C Poly ‐ caspase activity in THEMIS KD 1G4 ‐ CD8 cells . Cells were stimulated for 24 h with NY ‐ ESO ‐ 1 pMHC tetramers . Camptothecin was used as a positive control for caspase activation . A FAM ‐ FLICA detection probe was used to assess poly ‐ caspase activity by flow cytometry . Data shown are a representative example of three independent experiments . D Quantification of poly ‐ caspase activity of cells from ( C ) . Data from three independent experiments were used in the analysis . n = 3 , means ± SEM are shown ; two ‐ tailed unpaired Student ' s t ‐ test , * P 0 . 05 , * * P 0 . 01 ; ns , not significant .

O O O B-SUBCELLULAR O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Poly‐caspase activity in THEMIS KD 1G4‐CD8 cells. Cells were stimulated for 24 h with NY‐ESO‐1 pMHC tetramers. Camptothecin was used as a positive control for caspase activation. A FAM‐FLICA detection probe was used to assess poly‐caspase activity by flow cytometry. Data shown are a representative example of three independent experiments.D Quantification of poly‐caspase activity of cells from (C). Data from three independent experiments were used in the analysis. n = 3, means ± SEM are shown; two‐tailed unpaired Student's t‐test, *P 0.05, **P 0.01; ns, not significant.


A Lentiviral KD of SHP1 expression in 1G4 ‐ CD8 cells . KD efficiency was assessed using immunoblotting .

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A Lentiviral KD of SHP1 expression in 1G4‐CD8 cells. KD efficiency was assessed using immunoblotting.


B Annexin ‐ V staining of THEMIS and SHP1 KD 1G4 ‐ CD8 cells . Control , THEMIS and SHP1 knock ‐ down Jurkat 1G4 ‐ CD8 cells were stimulated for 24 h with plate ‐ bound 6V tetramers . Annexin ‐ V surface expression was analysed by flow cytometry and is shown as fold ‐ increase over non ‐ stimulated . Data from three independent experiments were used in the analysis . n = 3 , means ± SD are shown ; two ‐ way ANOVA , Bonferroni post ‐ test , * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 .

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B Annexin‐V staining of THEMIS and SHP1 KD 1G4‐CD8 cells. Control, THEMIS and SHP1 knock‐down Jurkat 1G4‐CD8 cells were stimulated for 24 h with plate‐bound 6V tetramers. Annexin‐V surface expression was analysed by flow cytometry and is shown as fold‐increase over non‐stimulated. Data from three independent experiments were used in the analysis. n = 3, means ± SD are shown; two‐way ANOVA, Bonferroni post‐test, *P 0.05, **P 0.01, ***P 0.001.


C Lentiviral knock ‐ down of THEMIS and SHP1 expression in primary humanCD4 + T cells . Following transduction , cells were expanded on CD3 / CD28 beads in the presence of exogenous IL ‐ 2 . Knock ‐ down efficiency was analysed by immunoblotting on day 4 post ‐ transduction .

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C Lentiviral knock‐down of THEMIS and SHP1 expression in primary humanCD4+T cells. Following transduction, cells were expanded on CD3/CD28 beads in the presence of exogenous IL‐2. Knock‐down efficiency was analysed by immunoblotting on day 4 post‐transduction.


D Expression of CD25 , CD69 and Annexin ‐ V surface markers of cells from ( C ) was assessed by flow cytometry . Data shown are representative of three independent experiments .

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D Expression of CD25, CD69 and Annexin‐V surface markers of cells from (C) was assessed by flow cytometry. Data shown are representative of three independent experiments.


E Annexin ‐ V staining of 1G4 ‐ CD8 cells re ‐ expressing a SHP1 tail tyrosine mutant . Jurkat 1G4 ‐ CD8 cells were transduced with Knock ‐ down / re ‐ expression constructs for HA ‐ SHP1 wild ‐ type ( wt ) or Tyr536 / 564 to Phe double mutant ( YYFF ) . Cells were stimulated and analysed by flow cytometry as in ( B ) . Data shown are representative of three independent experiments . n = 3 , means ± SD are shown .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD 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-EXP_ASSAY I-EXP_ASSAY O 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 Annexin‐V staining of 1G4‐CD8 cells re‐expressing a SHP1 tail tyrosine mutant. Jurkat 1G4‐CD8 cells were transduced with Knock‐down/re‐expression constructs for HA‐SHP1 wild‐type (wt) or Tyr536/564 to Phe double mutant (YYFF). Cells were stimulated and analysed by flow cytometry as in (B). Data shown are representative of three independent experiments. n = 3, means ± SD are shown.


A CD8 + T cells from OT ‐ I Rag2 − / − mice expressing wild ‐ type ( LCK ) or LCKS59A proteins were stimulated with H ‐ 2Kb ‐ positive antigen ‐ presenting cells pulsed with serial dilutions of the OVA ( 257 - 264 ) peptide , the OVA peptide variants Q4 and T4 , and VSV , a peptide that is not recognized by the OT ‐ I TCR . CD69 up ‐ regulation ( left ) and TCR down ‐ regulation ( right ) were analysed . Data are representative of three independent experiments .

O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O O O B-ORGANISM O O O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O

A CD8+T cells from OT‐I Rag2−/−mice expressing wild‐type (LCK) or LCKS59A proteins were stimulated with H‐2Kb‐positive antigen‐presenting cells pulsed with serial dilutions of the OVA (257-264) peptide, the OVA peptide variants Q4 and T4, and VSV, a peptide that is not recognized by the OT‐I TCR. CD69 up‐regulation (left) and TCR down‐regulation (right) were analysed. Data are representative of three independent experiments.


B CD8 + T cells from OT ‐ I Rag2 − / − mice expressing wild ‐ type ( LCK ) or LCKS59A proteins were labelled with CFSE and stimulated with serial dilution of H ‐ 2Kb ‐ OVA or H ‐ 2Kb ‐ Q4R7 peptide ‐ MHC tetramers . After 3 days , the absolute numbers of divided T cells were determined . Data are representative of three independent experiments .

O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O O O B-ORGANISM O O O O O B-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O

B CD8+T cells from OT‐I Rag2−/−mice expressing wild‐type (LCK) or LCKS59A proteins were labelled with CFSE and stimulated with serial dilution of H‐2Kb‐OVA or H‐2Kb‐Q4R7 peptide‐MHC tetramers. After 3 days, the absolute numbers of divided T cells were determined. Data are representative of three independent experiments.


( B ) Representative confocal microscopy sections of m6A - Tracer signal in HAP - 1 cells following pA - DamID with indicated antibodies . pA - Dam only : primary antibody was omitted , the dotted yellow line indicates DAPI segmentation . Free Dam : permeabilized cells were treated with freely diffusing pure Dam protein and SAM for 30 min . Scale bar corresponds to 2 µm . ( C ) Quantification of peripheral enrichment of antibody staining and m6A - Tracer signals after pA - DamID with indicated antibodies . The nuclear rim and interior were segmented using DAPI signal and the mean m6A or antibody signal was determined and tranformed to a log2 - ratio . The nuclear rim mask extends slightly beyond the NL , resulting in underestimation of the real enrichment .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O

(B) Representative confocal microscopy sections of m6A-Tracer signal in HAP-1 cells following pA-DamID with indicated antibodies. pA-Dam only: primary antibody was omitted, the dotted yellow line indicates DAPI segmentation. Free Dam: permeabilized cells were treated with freely diffusing pure Dam protein and SAM for 30 min. Scale bar corresponds to 2 µm. (C) Quantification of peripheral enrichment of antibody staining and m6A-Tracer signals after pA-DamID with indicated antibodies. The nuclear rim and interior were segmented using DAPI signal and the mean m6A or antibody signal was determined and tranformed to a log2-ratio. The nuclear rim mask extends slightly beyond the NL, resulting in underestimation of the real enrichment.


( D ) HT1080 cells expressing inducible Dam - Lamin B1 were treated with Shield1 to induce m6A methylation . Cells were either fixed immediately or processed according to the pA - DamID protocol ( as negative controls ) and then fixed on poly - L - lysine coated cover slips . Cells were imaged by confocal microscopy for the NL ( Lamin B2 antibody ) and methylation ( m6A - Tracer ) for both conditions . Laser settings were changed between images to optimize image quality . The mask for defining the nuclear rim was obtained by segmentation of the DAPI image . The scale bar corresponds to 2 µm .

O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SMALL_MOLECULE O O O O O O O O O O

(D) HT1080 cells expressing inducible Dam-Lamin B1 were treated with Shield1 to induce m6A methylation. Cells were either fixed immediately or processed according to the pA-DamID protocol (as negative controls) and then fixed on poly-L-lysine coated cover slips. Cells were imaged by confocal microscopy for the NL (Lamin B2 antibody) and methylation (m6A-Tracer) for both conditions. Laser settings were changed between images to optimize image quality. The mask for defining the nuclear rim was obtained by segmentation of the DAPI image.The scale bar corresponds to 2 µm.


( E ) For every cell , the 50 % decay distance from the nuclear periphery was determined for Lamin B2 and m6A - Tracer by fitting exponential decay functions from the nuclear rim as defined by segmentation of the DAPI image The difference in 50 % decay distance between Lamin B2 and m6A - Tracer was used as a measure of the thickness of the m6A - Tracer layer .

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(E) For every cell, the 50% decay distance from the nuclear periphery was determined for Lamin B2 and m6A-Tracer by fitting exponential decay functions from the nuclear rim as defined by segmentation of the DAPI image The difference in 50% decay distance between Lamin B2 and m6A-Tracer was used as a measure of the thickness of the m6A-Tracer layer.


( F ) Distribution of the m6A - Tracer layer thickness for HT1080 cells expressing Dam - Lamin B1 , visualized before and after the pA - DamID protocol . For comparison , a similar analysis was performed with HT1080 cells not expressing Dam - LaminB1 , subjected to Lamin B2 pA - DamID .

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(F) Distribution of the m6A-Tracer layer thickness for HT1080 cells expressing Dam-Lamin B1, visualized before and after the pA-DamID protocol. For comparison, a similar analysis was performed with HT1080 cells not expressing Dam-LaminB1, subjected to Lamin B2 pA-DamID.


( A ) Example of raw pA - DamID data tracks from 2 million HAP - 1 cells for Lamin B2 , H3K27me3 and the Dam control from a single experiment . Sequenced reads are counted in 20kb bins and normalized for library size . ( B ) Same pA - DamID tracks of Lamin B2 and H3K27me3 as in ( A ) but after normalization to the Dam - only control ( to correct for accessibility and amplification biases ) and log2 - transformation .

O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O

(A) Example of raw pA-DamID data tracks from 2 million HAP-1 cells for Lamin B2, H3K27me3 and the Dam control from a single experiment. Sequenced reads are counted in 20kb bins and normalized for library size. (B) Same pA-DamID tracks of Lamin B2 and H3K27me3 as in (A) but after normalization to the Dam-only control (to correct for accessibility and amplification biases) and log2-transformation.


( C ) Correlation between the median peripheral enrichment of m6A - Tracer determined by confocal microscopy and enrichment of sequencing reads within LADs in HAP - 1 cells . The LAD definition is based on conventional Lamin B1 DamID data . Every point represents a single pA - DamID experiment for which both microscopy and sequencing data were generated , colored by the antibody used . The blue line represents a linear model with a standard error confidence interval in grey . The red dashed line represents the diagonal . The Pearson correlation coefficient was converted to a t - statistic and a one - sided t - test with n - 2 degrees of freedom was used to determine statistical significance .

O O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Correlation between the median peripheral enrichment of m6A-Tracer determined by confocal microscopy and enrichment of sequencing reads within LADs in HAP-1 cells. The LAD definition is based on conventional Lamin B1 DamID data. Every point represents a single pA-DamID experiment for which both microscopy and sequencing data were generated, colored by the antibody used. The blue line represents a linear model with a standard error confidence interval in grey. The red dashed line represents the diagonal. The Pearson correlation coefficient was converted to a t-statistic and a one-sided t-test with n-2 degrees of freedom was used to determine statistical significance.


( D ) Comparisons of pA - DamID genome - wide data ( bin size 20 kb ) for different lamin antibodies in hTERT - RPE cells : Lamin B1 vs . Lamin B2 ( left panel ) and Lamin B2 vs . Lamin A / C ( right panel ) . n denotes the number of independent biological experiments that were averaged for each data track . The blue line represents a linear model . The red dashed line represents the diagonal .

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(D) Comparisons of pA-DamID genome-wide data (bin size 20 kb) for different lamin antibodies in hTERT-RPE cells: Lamin B1 vs. Lamin B2 (left panel) and Lamin B2 vs. Lamin A/C (right panel). n denotes the number of independent biological experiments that were averaged for each data track. The blue line represents a linear model. The red dashed line represents the diagonal.


( A ) Comparison of pA - DamID and DamID data for for various lamins across a representative genomic locus in HAP - 1 and hTERT - RPE cells . Arrow indicates a region with dissimilar pA - DamID and DamID scores .

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(A) Comparison of pA-DamID and DamID data for for various lamins across a representative genomic locus in HAP-1 and hTERT-RPE cells. Arrow indicates a region with dissimilar pA-DamID and DamID scores.


( B ) Genome - wide correlation of Lamin B1 pA - DamID and Lamin B1 DamID data . The blue line represents a linear model . The red dashed line represents the diagonal .

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(B) Genome-wide correlation of Lamin B1 pA-DamID and Lamin B1 DamID data. The blue line represents a linear model. The red dashed line represents the diagonal.


( D ) Multidimensional scaling ( MDS ) plot showing all individual replicates of DamID and pA - DamID experiments for various lamins and cell types .

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(D) Multidimensional scaling (MDS) plot showing all individual replicates of DamID and pA-DamID experiments for various lamins and cell types.


( B ) NL interactions in hTERT - RPE cells at indicated times after mitosis for chromosome 2 , as determined by Lamin B2 pA - DamID . Log2 - ratios were convered to z - scores to account for differences in dynamic range between replicates Average of two independent biological replicates . LADs are colored by their differential class ( down : red , stable : grey , up : green , The centromere is shown as grey box .

O O O B-SUBCELLULAR O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O

(B) NL interactions in hTERT-RPE cells at indicated times after mitosis for chromosome 2, as determined by Lamin B2 pA-DamID. Log2-ratios were convered to z-scores to account for differences in dynamic range between replicates Average of two independent biological replicates. LADs are colored by their differential class (down:red, stable:grey, up:green, The centromere is shown as grey box.


( C ) pA - DamID signal as a function of time after mitosis , for stable ( grey , n = 717 ) and dynamic LADs ( red and green : decreasing ( n = 234 ) and increasing ( n = 225 ) NL interactions , respectively ) . Lines represent mean signals of the three LAD classes and the shaded area one standard deviation on both sides . ( D ) Rate of changes in NL interactions ( average pA - DamID z - score per hour ) as function of time after mitosis for stable and dynamics LADs Boxplots : horizontal lines represent 25th , 50th and 75th percentiles ; whiskers extend to the smallest values no further than 1 . 5 times distance between 25th and 75th percentiles .

O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O 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-SUBCELLULAR O O O O O O O O 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-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O 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) pA-DamID signal as a function of time after mitosis, for stable (grey, n=717) and dynamic LADs (red and green: decreasing (n=234) and increasing (n=225) NL interactions, respectively). Lines represent mean signals of the three LAD classes and the shaded area one standard deviation on both sides. (D) Rate of changes in NL interactions (average pA-DamID z-score per hour) as function of time after mitosis for stable and dynamics LADs Boxplots: horizontal lines represent 25th, 50th and 75th percentiles; whiskers extend to the smallest values no further than 1.5 times distance between 25th and 75th percentiles.


( E - G ) LAD size distribution in basepairs ( E ) , density of active genes ( F ) and Repli - seq score distributions ( G ) for stable and dynamic LADs Statistical significance was determined using a two ‐ way Wilcoxin test with Benjamini - Hochberg p - value correction to account for multiple testing .

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(E-G) LAD size distribution in basepairs (E), density of active genes (F) and Repli-seq score distributions (G) for stable and dynamic LADs Statistical significance was determined using a two‐way Wilcoxin test with Benjamini-Hochberg p-value correction to account for multiple testing.


( H ) Difference in pA - DamID z - scores between 1h and 21h time points , plotted against the distance to telomeres ( leftpanel ) or centromeres ( rightpanel ) . Each dot represents a single LAD . Chromosomes are colored and ordered by size . Red line is a fitted loess curve . The Spearman correlation coefficient ( ρ ) was used to test for a significant monotonic , non - linear association .

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(H) Difference in pA-DamID z-scores between 1h and 21h time points, plotted against the distance to telomeres (leftpanel) or centromeres (rightpanel). Each dot represents a single LAD. Chromosomes are colored and ordered by size. Red line is a fitted loess curve. The Spearman correlation coefficient (ρ) was used to test for a significant monotonic, non-linear association.


Difference in pA - DamID z - scores plotted against the distance to telomeres ( leftpanel ) or centromeres ( rightpanel ) . Each dot represents a single LAD . Chromosomes are colored and ordered by size . Red line is a fitted loess curve . The Spearman correlation coefficient ( ρ ) was used to test for a significant monotonic , non - linear association . pA - DamID z - scores for individual time points .

O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O O O O O O

Difference in pA-DamID z-scores plotted against the distance to telomeres (leftpanel) or centromeres (rightpanel). Each dot represents a single LAD. Chromosomes are colored and ordered by size. Red line is a fitted loess curve. The Spearman correlation coefficient (ρ) was used to test for a significant monotonic, non-linear association. pA-DamID z-scores for individual time points.


( A ) Lamin B2 pA - DamID z - scores of a representative locus in HAP - 1 cells sorted for G1 , mid - S and G2 phases . Arrows point to regions that lose NL interaction from G1 to G2 . The red star indicates the end of the chromosome . Average of three biological replicates .

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(A) Lamin B2 pA-DamID z-scores of a representative locus in HAP-1 cells sorted for G1, mid-S and G2 phases. Arrows point to regions that lose NL interaction from G1 to G2. The red star indicates the end of the chromosome. Average of three biological replicates.


difference in pA - DamID z - scores between G2 and G1 sorted pools as function of distance to either telomere or centromere for HAP - 1 ( n = 3 ) , HCT116 ( n = 2 ) and K562 cells ( n = 2 ) .

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difference in pA-DamID z-scores between G2 and G1 sorted pools as function of distance to either telomere or centromere for HAP-1 (n=3), HCT116 (n=2) and K562 cells (n=2).


( C - E ) LAD size distribution in basepairs ( C ) , density of active genes ( D ) and Repli - seq scores ( E ) for stable and dynamic LADs . Repli - seq data are not available for HAP - 1 cells .

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(C-E) LAD size distribution in basepairs (C), density of active genes (D) and Repli-seq scores (E) for stable and dynamic LADs. Repli-seq data are not available for HAP-1 cells.


( F ) Overlapping fractions of stable and dynamic LADs and iLADs with Hi - C subcompartments in HAP - 1 and K562 cells , as imputed by SNIPER A permutation test with 1000 permutations ( R - package regioneR was used to test for a significant enrichment of B1 subcompartments in decreasing LADs , resulting in P - values of 1e - 3 and 1e - 3 for HAP - 1 and K562 , respectively .

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(F) Overlapping fractions of stable and dynamic LADs and iLADs with Hi-C subcompartments in HAP-1 and K562 cells, as imputed by SNIPER A permutation test with 1000 permutations (R-package regioneR was used to test for a significant enrichment of B1 subcompartments in decreasing LADs, resulting in P-values of 1e-3 and 1e-3 for HAP-1 and K562, respectively.


( A ) Representative K562 locus comparing Lamin B2 pA - DamID z - scores in cell cycle sorted cells with replication timing . Arrows point to regions with increased mid - S signal compared to G1 and G2 , which tend to overlap with regions that exhibit intermediate replication timing .

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(A) Representative K562 locus comparing Lamin B2 pA-DamID z-scores in cell cycle sorted cells with replication timing. Arrows point to regions with increased mid-S signal compared to G1 and G2, which tend to overlap with regions that exhibit intermediate replication timing.


( B ) Difference tracks in pA - DamID z - scores between the cell cycle stages Data were smoothed by a running mean of 5

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(B) Difference tracks in pA-DamID z-scores between the cell cycle stages Data were smoothed by a running mean of 5 bins


( C ) Scatterplots of Repli - seq vs . the difference in smoothed pA - DamID z - scores between mid - S sorted cells and either G1 or G2 sorted cells for K562 and HCT116 cells ( two replicates ) . Bins with pA - DamID differences between G1 and G2 bigger than 0 . 2 were filtered out to prevent artifacts from cell cycle dynamics . Blue line shows a fitted loess curve . The Spearman correlation coefficient ( ρ ) between the absolute Repli - seq signal and pA - DamID difference was used as a measure of the enriched signal at mid - S replicating DNA ( with a Repli - seq score around zero ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-CELL_LINE O B-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O

(C) Scatterplots of Repli-seq vs. the difference in smoothed pA-DamID z-scores between mid-S sorted cells and either G1 or G2 sorted cells for K562 and HCT116 cells (two replicates). Bins with pA-DamID differences between G1 and G2 bigger than 0.2 were filtered out to prevent artifacts from cell cycle dynamics. Blue line shows a fitted loess curve. The Spearman correlation coefficient (ρ) between the absolute Repli-seq signal and pA-DamID difference was used as a measure of the enriched signal at mid-S replicating DNA (with a Repli-seq score around zero).


( D ) Scatterplots of changes in pA - DamID signal versus the pA - DamID signal in G1 ( left - hand panels ) or G2 cells ( right - hand panels )

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(D) Scatterplots of changes in pA-DamID signal versus the pA-DamID signal in G1 (left-hand panels) or G2 cells (right-hand panels)


( A ) Ribbon diagram of the HsAtg4B - LC3 ( 1 - 120 ) complex . HsAtg4B is coloured salmon red , and LC3 is coloured green .

O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O

(A) Ribbon diagram of the HsAtg4B-LC3(1-120) complex. HsAtg4B is coloured salmon red, and LC3 is coloured green.


( B ) Structural comparison between free and HsAtg4B ‐ bound LC3 . Crystal structure of free LC3 ( 1 - 120 ) ( PDB code 1UGM ) is superimposed on the HsAtg4B ‐ bound LC3 . HsAtg4B ‐ bound LC3 is coloured green , and free LC3 is coloured grey .

O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O

(B) Structural comparison between free and HsAtg4B‐bound LC3. Crystal structure of free LC3(1-120) (PDB code 1UGM) is superimposed on the HsAtg4B‐bound LC3. HsAtg4B‐bound LC3 is coloured green, and free LC3 is coloured grey.


( C ) Structural comparison between free and LC3 ‐ bound HsAtg4B . Crystal structure of free HsAtg4B ( PDB code 2CY7 ) is superimposed on the LC3 ‐ bound HsAtg4B . LC3 ‐ bound HsAtg4B is coloured salmon red , and free HsAtg4B is coloured grey . The regions with large conformational differences between free and LC3 ‐ bound forms are coloured black ( free ) and red ( LC3 bound ) . All the figures representing molecular structures were generated with PyMOL ( DeLano , 2002 ) .

O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD 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-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

(C) Structural comparison between free and LC3‐bound HsAtg4B. Crystal structure of free HsAtg4B (PDB code 2CY7) is superimposed on the LC3‐bound HsAtg4B. LC3‐bound HsAtg4B is coloured salmon red, and free HsAtg4B is coloured grey. The regions with large conformational differences between free and LC3‐bound forms are coloured black (free) and red (LC3 bound). All the figures representing molecular structures were generated with PyMOL ( DeLano, 2002).


( A ) Interaction between HsAtg4B and the ubiquitin core of LC3 . HsAtg4B is coloured salmon red , and LC3 is coloured green . The side chains of the residues involved in the HsAtg4B - LC3 interaction are shown as stick models . Possible hydrophilic interactions are shown as broken lines .

O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O

(A) Interaction between HsAtg4B and the ubiquitin core of LC3. HsAtg4B is coloured salmon red, and LC3 is coloured green. The side chains of the residues involved in the HsAtg4B-LC3 interaction are shown as stick models. Possible hydrophilic interactions are shown as broken lines.


( B ) Interaction between HsAtg4B and the C ‐ terminal tail of LC3 . Trp142 and the regulatory loop of HsAtg4B are coloured blue , whereas the remainder is coloured as in ( A ) . As the side chain of Cys74 has two conformations , both conformations are shown . Possible hydrophilic interactions are shown as broken lines .

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(B) Interaction between HsAtg4B and the C‐terminal tail of LC3. Trp142 and the regulatory loop of HsAtg4B are coloured blue, whereas the remainder is coloured as in (A). As the side chain of Cys74 has two conformations, both conformations are shown. Possible hydrophilic interactions are shown as broken lines.


( C ) In vitro proteolysis assay . Left , LC3 mutants fused to GST were incubated with HsAtg4B . Right , LC3 - GST was incubated with HsAtg4B mutants . Procedures are described in detail in Materials and methods .

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(C) In vitro proteolysis assay. Left, LC3 mutants fused to GST were incubated with HsAtg4B. Right, LC3-GST was incubated with HsAtg4B mutants. Procedures are described in detail in Materials and methods.


( A ) Annealed Fo − Fc electron density map for the C ‐ terminal region ( residues 119 - 122 ) of LC3 ( 1 - 124 ) . Left , LC3 ( 1 - 124 ) bound to the HsAtg4B H280A mutant ; right , LC3 ( 1 - 124 ) bound to the HsAtg4B C74S mutant . Structure of the C ‐ terminal region of LC3 is also shown as a stick model , in which carbon , nitrogen and oxygen atoms are coloured green , blue and red , respectively .

O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O

(A) Annealed Fo−Fc electron density map for the C‐terminal region (residues 119-122) of LC3(1-124). Left, LC3(1-124) bound to the HsAtg4B H280A mutant; right, LC3(1-124) bound to the HsAtg4B C74S mutant. Structure of the C‐terminal region of LC3 is also shown as a stick model, in which carbon, nitrogen and oxygen atoms are coloured green, blue and red, respectively.


( B ) Structure of the C ‐ terminal tail of LC3 ( 1 - 124 ) and the catalytic site of HsAtg4B . Left , LC3 ( 1 - 124 ) bound to HsAtg4B H280A mutant , right , LC3 ( 1 - 124 ) bound to the HsAtg4B C74S mutant . HsAtg4B is shown as a ribbon model , whereas LC3 is shown as a stick model . The side chains of the residues comprising the catalytic triad and Tyr54 of HsAtg4B are also shown as a stick model . Carbon , nitrogen , oxygen and sulphur atoms are coloured green , blue , red and yellow , respectively . Mutated catalytic residues are coloured cyan .

O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

(B) Structure of the C‐terminal tail of LC3(1-124) and the catalytic site of HsAtg4B. Left, LC3(1-124) bound to HsAtg4B H280A mutant, right, LC3(1-124) bound to the HsAtg4B C74S mutant. HsAtg4B is shown as a ribbon model, whereas LC3 is shown as a stick model. The side chains of the residues comprising the catalytic triad and Tyr54 of HsAtg4B are also shown as a stick model. Carbon, nitrogen, oxygen and sulphur atoms are coloured green, blue, red and yellow, respectively. Mutated catalytic residues are coloured cyan.


( C ) Structural comparison between the HsAtg4B - LC3 precursor and SENP2 - SUMO ‐ 3 precursor complexes . Left , structure of the HsAtg4B - LC3 precursor complex ; right , structure of the SENP2 - SUMO ‐ 3 precursor complex . HsAtg4B and SENP2 are shown as a ribbon model , whereas LC3 and SUMO ‐ 3 precursors are shown as a stick model . The side chains of the important residues at the catalytic site are also shown as a stick model . Atom colouring is the same as in ( A , B ) .

O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O 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) Structural comparison between the HsAtg4B-LC3 precursor and SENP2-SUMO‐3 precursor complexes. Left, structure of the HsAtg4B-LC3 precursor complex; right, structure of the SENP2-SUMO‐3 precursor complex. HsAtg4B and SENP2 are shown as a ribbon model, whereas LC3 and SUMO‐3 precursors are shown as a stick model. The side chains of the important residues at the catalytic site are also shown as a stick model. Atom colouring is the same as in (A, B).


( A ) Surface model of free ( left ) and LC3 ( 1 - 124 ) ‐ bound ( right ) HsAtg4B . W142 and the regulatory loop are coloured blue , and the N ‐ terminal tail is coloured cyan . The catalytic Cys74 is coloured red . LC3 ( 1 - 124 ) is shown as a ribbon model .

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(A) Surface model of free (left) and LC3(1-124)‐bound (right) HsAtg4B. W142 and the regulatory loop are coloured blue, and the N‐terminal tail is coloured cyan. The catalytic Cys74 is coloured red. LC3(1-124) is shown as a ribbon model.


( B ) In vitro proteolysis assay using N ‐ terminal tail ‐ deleted HsAtg4B . LC3 - GST was incubated with either wild ‐ type or N ‐ terminally truncated HsAtg4B and was subjected to SDS – PAGE analysis .

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(B) In vitro proteolysis assay using N‐terminal tail‐deleted HsAtg4B. LC3-GST was incubated with either wild‐type or N‐terminally truncated HsAtg4B and was subjected to SDS–PAGE analysis.


( C ) Structural comparison between free and LC3 ( 1 - 124 ) ‐ bound HsAtg4B and SENP2 - SUMO ‐ 3 precursor complexes . Left , free HsAtg4B ; middle , the HsAtg4B - LC3 precursor complex ; right , the SENP2 - SUMO ‐ 3 precursor complex . The three structures are shown in the same orientation . Model colouring is the same as in Figure 3 .

O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

(C) Structural comparison between free and LC3(1-124)‐bound HsAtg4B and SENP2-SUMO‐3 precursor complexes. Left, free HsAtg4B; middle, the HsAtg4B-LC3 precursor complex; right, the SENP2-SUMO‐3 precursor complex. The three structures are shown in the same orientation. Model colouring is the same as in Figure 3.


( D ) urface model of LC3 ( 1 - 120 ) bound to HsAtg4B . Atom colouring is the same as in ( A ) .

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(D) urface model of LC3(1-120) bound to HsAtg4B. Atom colouring is the same as in (A).


( A ) Overall structure of Hs bound to both substrate and non ‐ substrate LC3 . Substrate LC3 is designated as LC3 ( S ) and coloured green , whereas non ‐ substrate LC3 is designated as LC3 ( N ) and coloured blue .

O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O

(A) Overall structure of Hs bound to both substrate and non‐substrate LC3. Substrate LC3 is designated as LC3(S) and coloured green, whereas non‐substrate LC3 is designated as LC3(N) and coloured blue.


( B ) Stereo ‐ view of the N ‐ terminal tail of HsAtg4B bound to the WXXL ‐ binding site of LC3 . The side chains of the residues involved in the interaction between HsAtg4B and LC3 are labelled and shown with a stick model . Model colouring is the same as in ( A ) .

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(B) Stereo‐view of the N‐terminal tail of HsAtg4B bound to the WXXL‐binding site of LC3. The side chains of the residues involved in the interaction between HsAtg4B and LC3 are labelled and shown with a stick model. Model colouring is the same as in (A).


( C ) Structural comparison of the N ‐ terminal tail - LC3 interaction with the p62 - LC3 interaction . The structure of LC3 complexed with a p62 peptide is superimposed on that complexed with the N ‐ terminal tail peptide . As the structure of LC3 is almost identical to each other , only that bound to the N ‐ terminal tail peptide of Hs is shown .

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(C) Structural comparison of the N‐terminal tail-LC3 interaction with the p62-LC3 interaction. The structure of LC3 complexed with a p62 peptide is superimposed on that complexed with the N‐terminal tail peptide. As the structure of LC3 is almost identical to each other, only that bound to the N‐terminal tail peptide of Hs is shown.


( D ) Chemical shift perturbations of the LC3 backbone amide groups upon complex formation with the N ‐ terminal ‐ tail peptide . The combined 1H and 15N chemical shift differences , calculated using the equation Δp . p . m . = [ ( ΔδHN ) 2 + ( ΔδN / 5 ) 2 ] 1 / 2 were plotted against residue numbers .

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(D) Chemical shift perturbations of the LC3 backbone amide groups upon complex formation with the N‐terminal‐tail peptide. The combined 1H and 15N chemical shift differences, calculated using the equation Δp.p.m.=[(ΔδHN)2+(ΔδN/5)2]1/2 were plotted against residue numbers.


( E ) Mapping of chemical shift perturbation results on the crystal structure of LC3 bound to HsAtg4B . The residues with Δp . p . m . > 0 . 4 are shown in blue , 0 . 4 > Δp . p . m . > 0 . 3 in cyan and 0 . 3 > Δp . p . m . > 0 . 2 in green .

O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mapping of chemical shift perturbation results on the crystal structure of LC3 bound to HsAtg4B. The residues with Δp.p.m. >0.4 are shown in blue, 0.4>Δp.p.m.>0.3 in cyan and 0.3>Δp.p.m. >0.2 in green.


( A ) Representative images ( and details from boxed regions ) of WT MEF cells transfected with YFPParkin ( green ) and treated with FCCP ( 10 μM ) to induce mitophagy . Cells were immunostained with anti - Tom20 ( magenta ) to reveal the mitochondrial network . Each row represents one stage in the process of mitophagy that has been used to score the progression of mitophagy at different time - points for the different conditions tested ( the specific time - point of each example is indicated in the image ) . From top to bottom : i ) " Diffuse Parkin " defines cells in where Parkin expression is homogenously distributed throughout the cytoplasm ( quantified in B ) . ii ) Soon after mitochondrial damage Parkin appears enriched in small " puncta " throughout the cytoplasm localizing within isolated mitochondria ( quantified in C ) . iii ) Later in the process these puncta structures aggregate in bigger structures accumulating in perinuclear regions ( quantified in D ) . iv ) The process of Parkin translocation eventually affects all the mitochondrial population and usually collapses in few large structures possibly forming part of big autolysosomes in the perinuclear region ( quantified in D ) . Scoring was performed from 3 independent experiments ( n = 3 ; one way ANOVA with Dunnett post test ) . Error bars represent s . e . m . Significance : * p < 0 . 05 and * * p < 0 .

O O O O O O O O O O O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O B-GENEPROD B-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images (and details from boxed regions) of WT MEF cells transfected with YFPParkin (green) and treated with FCCP (10 μM) to induce mitophagy. Cells were immunostained with anti-Tom20 (magenta) to reveal the mitochondrial network. Each row represents one stage in the process of mitophagy that has been used to score the progression of mitophagy at different time-points for the different conditions tested (the specific time-point of each example is indicated in the image). From top to bottom: i) "Diffuse Parkin" defines cells in where Parkin expression is homogenously distributed throughout the cytoplasm (quantified in B). ii) Soon after mitochondrial damage Parkin appears enriched in small "puncta" throughout the cytoplasm localizing within isolated mitochondria (quantified in C). iii) Later in the process these puncta structures aggregate in bigger structures accumulating in perinuclear regions (quantified in D). iv) The process of Parkin translocation eventually affects all the mitochondrial population and usually collapses in few large structures possibly forming part of big autolysosomes in the perinuclear region (quantified in D). Scoring was performed from 3 independent experiments (n=3; one way ANOVA with Dunnett post test). Error bars represent s.e.m. Significance: *p<0.05 and **p<0.01


( F ) Representative images ( and details from boxed regions ) of WT MEFs cells transfected with GFPPINK1 ( green ) and treated with FCCP ( 10 μM ) to induce mitophagy . Cells were immunostained with anti - Tom20 ( magenta ) to reveal the mitochondrial network . Each row represents one of the three categories of GFP signal used to describe PINK1 translocation to mitochondria after mitochondrial damage ( cytoplasmic , intermediate and mitochondrial ) . The scoring of these categories was used to describe PINK1 translocation to mitochondria in WT and MiroDKO cells at different time - points shortly after mitochondrial insult ( G ) . Data collected from 3 independent experiments ( n = 3 ) . Error bars represent s . e . m . Significance : * p < 0 . 05 and * * p < 0 . 01

O O O O O O O O O O O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O 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) Representative images (and details from boxed regions) of WT MEFs cells transfected with GFPPINK1 (green) and treated with FCCP (10 μM) to induce mitophagy. Cells were immunostained with anti-Tom20 (magenta) to reveal the mitochondrial network. Each row represents one of the three categories of GFP signal used to describe PINK1 translocation to mitochondria after mitochondrial damage (cytoplasmic, intermediate and mitochondrial). The scoring of these categories was used to describe PINK1 translocation to mitochondria in WT and MiroDKO cells at different time-points shortly after mitochondrial insult (G). Data collected from 3 independent experiments (n=3). Error bars represent s.e.m. Significance: *p<0.05 and **p<0.01


B ) Representative images showing full length control and selected Miro1 lysine mutants ( Miro15R , Miro1allR and Miro1R572K ) expressing in MiroDKO MEF cells . All constructs ( myc - tag , green ) localise to mitochondria ( Tom20 , red ) .

O O O O O O O O O O B-GENEPROD B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD B-CELL_TYPE O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-SUBCELLULAR O B-GENEPROD O O O O

B) Representative images showing full length control and selected Miro1 lysine mutants (Miro15R, Miro1allR and Miro1R572K) expressing in MiroDKO MEF cells. All constructs (myc-tag, green) localise to mitochondria (Tom20, red).


C ) Immunoblot showing efficient expression of Miro1 lysine mutants in FlagParkin expressing SH - SY5Y cells .

O O B-EXP_ASSAY O O O O B-GENEPROD B-SMALL_MOLECULE O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O

C) Immunoblot showing efficient expression of Miro1 lysine mutants in FlagParkin expressing SH-SY5Y cells.


D ) Ubiquitination assay showing that Miro1 lysine mutants have reduced ubiquitination upon FCCP treatment ( 1h , 10 μM ) in FlagParkin overexpressing SH - SY5Y . The effect is quantified in ( E ) ( n = 4 independent experiments for Miro1WT and Miro15R and n = 3 for Miro1allR and Miro1R572K , ANOVA with Sidak ' s post hoc test ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 .

O O B-EXP_ASSAY O O O B-GENEPROD B-SMALL_MOLECULE O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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) Ubiquitination assay showing that Miro1 lysine mutants have reduced ubiquitination upon FCCP treatment (1h, 10 μM) in FlagParkin overexpressing SH-SY5Y. The effect is quantified in (E) (n=4 independent experiments for Miro1WT and Miro15R and n=3 for Miro1allR and Miro1R572K, ANOVA with Sidak's post hoc test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.


F ) Representative western blot showing a degradation assay in FlagParkin overexpressing SH - SY5Y cells transfected with Miro1WT , Miro15R or Miro1allR constructs and treated with FCCP ( 10 μM ) for 3 or 6 hours . Quantification of Miro1 levels ( G ) the matrix protein PDH E1α in ( H ) and Parkin levels ( I ) ( n = 4 independent experiments ; ANOVA with Sidak ' s post hoc test ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative western blot showing a degradation assay in FlagParkin overexpressing SH-SY5Y cells transfected with Miro1WT, Miro15R or Miro1allR constructs and treated with FCCP (10 μM) for 3 or 6 hours. Quantification of Miro1 levels (G) the matrix protein PDH E1α in (H) and Parkin levels (I) (n=4 independent experiments; ANOVA with Sidak's post hoc test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.


( A ) Representative images at 3 hours of FCCP treatment ( 10 μM ) of YFPParkin ( green ) expressing MiroDKO cells co - transfected with the specified myc - tagged versions of Miro1 ( cyan ) . Tom20 ( red ) was used to reveal the mitochondrial network . A detail of YFPParkin translocation onto the mitochondrial network is also shown for each condition .

O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O

(A) Representative images at 3 hours of FCCP treatment (10 μM) of YFPParkin (green) expressing MiroDKO cells co-transfected with the specified myc-tagged versions of Miro1 (cyan). Tom20 (red) was used to reveal the mitochondrial network. A detail of YFPParkin translocation onto the mitochondrial network is also shown for each condition.


( B and C ) Quantification of the fraction of cells showing advanced stages of the mitophagic process ( cells showing mitochondrial aggregation due to advanced Parkin translocation or the complete translocation of Parkin onto all the mitochondrial network ) at 1 hour ( B ) or 3 hours ( C ) after FCCP treatment ( data collected from at least 3 independent experiments : WT n = 7 , MiroDKO n = 8 , + mycMiro1WT = 7 , mycMiro15R = 3 , mycMiro1allR = 3 ; mycMiro2 = 3 ; one way ANOVA with Dunnett post test ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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 and C) Quantification of the fraction of cells showing advanced stages of the mitophagic process (cells showing mitochondrial aggregation due to advanced Parkin translocation or the complete translocation of Parkin onto all the mitochondrial network) at 1 hour (B) or 3 hours (C) after FCCP treatment (data collected from at least 3 independent experiments: WT n=7, MiroDKO n=8, +mycMiro1WT=7, mycMiro15R=3, mycMiro1allR=3; mycMiro2=3; one way ANOVA with Dunnett post test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001


D ) Western blots and quantification of Miro1 degradation in MiroDKO cells co - expressing YFPParkin and the indicated myc - tagged Miro1 constructs and treated with FCCP ( 10 μM ) for the indicated times ( n = 3 different experiments ; 2 - way - ANOVA with Dunnett post test ; comparisons between Miro1WT and Miro1allR are represented with yellow asteriscs , comparisons between Miro1WT and Miro15R are represented in green and comparisons between Miro15R and Miro1allR are represented in black ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O 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-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O

D) Western blots and quantification of Miro1 degradation in MiroDKO cells co-expressing YFPParkin and the indicated myc-tagged Miro1 constructs and treated with FCCP (10 μM) for the indicated times (n=3 different experiments; 2-way-ANOVA with Dunnett post test; comparisons between Miro1WT and Miro1allR are represented with yellow asteriscs, comparisons between Miro1WT and Miro15R are represented in green and comparisons between Miro15R and Miro1allR are represented in black).


E ) . Representative images at 24 hours of FCCP treatment ( 10 μM ) of YFPParkin ( green ) expressing MiroDKO cells co - transfected with the specified myc - tagged versions of Miro1 ( cyan ) . Tom20 ( red ) was used to reveal the mitochondrial network . Expression of both ubiquitin mutants ( Miro15R and Miro1allR - arrowheads ) protects from the mitophagic clearance of mitochondria ( arrows ) after damage . ( F ) Quantification of the fraction of cells showing the complete loss of Tom20 signal of mitochondria after 24 hours of FCCP treatment ( data collected from at least 3 independent experiments : MiroDKO n = 8 , mycMiro1WT = 7 , mycMiro15R = 3 , mycMiro1allR = 3 ; one way ANOVA with Dunnett post test ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001

O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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). Representative images at 24 hours of FCCP treatment (10 μM) of YFPParkin (green) expressing MiroDKO cells co-transfected with the specified myc-tagged versions of Miro1 (cyan). Tom20 (red) was used to reveal the mitochondrial network. Expression of both ubiquitin mutants (Miro15R and Miro1allR - arrowheads) protects from the mitophagic clearance of mitochondria (arrows) after damage. (F) Quantification of the fraction of cells showing the complete loss of Tom20 signal of mitochondria after 24 hours of FCCP treatment (data collected from at least 3 independent experiments: MiroDKO n=8, mycMiro1WT=7, mycMiro15R=3, mycMiro1allR=3; one way ANOVA with Dunnett post test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001


( A ) Representative confocal images of the soma of WT and Miro1KO cortical neurons expressing YFPParkin and MtDsRed after valinomycin treatment at the indicated time points ( scale bars = 5 µm ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O O O O

(A) Representative confocal images of the soma of WT and Miro1KO cortical neurons expressing YFPParkin and MtDsRed after valinomycin treatment at the indicated time points (scale bars = 5 µm).


( B ) Airyscan confocal images of Parkin recruitment after 5 hours of valinomycin treatment in WT and Miro1KO neuronal processes ( scale bars = 2 . 5 µm ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD B-CELL_TYPE O O O O O O O O O O O

(B) Airyscan confocal images of Parkin recruitment after 5 hours of valinomycin treatment in WT and Miro1KO neuronal processes (scale bars = 2.5 µm).


( C ) Fluorescent linescans of YFPParkin and MtDsRed signal in WT and Miro1KO neurons after 5 hours of valinomycin treatment ( lines shown in A ) .

O O O B-EXP_ASSAY O O B-GENEPROD O B-SUBCELLULAR O O O O B-GENEPROD B-CELL_TYPE O O O O B-SMALL_MOLECULE O O O O O O O O

(C) Fluorescent linescans of YFPParkin and MtDsRed signal in WT and Miro1KO neurons after 5 hours of valinomycin treatment (lines shown in A).


( D ) Quantification of Parkin recruitment to mitochondria ( YFPParkin signal overlapping MtDsRed signal - intensity - adjusted and normalised to t = 0 ) in WT and Miro1KO neurons following valinomycin treatment ( n = 15 cells all conditions per genotype over 3 neuronal preparations ; two - way ANOVA ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 .

O O O O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O 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) Quantification of Parkin recruitment to mitochondria (YFPParkin signal overlapping MtDsRed signal - intensity-adjusted and normalised to t=0) in WT and Miro1KO neurons following valinomycin treatment (n=15 cells all conditions per genotype over 3 neuronal preparations; two-way ANOVA). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001


( E ) Quantification of mitochondrial occupancy ( area of MtDsRed signal in soma / entire area of soma ) in the somas of WT and Miro1KO neurons following valinomycin treatment ( n = 15 cells all conditions per genotype over 3 neuronal preparations ; two - way ANOVA ) .

O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O B-GENEPROD B-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O

(E) Quantification of mitochondrial occupancy (area of MtDsRed signal in soma / entire area of soma) in the somas of WT and Miro1KO neurons following valinomycin treatment (n=15 cells all conditions per genotype over 3 neuronal preparations; two-way ANOVA).


( F ) Quantification of mitochondrial clearance in somas of WT and Miro1KO neurons between 2 and 5 hours of valinomycin treatment ( n = 15 cells all conditions per genotype over 3 neuronal preparations ; Unpaired t - test ) .

O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O B-GENEPROD B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O

(F) Quantification of mitochondrial clearance in somas of WT and Miro1KO neurons between 2 and 5 hours of valinomycin treatment (n=15 cells all conditions per genotype over 3 neuronal preparations; Unpaired t-test).


( G ) Representative confocal images of WT , Miro1KO and PINK1KO cortical neurons immunostained with MAP2 ( cyan ) and s65 - phospho - ubiquitin ( green ) after valinomycin treatment ( Scale bars = 20µm ) . ( H ) Quantification of s65 - phospho - ubiquitin signal intensity within MAP2 signal ( normalised to MAP2 area and t = 0 ) in Miro1WT , Miro1KO and PINK1KO neurons following valinomycin treatment n = 3 , 4 and 3 embryos from WT , Miro1KO and PINK1KO embryos , respectively ( 6 ROIs per condition ; two - way ANOVA ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O B-GENEPROD O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD B-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Representative confocal images of WT, Miro1KO and PINK1KO cortical neurons immunostained with MAP2 (cyan) and s65-phospho-ubiquitin (green) after valinomycin treatment (Scale bars = 20µm). (H) Quantification of s65-phospho-ubiquitin signal intensity within MAP2 signal (normalised to MAP2 area and t=0) in Miro1WT, Miro1KO and PINK1KO neurons following valinomycin treatment n= 3, 4 and 3 embryos from WT, Miro1KO and PINK1KO embryos, respectively (6 ROIs per condition; two-way ANOVA). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001


( I ) Representative confocal images of the soma of Miro1KO cortical neurons expressing Miro1WT and mutant forms of Miro1 , YFPParkin and MtDsRed without valinomycin treatment ( Scale bars = 10 µm ) . Quantification of parkin colocalization with MtDsRed ( YFPParkin signal overlapping MtDsRed signal - intensity - adjusted ) in the soma of Miro1KO and PINK1KO cortical neurons ( n = 12 cells all conditions per genotype over 3 neuronal preparations ; one - way ANOVA ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) Representative confocal images of the soma of Miro1KO cortical neurons expressing Miro1WT and mutant forms of Miro1, YFPParkin and MtDsRed without valinomycin treatment (Scale bars = 10 µm). Quantification of parkin colocalization with MtDsRed (YFPParkin signal overlapping MtDsRed signal - intensity-adjusted) in the soma of Miro1KO and PINK1KO cortical neurons (n=12 cells all conditions per genotype over 3 neuronal preparations; one-way ANOVA). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001


( J ) Representative confocal images of the soma of Miro1KO cortical neurons expressing WT and mutant forms of Miro1 , YFPParkin and MtDsRed after 5 hours of valinomycin treatment ( Scale bars = 10 µm ) . Quantification of Parkin recruitment to mitochondria ( Normalised to t = 0 ) in Miro1KO and PINK1KO cortical neurons following valinomycin treatment ( n = 12 cells all conditions per genotype over 3 neuronal preparations , two - way ANOVA ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O B-GENEPROD B-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) Representative confocal images of the soma of Miro1KO cortical neurons expressing WT and mutant forms of Miro1, YFPParkin and MtDsRed after 5 hours of valinomycin treatment (Scale bars = 10 µm). Quantification of Parkin recruitment to mitochondria (Normalised to t=0) in Miro1KO and PINK1KO cortical neurons following valinomycin treatment (n=12 cells all conditions per genotype over 3 neuronal preparations, two-way ANOVA). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001


( A ) Representative blots of 4 and 12 months old hippocampal lysates from control , Miro1CKO and Miro2KO . B ) Quantification of protein levels in 4 month lysates and 12 month lysates ( n = 3 animals / genotype at 4 months and n = 4 animals / genotype at 12 months ; one - way ANOVA with post - hoc Dunnett ' s test ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 .

O O O O B-EXP_ASSAY O O O O O O B-TISSUE O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative blots of 4 and 12 months old hippocampal lysates from control, Miro1CKO and Miro2KO. B) Quantification of protein levels in 4 month lysates and 12 month lysates (n=3 animals / genotype at 4 months and n=4 animals / genotype at 12 months; one-way ANOVA with post-hoc Dunnett's test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.


C ) Representative images of hippocampal regions from control and Miro1CKO crossed with MitoDendra animals .

O O O O O B-TISSUE O O O O B-GENEPROD O O B-SMALL_MOLECULE O O

C) Representative images of hippocampal regions from control and Miro1CKO crossed with MitoDendra animals.


( D ) Details from cortical regions of control , Miro1CKO and Miro2KO animals at 12 months of age showing large mitochondrial structures occurring only in the case of Miro1CKO animals .

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(D) Details from cortical regions of control, Miro1CKO and Miro2KO animals at 12 months of age showing large mitochondrial structures occurring only in the case of Miro1CKO animals.


( E ) Electron microcopy images of control and Miro1CKO brains showing the ultrastructure of the mitochondrial compartment . The mitochondrial units in Miro1CKO cells are enlarged and show altered cristae structure at 4 months of age . This process is progressive as mitochondrial structures appear larger and with reduced intramitochondrial complexity at 12 months of age .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD B-TISSUE O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O

(E) Electron microcopy images of control and Miro1CKO brains showing the ultrastructure of the mitochondrial compartment. The mitochondrial units in Miro1CKO cells are enlarged and show altered cristae structure at 4 months of age. This process is progressive as mitochondrial structures appear larger and with reduced intramitochondrial complexity at 12 months of age.


Representative images ( F ) and quantification ( G ) of Mfn1 and Mfn2 staining in cortical slices from 12 month WT and Miro1CKO mice ( n = 4 animals / genotype ; student ' s t - test ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 .

O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-TISSUE O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images (F) and quantification (G) of Mfn1 and Mfn2 staining in cortical slices from 12 month WT and Miro1CKO mice (n=4 animals / genotype; student's t-test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.


( A ) Representative western blot images of brain lysates from 12 - month WT , Miro1CKO and Miro2KO mice immunoblotted with the antibodies stated . ( B ) Quantification of eIF2α and P - eIF2α band intensity ( normalised to actin band intensity ) from 12 - month WT , Miro1CKO and Miro2KO brain lysates . Phosphorylation of eIF2α is defined as P - eIF2α / eIF2α ( n = 4 mice per genotype , unpaired t - test ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O O O O B-GENEPROD O B-GENEPROD B-ORGANISM B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD B-TISSUE O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O 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) Representative western blot images of brain lysates from 12-month WT, Miro1CKO and Miro2KO mice immunoblotted with the antibodies stated. (B) Quantification of eIF2α and P-eIF2α band intensity (normalised to actin band intensity) from 12-month WT, Miro1CKO and Miro2KO brain lysates. Phosphorylation of eIF2α is defined as P-eIF2α/eIF2α (n=4 mice per genotype, unpaired t-test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.


( C ) Representative confocal images of cortical regions of 12 - month mitoDendra - crossed WT and Miro1CKO mice stained with MAP2 and P - eIF2α . ( D ) Quantification of P - eIF2α signal intensity from cortical regions of 12 - month mitoDendra - crossed WT and Miro1CKO mice ( n = 4 mice per genotype ; unpaired t - test ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD B-ORGANISM B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-TISSUE O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD B-ORGANISM O O O O B-ORGANISM O O O O O O O O O

(C) Representative confocal images of cortical regions of 12-month mitoDendra-crossed WT and Miro1CKO mice stained with MAP2 and P-eIF2α. (D) Quantification of P-eIF2α signal intensity from cortical regions of 12-month mitoDendra-crossed WT and Miro1CKO mice (n=4 mice per genotype; unpaired t-test).


( E ) Representative confocal images ( 63x ) of cortical regions of 12 - month mitoDendra - crossed WT and Miro1CKO mice stained with MAP2 and P - eIF2α . Arrows indicate the presence of megamitochondria ( > 2 . 5 µm2 ) , stars indicate neuronal somas without megamitochondria .

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(E) Representative confocal images (63x) of cortical regions of 12-month mitoDendra -crossed WT and Miro1CKO mice stained with MAP2 and P-eIF2α. Arrows indicate the presence of megamitochondria (>2.5 µm2), stars indicate neuronal somas without megamitochondria.


( F ) Example zoom images of neuronal somas with and without megamitochondria from cortical regions of 12 - month mitoDendra - crossed Miro1CKO mice stained with P - eIF2α . ( G ) Quantification P - eIF2α signal intensity ( a . u . ) from neuronal somas with and without megamitochondria from cortical regions of 12 - month mitoDendra - crossed Miro1CKO ( n = 16 sections from 4 mice ; paired t - test ) .

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(F) Example zoom images of neuronal somas with and without megamitochondria from cortical regions of 12-month mitoDendra -crossed Miro1CKO mice stained with P-eIF2α. (G) Quantification P-eIF2α signal intensity (a.u.) from neuronal somas with and without megamitochondria from cortical regions of 12-month mitoDendra-crossed Miro1CKO (n=16 sections from 4 mice; paired t-test).


A Mpc1 and Mpc2 are present under fermentative conditions , while Mpc1 and Mpc3 are present under respiratory conditions . MPC subunits were detected by Western blot in mitochondria isolated from yeast cells grown in glucose ( YPD ) or glycerol ( YPG ) . Mitochondrial matrix protein aconitase ( Aco1 ) , 23 ‐ kDa translocase of the inner membrane subunit ( Tim23 ) , and 70 ‐ kDa translocase of the outer membrane subunit ( Tom70 ) were used as loading controls .

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A Mpc1 and Mpc2 are present under fermentative conditions, while Mpc1 and Mpc3 are present under respiratory conditions. MPC subunits were detected by Western blot in mitochondria isolated from yeast cells grown in glucose (YPD) or glycerol (YPG). Mitochondrial matrix protein aconitase (Aco1), 23‐kDa translocase of the inner membrane subunit (Tim23), and 70‐kDa translocase of the outer membrane subunit (Tom70) were used as loading controls.


B Kinetics of the switch from Mpc2 to Mpc3 . Yeast cells were grown in YPD until mid ‐ log phase and then shifted to YPG medium . Whole cell extracts were taken at the indicated time points after the shift , and HA ‐ tagged Mpc2 and Mpc3 detected by Western blotting . Mpc3 is induced very rapidly by switch from YPD to YPG , while Mpc2 levels decrease only gradually .

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B Kinetics of the switch from Mpc2 to Mpc3. Yeast cells were grown in YPD until mid‐log phase and then shifted to YPG medium. Whole cell extracts were taken at the indicated time points after the shift, and HA‐tagged Mpc2 and Mpc3 detected by Western blotting. Mpc3 is induced very rapidly by switch from YPD to YPG, while Mpc2 levels decrease only gradually.


C Alternative pyruvate carrier complexes MPCFERM and MPCOX are detected by BN ‐ PAGE . Isolated mitochondria expressing HA ‐ tagged MPC proteins were run on blue native gels , and MPC complexes detected with an HA ‐ specific antibody . MPCFERM consisting of Mpc1 and Mpc2 is present under fermentative conditions , and MPCOX consisting of Mpc1 and Mpc3 is present under respiratory conditions . In addition , a complex of ˜ 300 kDa is observed under respiratory conditions that contains Mpc1 and Mpc3 . Non ‐ specific reactivity of the anti ‐ HA antibody toward respiratory chain complexes that are highly abundant in oxidative growth conditions is denoted by an asterisk ( * ) .

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C Alternative pyruvate carrier complexes MPCFERM and MPCOX are detected by BN‐PAGE. Isolated mitochondria expressing HA‐tagged MPC proteins were run on blue native gels, and MPC complexes detected with an HA‐specific antibody. MPCFERM consisting of Mpc1 and Mpc2 is present under fermentative conditions, and MPCOX consisting of Mpc1 and Mpc3 is present under respiratory conditions. In addition, a complex of ˜300 kDa is observed under respiratory conditions that contains Mpc1 and Mpc3. Non‐specific reactivity of the anti‐HA antibody toward respiratory chain complexes that are highly abundant in oxidative growth conditions is denoted by an asterisk (*).


A Constitutive expression of either MPCFERM or MPCOX can rescue the slow growth phenotype of the mpc1Δmpc2Δmpc3Δ triple deletion mutant . Growth tests were performed with cells transformed with MPC1 , MPC2 , or MPC3 expression plasmids or with empty vectors in indicated combinations . A serial dilution of yeast cells was spotted on agar plates with glucose ‐ containing synthetic minimal medium with ( SD + AA ) or without ( SD − AA ) amino acids .

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A Constitutive expression of either MPCFERM or MPCOX can rescue the slow growth phenotype of the mpc1Δmpc2Δmpc3Δ triple deletion mutant. Growth tests were performed with cells transformed with MPC1, MPC2, or MPC3 expression plasmids or with empty vectors in indicated combinations. A serial dilution of yeast cells was spotted on agar plates with glucose‐containing synthetic minimal medium with (SD+AA) or without (SD−AA) amino acids.


B Plasmid ‐ encoded MPC proteins can be detected in isolated mitochondria when Mpc1 is co ‐ expressed with either Mpc2 or Mpc3 . Cells were grown in selective medium containing glycerol as a carbon source . MPC subunits were constitutively expressed from plasmids in indicated combinations in the mpc1Δmpc2Δmpc3Δ background and detected by Western blot using anti ‐ HA or anti ‐ Flag antibodies . Mitochondrial enzymes aconitase ( Aco1 ) and malate dehydrogenase ( Mdh1 ) were used as loading controls .

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B Plasmid‐encoded MPC proteins can be detected in isolated mitochondria when Mpc1 is co‐expressed with either Mpc2 or Mpc3. Cells were grown in selective medium containing glycerol as a carbon source. MPC subunits were constitutively expressed from plasmids in indicated combinations in the mpc1Δmpc2Δmpc3Δ background and detected by Western blot using anti‐HA or anti‐Flag antibodies. Mitochondrial enzymes aconitase (Aco1) and malate dehydrogenase (Mdh1) were used as loading controls.


C BN ‐ PAGE confirms the formation of MPCFERM and MPCOX complexes after constitutive plasmid ‐ based protein expression . Non ‐ specific reactivity of the antibody is denoted by an asterisk ( * ) . It has to be noted that all complexes migrate slightly faster than in Fig C , most likely because MPC proteins are tagged with a single epitope tag as opposed to the 3HA ‐ tag in Fig C .

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C BN‐PAGE confirms the formation of MPCFERM and MPCOX complexes after constitutive plasmid‐based protein expression. Non‐specific reactivity of the antibody is denoted by an asterisk (*). It has to be noted that all complexes migrate slightly faster than in Fig C, most likely because MPC proteins are tagged with a single epitope tag as opposed to the 3HA‐tag in Fig C.


D Mpc1 is co ‐ immunoprecipitated with Mpc2 or Mpc3 . Mitochondria isolated from cells harboring appropriate expression plasmids and grown in glycerol ‐ containing medium were lysed in 1 % digitonin , and either Mpc2 ‐ Flag or Mpc3 ‐ Flag was immunoprecipitated . Mpc1 ‐ HA was detected by Western blot with an antibody directed against the epitope tag . The matrix protein aconitase ( Aco1 ) and 40 ‐ kDa translocase of the outer membrane subunit ( Tom40 ) were not co ‐ immunoprecipitated .

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D Mpc1 is co‐immunoprecipitated with Mpc2 or Mpc3. Mitochondria isolated from cells harboring appropriate expression plasmids and grown in glycerol‐containing medium were lysed in 1% digitonin, and either Mpc2‐Flag or Mpc3‐Flag was immunoprecipitated. Mpc1‐HA was detected by Western blot with an antibody directed against the epitope tag. The matrix protein aconitase (Aco1) and 40‐kDa translocase of the outer membrane subunit (Tom40) were not co‐immunoprecipitated.


E , F Stoichiometry of Mpc1 complexes . Chemical cross ‐ linking indicates that Mpc1 / Mpc2 and Mpc1 / Mpc3 form heterodimers of the predicted molecular weight . DSG , disuccimidyl glutarate . Cross ‐ linking products with unknown partners are labeled with a question mark ( ? ) .

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E, F Stoichiometry of Mpc1 complexes. Chemical cross‐linking indicates that Mpc1/Mpc2 and Mpc1/Mpc3 form heterodimers of the predicted molecular weight. DSG, disuccimidyl glutarate. Cross‐linking products with unknown partners are labeled with a question mark (?).


A , B Determination of MPC membrane topology by IASD labeling of isolated mitochondria expressing the indicated single cysteine variants of Mpc1 ( A ) and Mpc3 ( B ) . Mitochondria were treated with IASD as indicated . Negative [ quenching by pretreatment with DTT ( pre ‐ DTT ) ] and positive [ lysis with 0 . 5 % Triton X ‐ 100 ( T ‐ X100 ) ] controls for the labeling reaction are shown . As a further control , also translocase of the inner membrane subunit Tim23 is detected by Western blot , which contains three endogenous cysteines , all of which are located in membrane ‐ embedded regions . Labeling of an IMS ‐ exposed cysteine with IASD is indicated by a mobility shift on SDS - PAGE corresponding to the molecular weight of IASD ( ˜ 500 Da ) .

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A, B Determination of MPC membrane topology by IASD labeling of isolated mitochondria expressing the indicated single cysteine variants of Mpc1 (A) and Mpc3 (B). Mitochondria were treated with IASD as indicated. Negative [quenching by pretreatment with DTT (pre‐DTT)] and positive [lysis with 0.5% Triton X‐100 (T‐X100)] controls for the labeling reaction are shown. As a further control, also translocase of the inner membrane subunit Tim23 is detected by Western blot, which contains three endogenous cysteines, all of which are located in membrane‐embedded regions. Labeling of an IMS‐exposed cysteine with IASD is indicated by a mobility shift on SDS-PAGE corresponding to the molecular weight of IASD (˜500 Da).


C Protease protection assay in mitochondria expressing Mpc1 ‐ GFP , Mpc2 ‐ GFP , or Mpc3 ‐ GFP . Intact mitochondria ( M ) , mitoplasts with a ruptured outer membrane after hypo ‐ osmotic swelling ( Sw ) , or mitochondrial lysates with 0 . 5 % Triton X ‐ 100 ( Tr ) were treated with proteinase K ( PK ) . Loading controls are Tom70 ( outer membrane ) , Tim23 ( inner membrane ) , and Mdh1 ( matrix ) . GFP ‐ fused Mpc2 and Mpc3 are degraded in mitoplasts , whereas Mpc1 ‐ GFP is not .

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C Protease protection assay in mitochondria expressing Mpc1‐GFP, Mpc2‐GFP, or Mpc3‐GFP. Intact mitochondria (M), mitoplasts with a ruptured outer membrane after hypo‐osmotic swelling (Sw), or mitochondrial lysates with 0.5% Triton X‐100 (Tr) were treated with proteinase K (PK). Loading controls are Tom70 (outer membrane), Tim23 (inner membrane), and Mdh1 (matrix). GFP‐fused Mpc2 and Mpc3 are degraded in mitoplasts, whereas Mpc1‐GFP is not.


A MPCOX has higher transport activity than MPCFERM . The uptake of 14C ‐ labeled pyruvate into intact mitochondria was measured in vitro . Mitochondria had been isolated from cells grown in glycerol ‐ containing medium and expressing no subunit ( vectors ) , MPCFERM , MPCOX , Mpc2 , or Mpc3 . Imported pyruvate was quantified by re ‐ isolation of mitochondria and subsequent scintillation counting after 1 , 2 , or 5 min of incubation with [ 14C ] ‐ pyruvate . B Imported [ 14C ] ‐ pyruvate as in ( A ) after 5 ‐ min incubation . The difference between MPCOX and MPCFERM ( * * P = 0 . 0037 ) was significant ( unpaired t ‐ test ) .

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A MPCOX has higher transport activity than MPCFERM. The uptake of 14C‐labeled pyruvate into intact mitochondria was measured in vitro. Mitochondria had been isolated from cells grown in glycerol‐containing medium and expressing no subunit (vectors), MPCFERM, MPCOX, Mpc2, or Mpc3. Imported pyruvate was quantified by re‐isolation of mitochondria and subsequent scintillation counting after 1, 2, or 5 min of incubation with [14C]‐pyruvate.B Imported [14C]‐pyruvate as in (A) after 5‐min incubation. The difference between MPCOX and MPCFERM (**P = 0.0037) was significant (unpaired t‐test).


C Pyruvate dehydrogenase activity was measured in lysates of mitochondria expressing no subunit ( vectors ) , MPCFERM , or MPCOX .

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C Pyruvate dehydrogenase activity was measured in lysates of mitochondria expressing no subunit (vectors), MPCFERM, or MPCOX.


The chimeric construct Mpc2C3 is expressed in isolated mitochondria , while Mpc3C2 is not . Mpc1 ‐ HA and the Flag ‐ tagged chimeric proteins were detected by Western blotting in isolated mitochondria .

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The chimeric construct Mpc2C3 is expressed in isolated mitochondria, while Mpc3C2 is not. Mpc1‐HA and the Flag‐tagged chimeric proteins were detected by Western blotting in isolated mitochondria.


Mpc2C3 forms a carrier complex with Mpc1 , as analyzed by BN ‐ PAGE . When co ‐ expressed with Mpc1 , Mpc2C3 shows the same complex pattern as Mpc3 , including the 300K complex .

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Mpc2C3 forms a carrier complex with Mpc1, as analyzed by BN‐PAGE. When co‐expressed with Mpc1, Mpc2C3 shows the same complex pattern as Mpc3, including the 300K complex.


Pyruvate transport activity of the Mpc1 / Mpc2C3 complex reaches ˜ 85 % of MPCOX activity . Uptake of [ 14C ] ‐ pyruvate into mitochondria isolated from yeastcells expressing the indicated MPC protein combinations was measured in vitro . Difference between MPCFERM and Mpc1 / Mpc2C3 is significant ( unpaired t ‐ test ; * P = 0 . 02 ) . Means of n = 4 experiments are shown , with error bars representing SEM .

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Pyruvate transport activity of the Mpc1/Mpc2C3 complex reaches ˜85% of MPCOX activity. Uptake of [14C]‐pyruvate into mitochondria isolated from yeastcells expressing the indicated MPC protein combinations was measured in vitro. Difference between MPCFERM and Mpc1/Mpc2C3 is significant (unpaired t‐test; *P = 0.02). Means of n = 4 experiments are shown, with error bars representing SEM.


a , A TRPML1Va - expressing HEK293T cell showed a large inwardly rectifying whole - cell current elicited by voltage steps ( from - 140 mV to + 80 mV in increments of 20 mV ) in the standard extracellular ( Tyrode ' s ) bath solution . Step duration , 90 ms ; holding potential , 0 mV ; Vm , membrane potential .

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a, A TRPML1Va-expressing HEK293T cell showed a large inwardly rectifying whole-cell current elicited by voltage steps (from -140 mV to +80 mV in increments of 20 mV) in the standard extracellular (Tyrode's) bath solution. Step duration, 90 ms; holding potential, 0 mV; Vm, membrane potential.


b , No significant inward current was detected in NMDG + ( Na + - free , Ca2 + - free , pH 4 . 6 ) solution .

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b, No significant inward current was detected in NMDG+ (Na+-free, Ca2+-free, pH 4.6) solution.


c , Inwardly rectifying step currents were evoked by 30 mM Fe2 + solution ( pH 4 . 6 ) in the same cell as shown in a and b .

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c, Inwardly rectifying step currents were evoked by 30 mM Fe2+ solution (pH 4.6) in the same cell as shown in a and b.


d , pH - dependence of IFe / TRPML1Va . Whole - cell currents were elicited by repeated voltage ramps ( - 100 to + 100 mV ; 400 ms ) with a 4 - s interval between ramps . Only a portion of the voltage protocol is shown . Holding potential , 0 mV .

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d, pH-dependence of IFe/TRPML1Va. Whole-cell currents were elicited by repeated voltage ramps (-100 to +100 mV; 400 ms) with a 4-s interval between ramps. Only a portion of the voltage protocol is shown. Holding potential, 0 mV.


e , Large IFe / TRPML2Va was seen in the presence of 30 and 105 mM Fe2 + ( pH 4 . 6 ) .

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e, Large IFe/TRPML2Va was seen in the presence of 30 and 105 mM Fe2+ (pH 4.6).


f , Little or no IFe was seen in a TRPML3Va - expressing cell .

f, Little or no IFe was seen in a TRPML3Va-expressing cell.


a , Current densities ( mean ± s . e . m . , n = 4 - 10 ) of IFe ( 30 mM Fe2 + , pH 4 . 6 ) for TRPML1Va and ML4 mutant TRPML1Va channels . Asterisk indicates statistical difference ( P < 0 . 01 ) compared to TRPML1Va .

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a, Current densities (mean ± s.e.m., n = 4-10) of IFe (30 mM Fe2+, pH 4.6) for TRPML1Va and ML4 mutant TRPML1Va channels. Asterisk indicates statistical difference (P < 0.01) compared to TRPML1Va.


b , 55Fe2 + uptake ( normalized ) in HEK293T cells transfected with vector control , with TRPML1Va , with F408D - TRPML1Va and with T232P - TRPML1Va constructs . Error bars indicate the standard deviation on the basis of two independent triplicate experiments .

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b, 55Fe2+uptake (normalized) in HEK293T cells transfected with vector control, with TRPML1Va, with F408D-TRPML1Va and with T232P-TRPML1Va constructs. Error bars indicate the standard deviation on the basis of two independent triplicate experiments.


c , [ Fe2 + ] o - dependent quenching of Fura - 2 fluorescence in TRPML1Va - transfected cells ( arrows ) , but not in non - transfected control cells ( arrowheads ) or T232P - TRPML1Va - transfected cells ( bottom row ) . The fluorescence intensity was measured at an excitation wavelength of 360 nm ( F360 ) . The original magnification used for all micrographs was × 200 .

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c, [Fe2+]o-dependent quenching of Fura-2 fluorescence in TRPML1Va-transfected cells (arrows), but not in non-transfected control cells (arrowheads) or T232P-TRPML1Va-transfected cells (bottom row). The fluorescence intensity was measured at an excitation wavelength of 360 nm (F360). The original magnification used for all micrographs was ×200.


d , Average normalized responses of EGFP - positive TRPML1Va - transfected cells ( typically n = 20 - 40 cells ) to 1 or 10 mM Fe2 + ( pH 4 . 6 ) .

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d, Average normalized responses of EGFP-positive TRPML1Va-transfected cells (typically n = 20-40 cells) to 1 or 10 mM Fe2+ (pH 4.6).


e , No significant quenching was seen in T232P - TRPML1Va - transfected cells .

e, No significant quenching was seen in T232P-TRPML1Va-transfected cells.


f , Slightly less quenching was observed for the F408Dgr ; - TRPML1Va - expressing cells .

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f, Slightly less quenching was observed for the F408Dgr;-TRPML1Va-expressing cells.


g , A small but significant quenching reaction was detected ( with 10 mM Fe2 + ) in R403C - TRPML1Va - expressing cells .

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g, A small but significant quenching reaction was detected (with 10 mM Fe2+) in R403C-TRPML1Va-expressing cells.


a , Co - localization of mCherry - TRPML1 and EGFP - LAMP1 at the membrane of an isolated enlarged LEL ( see Methods ) . The patch pipette was filled with red rhodamine B dye ( shown in blue for the purpose of illustration ) .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O

a, Co-localization of mCherry-TRPML1 and EGFP-LAMP1 at the membrane of an isolated enlarged LEL (see Methods). The patch pipette was filled with red rhodamine B dye (shown in blue for the purpose of illustration).


c , Lysosomal ITRPML1Va . Switching from lysosome - attached to ( lysosome ) luminal - side - out configuration significantly reduced the amplitude of the current . The luminal - side - out patch was exposed to the Tyrode ' s solution . A Cs + - based solution ( 147 mM Cs - methanesulphonate ( Cs - MSA ) ) was used as a pipette solution for both configurations .

O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

c, Lysosomal ITRPML1Va. Switching from lysosome-attached to (lysosome) luminal-side-out configuration significantly reduced the amplitude of the current. The luminal-side-out patch was exposed to the Tyrode's solution. A Cs+-based solution (147 mM Cs-methanesulphonate (Cs-MSA)) was used as a pipette solution for both configurations.


d , NMDG + - impermeable lysosomal ITRPML1Va was much larger in the absence of divalent cations ( nominal divalent - free ) . e , Lowering pH potentiated lysosomal ITRPML1Va . f , IFe / TRPML1Va induced by 30 mM and 105 mM Fe2 + .

O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O

d, NMDG+-impermeable lysosomal ITRPML1Va was much larger in the absence of divalent cations (nominal divalent-free). e, Lowering pH potentiated lysosomal ITRPML1Va. f, IFe/TRPML1Va induced by 30 mM and 105 mM Fe2+.