 . 05 .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Western blotting of Hep3B lysates after treatment for 2 h with DMSO or 80 µM Dynasore, in the presence or absence of 50 µM leupeptin. Quantitation of LC3-II levels relative to control are shown below the blots. The data are represented as mean ± SE; *, P ≤ 0.05.


Dyn2 knockdown results in the formation of enlarged autophagic structures . ( A - D ) Transmission electron micrographs ( TEMs ) of oleate - loaded Hep3B hepatocytes treated with nontargeting control ( siNT ) or Dyn2 ( siDyn2 ) siRNA for 72 h . Bars : ( A and B ) 2 µm ; ( A ′ and B ′ ) 1 µm ; ( C and D ) 0 . 5 µm . Insets in A and B show fluorescent micrographs of LAMP1 - stained cells ( bars , 10 µM ) . Control cells ( A ) contain an abundance of small ( < 1 µm ) electron - dense lysosomes and autolysosomes ( arrowheads ) . Under conditions in which Dyn2 expression is suppressed ( B - D ) , far fewer small lysosomes are observed . Instead , the cells are populated by larger autolysosomes ( ≥ 1 µm ) with aberrant morphologies and containing putative LDs ( * ) . ( E ) Quantitative measure of autolysosomal size from cells in which Dyn2 was knocked down as compared with control cells . Values represent the fold - increase in the number of autophagic structures of a given diameter ( measured in microns ) observed over siNT - treated control cells . Data were obtained from a single experiment with n = 6 cells examined by electron microscopy from each condition .

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Dyn2 knockdown results in the formation of enlarged autophagic structures. (A-D) Transmission electron micrographs (TEMs) of oleate-loaded Hep3B hepatocytes treated with nontargeting control (siNT) or Dyn2 (siDyn2) siRNA for 72 h. Bars: (A and B) 2 µm; (A′ and B′) 1 µm; (C and D) 0.5 µm. Insets in A and B show fluorescent micrographs of LAMP1-stained cells (bars, 10 µM). Control cells (A) contain an abundance of small (<1 µm) electron-dense lysosomes and autolysosomes (arrowheads). Under conditions in which Dyn2 expression is suppressed (B-D), far fewer small lysosomes are observed. Instead, the cells are populated by larger autolysosomes (≥1 µm) with aberrant morphologies and containing putative LDs (*). (E) Quantitative measure of autolysosomal size from cells in which Dyn2 was knocked down as compared with control cells. Values represent the fold-increase in the number of autophagic structures of a given diameter (measured in microns) observed over siNT-treated control cells. Data were obtained from a single experiment with n = 6 cells examined by electron microscopy from each condition.


Hep3B cells treated with either a nontargeting control siRNA ( A and B , siNT ) or an siRNA targeting human Dyn2 ( C and D , siDyn2 ) were fixed and stained with an antibody specific for LAMP1 after a 24 - h starvation period in media containing 0 . 1 % FBS . Control cells displayed normal lysosomal compartments , with LAMP1 - stained structures dispersed throughout the cytosol , some containing short reformation tubules ( B ′ ) . After Dyn2 knockdown , many cells contained enlarged autolysosomes with a noticeable increase in persistent and lengthy reformation tubules emanating from LAMP1 - positive compartments ( C ′ and D ′ ) . Bars : ( A - D ) 20 µM ; ( A ′ and B ′ ) 5 µM ; ( C ′ and D ′ ) 10 µM . ( E and F ) Measurements of the length ( E ) and distribution ( F ) of LAMP1 - stained ALR tubules in control ( siNT ) and siDyn2 - treated cells . Data represent the mean derived from measurements of tubules in a minimum of 40 cells for each experimental group over 6 independent experiments . Error bars represent SE ; * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Hep3B cells treated with either a nontargeting control siRNA (A and B, siNT) or an siRNA targeting human Dyn2 (C and D, siDyn2) were fixed and stained with an antibody specific for LAMP1 after a 24-h starvation period in media containing 0.1% FBS. Control cells displayed normal lysosomal compartments, with LAMP1-stained structures dispersed throughout the cytosol, some containing short reformation tubules (B′). After Dyn2 knockdown, many cells contained enlarged autolysosomes with a noticeable increase in persistent and lengthy reformation tubules emanating from LAMP1-positive compartments (C′ and D′). Bars: (A-D) 20 µM; (A′ and B′) 5 µM; (C′ and D′) 10 µM. (E and F) Measurements of the length (E) and distribution (F) of LAMP1-stained ALR tubules in control (siNT) and siDyn2-treated cells. Data represent the mean derived from measurements of tubules in a minimum of 40 cells for each experimental group over 6 independent experiments. Error bars represent SE; *, P ≤ 0.05; **, P ≤ 0.01.


( G ) TEM of siDyn2 - treated Hep3B hepatocyte exhibiting enlarged autolysosomal structures with extensive tubulation ( arrows ) . Bar , 2 µm . ( G ′ ) Inset showing enlargement of autolysosomal tubule ( arrows ) . Bar , 1 µm . ( H ) Tubulating autolysosome with engulfed lipid droplet . Bar , 0 . 5 µm .

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(G) TEM of siDyn2-treated Hep3B hepatocyte exhibiting enlarged autolysosomal structures with extensive tubulation (arrows). Bar, 2 µm. (G′) Inset showing enlargement of autolysosomal tubule (arrows). Bar, 1 µm. (H) Tubulating autolysosome with engulfed lipid droplet. Bar, 0.5 µm.


Acute inhibition of Dyn2 reversibly disrupts autophagic lysosomal reformation ( ALR ) and lysosomal tubule scission . ( A - D ) Still frames from time - lapse movies of Hep3B cells expressing LAMP1 - mCherry . Cells were starved for 2 h in HBSS and subsequently treated for 30 min with either DMSO ( A and B ) or 40 µM Dynasore ( C and D ) , which induced extensive tubulation of LAMP1 - positive compartments . Bars ( A - D ) : 20 µM ; ( A ′ - B ′ ) 2 µM ; ( C ′ - D ′ ) 10 µM . ( E - G ) To demonstrate the reversibility of this tubulation , Dynasore - treated cells were washed extensively with drug - free media containing 10 % FBS and monitored by time - lapse microscopy for 45 min . Frequently , after drug washout , LAMP1 - positive tubules exhibited noticeable varicosities ( E and F , arrows ; bars , 10 µM ) along their length . These sites are suggestive of areas of scission and resumed budding of nascent protolysosomes from the reformation tubules ( G ; bars , 10 µM ) . ( H ) Tubules from cells undergoing drug washout were quantified by tracing their lengths at the beginning and end of these movies . Still frames from a representative movie show tubule content at t = 10 and 45 min after drug washout . Bars , 20 µM . ( I ) Analysis of five independent movies showed an average decrease in total tubulation of ∼ 50 % after drug washout . Data represent the average relative change in total tubule length between the first and last frames of the time - lapse movies . Error bars represent SE ; * , P < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Acute inhibition of Dyn2 reversibly disrupts autophagic lysosomal reformation (ALR) and lysosomal tubule scission. (A-D) Still frames from time-lapse movies of Hep3B cells expressing LAMP1-mCherry. Cells were starved for 2 h in HBSS and subsequently treated for 30 min with either DMSO (A and B) or 40 µM Dynasore (C and D), which induced extensive tubulation of LAMP1-positive compartments. Bars (A-D): 20 µM; (A′-B′) 2 µM; (C′-D′) 10 µM. (E-G) To demonstrate the reversibility of this tubulation, Dynasore-treated cells were washed extensively with drug-free media containing 10% FBS and monitored by time-lapse microscopy for 45 min. Frequently, after drug washout, LAMP1-positive tubules exhibited noticeable varicosities (E and F, arrows; bars, 10 µM) along their length. These sites are suggestive of areas of scission and resumed budding of nascent protolysosomes from the reformation tubules (G; bars, 10 µM). (H) Tubules from cells undergoing drug washout were quantified by tracing their lengths at the beginning and end of these movies. Still frames from a representative movie show tubule content at t = 10 and 45 min after drug washout. Bars, 20 µM. (I) Analysis of five independent movies showed an average decrease in total tubulation of ∼50% after drug washout. Data represent the average relative change in total tubule length between the first and last frames of the time-lapse movies. Error bars represent SE; *, P < 0.05.


( A and B ) Subcellular density gradient fractionation of Hep3B hepatocytes starved for 2 h in HBSS and treated with 40 µM Dynasore , to induce tubule formation , as in Fig . 5 . Cells were lysed ( WCL ) , and the post - nuclear supernatant ( PNS ) was pelleted by centrifugation to produce a crude lysosomal fraction ( CLF ) and high - speed supernatant ( HSS ) . The CLF was subsequently loaded onto an 8 - 27 % discontinuous iodixanol ( OptiPrep ) gradient for separation by ultracentrifugation . Nine fractions were collected from the top of the gradient and blotted for Dyn2 and the LAMP1 resident protein , LAMP1 . Lysosomal acid phosphatase activity roughly correlates with LAMP1 protein levels in each fraction . Levels of Dyn2 are highest in fraction 2 , corresponding with both the peak levels of LAMP1 and lysosomal activity . ( A ) The data shown are from a single representative experiment out of three repeats . ( B ) Blotting for subcellular components shows that Dyn2 is specific for these same fractions , unlike other organelle markers such as EEA1 ( early endosomes ) and COXIV ( mitochondria ) .

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(A and B) Subcellular density gradient fractionation of Hep3B hepatocytes starved for 2 h in HBSS and treated with 40 µM Dynasore, to induce tubule formation, as in Fig. 5. Cells were lysed (WCL), and the post-nuclear supernatant (PNS) was pelleted by centrifugation to produce a crude lysosomal fraction (CLF) and high-speed supernatant (HSS). The CLF was subsequently loaded onto an 8-27% discontinuous iodixanol (OptiPrep) gradient for separation by ultracentrifugation. Nine fractions were collected from the top of the gradient and blotted for Dyn2 and the LAMP1 resident protein, LAMP1. Lysosomal acid phosphatase activity roughly correlates with LAMP1 protein levels in each fraction. Levels of Dyn2 are highest in fraction 2, corresponding with both the peak levels of LAMP1 and lysosomal activity. (A) The data shown are from a single representative experiment out of three repeats. (B) Blotting for subcellular components shows that Dyn2 is specific for these same fractions, unlike other organelle markers such as EEA1 (early endosomes) and COXIV (mitochondria).


( C and D ) Dyn2 localizes to the surface of LAMP1 - positive compartments . Fluorescence imaging of Hep3B hepatocytes transfected with Dyn2 - GFP and LAMP1 - mCherry under resting ( C and C ′ ) or starvation ( D ) conditions . ( C ′ ′ ) Dyn2 - GFP localizing to LAMP1 - labeled lysosome structures under starvation conditions . Arrows indicate regions of protein colocalization . Dyn2 ( arrows ) is present at the site of scission of LAMP1 - positive tubules ( arrowhead ) from large autolysosomal structures . Bars : ( C ) 5 µM ; ( C ′ , C ′ ′ , and D ) 3 µM .

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(C and D) Dyn2 localizes to the surface of LAMP1-positive compartments. Fluorescence imaging of Hep3B hepatocytes transfected with Dyn2-GFP and LAMP1-mCherry under resting (C and C′) or starvation (D) conditions. (C′′) Dyn2-GFP localizing to LAMP1-labeled lysosome structures under starvation conditions. Arrows indicate regions of protein colocalization. Dyn2 (arrows) is present at the site of scission of LAMP1-positive tubules (arrowhead) from large autolysosomal structures. Bars: (C) 5 µM; (C′, C′′, and D) 3 µM.


( b ) RT - PCR analysis using the blue primer set ( left ) and green primer set ( right ) from a . Whereas control cDNA samples showed a single product corresponding to the wild - type allele ( WT ) , an apparently longer product was observed in subjects 1 , 2 and 5 , indicating that only the transcripts from the mutant allele were expressed . In subject 3 , both wild - type and mutant alleles were expressed . Template without reverse transcriptase was used as a negative control , RT ( - ) .

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(b) RT-PCR analysis using the blue primer set (left) and green primer set (right) from a. Whereas control cDNA samples showed a single product corresponding to the wild-type allele (WT), an apparently longer product was observed in subjects 1, 2 and 5, indicating that only the transcripts from the mutant allele were expressed. In subject 3, both wild-type and mutant alleles were expressed. Template without reverse transcriptase was used as a negative control, RT(-).


( a - e ) T1 - weighted imaging shows hyperintensity of the substantia nigra with a central band of T1 - weighted hypointensity ( arrowheads ) . Images are shown for subject 1 at 33 years ( a ) , subject 2 at 25 years ( b ) , subject 3 at 39 years ( c ) , subject 4 at 46 years ( d ) and subject 5 at 33 years ( e ) . ( f - h ) T2 - weighted imaging shows marked hypointensity of the globus pallidus ( arrows ) , suggesting iron deposition . Cerebral atrophy and mild cerebellar atrophy are also seen . Images are shown for subject 1 ( f ) , subject 2 ( g ) and subject 3 ( h ) . ( i , j ) The fluid attenuated inversion recovery ( FLAIR ) image of subject 1 ( i ) and the T1 - weighted FLAIR coronal image of subject 2 ( j ) also show cerebral atrophy .

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(a-e) T1-weighted imaging shows hyperintensity of the substantia nigra with a central band of T1-weighted hypointensity (arrowheads). Images are shown for subject 1 at 33 years (a), subject 2 at 25 years (b), subject 3 at 39 years (c), subject 4 at 46 years (d) and subject 5 at 33 years (e). (f-h) T2-weighted imaging shows marked hypointensity of the globus pallidus (arrows), suggesting iron deposition. Cerebral atrophy and mild cerebellar atrophy are also seen. Images are shown for subject 1 (f), subject 2 (g) and subject 3 (h). (i,j) The fluid attenuated inversion recovery (FLAIR) image of subject 1 (i) and the T1-weighted FLAIR coronal image of subject 2 (j) also show cerebral atrophy.


( a ) Immunoblot analysis of the WIPI4 protein ( apparent mobility at ∼ 35 kDa ) in LCLs ( top ) . Truncated forms were not detected ( middle ) . HSP90 was used as a loading control ( bottom ) . An asterisk indicates nonspecific immunoreactive bands .

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(a) Immunoblot analysis of the WIPI4 protein (apparent mobility at ∼35 kDa) in LCLs (top). Truncated forms were not detected (middle). HSP90 was used as a loading control (bottom). An asterisk indicates nonspecific immunoreactive bands.


( b ) Cells were treated with 250 nM Torin1 in the presence or absence of 20 μM chloroquine for 2 h . Cell lysates were analyzed by SDS - PAGE and immunoblotting using antibodies to LC3 , WIPI4 and HSP90 . The positions of LC3 - I ( cytosolic ) and LC3 - II ( membrane bound ) are indicated .

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(b) Cells were treated with 250 nM Torin1 in the presence or absence of 20 μM chloroquine for 2 h. Cell lysates were analyzed by SDS-PAGE and immunoblotting using antibodies to LC3, WIPI4 and HSP90. The positions of LC3-I (cytosolic) and LC3-II (membrane bound) are indicated.


( c - e ) Cells were cultured in the presence of Torin1 for 2 h . ( c ) Cytospun cells were fixed and analyzed by immunofluorescence microscopy using antibodies to LC3 and ATG9A . Abnormal colocalization of LC3 with ATG9A was observed in the LCLs of affected subjects . Scale bars , 10 μm and 1 μm in the inset . ( d , e ) The numbers of LC3 + ( d ) and LC3 + ATG9A + ( e ) foci were quantified from more than 20 images from 3 independent samples ( Online Methods ) . Data are presented as mean ± s . e . m . * P 0 . 05 , ANOVA followed by Bonferroni - Dunn post - hoc test .

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(c-e) Cells were cultured in the presence of Torin1 for 2 h. (c) Cytospun cells were fixed and analyzed by immunofluorescence microscopy using antibodies to LC3 and ATG9A. Abnormal colocalization of LC3 with ATG9A was observed in the LCLs of affected subjects. Scale bars, 10 μm and 1 μm in the inset. (d,e) The numbers of LC3+ (d) and LC3+ATG9A+ (e) foci were quantified from more than 20 images from 3 independent samples (Online Methods). Data are presented as mean ± s.e.m. *P 0.05, ANOVA followed by Bonferroni-Dunn post-hoc test.


B Ub chain interaction of the LUBEL - NZF and UBA2 domains . Immobilized GST - NZF or GST - UBA2 was incubated with Lys ( K ) 48 - , K 63 - linked , or linear di - Ub ( Ub2 ) chains and bound Ub chains were subjected to immunoblotting using anti - Ub antibody or a linear linkage - specific Ub antibody ( anti - linear Ub antibody ) . Loading of GST - fusion proteins was visualized by Ponceau S staining .

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B Ub chain interaction of the LUBEL-NZF and UBA2 domains. Immobilized GST-NZF or GST-UBA2 was incubated with Lys (K) 48-, K 63-linked, or linear di-Ub (Ub2) chains and bound Ub chains were subjected to immunoblotting using anti-Ub antibody or a linear linkage-specific Ub antibody (anti-linear Ub antibody). Loading of GST-fusion proteins was visualized by Ponceau S staining.


A In vitro ubiquitination assays of LUBEL - RBR - C WT or catalytically dead C2704A in combination with Ube1 and two different Drosophila E2s , UbcD10 or Effete / UbcD1 . Reactions were terminated at indicated times and synthesized Ub chains were detected by immunoblotting using anti - linear Ub antibody . Total protein loading was visualized by Ponceau S staining . * : nonspecific band .

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A In vitro ubiquitination assays of LUBEL-RBR-C WT or catalytically dead C2704A in combination with Ube1 and two different Drosophila E2s, UbcD10 or Effete/UbcD1. Reactions were terminated at indicated times and synthesized Ub chains were detected by immunoblotting using anti-linear Ub antibody. Total protein loading was visualized by Ponceau S staining. * : nonspecific band.


B In vitro deubiquitination of Ub chains synthesized by HOIP - and LUBEL - RBR - C . Ub chains produced by RBR - C ( Human or Drosophila ) with UbcH7 , UbcD10 or Effete were incubated with a linear linkage - specific DUB , OTULIN ( WT or catalytically dead C129A ( C / A ) mutant ) or a Lys - linkage - specific DUB , vOTU . The DUB - treated samples were subjected to Coomassie staining or immunoblotting using anti - Ub antibody .

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B In vitro deubiquitination of Ub chains synthesized by HOIP- and LUBEL-RBR-C. Ub chains produced by RBR-C (Human or Drosophila) with UbcH7, UbcD10 or Effete were incubated with a linear linkage-specific DUB, OTULIN (WT or catalytically dead C129A (C/A) mutant) or a Lys-linkage-specific DUB, vOTU. The DUB-treated samples were subjected to Coomassie staining or immunoblotting using anti-Ub antibody.


C Thioester formation assay using Atto647 - labelled Ub . Ubiquitination assay using LUBEL - RBR - C WT ( left panel ) or C2704A ( right panel ) : lanes 1 / 2 Atto - Ub + E1 , lanes 3 / 4 + ATP , lanes 5 / 6 + E2 , lanes 7 / 8 + E3 , and lanes 9 / 10 + Ub - His6 . In the presence of N - terminally tagged Atto - Ub and C - terminally tagged Ub - His6 , only Ub2 product can be synthesized and longer chain formation is restricted . Samples are run without or with DTT in odd or even numbered lanes , respectively . The gels monitor the Atto - labelled Ub .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

C Thioester formation assay using Atto647-labelled Ub. Ubiquitination assay using LUBEL-RBR-C WT (left panel) or C2704A (right panel): lanes 1/2 Atto-Ub + E1, lanes 3/4 + ATP, lanes 5/6 + E2, lanes 7/8 + E3, and lanes 9/10 + Ub-His6. In the presence of N-terminally tagged Atto-Ub and C-terminally tagged Ub-His6, only Ub2 product can be synthesized and longer chain formation is restricted. Samples are run without or with DTT in odd or even numbered lanes, respectively. The gels monitor the Atto-labelled Ub.


D The linear Ub chain formation activity of LUBEL - LDD mutants . Recombinant proteins of RBR - C WT , C2704A mutant , and LDD mutants ( R2754A and D2755A ) were assessed for their activity by in vitro ubiquitination assay . The samples were resolved on a gel and stained with Coomassie or immunoblotted using anti - linear Ub antibody .

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D The linear Ub chain formation activity of LUBEL-LDD mutants. Recombinant proteins of RBR-C WT, C2704A mutant, and LDD mutants (R2754A and D2755A) were assessed for their activity by in vitro ubiquitination assay. The samples were resolved on a gel and stained with Coomassie or immunoblotted using anti-linear Ub antibody.


E Linear Ub chain formation by full - length LUBEL transient expression in insect cells . Full - length Myc - LUBEL was transiently expressed in Drosophila Schneider 2 ( S2 ) cells , and Myc - HOIP alone or with HA - HOIL - 1L in HEK293T cells . Total cell lysates ( TCL ) of control and transfected samples were incubated with immobilized GST - Linear - Tandem Ub binding entity ( Linear - TUBE ) containing three tandem repeats of ABIN - 1 - UBAN . Pulldown samples were blotted with anti - linear Ub antibody , while TCL were blotted with anti - Myc antibody for exogenous LUBEL and HOIP , anti - HA antibody for HOIL - 1L , and anti - Tubulin antibody for loading . Input of GST proteins was analyzed by Ponceau S staining . * : nonspecific band .

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E Linear Ub chain formation by full-length LUBEL transient expression in insect cells. Full-length Myc-LUBEL was transiently expressed in Drosophila Schneider 2 (S2) cells, and Myc-HOIP alone or with HA-HOIL-1L in HEK293T cells. Total cell lysates (TCL) of control and transfected samples were incubated with immobilized GST-Linear-Tandem Ub binding entity (Linear-TUBE) containing three tandem repeats of ABIN-1-UBAN. Pulldown samples were blotted with anti-linear Ub antibody, while TCL were blotted with anti-Myc antibody for exogenous LUBEL and HOIP, anti-HA antibody for HOIL-1L, and anti-Tubulin antibody for loading. Input of GST proteins was analyzed by Ponceau S staining. * : nonspecific band.


A Endogenous linear Ub chains enriched in total protein extracts ( input , lane 1 ) from adult w1118 ( w - ) flies by GST - Linear - TUBE ( lane 3 ) . The enriched samples were further treated with recombinant DUBs , vOTU ( lane 4 ) or OTULIN ( lane 5 ) . Ub chains were visualized by immunoblotting using anti - linear Ub antibody or anti - Ub antibody . Loading of GST proteins was analyzed by Ponceau S staining .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Endogenous linear Ub chains enriched in total protein extracts (input, lane 1) from adult w1118 (w-) flies by GST-Linear-TUBE (lane 3). The enriched samples were further treated with recombinant DUBs, vOTU (lane 4) or OTULIN (lane 5). Ub chains were visualized by immunoblotting using anti-linear Ub antibody or anti-Ub antibody. Loading of GST proteins was analyzed by Ponceau S staining.


B Endogenous linear Ub chains in total cell lysate ( TCL ) of S2 cells expressing human Flag - OTULIN WT or catalytically inactive C129A mutant . TCLs enriched with GST - Linear - TUBE were examined by immunoblotting using anti - linear Ub antibody . Expression of OTULIN was analyzed by anti - Flag antibody and loading of TCL was detected by anti - Tubulin antibody . Input of GST proteins was analyzed by Ponceau S staining . * : nonspecific band .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Endogenous linear Ub chains in total cell lysate (TCL) of S2 cells expressing human Flag-OTULIN WT or catalytically inactive C129A mutant. TCLs enriched with GST-Linear-TUBE were examined by immunoblotting using anti-linear Ub antibody. Expression of OTULIN was analyzed by anti-Flag antibody and loading of TCL was detected by anti-Tubulin antibody. Input of GST proteins was analyzed by Ponceau S staining. * : nonspecific band.


C Mass spectrometry analysis using an identical sample as Figure 3B lane 6 . MS / MS spectra of the prototypic linear Ub chain peptide ( GGMQIFVK ) is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Mass spectrometry analysis using an identical sample as Figure 3B lane 6. MS/MS spectra of the prototypic linear Ub chain peptide (GGMQIFVK) is shown.


C In vitro deubiquitination assay of dCYLD . dCYLD was incubated with all eight linkage types of Ub2 chains . dCYLD catalytically dead C284S mutant was incubated with K 63 - or linear Ub2 chains . All the proteins in the reactions were resolved on Coomassie - stained SDS - PAGE gel . * : nonspecific band .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C In vitro deubiquitination assay of dCYLD. dCYLD was incubated with all eight linkage types of Ub2 chains. dCYLD catalytically dead C284S mutant was incubated with K 63- or linear Ub2 chains. All the proteins in the reactions were resolved on Coomassie-stained SDS-PAGE gel. * : nonspecific band.


D Stabilization of linear Ub chains by dCYLD C284S mutant transient expression in S2 cells . Myc - tagged dCYLD WT and C284S were transfected in S2 cells and linear Ub chains were enriched with GST - Linear - TUBE and immunoblotted by anti - linear Ub or anti - Ub antibodies . Input of GST proteins was visualized by Ponceau S staining . Expression of Myc - dCYLD was examined by anti - Myc antibody , and anti - Tubulin antibody was used for the loading control of TCL .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Stabilization of linear Ub chains by dCYLD C284S mutant transient expression in S2 cells. Myc-tagged dCYLD WT and C284S were transfected in S2 cells and linear Ub chains were enriched with GST-Linear-TUBE and immunoblotted by anti-linear Ub or anti-Ub antibodies. Input of GST proteins was visualized by Ponceau S staining. Expression of Myc-dCYLD was examined by anti-Myc antibody, and anti-Tubulin antibody was used for the loading control of TCL.


E Linear Ub chains in dCYLD mutant flies . Endogenous levels of linear Ub chains in dCYLD mutant flies were compared with a control w - fly strain by performing GST - Linear - TUBE pulldown . Enriched linear Ub chains were immunoblotted with anti - linear Ub antibody . Input of GST proteins was visualized by Ponceau S staining . Tubulin was used for the loading control of the input .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Linear Ub chains in dCYLD mutant flies. Endogenous levels of linear Ub chains in dCYLD mutant flies were compared with a control w- fly strain by performing GST-Linear-TUBE pulldown. Enriched linear Ub chains were immunoblotted with anti-linear Ub antibody. Input of GST proteins was visualized by Ponceau S staining. Tubulin was used for the loading control of the input.


B Linear Ub chains in w - and LUBEL mutant fly lysates . w - , CC / SS and delR2 flies were analyzed for the level of endogenous linear Ub chains by enriching the linear ubiquitin chains by GST - Linear - TUBE . Pulldown samples were immunoblotted by anti - linear Ub antibody , while GST - proteins were analyzed by Ponceau S staining . Anti - Tubulin antibody was used for the loading control of the input . * : nonspecific band .

B Linear Ub chains in w- and LUBEL mutant fly lysates. w-, CC/SS and delR2 flies were analyzed for the level of endogenous linear Ub chains by enriching the linear ubiquitin chains by GST-Linear-TUBE. Pulldown samples were immunoblotted by anti-linear Ub antibody, while GST-proteins were analyzed by Ponceau S staining. Anti-Tubulin antibody was used for the loading control of the input. * : nonspecific band.


C The lifespans of female w - and catalytically dead LUBEL mutant flies ( two clones of CC / SS , indicated as # 1 and # 2 , and delR2 ) . Survival of three independent cohorts with approximately 80 flies each was monitored over time . Median survival time ( days ) : w - = 78 , CC / SS # 1 = 68 , CC / SS # 2 = 65 , and delR2 = 71 . Total sample sizes are as follow ; w - = 215 , CC / SS # 1 = 242 , CC / SS # 2 = 233 , delR2 = 273 . Log - rank ( Mantel - cox ) test between fly lines : w - and CC / SS # 1 > 0 . 0001 , w - and CC / SS # 2 > 0 . 0001 , w - and delR2 > 0 . 0001 , CC / SS # 1 and CC / SS # 2 = 0 . 0007 , CC / SS # 1 and delR2 = not significant , CC / SS # 2 and delR2 = not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C The lifespans of female w- and catalytically dead LUBEL mutant flies (two clones of CC/SS, indicated as #1 and #2, and delR2). Survival of three independent cohorts with approximately 80 flies each was monitored over time. Median survival time (days): w- = 78, CC/SS #1 = 68, CC/SS #2 = 65, and delR2 = 71. Total sample sizes are as follow; w- = 215, CC/SS #1 = 242, CC/SS #2 = 233, delR2 = 273. Log-rank (Mantel-cox) test between fly lines: w- and CC/SS #1 > 0.0001, w- and CC/SS #2 > 0.0001, w- and delR2 > 0.0001, CC/SS #1 and CC/SS #2 = 0.0007, CC/SS #1 and delR2 = not significant, CC/SS #2 and delR2 = not significant.


D Histological analysis of muscle in LUBEL mutant flies . Hematoxylin and eosin ( H & amp ; amp ; E ) ( top panels ) and actin immunofluorescent staining ( Actin - IF ) ( bottom panels ) of thorax muscles from young ( Day 3 ) or aged ( Day 60 ) w - or CC / SS female flies . Scale bars : 50 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Histological analysis of muscle in LUBEL mutant flies. Hematoxylin and eosin (H&amp;E) (top panels) and actin immunofluorescent staining (Actin-IF) (bottom panels) of thorax muscles from young (Day 3) or aged (Day 60) w- or CC/SS female flies. Scale bars: 50 μm.


A The level of poly - Ub chains in w - flies after heat shock . w - male flies were incubated in a 36 ° C water bath for indicated times and the levels of poly - Ub chains in total protein extracts were compared by Ub using antibodies against linear , K 48 - , and K 63 - linked Ub chains . Ponceau S staining was used to visualize the protein loading .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A The level of poly-Ub chains in w- flies after heat shock. w- male flies were incubated in a 36 °C water bath for indicated times and the levels of poly-Ub chains in total protein extracts were compared by Ub using antibodies against linear, K 48-, and K 63-linked Ub chains. Ponceau S staining was used to visualize the protein loading.


B Survival of catalytically dead LUBEL mutant flies upon heat shock . Young adult flies ( 15 males and 15 females ) were incubated in a 36 ° C water bath and immobilized flies were counted over the time indicated . Median survival time ( hours ) : w - = 9 . 25 , CC / SS # 1 = 5 . 5 , CC / SS # 2 = 5 , delR2 = 5 . 75 , CC / SS # 1 / + = 8 . 5 , CC / SS # 2 / + = 10 , delR2 / + = 8 . p - values calculated by Gehan - Breslow - Wilcoxon test between fly lines : w - and CC / SS # 1 , CC / SS # 2 , or delR2 > 0 . 0001 , w - and CC / SS # 1 / + = 0 . 0134 , w - and CC / SS # 2 / + = ns , w - and delR2 / + = 0 . 0002 , CC / SS # 1 and CC / SS # 1 / + > 0 . 0001 , CC / SS # 2 and CC / SS # 2 / + > 0 . 0001 , delR2 and delR2 / + > 0 . 0001 . Representative data are shown from six independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Survival of catalytically dead LUBEL mutant flies upon heat shock. Young adult flies (15 males and 15 females) were incubated in a 36 °C water bath and immobilized flies were counted over the time indicated. Median survival time (hours): w- = 9.25, CC/SS #1 = 5.5, CC/SS #2 = 5, delR2 = 5.75, CC/SS #1/+ = 8.5, CC/SS #2/+ = 10, delR2/+ = 8. p-values calculated by Gehan-Breslow-Wilcoxon test between fly lines: w- and CC/SS #1, CC/SS#2, or delR2 > 0.0001, w- and CC/SS #1/+ = 0.0134, w- and CC/SS #2/+ = ns, w- and delR2/+ = 0.0002, CC/SS #1 and CC/SS #1/+ > 0.0001, CC/SS #2 and CC/SS #2/+ > 0.0001, delR2 and delR2/+ > 0.0001. Representative data are shown from six independent experiments.


C Survival of whole body or muscle - specific LUBEL knockdown flies upon heat shock . shRNA - based knockdown ( KD ) of LUBEL were driven by Tub - Gal4 or Mef2 - Gal4 flies . Control fly lines ( Tub - Gal4 / + and Mef2 - Gal4 / + ) were used to compare with the KD flies . 15 males and 15 females per each line were used in this assay . Median survival time ( hours ) : Tub - Gal4 / + = 7 , Tub - Gal4 > LUBEL = 3 . 5 , Mef2 - Gal4 / + = 7 . 5 , Mef2 - Gal4 > LUBEL = 7 . p - values calculated by Gehan - Breslow - Wilcoxon test : Tub - Gal4 < 0 . 0001 ( * * * * ) , Mef2 - Gal4 = 0 . 0029 ( * * ) . Representative data are shown from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Survival of whole body or muscle-specific LUBEL knockdown flies upon heat shock. shRNA-based knockdown (KD) of LUBEL were driven by Tub-Gal4 or Mef2-Gal4 flies. Control fly lines (Tub-Gal4/+ and Mef2-Gal4/+) were used to compare with the KD flies. 15 males and 15 females per each line were used in this assay. Median survival time (hours): Tub-Gal4/+ = 7, Tub-Gal4>LUBEL = 3.5, Mef2-Gal4/+ = 7.5, Mef2-Gal4>LUBEL= 7. p-values calculated by Gehan-Breslow-Wilcoxon test: Tub-Gal4 <0.0001 (****), Mef2-Gal4 = 0.0029 (**). Representative data are shown from three independent experiments.


D Heat - induced mRNA expression of HSP70 . w - , CC / SS # 2 and delR2 flies were heat treated for 60 min and recovered for 1 hour , and mRNA HSP70 was quantified by qPCR . Rp49 was used as a reference and w - untreated sample was used as calibrator to calculate the expression ratio . Data are presented as meanSD ( * < 0 . 01 , * * * < 0 . 001 , * * * * < 0 . 0001 ) . Representative data are shown from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Heat-induced mRNA expression of HSP70. w-, CC/SS #2 and delR2 flies were heat treated for 60 min and recovered for 1 hour, and mRNA HSP70 was quantified by qPCR. Rp49 was used as a reference and w- untreated sample was used as calibrator to calculate the expression ratio. Data are presented as meanSD (*<0.01, ***<0.001, ****<0.0001). Representative data are shown from three independent experiments.


Representative still images of control , Rod depleted cells and Rod depleted cells supplemented with RNAi resistant Venus - Rod treated with nocodazole and time in minutes indicated . Scale bar , 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative still images of control, Rod depleted cells and Rod depleted cells supplemented with RNAi resistant Venus-Rod treated with nocodazole and time in minutes indicated. Scale bar, 5 μm.


Time from NEBD to mitotic exit for the indicated conditions with each circle representing a single cell analyzed and mean time indicated by red line and standard error of mean shown . The number of cells counted and mean time indicated above .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Time from NEBD to mitotic exit for the indicated conditions with each circle representing a single cell analyzed and mean time indicated by red line and standard error of mean shown. The number of cells counted and mean time indicated above.


Immunofluorescence analysis of ZW10 and Mad1 upon depletion of Rod . Scale bar , 5 μm .

Immunofluorescence analysis of ZW10 and Mad1 upon depletion of Rod. Scale bar, 5 μm.


Quantification of kinetochore intensities of the indicated proteins in control or Rod depleted cells with the signal normalized to CREST levels and Bub1 pSpT normalized to Bub1 . Bar indicates mean and standard error of mean is shown by line . At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of kinetochore intensities of the indicated proteins in control or Rod depleted cells with the signal normalized to CREST levels and Bub1 pSpT normalized to Bub1. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments is shown.


Time from NEBD to mitotic exit in control depleted , Rod depleted , Bub1 depleted and Rod and Bub1 co - depleted HeLa , U2OS and RPE1 cells . Cells were treated with nocodazole .

Time from NEBD to mitotic exit in control depleted, Rod depleted, Bub1 depleted and Rod and Bub1 co-depleted HeLa, U2OS and RPE1 cells. Cells were treated with nocodazole.


Mad1 kinetochore levels in the indicated conditions normalized to level of CREST in HeLa cells . Cells were fixed at 45 min after releasing from RO3306 arrest into nocodazole . Bar indicates mean and standard error of mean is shown by line . At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mad1 kinetochore levels in the indicated conditions normalized to level of CREST in HeLa cells. Cells were fixed at 45 min after releasing from RO3306 arrest into nocodazole. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments is shown.


Western blot analysis of whole cell extract for the indicated conditions . Quantitative analysis of Rod levels using LiCor technology is indicated below . The asterisk indicates an unspecific band .

Western blot analysis of whole cell extract for the indicated conditions. Quantitative analysis of Rod levels using LiCor technology is indicated below. The asterisk indicates an unspecific band.


Analysis of ZW10 and Mad1 kinetochore levels in the indicated cells and conditions . Kinetochore signal is normalized to CREST signal . Bar indicates mean and standard error of mean is shown by line . At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Analysis of ZW10 and Mad1 kinetochore levels in the indicated cells and conditions. Kinetochore signal is normalized to CREST signal. Bar indicates mean and standard error of mean is shown by line. At least 200 kinetochores from 10 cells were analyzed and representative result from at least 2 independent experiments shown.


Representative still images of unperturbed mitosis for parental cells , Rod C cells and Rod CR cells with time in minutes indicated . CFP - Histone 3 was used here as the chromosome marker . Scale bar , 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative still images of unperturbed mitosis for parental cells, Rod C cells and Rod CR cells with time in minutes indicated. CFP-Histone 3 was used here as the chromosome marker. Scale bar, 5 μm.


Time from NEBD to exit of indicated conditions with cells treated with nocodazole Red circles represent cells still arrested in mitosis when the filming stopped .

O O O O O O O O O O O O O O O O O O O O O O O O O O

Time from NEBD to exit of indicated conditions with cells treated with nocodazole Red circles represent cells still arrested in mitosis when the filming stopped.


Time from NEBD to exit of indicated conditions with cells treated with taxol . Red circles represent cells still arrested in mitosis when the filming stopped .

O O O O O O O O O O O O O O O O O O O O O O O O O O O

Time from NEBD to exit of indicated conditions with cells treated with taxol. Red circles represent cells still arrested in mitosis when the filming stopped.


Time from NEBD to exit in indicated conditions with cells treated with nocodazole Red circles represent cells still arrested in mitosis when the filming stopped .

Time from NEBD to exit in indicated conditions with cells treated with nocodazole Red circles represent cells still arrested in mitosis when the filming stopped.


Time from NEBD to exit in indicated conditions with cells treated with taxol . Red circles represent cells still arrested in mitosis when the filming stopped .

Time from NEBD to exit in indicated conditions with cells treated with taxol. Red circles represent cells still arrested in mitosis when the filming stopped.


Mass spectrometry analysis of BubR1 IPs from the indicated cell lines . Relative protein quantification values ( MaxLFQ , log10 ) are plotted across conditions . Data from analysis of 3 technical repeats of BubR1 purifications with standard deviation indicated .

Mass spectrometry analysis of BubR1 IPs from the indicated cell lines. Relative protein quantification values (MaxLFQ, log10) are plotted across conditions. Data from analysis of 3 technical repeats of BubR1 purifications with standard deviation indicated.


Estimated protein levels of Bub1 in the indicated conditions relative to parental cell lines . In Bub1 CR we only detected one peptide from three purifications . Standard deviation indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Estimated protein levels of Bub1 in the indicated conditions relative to parental cell lines. In Bub1 CR we only detected one peptide from three purifications. Standard deviation indicated.


Representative still images of unperturbed mitosis for parental cells , Bub1 C cells and Bub1 CR cells with time in minutes indicated . CFP - Histone 3 was used as the chromosome marker . Scale bar , 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative still images of unperturbed mitosis for parental cells, Bub1 C cells and Bub1 CR cells with time in minutes indicated. CFP-Histone 3 was used as the chromosome marker. Scale bar, 5 μm.