( E , F ) Representative immunoblots ( E ) of of different Rab ( F ) in hippocampus of GC - treated CON . of Rab35 , but not other Rabs , are decreased in GC - treated compared to CON ones ( n = 5 / condition , unpaired student ' s t - , * p = 0 . 0257 )

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 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

(E, F) Representative immunoblots (E) and quantification of levels of different Rab proteins (F) in hippocampus of GC-treated and CON animals. Protein levels of Rab35, but not other Rabs, are decreased in GC-treated animals compared to CON ones (n=5 animals/condition, unpaired student's t-test, *p=0.0257)


( G , H ) Immunofluorescence of Rab35 ( green ) DAPI ( blue ) ( G ) showing that Rab35 is reduced in hippocampal ( CA1 ) of GC - treated aniamls ( H ) ( n = 15 slices / condition ; unpaired student ' s t - , * * * p = 0 . 0003 ) . Data information : numeric data represent mean ±

O O O O O O 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

(G, H) Immunofluorescence staining of Rab35 (green) and DAPI (blue) (G) showing that Rab35 fluorescence intensity is reduced in hippocampal area (CA1) of GC-treated aniamls (H) (n=15 slices/condition; unpaired student's t-test, ***p=0.0003). Data information: All numeric data represent mean ± SEM


( A , B ) Representative immunoblots ( A ) of Tau degradation ( B ) in 14 DIV neurons expressing mCh - Rab35 or mCh , treated for 24 h with cycloheximide ( CHX ) or DMSO ( CON ) under GC conditions . GC significantly decreases Tau degradation , whereas Rab35 overexpression blocks this effect ( n = 12 - 14 / condition ; 1 - way ANOVA , Dunnet post - hoc , * pmCh vs mCh GC = 0 , 0319 , * pmCh vs Rab35 GC = 0 , 047 )

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

(A, B) Representative immunoblots (A) and quantification of Tau degradation (B) in 14 DIV neurons expressing mCh-Rab35 or mCh, treated for 24 h with cycloheximide (CHX) or DMSO (CON) under GC conditions. GC significantly decreases Tau degradation, whereas Rab35 overexpression blocks this effect (n=12-14/condition; 1-way ANOVA, Dunnet post-hoc analysis, *pmCh vs mCh GC=0,0319, *pmCh vs Rab35 GC=0,047)


( D , E ) Representative immunoblots ( D of Tau ( E ) in hippocampal synaptosomes reveal that GC increases total Tau in expressing EGFP , but not Rab35 ( n = 5 - 6 / group , 2 replicates , 2 - way ANOVA , GC x Rab35 interaction F1 , 39 = 10 , 51 p = 0 . 002 ; Sidak posthoc * p = 0 . 0168 )

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 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

(D, E) Representative immunoblots (D of Tau levels (E) in hippocampal synaptosomes reveal that GC increases total Tau levels in animals expressing EGFP, but not Rab35 (n=5-6 animals/group, 2 replicates, 2-way ANOVA, GC x Rab35 interaction F1, 39=10,51 p=0.002; Sidak posthoc analysis *p=0.0168)


( F ) Golgi - 3D neuronal reconstruction of hippocampal pyramidal neurons ( CA1 )

(F) Golgi-based 3D neuronal reconstruction of hippocampal pyramidal neurons (CA1 area)


( G ) GC treatment reduces of apical dendrites in expressing EGFP , but not EGFP - Rab35 ( n = 6 - 7 / group ; 6 - 8 neurons / , 2 - way ANOVA , GC x Rab35 interaction F1 , 155 = 3 . 969 p = 0 . 0481 , overall GC effect F1 , 155 = 8 . 998 p = 0 . 0031 , Sidak posthoc * * p = 0 . 0021 )

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 B-CONTROLLED_VAR 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

(G) GC treatment reduces the length of apical dendrites in animals expressing EGFP, but not EGFP-Rab35 (n=6-7 animals/group; 6-8 neurons/animal, 2-way ANOVA , GC x Rab35 interaction F1, 155=3.969 p=0.0481, overall GC effect F1, 155=8.998 p=0.0031, Sidak posthoc analysis **p=0.0021)


( H ) GC treatment reduces mature spine density in expressing EGFP , but not EGFP - Rab35 ( 2 - way ANOVA , GC x Rab35 interaction F1 , 499 = 12 . 33 p = 0 . 0005 , overall GC effect F1 , 499 = 4 . 373 p = 0 . 0370 , Sidak posthoc * * * p = 0 . 0003 ; n = 6 - 7 / group ; 6 - 8 neurons per )

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 B-CONTROLLED_VAR 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

(H) GC treatment reduces mature spine density in animals expressing EGFP, but not EGFP-Rab35 (2-way ANOVA, GC x Rab35 interaction F1, 499=12.33 p=0.0005, overall GC effect F1, 499=4.373 p=0.0370, Sidak posthoc analysis *** p=0.0003; n=6-7 animals/group; 6-8 neurons per animal)


( I , J ) Sholl of apical dendrites in rat hippocampus shows reduced dendritic intersections after GC treatment in EGFP - expressing ; however , this effect is not seen in EGFP - Rab35 - expressing ( 3 - way ANOVA , GC x Rab35 interaction F1 , 4212 = 14 , 926 p < 0 . 0001 , Simple effect , Sidak for multiple comparisons * p120 = 0 . 012 , * * p140 = 0 . 001 , * * p220 = 0 . 002 , * * * p240 < 0 . 001 , * * * p260 < 0 . 001 , * * p280 = 0 . 001 , * * p300 = 0 . 001 , * p320 = 0 . 021 , * p340 = 0 . 047 , n = 6 - 7 / group , 6 - 8 neurons per )

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 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

(I, J) Sholl analysis of apical dendrites in rat hippocampus shows reduced dendritic intersections after GC treatment in EGFP-expressing animals; however, this effect is not seen in EGFP-Rab35-expressing animals (3-way ANOVA, GC x Rab35 interaction F1, 4212= 14,926 p<0.0001, Simple effect analysis, Sidak test for multiple comparisons *p120=0.012, **p140=0.001, **p220=0.002, ***p240<0.001, ***p260<0.001, **p280=0.001, **p300=0.001, *p320=0.021, *p340=0.047, n=6-7 animals/group, 6-8 neurons per animal)


( A ) Volcano plot representation of PARL interaction partners . Mitochondria isolated from PARL - / - FITR293Tcells expressing PARL - FLAG were subjected to co - immunoprecipitation ( IP ) . Co - purifying were separated by SDS - PAGE identified by quantitative MS ( n = 3 ) ( Dataset EV1 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Volcano plot representation of PARL interaction partners. Mitochondria isolated from PARL-/- FITR293Tcells expressing PARL-FLAG were subjected to co-immunoprecipitation (IP). Co-purifying proteins were separated by SDS-PAGE and identified by quantitative MS (n=3)(Dataset EV1).


( B ) Heat map of Log2 transformed LFQ intensities for three independent experiments . significant ( FDR < 0 . 05 ) showing a positive between PARLIP control in ( A ) are shown . Grey color shows missing quantitative information . Clustering was performed using complete method with Euclidean .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Heat map of Log2 transformed LFQ intensities for three independent experiments. All significant (FDR < 0.05) proteins showing a positive ratio between PARLIP and control in (A) are shown. Grey color shows missing quantitative information. Clustering was performed using the complete method with Euclidean distance.


( C ) Complexome of MEFmitochondria ( n = 3 ) . Heat maps of relative abundances of significantly enriched in eluate of PARLimmunoprecipitates at a FDR level of 0 . 05 ( A ) identified by mass spectrometry after BN - PAGE are shown after hierarchical clustering .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Complexome analysis of MEFmitochondria (n=3). Heat maps of the relative abundances of proteins significantly enriched in the eluate of PARLimmunoprecipitates at a FDR level of 0.05 (A) and identified by mass spectrometry after BN-PAGE analysis are shown after hierarchical clustering.


( D ) BN - PAGE of mitochondria lacking SLP2 , YME1L or PARL . Mitochondria isolated from corresponding MEFs were solubilized using 1 . 5 % ( w / v ) digitonin at a of 2 . 5 mg / ml . Solubilized were separated by a 3 - 13 % blue native gradient gel analyzed using SLP2 - , YME1L - PARL - specific antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) BN-PAGE analysis of mitochondria lacking SLP2, YME1L or PARL. Mitochondria isolated from corresponding MEFs were solubilized using 1.5% (w/v) digitonin at a protein concentration of 2.5 mg/ml. Solubilized proteins were separated by a 3-13% blue native gradient gel and analyzed using SLP2-, YME1L- and PARL-specific antibodies.


( E ) High resolution complexome of SPY members SLP2 , YME1L PARL using MEFmitochondria ( n = 3 ) . Heat maps migration profiles are shown after separation using a 3 - 9 % BN - PAGE . SLP2 , YME1L PARL migrate in a high molecular ( 2 MDa ) , whereas SLP2 additionally is present in complexes of 1 . 6 MDa ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) High resolution complexome analysis of SPY complex members SLP2, YME1L and PARL using MEFmitochondria (n=3). Heat maps and migration profiles are shown after separation using a 3-9% BN-PAGE. SLP2, YME1L and PARL migrate in a high molecular weight complex (2 MDa), whereas SLP2 additionally is present in complexes of 1.6 MDa).


( A ) Co - immunoprecipitation of endogenous SLP2 , PARL YME1L in mitochondria isolated from human FITR293T using either SLP2 - or PARL - specific antibodies . IgG was used as a negative control . In , input ( 10 % ) .

O O O O O O O O O O O O O O O O O O O O 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) Co-immunoprecipitation of endogenous SLP2, PARL and YME1L in mitochondria isolated from human FITR293T cells using either SLP2- or PARL-specific antibodies. IgG was used as a negative control. In, input (10%).


( B , C ) Co - immunoprecipitation of endogenous SLP2 YME1L with PARL - specific antibodies in mitochondria isolated from wild type MEFs ( WT ) MEFs lacking YME1L , SLP2 or PARL . In , input ( 10 % ) .

O O O O O O O O O O O O O O O O O O O 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, C) Co-immunoprecipitation of endogenous SLP2 and YME1L with PARL-specific antibodies in mitochondria isolated from wild type MEFs (WT) and MEFs lacking YME1L, SLP2 or PARL. In, input (10%).


( D ) Assembly of SLP2 , PARL YME1L into a high molecular . Mitochondria isolated from FITR293T ( Con ) inducibly expressing SLP2 - FLAG ( FLAG ) were solubilized in digitonin subjected to immunoprecipitation using FLAG - specific antibodies . Native eluates of precipitate were analyzed by BN - PAGE immunoblotting using SLP2 - , PARL - , YME1L - PHB2 - specific antibodies . In , input ( 8 % ) ; E , eluate ( 100 % ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Assembly of SLP2, PARL and YME1L into a high molecular weight complex. Mitochondria isolated from FITR293T cells (Con) and cells inducibly expressing SLP2-FLAG (FLAG) were solubilized in digitonin and subjected to immunoprecipitation using FLAG-specific antibodies. Native eluates of the precipitate were analyzed by BN-PAGE and immunoblotting using SLP2-, PARL-, YME1L- and PHB2-specific antibodies. In, input (8%); E, eluate (100%).


( E ) High resolution BN - PAGE of mitochondria lacking PARL , YME1L or SLP2 . Mitochondria isolated from corresponding MEFs were solubilized using 1 . 5 % ( w / v ) digitonin at a of 2 . 5 mg / ml . Solubilized were separated by a 3 - 9 % gradient gel containing 0 - 10 % glycerol analyzed using SLP2 - specific antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) High resolution BN-PAGE analysis of mitochondria lacking PARL, YME1L or SLP2. Mitochondria isolated from corresponding MEFs were solubilized using 1.5% (w/v) digitonin at a protein concentration of 2.5 mg/ml. Solubilized proteins were separated by a 3-9% gradient gel containing 0-10% glycerol and analyzed using SLP2-specific antibodies.


( F , G ) Submitochondrial localization of SLP2 . ( F ) Mitochondria were isolated from MEFs fractionated in presence or absence of proteinase K as indicated . Fractions were analyzed by SDS - PAGE immunoblotting . MFN2 served as OM marker , TIMM23 , SMAC / DIABLO , YME1L as IMS markers , AFG3L1 AFG3L2 as matrix marker .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G) Submitochondrial localization of SLP2. (F) Mitochondria were isolated from MEFs and fractionated in the presence or absence of proteinase K as indicated. Fractions were analyzed by SDS-PAGE and immunoblotting. MFN2 served as OM marker, TIMM23, SMAC/DIABLO, and YME1L as IMS markers, and AFG3L1 and AFG3L2 as matrix marker proteins.


( F , G ) Submitochondrial localization of SLP2 . ( G ) Mitochondrial membranes were extracted with sodium carbonate at indicated pH separated into pellet ( P ) supernatant ( S ) fraction by centrifugation . Fractions were analyzed by SDS - PAGE immunoblotting .

O O O O O O O O O O O O O O O O O O O O O O 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, G) Submitochondrial localization of SLP2. (G) Mitochondrial membranes were extracted with sodium carbonate at the indicated pH and separated into pellet (P) and supernatant (S) fraction by centrifugation. Fractions were analyzed by SDS-PAGE and immunoblotting.


( A ) Steady state of proteolytic substrates of YME1L . Whole extracts of Yme1l - / - , Parl - / - , Slp2 - / - MEFs were analyzed by SDS - PAGE immunoblotting using indicated antibodies .

O O O O O O O O O O O O O O O O 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) Steady state levels of proteolytic substrates of YME1L. Whole cell extracts of Yme1l-/-, Parl-/-, and Slp2-/- MEFs were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies.


( B ) PGAM5 processing depends on proteolytically active PARL . PARL catalytic inactive PARLS277A harboring C - terminal FLAG - epitopes were expressed under control of a tetracycline ( tet ) - inducible promoter in wild type ( WT ) PARL - / - FITR293T as indicated . Processing of L - PGAM5 to S - PGAM5 was monitored by immunoblotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) PGAM5 processing depends on proteolytically active PARL. PARL and catalytic inactive PARLS277A harboring C-terminal FLAG-epitopes were expressed under the control of a tetracycline (tet)-inducible promoter in wild type (WT) and PARL-/- FITR293T cells as indicated. Processing of L-PGAM5 to S-PGAM5 was monitored by immunoblotting.


( C ) Accelerated processing of L - PGAM5 in Slp2 - / - . Processing of L - PGAM5 was analyzed by immunoblotting in wild type ( WT ) , Yme1l - / - , Parl - / - Slp2 - / - MEFs after inhibition of synthesis by cycloheximide ( CHX ) . A of L - PGAM5 at different time points is shown in lower panel . Two - way ANOVA ( n = 3 ; * * * * , p < 0 . 0001 ) .

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

(C) Accelerated processing of L-PGAM5 in Slp2-/- cells. Processing of L-PGAM5 was analyzed by immunoblotting in wild type (WT), Yme1l-/-, Parl-/- and Slp2-/-MEFs after inhibition of cytosolic protein synthesis by cycloheximide (CHX). A quantification of L-PGAM5 levels at different time points is shown in the lower panel. Two-way ANOVA analysis (n=3; ****, p<0.0001).


( D ) accelerated processing of L - PGAM5 in Slp2 - / - is mediated by PARL . PARL was depleted from Slp2 - / - by RNAi prior to CHX treatment . A of L - PGAM5 at different time points is shown . Two - way ANOVA ( n = 3 ; * * , p < 0 . 01 . * * * * , p < 0 . 0001 ) .

O O O O O O O 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

(D) The accelerated processing of L-PGAM5 in Slp2-/- cells is mediated by PARL. PARL was depleted from Slp2-/- cells by RNAi prior to CHX treatment. A quantification of L-PGAM5 levels at different time points is shown. Two-way ANOVA analysis (n=3; **, p<0.01. ****, p<0.0001).


( E ) PGAM5 associates with SPY harboring proteolytically inactive PARL . Mitochondria isolated from FITR293T ( WT ) or PARL - / - FITR293T expressing PARL - FLAG ( PARL - WT ) or PARLS277A - FLAG ( PARL - S277A ) were solubilized in digitonin were analyzed by BN - PAGE immunoblotting using FLAG - PGAM5 - specific antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) PGAM5 associates with the SPY complex harboring proteolytically inactive PARL. Mitochondria isolated from FITR293T cells (WT) or PARL-/- FITR293T cells expressing PARL-FLAG (PARL-WT) or PARLS277A-FLAG (PARL-S277A) were solubilized in digitonin were analyzed by BN-PAGE and immunoblotting using FLAG- and PGAM5-specific antibodies.


( A ) PARL dependent cleavage of PINK1 - HA is reduced in Slp2 - / - Yme1l - / - MEFs . Whole extracts of Slp2 - / - , Parl - / - Yme1l - / - MEFs expressing PINK1 - HA were analysed by SDS - PAGE immunoblotting using indicated antibodies . were treated with 20 µM MG132 or 20 µM CCCP for 4 h . ( B ) of ( Log2 ) PINK1 - HA 52 kDa / 63 kDa ( n = 3 ; * , p < 0 . 05 ; One - way ANOVA ) . n . s . , not significant . Error bars indicate .

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

(A) PARL dependent cleavage of PINK1-HA is reduced in Slp2-/- and Yme1l-/- MEFs. Whole cell extracts of Slp2-/-, Parl-/ -and Yme1l-/- MEFs expressing PINK1-HA were analysed by SDS-PAGE and immunoblotting using the indicated antibodies. Cells were treated with 20 µM MG132 or 20 µM CCCP for 4 h.(B) Quantification of the protein ratio (Log2) PINK1-HA 52 kDa/63 kDa (n=3; *, p<0.05; One-way ANOVA). n.s., not significant. Error bars indicate SEM.


( C ) PINK1 - HA associates with SPY harboring proteolytically inactive PARL . Mitochondria isolated from FITR293T ( WT ) or PARL - / - FITR293T expressing PARL - FLAG ( PARL - WT ) or PARLS277A - FLAG ( PARL - S277A ) were solubilized in digitonin analysed by BN - PAGE immunoblotting using FLAG - , HA - SLP2 - specific antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) PINK1-HA associates with the SPY complex harboring proteolytically inactive PARL. Mitochondria isolated from FITR293T cells (WT) or PARL-/- FITR293T cells expressing PARL-FLAG (PARL-WT) or PARLS277A-FLAG (PARL-S277A) were solubilized in digitonin and analysed by BN-PAGE and immunoblotting using FLAG-, HA- and SLP2-specific antibodies.


( D ) PINK1 associates with SLP2 . Mitochondria isolated from FITR293T ( WT ) or PARL - / - FITR293T , depleted of SLP2 by siRNA as indicated , were solubilized in digitonin analysed by BN - PAGE immunoblotting using SLP2 - PINK1 - specific antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O 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) PINK1 associates with SLP2. Mitochondria isolated from FITR293T cells (WT) or PARL-/- FITR293T cells, depleted of SLP2 by siRNA as indicated, were solubilized in digitonin and analysed by BN-PAGE and immunoblotting using SLP2- and PINK1-specific antibodies.


( A ) Processing of L - PGAM5 to S - PGAM5 in depolarized mitochondria of wild type ( WT ) , Slp2 - / - , Yme11 - / - , Parl - / - MEFs . PGAM5 processing was monitored by immunoblotting at indicated time points after inhibition of synthesis with cycloheximide ( CHX ) . A of L - PGAM5 at different time points is shown . L - PGAM5 at t = 0 was set to 100 % . Two - way ANOVA ( n = 3 ; * , p < 0 . 05 , * * , p < 0 . 01 . * * * * , p < 0 . 0001 ) . Arrowheads denote intermediate PGAM5 cleavage products .

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

(A) Processing of L-PGAM5 to S-PGAM5 in depolarized mitochondria of wild type (WT), Slp2-/-, Yme11-/-, and Parl-/- MEFs. PGAM5 processing was monitored by immunoblotting at the indicated time points after inhibition of cytosolic protein synthesis with cycloheximide (CHX). A quantification of L-PGAM5 levels at different time points is shown. L-PGAM5 levels at t=0 was set to 100%. Two-way ANOVA analysis (n=3; *, p<0.05, **, p<0.01. ****, p<0.0001). Arrowheads denote intermediate PGAM5 cleavage products.


( B ) OMA1 - mediated processing of PGAM5 in depolarized Parl - / - mitochondria . Processing of L - PGAM5 to S - PGAM5 in Parl - / mitochondria was assessed as in ( A ) in presence or absence of CCCP ( 20 µM ) , which were depleted of OMA1 by siRNA as indicated . arrowhead denotes an intermediate PGAM5 cleavage product . SCR , scrambled .

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

(B) OMA1-mediated processing of PGAM5 in depolarized Parl-/-mitochondria. Processing of L-PGAM5 to S-PGAM5 in Parl-/ mitochondria was assessed as in (A) in the presence or absence of CCCP (20 µM), which were depleted of OMA1 by siRNA as indicated. The arrowhead denotes an intermediate PGAM5 cleavage product. SCR, scrambled.


( C ) Impaired PGAM5 processing in depolarized Oma1 - / - mitochondria lacking PARL . Processing of L - PGAM5 to S - PGAM5 was examined as in ( A ) after 2 h in wild type ( WT ) Oma1 - / - mitochondria in presence or absence of CCCP ( 20 µM , 2 h ) , which were depleted of PARL by siRNA as indicated . * unspecific cross - reaction .

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

(C) Impaired PGAM5 processing in depolarized Oma1-/- mitochondria lacking PARL. Processing of L-PGAM5 to S-PGAM5 was examined as in (A) after 2 h in wild type (WT) and Oma1-/- mitochondria in the presence or absence of CCCP (20 µM, 2 h), which were depleted of PARL by siRNA as indicated. * unspecific cross-reaction.


( D ) OMA1 mediates accelerated processing of L - OPA1 in Slp2 - / - MEFs . Processing of OPA1 PGAM5 was monitored in Slp2 - / - , Oma1 - / - , Slp2 - / - Oma1 - / - by immunoblotting after inhibition of synthesis with cycloheximide ( CHX ) .

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

(D) OMA1 mediates accelerated processing of L-OPA1 in Slp2-/- MEFs. Processing of OPA1 and PGAM5 was monitored in Slp2-/-, Oma1-/-, and Slp2-/-Oma1-/- cells by immunoblotting after inhibition of cytosolic protein synthesis with cycloheximide (CHX).


( E ) OMA1 interacts with SLP2 . OMA1 - / - FITR293T ectopically expressing OMA1 - myc were transfected with pcDNA5 ( Control ) or pcDNA5 - SLP2 - FLAG ( FLAG ) . Isolated mitochondria were solubilized in digitonin subjected to immunoprecipitation using FLAG - specific antibodies . Native eluates of precipitate were analyzed by BN - PAGE immunoblotting using SLP2 - , YME1L - , PARL - , myc - specific antibodies . In , input ( 8 % ) ; E , eluate ( 100 % ) . Samples were analyzed by SDS - PAGE in parallel ( lower panel ) . ( F ) of immunoprecipitation efficiencies in ( E ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) OMA1 interacts with SLP2. OMA1-/-FITR293T cells ectopically expressing OMA1-myc were transfected with pcDNA5 (Control) or pcDNA5-SLP2-FLAG (FLAG). Isolated mitochondria were solubilized in digitonin and subjected to immunoprecipitation using FLAG-specific antibodies. Native eluates of the precipitate were analyzed by BN-PAGE and immunoblotting using SLP2-, YME1L-, PARL-, and myc-specific antibodies. In, input (8%); E, eluate (100%). Samples were analyzed by SDS-PAGE in parallel (lower panel).(F) Quantification of immunoprecipitation efficiencies in (E).


( a ) Western blot showing replacement of endogenous Beclin 1 with Beclin 1 - EGFP in Becn1 - / - ; Becn1 - EGFP / + mice , as detected by an anti - Beclin 1 antibody .

O O O O O O O O O O O O O O O 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) Western blot analysis showing the replacement of endogenous Beclin 1 with Beclin 1-EGFP in Becn1-/-;Becn1-EGFP/+ mice, as detected by an anti-Beclin 1 antibody.


( b ) Coomassie - stained SDS - PAGE showing Beclin 1 - interacting immuno - isolated from brain liver of ' rescued ' mice ( lanes 2 4 ) of control Becn1 + / - ( lanes 1 3 ) , using an anti - GFP antibody . in gel bands were extracted identified by mass spectrometry as Vps15 / p150 ( band 1 ) , Vps34 / class III PI ( 3 ) K ( band 3 ) , UVRAG ( band 4 ) , Beclin 1 - EGFP ( band 5 ) , Atg14L ( band 6 , asterisk , gi | 27369860 ) Rubicon ( band 2 , asterisk , gi | 45708948 ) . UVRAG varied with different affinity - purification conditions , suggesting an unstable association of UVRAG with .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Coomassie-stained SDS-PAGE showing the Beclin 1-interacting proteins immuno-isolated from brain and liver of the 'rescued' mice (lanes 2 and 4) and of control Becn1+/- littermates (lanes 1 and 3), using an anti-GFP antibody. Proteins in the gel bands were extracted and identified by mass spectrometry as Vps15/p150 (band 1), Vps34/class III PI(3)K (band 3), UVRAG (band 4), Beclin 1-EGFP (band 5), Atg14L (band 6, asterisk, gi|27369860) and Rubicon (band 2, asterisk, gi|45708948). UVRAG levels varied with different affinity-purification conditions, suggesting an unstable association of UVRAG with the complex.


( d ) Western blot of Atg14L , Rubicon , Vps34 Beclin 1 in gel filtration fractions from wild - type mouse liver extract showed co - elution of these in fractions 38 - 45 . Atg14L was also eluted in later fractions 51 - 56 . fractions for peak elution of thyroglobulin ( 670K ) γ - globulin ( 158K ) are indicated by arrows . Control siRNA - transfected NIH 3T3 lysate was loaded as a positive control ( labelled as 1 ) for migration position of Atg14L on SDS - PAGE ; Atg14L siRNA - transfected NIH 3T3 lysate was loaded as a negative control ( labelled as 2 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of Atg14L, Rubicon, Vps34 and Beclin 1 in gel filtration fractions from wild-type mouse liver extract showed co-elution of these proteins in fractions 38-45. Atg14L was also eluted in later fractions 51-56. The fractions for the peak elution of thyroglobulin (670K) and γ-globulin (158K) are indicated by arrows. Control siRNA-transfected NIH 3T3 cell lysate was loaded as a positive control (labelled as 1) for the migration position of the Atg14L protein on SDS-PAGE; Atg14L siRNA-transfected NIH 3T3 cell lysate was loaded as a negative control (labelled as 2).


( e , f ) Co - immunoprecipitation confirmed - interaction between Atg14L Rubicon . HEK 293 were co - transfected with Atg14L - EGFP Flag - Rubicon ( e ) or Rubicon - EGFP Flag - Atg14L ( f ) . lysates were used for immunoprecipitation with an anti - GFP antibody resulting immunoprecipitates were blotted with an anti - Flag antibody . Our results show immunoprecipitation of Rubicon by Atg14L ( e ) vice versa ( f ) . WCL , whole lysate ; IP , immunoprecipitated . See Supplementary Information , Fig . S6 for full scans of blots in a , d , e f .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Co-immunoprecipitation confirmed protein-protein interaction between Atg14L and Rubicon. HEK 293 cells were co-transfected with Atg14L-EGFP and Flag-Rubicon (e) or Rubicon-EGFP and Flag-Atg14L (f). Cell lysates were used for immunoprecipitation with an anti-GFP antibody and the resulting immunoprecipitates were blotted with an anti-Flag antibody. Our results show immunoprecipitation of Rubicon by Atg14L (e) and vice versa (f). WCL, whole cell lysate; IP, immunoprecipitated. See Supplementary Information, Fig. S6 for full scans of blots in a, d, e and f.


( a ) Beclin 1 or Atg14L siRNA reduced Atg14L increased p62 / SQSTM1 LC3 II under normal nutrient - starvation conditions in NIH 3T3 .

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

(a) Beclin 1 or Atg14L siRNA reduced Atg14L levels and increased p62/SQSTM1 and LC3 II levels under normal and nutrient-starvation conditions in NIH 3T3 cells.


( b ) Compared with control siRNA , Atg14L siRNA decreased long - lived degradation in NIH 3T3 under normal ( * P = 0 . 007 ) starvation ( * P = 5 × 10 − 6 ) conditions ( one - Student ' s t - with equal variances , n = 4 ) . This difference was diminished when starved were treated with 3 - methyladenine ( 3MA , 10 mM ) , a PI ( 3 ) K inhibitor .

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

(b) Compared with control siRNA, Atg14L siRNA decreased long-lived protein degradation in NIH 3T3 cells under normal (*P = 0.007) and starvation (*P = 5 × 10−6) conditions (one-tailed Student's t-test with equal variances, n = 4). This difference was diminished when the starved cells were treated with 3-methyladenine (3MA, 10 mM), a PI(3)K inhibitor.


( c ) Vps34 kinase . HEK 293T were co - transfected with Myc - Vps34 - Vps15 Flag - Atg14L or Flag vector , either in absence or in presence of Beclin 1 - EGFP . Myc - Vps34 - Vps15 was immunoprecipitated by anti - Myc antibody for in vitro kinase . resulting radioactive PI ( 3 ) P was separated by thin - layer chromatography ( TLC ) , quantified normalized against amount of immunoprecipitated Myc - tagged Vps34 as measured by western blot ( upper panel ) . quantified results ( lower panel ) show that overexpressing Atg14L significantly upregulated Vps34 kinase activity by 2 . 5 - fold , but only when Beclin 1 was also overexpressed ( * P = 0 . 04 , one - Student ' s t - with unequal variances , n = 5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-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 O O O O O O O O O O O O O O O O O O O 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) Vps34 kinase assay. HEK 293T cells were co-transfected with Myc-Vps34-Vps15 and Flag-Atg14L or Flag vector, either in the absence or in the presence of Beclin 1-EGFP. Myc-Vps34-Vps15 was immunoprecipitated by anti-Myc antibody for the in vitro kinase assay. The resulting radioactive PI(3)P was separated by thin-layer chromatography (TLC), quantified and normalized against the amount of immunoprecipitated Myc-tagged Vps34 as measured by western blot (upper panel). The quantified results (lower panel) show that overexpressing Atg14L significantly upregulated Vps34 kinase activity by 2.5-fold, but only when Beclin 1 was also overexpressed (*P = 0.04, one-tailed Student's t-test with unequal variances, n = 5).


( d ) Colocalization of co - expressed Atg14L - EGFP ( green ) Beclin 1 - AsRed ( red ) in punctate structures in transiently transfected HeLa . Scale bar , 10 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

(d) Colocalization of co-expressed Atg14L-EGFP (green) and Beclin 1-AsRed (red) in punctate structures in transiently transfected HeLa cells. Scale bar, 10 μm.


( e ) Electron microscopy show large structures ( asterisks ) that are often enwrapped with double membranes in HEK 293T co - transfected with Atg14L - EGFP Beclin 1 - AsRed : concentric ' rings ' ( panel 1 ) ; two large structures ( 3 - 5 μm in diameter , panel 2 ) containing material with high electron density ( inset , enwrapping double membranes ) ; numerous autophagosomes ( arrows , panel 3 ) in cytoplasm ; immuno - electron microscopy image of a Atg14L - Beclin 1 structure ( labelled with anti - GFP antibody developed by DAB , panel 4 ) enwrapped with concentric ' rings ' . M , mitochondria ; N , nucleus . Scale bar , 500 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Electron microscopy images show large structures (asterisks) that are often enwrapped with double membranes in the HEK 293T cells co-transfected with Atg14L-EGFP and Beclin 1-AsRed: concentric membrane 'rings' (panel 1); two large structures (3-5 μm in diameter, panel 2) containing material with high electron density (inset, enwrapping double membranes); numerous autophagosomes (arrows, panel 3) in the cytoplasm; immuno-electron microscopy image of a Atg14L-Beclin 1 structure (labelled with anti-GFP antibody and developed by DAB, panel 4) enwrapped with concentric membrane 'rings'. M, mitochondria; N, nucleus. Scale bar, 500 nm.


( f , g ) EGFP - Atg12 ( f ) or EGFP - Atg5 ( g ) ( green ) was colocalized with large structures ( arrows ) that were labelled by Atg14L - AsRed ( red ) Beclin 1 - Myc ( blue ) in transfected HeLa . Some of these structures seemed to be ' ring ' - shaped ( yellow arrows inset ) . Scale bar , 10 μm . See Supplementary Information , Fig . S6 for full scans of blots in a c .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, g) EGFP-Atg12 (f) or EGFP-Atg5 (g) (green) was colocalized with the large structures (arrows) that were labelled by Atg14L-AsRed (red) and Beclin 1-Myc (blue) in transfected HeLa cells. Some of these structures seemed to be 'ring'-shaped (yellow arrows and inset). Scale bar, 10 μm. See Supplementary Information, Fig. S6 for full scans of blots in a and c.


( a ) Rubicon siRNA treatment of NIH 3T3 led to decreased of p62 LC3 II under both normal nutrient - starvation conditions .

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

(a) Rubicon siRNA treatment of the NIH 3T3 cells led to decreased levels of p62 and LC3 II under both normal and nutrient-starvation conditions.


( b ) Overexpression of Rubicon resulted in increased of p62 under both normal nutrient - starved conditions in HEK 293 either stably expressing ( upper rows ) or transiently transfected with ( lower rows ) Rubicon - EGFP . control were either stably expressing or transiently transfected with EGFP - N3 vector .

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

(b) Overexpression of Rubicon resulted in increased levels of p62 under both normal and nutrient-starved conditions in HEK 293 cells either stably expressing (upper rows) or transiently transfected with (lower rows) Rubicon-EGFP. The control cells were either stably expressing or transiently transfected with the EGFP-N3 vector.


( c ) Vps34 kinase activity . HEK 293T were co - transfected with Myc - Vps34 - Vps15 Flag - Rubicon or Flag vector , either in absence or in presence of Beclin 1 - EGFP . Myc - Vps34 - Vps15 was immunoprecipitated by an anti - Myc antibody used for in vitro kinase . resulting radioactive PI ( 3 ) P was separated by TLC , quantified normalized against amount of immunoprecipitated Myc - tagged Vps34 , as measured by western blotting ( upper panel ) . quantified results ( lower panel ) show that overexpressing Rubicon significantly downregulated Vps34 kinase activity to 0 . 58 - fold , but only without Beclin 1 overexpression ( * P = 0 . 04 , one - Student ' s t - with unequal variances , n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-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 O O O O O O O O O O O O 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) Vps34 kinase activity. HEK 293T cells were co-transfected with Myc-Vps34-Vps15 and Flag-Rubicon or Flag vector, either in the absence or in the presence of Beclin 1-EGFP. Myc-Vps34-Vps15 was immunoprecipitated by an anti-Myc antibody and used for the in vitro kinase assay. The resulting radioactive PI(3)P was separated by TLC, quantified and normalized against the amount of immunoprecipitated Myc-tagged Vps34, as measured by western blotting (upper panel). The quantified results (lower panel) show that overexpressing Rubicon significantly downregulated the Vps34 kinase activity to 0.58-fold, but only without Beclin 1 overexpression (*P = 0.04, one-tailed Student's t-test with unequal variances, n = 4).


( d ) Effect of overexpressing Flag - Rubicon on autophagosome acidification , as monitored by mCherry - GFP - LC3 . HeLa were transiently co - transfected with mCherry - GFP - LC3 Flag - Rubicon ( or control Flag vector ) . co - expressing mCherry - GFP - LC3 control Flag vector contained many red - only along with yellow ( indicating presence of both red green ) , suggesting presence of both autolysosomes nascent autophagosomes ( upper panel ) . In contrast , co - expressing mCherry - GFP - LC3 Flag - Rubicon contained primarily yellow or , suggesting presence of only nascent autophagosomes ( lower panel , arrows ) . Notably , some , which were co - transfected with mCherry - GFP - LC3 Flag - Rubicon but expressed high of mCherry - GFP - LC3 undetectable of FLAG - Rubicon , contained many red - only ( lower panel , yellow arrows ) . ( e ) of results in d show that overexpressing Flag - Rubicon markedly reduced of red - only ( mCherry - LC3 ) from 39 % in control Flag vector - transfected to 2 % in Flag - Rubicon - transfected ( * P = 2 × 10 − 26 , one - Student ' s t - with unequal variances , n = 30 ) , indicating that overexpression of Rubicon blocks autophagosome acidification or maturation . See Supplementary Information , Fig . S6 for full scans of blots in a - c .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Effect of overexpressing Flag-Rubicon on autophagosome acidification, as monitored by mCherry-GFP-LC3 fluorescence. HeLa cells were transiently co-transfected with mCherry-GFP-LC3 and Flag-Rubicon (or control Flag vector). Cells co-expressing mCherry-GFP-LC3 and control Flag vector contained many red-only puncta along with yellow (indicating the presence of both red and green) puncta, suggesting the presence of both autolysosomes and nascent autophagosomes (upper panel). In contrast, cells co-expressing mCherry-GFP-LC3 and Flag-Rubicon contained primarily yellow or white puncta, suggesting the presence of only nascent autophagosomes (lower panel, white arrows). Notably, some cells, which were co-transfected with mCherry-GFP-LC3 and Flag-Rubicon but expressed high levels of mCherry-GFP-LC3 and undetectable levels of FLAG-Rubicon, contained many red-only puncta (lower panel, yellow arrows). (e) Quantification of the results in d show that overexpressing Flag-Rubicon markedly reduced the percentage of red-only puncta (mCherry-LC3) from 39% in the control Flag vector-transfected cells to 2% in the Flag-Rubicon-transfected cells (*P = 2 × 10−26, one-tailed Student's t-test with unequal variances, n = 30), indicating that overexpression of Rubicon blocks autophagosome acidification or maturation. See Supplementary Information, Fig. S6 for full scans of blots in a-c.


( a ) Colocalization of Rubicon - EGFP - associated structures with late endosome / lysosome marker Lamp1 ( arrows ) in HeLa transfected with Rubicon - EGFP . Note that some of Rubicon - EGFP - associated structures show a ' ring ' shape ( yellow arrows ) . Scale bar , 10 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

(a) Colocalization of Rubicon-EGFP-associated structures with the late endosome/lysosome marker Lamp1 (arrows) in HeLa cells transfected with Rubicon-EGFP. Note that some of the Rubicon-EGFP-associated structures show a 'ring' shape (yellow arrows). Scale bar, 10 μm.


( b ) Partial colocalization of Rubicon - EGFP - associated structures with MVB marker LBPA ( arrows ) in HeLa transfected with Rubicon - EGFP . Scale bar , 10 μm .

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

(b) Partial colocalization of Rubicon-EGFP-associated structures with the MVB marker LBPA (arrows) in HeLa cells transfected with Rubicon-EGFP. Scale bar, 10 μm.


( c ) Representative ultrastructural show aberrant expansion of late endosomal / lysosomal structures in HEK 293T overexpressing Rubicon - EGFP . These abnormal organelles are large in , with high ( orange arrows ) or low ( black arrows ) electron density . Some enclose small vesicles ( purple arrows ) some resemble MVB ( blue arrows ) . Scale bars , 500 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative ultrastructural images show aberrant expansion of late endosomal/lysosomal structures in HEK 293T cells overexpressing Rubicon-EGFP. These abnormal organelles are large in size, with high (orange arrows) or low (black arrows) electron density. Some enclose small vesicles (purple arrows) and some resemble the MVB (blue arrows). Scale bars, 500 nm.


( d ) Representative ultrastructural show late endosome / lysosome - like structures that are labelled with anti - GFP gold particles ( panels 3 , 4 ) in HEK 293T transiently transfected with Rubicon - EGFP . These structures are enwrapped by double membranes ( panel 4 inset ) co - labelled by anti - GFP ( developed by DAB ) anti - Lamp1 ( gold enhanced ) ( panels 5 - 7 ) antibodies . Note that mitochondria are mostly negative for Rubicon - EGFP ( panel 4 ) . negative controls are without antibody ( panels 1 - 2 ) . M , mitochondria . Scale bars , 200 nm .

(d) Representative ultrastructural images show late endosome/lysosome-like structures that are labelled with anti-GFP gold particles (panels 3, 4) in HEK 293T cells transiently transfected with Rubicon-EGFP. These structures are enwrapped by double membranes (panel 4 inset) and co-labelled by anti-GFP (developed by DAB) and anti-Lamp1 (gold enhanced) (panels 5-7) antibodies. Note that mitochondria are mostly negative for Rubicon-EGFP (panel 4). The negative controls are without antibody (panels 1-2). M, mitochondria. Scale bars, 200 nm.


( b ) Colocalization of PI ( 3 ) P - enriched lipid domain marker p40 ( phox ) - PX - EGFP ( green ) Rubicon - AsRed ( red ) on large punctate structures ( arrows ) in co - transfected HeLa ( upper panels ) . Treatment with PI ( 3 ) K inhibitor wortmannin ( 75 nM ) for 1 h caused disappearance of PI ( 3 ) P - enriched lipid domains , whereas Rubicon - AsRed - positive structures were maintained ( lower panels ) . Scale bars , 10 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 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

(b) Colocalization of the PI(3)P-enriched lipid domain marker p40 (phox)-PX-EGFP (green) and Rubicon-AsRed (red) on large punctate structures (arrows) in the co-transfected HeLa cells (upper panels). Treatment with the PI(3)K inhibitor wortmannin (75 nM) for 1 h caused disappearance of the PI(3)P-enriched lipid domains, whereas the Rubicon-AsRed-positive structures were maintained (lower panels). Scale bars, 10 μm.


( c ) Subcellular localization of transiently transfected Rubicon - EGFP , RubiconΔRUN - EGFP , RubiconΔC - EGFP or RubiconΔRUNΔC - EGFP in HeLa . In contrast to punctate Rubicon - EGFP RubiconΔRUN - EGFP , RubiconΔC - EGFP RubiconΔRUNΔC - EGFP were dispersed in cytoplasm . ΔRUN , RUN domain deletion ; ΔC , cysteine - rich domain deletion . Scale bars , 10 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

(c) Subcellular localization of transiently transfected Rubicon-EGFP, RubiconΔRUN-EGFP, RubiconΔC-EGFP or RubiconΔRUNΔC-EGFP in HeLa cells. In contrast to punctate Rubicon-EGFP and RubiconΔRUN-EGFP, RubiconΔC-EGFP and RubiconΔRUNΔC-EGFP were dispersed in the cytoplasm. ΔRUN, RUN domain deletion; ΔC, cysteine-rich domain deletion. Scale bars, 10 μm.


( d , e ) Absence of full - Beclin 1 ( d ) or Beclin 1 - CE mutant ( e ) ( red ) on Rubicon - EGFP - positive structures ( green ) in HEK 293 stably expressing Rubicon - EGFP . These were transiently transfected with either Beclin 1 - AsRed ( d ) or Flag - Beclin 1 - CE ( e ; that is , Flag - tagged Beclin 1 mutant containing both CCD ECD , which mediate Beclin 1 - Rubicon interaction as shown in Supplementary Information , Fig . S3h ) . Scale bars , 10 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 O O O O O O O O O O O O O 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) Absence of full-length Beclin 1 (d) or Beclin 1-CE mutant (e) (red) on the Rubicon-EGFP-positive structures (green) in HEK 293 cells stably expressing Rubicon-EGFP. These cells were transiently transfected with either Beclin 1-AsRed (d) or Flag-Beclin 1-CE (e; that is, the Flag-tagged Beclin 1 mutant containing both CCD and ECD, which mediate the Beclin 1-Rubicon interaction as shown in Supplementary Information, Fig. S3h). Scale bars, 10 μm.


( f ) formation of Rubicon - EGFP - positive structures was not affected by siRNA knockdown of Beclin 1 in HEK 293 stably expressing Rubicon - EGFP .

O O O O O 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) The formation of the Rubicon-EGFP-positive structures was not affected by siRNA knockdown of Beclin 1 in HEK 293 cells stably expressing Rubicon-EGFP.


( a ) Beeswarm plot with Factor 1 values for each sample with colors corresponding to three clusters found by 3 - means clustering with low factor values ( LZ ) , intermediate factor values ( IZ ) high factor values ( HZ )

O O O O O O O O O O O O O O O O O O O O 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) Beeswarm plot with Factor 1 values for each sample with colors corresponding to three clusters found by 3-means clustering with low factor values (LZ), intermediate factor values (IZ) and high factor values (HZ)


( b ) Absolute loadings for genes with largest absolute weights in mRNA data . Plus or minus symbols on right indicate sign of loading . Genes highlighted in orange were previously described as prognostic markers in CLL associated with IGHV status ( Maloum et al , 2009 ; Morabito et al , 2015 ; Plesingerova et al , 2017 ; Trojani et al , 2012 ; Vasconcelos et al , 2005 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Absolute loadings for the genes with the largest absolute weights in the mRNA data. Plus or minus symbols on the right indicate the sign of the loading. Genes highlighted in orange were previously described as prognostic markers in CLL and associated with IGHV status (Maloum et al, 2009; Morabito et al, 2015; Plesingerova et al, 2017; Trojani et al, 2012; Vasconcelos et al, 2005)


( c ) of gene expression values for genes with largest weights as in

(c) Heatmap of gene expression values for genes with the largest weights as in b


( d ) Absolute loadings of drugs with largest weights , annotated by target

(d) Absolute loadings of the drugs with the largest weights, annotated by target category


( e ) Drug response curves for two of drugs , stratified by clusters as in

(e) Drug response curves for two of the drugs, stratified by the clusters as in a


B . of PLAs between BRCA2 DPP9 in control HeLa WT treated with non - targeting siRNA ( siNT ) or silenced with indicated oligos . 300 ng / mL MMC was added for 24 h . Each dot represents number of PLA events in a single , from two to seven biological replicates ( siNT - MMC ( n = 6 ) , siNT + MMC ( n = 7 ) , siDPP9 - MMC ( n = 4 ) , siDPP9 + MMC ( n = 5 ) , siBRCA2 - MMC ( n = 2 ) , siBRCA2 + MMC ( n = 3 ) , siFLNA - MMC ( n = 5 ) , siFLNA + MMC ( n = 6 ) , NgtCntrl - MMC ( n = 3 ) , NgtCntrl + MMC ( n = 6 ) ) . number of foci are shown on their localisation . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison . Shown are Mean ± ( * = p ≤ 0 . 05 , * * * * = p ≤ 0 . 0001 ) . C . Representative PLA showing close proximity between endogenous DPP9 endogenous BRCA2 in HeLa WT . Exposure of to MMC triggers more PLA events ( ) . Phalloidin ( green ) stains actin filaments , DAPI ( blue ) stains nucleus . Scale bar 10 µm . Anti - BRCA2 : RRID : AB _ 2259370 , anti - DPP9 : RRID : AB _

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

B. Quantification of PLAs between BRCA2 and DPP9 in control HeLa WT cells treated with non-targeting siRNA (siNT) or silenced with the indicated oligos. 300 ng / mL MMC was added for 24 h. Each dot represents the number of PLA events in a single cell, from two to seven biological replicates (siNT-MMC (n = 6), siNT+MMC (n = 7), siDPP9-MMC (n = 4), siDPP9+MMC (n = 5), siBRCA2-MMC (n = 2), siBRCA2+MMC (n = 3), siFLNA-MMC (n = 5), siFLNA+MMC (n = 6), NgtCntrl-MMC (n = 3), NgtCntrl+MMC (n = 6)). The number of foci are shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (* = p ≤ 0.05, **** = p ≤ 0.0001). C. Representative PLA images showing close proximity between endogenous DPP9 and endogenous BRCA2 in HeLa WT cells. Exposure of cells to MMC triggers more PLA events (white). Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_2889071


D . of PLAs between BRCA2 DPP9 in asynchronous HeLa WT or in in S - Phase . Each dot represents number of PLA events in a single , from three biological replicates . were synchronised with a double thymidine block released for 3 h before addition of NCS ( 250 ng / mL for 30 min ) . were sampled prior to NCS treatment , after NCS treatment with 1 . 5 h 3 h of recovery after NCS treatment . number of foci is shown on their localisation . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison . Shown are Mean ± ( * = p ≤ 0 . 05 , * * = p ≤ 0 . 01 , * * * * = p ≤ 0 . 0001 ) . E . Representative PLA showing close proximity between endogenous DPP9 endogenous BRCA2 in HeLa . Exposure of to NCS triggers more PLA events ( ) , which increase in time . in S - Phase show a greater fold - increase in number of PLA events in comparison to asynchronous . Phalloidin ( green ) stains actin filaments , DAPI ( blue ) stains nucleus . Scale bar 10 µm . Antibodies as in ( C ) .

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

D. Quantification of PLAs between BRCA2 and DPP9 in asynchronous HeLa WT cells or in cells in S-Phase. Each dot represents the number of PLA events in a single cell, from three biological replicates. Cells were synchronised with a double thymidine block and released for 3 h before addition of NCS (250 ng / mL for 30 min). Cells were sampled prior to NCS treatment, after NCS treatment and with 1.5 h and 3 h of recovery after NCS treatment. The number of foci is shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (* = p ≤ 0.05, ** = p ≤ 0.01, **** = p ≤ 0.0001). E. Representative PLA images showing close proximity between endogenous DPP9 and endogenous BRCA2 in HeLa cells. Exposure of cells to NCS triggers more PLA events (white), which increase in time. Cells in S-Phase show a greater fold-increase in the number of PLA events in comparison to asynchronous cells. Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Antibodies as in (C).


F . Co - immunoprecipitation assays showing binding of BRCA2 DPP9 - SWT . HEK293 DPP9 KO + DPP9WT , were treated with 1µg / mL Dox ( 24 h ) to induce expression of DPP9 - FLAG . damage was induced with 300 nM MMC treatment for 24h . Control do not express DPP9 ( - Dox ) . Bound were eluted with a FLAG peptide analysed by Western Blotting ( anti - BRCA2 : RRID : AB _ 2259370 , anti - DPP9 : RRID : AB _ 731947 , anti - FLAG RRID : AB _ 262044 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Co-immunoprecipitation assays showing binding of BRCA2 and DPP9-SWT. HEK293 DPP9 KO + DPP9WT cells, were treated with 1µg/mL Dox (24 h) to induce the expression of DPP9-FLAG. DNA damage was induced with 300 nM MMC treatment for 24h. Control cells do not express DPP9 (- Dox). Bound proteins were eluted with a FLAG peptide and analysed by Western Blotting (anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_731947, anti-FLAG RRID:AB_262044).


B . Pull - down showing direct binding of purified recombinant DPP9 to a BRCA2 N - terminal1 - 40 fragment immobilized on HA - beads . DPP9 inhibitors 1G244 or SLRFLYEG compete with BRCA21 - 40HA for interaction with DPP9 . Representative data of three technical replicates is shown . Anti - HA : RRID : AB _ 2565334 , anti - DPP9 : RRID : AB _ 2889071 .

O O O O O 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 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

B. Pull-down assay showing direct binding of purified recombinant DPP9 to a BRCA2 N-terminal1-40 fragment immobilized on HA-beads. The DPP9 inhibitors 1G244 or SLRFLYEG compete with BRCA21-40HA for interaction with DPP9. Representative data of three technical replicates is shown. Anti-HA: RRID:AB_2565334, anti-DPP9: RRID:AB_2889071.


C . of PLAs showing fewer MMC - induced DPP9 - BRCA2 PLA events in HeLa treated with 10µM 1G244 . Each dot represents number of PLA events in a single , from three biological replicates . number of foci is shown on their localisation . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison . Shown are Mean ± ( * * * = p ≤ 0 . 001 , * * * * = p ≤ 0 . 0001 ) . D . Representative of DPP9 - BRCA2 PLA in presence of 1G244 - a competitive inhibitor of DPP9 . Control were mock treated with DMSO . Phalloidin ( green ) stains actin filaments , DAPI ( blue ) stains nucleus . Scale bar 10 µm . Anti - BRCA2 : RRID : AB _ 2259370 , anti - DPP9 : RRID : AB _

O O O O O O B-CONTROLLED_VAR 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 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

C. Quantification of PLAs showing fewer MMC-induced DPP9-BRCA2 PLA events in HeLa cells treated with 10µM 1G244. Each dot represents the number of PLA events in a single cell, from three biological replicates. The number of foci is shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (*** = p ≤ 0.001, **** = p ≤ 0.0001). D. Representative images of DPP9-BRCA2 PLA in the presence of 1G244 - a competitive inhibitor of DPP9. Control cells were mock treated with DMSO. Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_2889071


F . Pull - down showing a competition between DPP9 PALB2 for binding to BRCA2 N - terminus . While each can bind to BRCA2 N - terminal peptide , in presence of DPP9 , PALB2 - BRCA2 binding is negatively affected . Representative data of three technical replicates is shown . Anti - PALB2 : RRID : AB _ 890607 , anti - HA : RRID : AB _ 2565334 , anti - DPP9 : RRID : AB _ 2889071 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Pull-down assay showing a competition between DPP9 and PALB2 for binding to the BRCA2 N-terminus. While each protein can bind to the BRCA2 N-terminal peptide, in the presence of DPP9, the PALB2-BRCA2 binding is negatively affected. Representative data of three technical replicates is shown. Anti-PALB2: RRID:AB_890607, anti-HA: RRID:AB_2565334, anti-DPP9: RRID:AB_2889071.


A - B . Representative Western Blots accompanying graph from more than three biological replicates show that MMC ( 300 nM , 24 h ) induces a rapid turnover of endogenous BRCA2 in HeLa WT , which is less pronounced in HeLa WT treated with proteasome inhibitor MG132 ( 100µM ) , in HeLa DPP9 KD in transiently silenced for DPP9 ( siDPP9 ) . RAD51 stability is not altered in HeLa DPP9 KD . Vinculin is a loading control for BRCA2 , Tubulin is a loading control for RAD51 . Shown originate from one representative cycloheximide ( CHX ) chase . ratios of BRCA2 to Vinculin RAD51 to Tubulin are defined as 100 % at time 0 h . Mean ± , data were analysed by a paired two - t - . ( * = p ≤ 0 . 03 ) . Anti - BRCA2 : RRID : AB _ 2259370 , anti - DPP9 : RRID : AB _ 731947 , anti - Vinculin : RRID : AB _ 477629 , anti - Tubulin : RRID : AB _ 628412 , anti - RAD51 : RRID : AB _ 1142428 .

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 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 B-CONTROLLED_VAR 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

A-B. Representative Western Blots and accompanying graph from more than three biological replicates show that MMC (300 nM, 24 h) induces a rapid turnover of endogenous BRCA2 in HeLa WT cells, which is less pronounced in HeLa WT cells treated with the proteasome inhibitor MG132 (100µM), in HeLa DPP9 KD cells and in cells transiently silenced for DPP9 (siDPP9). RAD51 stability is not altered in HeLa DPP9 KD cells. Vinculin is a loading control for BRCA2, Tubulin is a loading control for RAD51. Shown images originate from one representative cycloheximide (CHX) chase assay. The ratios of BRCA2 to Vinculin and RAD51 to Tubulin are defined as 100% at time 0 h. Mean ± SEM, data were analysed by a paired two-tailed t-test. (*= p ≤ 0.03). Anti-BRCA2: RRID:AB_2259370, anti-DPP9: RRID:AB_731947, anti-Vinculin: RRID:AB_477629, anti-Tubulin: RRID:AB_628412, anti-RAD51: RRID:AB_1142428.


C - D . Representative Western Blots graph summarizing results of more than three biological replicates . CHX chase assays show that MMC - induced degradation of BRCA2 is less pronounced in HEK293 DPP9 KO as well as in HEK293 DPP9 KO + DPP9 - SS729A overexpressing an inactive DPP9 - S mutant . Expression of DPP9 was induced ( + Dox , 1µg / ml ) simultaneously with MMC ( 300 nM ) , 24 h . that were overexpressing active variant ( DPP9 - SWT ) show similar of MMC - induced BRCA2 degradation . RAD51 stability is not altered by MMC is similar in lines . Vinculin is a loading control for BRCA2 , Tubulin is a loading control for RAD51 . Shown originate from one representative cycloheximide ( CHX ) chase . ratios of BRCA2 to Vinculin are defined as 100 % at time 0 h . Mean ± , data were analysed by a paired two - t - ( * = p ≤ 0 . 03 , * * = p ≤ 0 . 002 ) . Antibodies as described in ( A - B ) .

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 B-CONTROLLED_VAR 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 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 B-CONTROLLED_VAR 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

C-D. Representative Western Blots and graph summarizing results of more than three biological replicates. CHX chase assays show that the MMC-induced degradation of BRCA2 is less pronounced in HEK293 DPP9 KO cells as well as in HEK293 DPP9 KO + DPP9-SS729A cells overexpressing an inactive DPP9-S mutant. Expression of DPP9 was induced (+ Dox, 1µg/ml) simultaneously with MMC (300 nM), 24 h. Cells that were overexpressing the active variant (DPP9-SWT) show similar levels of MMC-induced BRCA2 degradation. RAD51 stability is not altered by MMC and is similar in all cell lines. Vinculin is a loading control for BRCA2, Tubulin is a loading control for RAD51. Shown images originate from one representative cycloheximide (CHX) chase assay. The ratios of BRCA2 to Vinculin are defined as 100% at time 0 h. Mean ± SEM, data were analysed by a paired two-tailed t-test (*= p ≤ 0.03, **= p ≤ 0.002). Antibodies as described in (A-B).


F - G . Representative Western Blots graphs summarizing CHX assays from three biological replicates showing that BRCA2 ∆ MP3 - 1000 truncation mutant is degraded at a higher rate compared to untruncated BRCA21 - 1000 . were co - transfected with a GFP expressing plasmid , as transfection loading control . Control were transfected with GFP only ( U . T ) . Image originates from one representative experiment . BRCA2 - FLAG signals are related to transfection loading control GFP . BRCA2 in relation to GFP were defined as 100 % at time 0 h . Mean ± , data were analysed by a paired two - t - ( * = p ≤ 0 . 03 ) . Anti - FLAG : RRID : AB _ 262044 , anti - GFP : RRID : AB _ 641123 .

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

F-G. Representative Western Blots and graphs summarizing CHX assays from three biological replicates showing that the BRCA2∆MP3-1000 truncation mutant is degraded at a higher rate compared to the untruncated BRCA21-1000. Cells were co-transfected with a GFP expressing plasmid, as transfection and loading control. Control cells were transfected with GFP only (U.T). Image originates from one representative experiment. BRCA2-FLAG signals are related to the transfection and loading control GFP. BRCA2 levels in relation to GFP were defined as 100 % at time 0 h. Mean ± SEM, data were analysed by a paired two-tailed t-test (*= p ≤ 0.03). Anti-FLAG: RRID:AB_262044, anti-GFP: RRID:AB_641123.


A . Representative immunofluorescence of γH2AX signals ( , RRID : AB _ 309864 ) in gki MEF DPP9S729A , showing more γH2AX in gki MEF DPP9S729A following removal of Neocarzinostatin ( NCS ) , in ( B ) . MEF DPP9S729A control MEF were treated with 250 ng / mL NCS for 30 min allowed to recover for indicated time - points . γH2AX signals at time 0 , reflect 30 min of NCS no recovery time . Nuclei are shown in blue ( DAPI ) . Scale bar 10 µm . B . of γH2AX in gki MEF DPP9S729A control MEF as described in ( A ) . Signals from more than 1700 were quantified per condition per experiment . Mean ± from six biological replicates , each in technical duplicates . Data were analysed by an unpaired two - way ANOVA with Sidak ' s multiple comparison ( * = 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

A. Representative immunofluorescence images of γH2AX signals (white, RRID:AB_309864) in gki MEF DPP9S729A, showing more γH2AX in gki MEF DPP9S729A cells following removal of Neocarzinostatin (NCS), quantification in (B). MEF DPP9S729A and control MEF cells were treated with 250 ng/mL NCS for 30 min and allowed to recover for the indicated time-points. γH2AX signals at time 0, reflect 30 min of NCS and no recovery time. Nuclei are shown in blue (DAPI). Scale bar 10 µm. B. Quantification of γH2AX in gki MEF DPP9S729A and control MEF cells as described in (A). Signals from more than 1700 cells were quantified per condition per experiment. Mean ± SEM from six biological replicates, each in technical duplicates. Data were analysed by an unpaired two-way ANOVA with Sidak's multiple comparison test (* = p ≤ 0.05, ** = p ≤ 0.01).


C . Representative PLA of BRCA2 - PALB2 PLA experiments showing more PLA events ( ) in HeLa silenced for DPP9 ( siDPP9 ) with respect to treated with non - targeting siRNA ( siNT ) . Phalloidin ( green ) stains actin filaments , DAPI ( blue ) stains nucleus . Scale bar 10 µm . Anti - PALB2 : RRID : AB _ 890607 , anti - BRCA2 : RRID : AB _ 2259370 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative PLA images of BRCA2-PALB2 PLA experiments showing more PLA events (white) in HeLa cells silenced for DPP9 (siDPP9) with respect to cells treated with non-targeting siRNA (siNT). Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Anti-PALB2: RRID:AB_890607, anti-BRCA2: RRID:AB_2259370.


D . of PLAs experiments showing more BRCA2 - PALB2 PLA events in silenced for DPP9 ( siDPP9 ) in comparison to non - targeting controls ( siNT ) . Each dot represents number of PLA events in a single , from four biological replicates . technical control samples ( NgtCntrl ) omitted BRCA2 antibody . number of foci is shown on their localisation . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison . Shown are Mean ± ( * * * * = p ≤ 0 . 0001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 PLAs experiments showing more BRCA2-PALB2 PLA events in cells silenced for DPP9 (siDPP9) in comparison to non-targeting controls (siNT). Each dot represents the number of PLA events in a single cell, from four biological replicates. The technical control samples (NgtCntrl) omitted the BRCA2 antibody. The number of foci is shown based on their cellular localisation. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001).


E . Representative PLA of γH2Ax - BRCA2 PLA experiment showing a reduction in number of PLA events ( ) between γH2Ax BRCA2 in HeLa DPP9 KD . Phalloidin ( green ) stains actin filaments , DAPI ( blue ) stains nucleus . Scale bar 10 µm . Anti - γH2Ax : RRID : AB _ 2118009 , anti - BRCA2 : RRID : AB _ 2259370 F . of PLAs showing fewer MMC - induced γH2Ax - BRCA2 PLA events in HeLa DPP9 KD , in comparison to HeLa WT . Each dot represents number of PLA events in a single , from three biological replicates . technical control samples ( NgtCntrl ) omitted γH2Ax antibody . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison . Shown are Mean ± ( * * * * = p ≤ 0 . 0001 ) .

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

E. Representative PLA images of γH2Ax-BRCA2 PLA experiment showing a reduction in the number of PLA events (white) between γH2Ax and BRCA2 in HeLa DPP9 KD cells. Phalloidin (green) stains actin filaments, DAPI (blue) stains the nucleus. Scale bar 10 µm. Anti-γH2Ax: RRID:AB_2118009, anti-BRCA2: RRID:AB_2259370 F. Quantification of PLAs showing fewer MMC-induced γH2Ax-BRCA2 PLA events in HeLa DPP9 KD cells, in comparison to HeLa WT cells. Each dot represents the number of PLA events in a single cell, from three biological replicates. The technical control samples (NgtCntrl) omitted the γH2Ax antibody. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001).


A . Representative immunofluorescence showing that re - expression of DPP9 - SWT leads to an increase in number of RAD51 foci formed following exposure to MMC in HEK293 DPP9 KO + DPP9 - SWT . Expression of DPP9 was induced ( + Dox , 1µg / ml ) simultaneously with MMC ( 300 nM ) , 24 h . RAD51 foci are shown in , nuclei ( DAPI ) are shown in blue . Scale bar 10 µm . Anti - RAD51 : RRID : AB _ 1142428 . B . Graph showing number of RAD51 foci following induction of DPP9 - SWT expression , compared to uninduced HEK293 DPP9 KO + DPP9WT ( - Dox ) . Induction of HEK293 DPP9 KO + DPP9S729A for expression of DPP9 - SS729A did not result in more RAD51 foci . Each dot represents number of RAD51 foci in a single , from three biological replicates . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison . Shown are Mean ± ( * * * * = p ≤ 0 . 0001 ) .

O O 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 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

A. Representative immunofluorescence images showing that re-expression of DPP9-SWT leads to an increase in the number of RAD51 foci formed following exposure to MMC in HEK293 DPP9 KO + DPP9-SWT cells. Expression of DPP9 was induced (+ Dox, 1µg/ml) simultaneously with MMC (300 nM), 24 h. RAD51 foci are shown in white, nuclei (DAPI) are shown in blue. Scale bar 10 µm. Anti-RAD51: RRID:AB_1142428. B. Graph showing the number of RAD51 foci following induction of DPP9-SWT expression, compared to uninduced HEK293 DPP9 KO + DPP9WT cells (-Dox). Induction of HEK293 DPP9 KO + DPP9S729A for expression of DPP9-SS729A did not result in more RAD51 foci. Each dot represents the number of RAD51 foci in a single cell, from three biological replicates. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001).


Representative ( C , summarizing graph ( D showing number of RAD51 foci per nucleus in HeLa WT ( C , D ) Where stated , were treated with control siRNA ( siNT ) or silenced for BRCA2 , transiently transfected with BRCA21 ‑ 3418 or BRCA23 - 3418 constructs . Both BRCA2 constructs can rescue RAD51 foci formation phenotype to control in HeLa WT ( C , D ) . Each dot represents number of RAD51 foci in a single , from three ( D ) biological replicates . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison . Shown are Mean ± ( * * * * = p ≤ 0 . 0001 ) . RAD51 foci are shown in , nuclei ( DAPI ) are shown in blue . Scale bar 10 µm . Anti - RAD51 : RRID : AB _ 1142428 ; ; anti - BRCA2 : RRID : AB _ 2259370 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (C, and summarizing graph (D showing the number of RAD51 foci per nucleus in HeLa WT cells (C, D) Where stated, cells were treated with control siRNA (siNT) or silenced for BRCA2, and transiently transfected with the BRCA21‑3418 or BRCA23-3418 constructs. Both BRCA2 constructs can rescue the RAD51 foci formation phenotype to the control levels in HeLa WT cells (C, D). Each dot represents the number of RAD51 foci in a single cell, from three (D) biological replicates. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001). RAD51 foci are shown in white, nuclei (DAPI) are shown in blue. Scale bar 10 µm. Anti-RAD51: RRID:AB_1142428; ; anti-BRCA2: RRID:AB_2259370.


Representative , E ) summarizing graph F ) showing number of RAD51 foci per nucleus in HeLa DPP9 KD ( E , F ) . In HeLa DPP9 KD significantly more RAD51 foci were present in transfected with BRCA2 ∆ MP3 - 3418 compared to BRCA2 silenced expressing untruncated BRCA21 - 3418 ( E , F ) . Each dot represents number of RAD51 foci in a single , from four ( F ) biological replicates . Data were analysed by a two - way ANOVA , with Tukey ' s Multiple Comparison . Shown are Mean ± ( * * * * = p ≤ 0 . 0001 ) . RAD51 foci are shown in , nuclei ( DAPI ) are shown in blue . Scale bar 10 µm . Anti - RAD51 : RRID : AB _ 1142428 ; anti - BRCA2 : RRID : AB _ 2259370 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 , E) and summarizing graph F) showing the number of RAD51 foci per nucleus in HeLa DPP9 KD cells (E, F). In HeLa DPP9 KD cells significantly more RAD51 foci were present in cells transfected with BRCA2∆MP3-3418 compared to BRCA2 silenced cells and cells expressing the untruncated BRCA21-3418 (E, F). Each dot represents the number of RAD51 foci in a single cell, from four (F) biological replicates. Data were analysed by a two-way ANOVA, with Tukey's Multiple Comparison test. Shown are Mean ± SEM (**** = p ≤ 0.0001). RAD51 foci are shown in white, nuclei (DAPI) are shown in blue. Scale bar 10 µm. Anti-RAD51: RRID:AB_1142428; anti-BRCA2: RRID:AB_2259370.


B , C CDKL51 - 352 autophosphorylation is shown . WT CDKL5 uses ATPγS , KD is inactive , F89A uses benzyl - ATPγS with reduced efficiency , F89A / C152A mutant has rescued activity . of Thio - P signal is normalized for CDKL5 ( HA ) KD background is subtracted . Fisher ' s LSD : n = 4 replicates . bn = benzyl

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C CDKL51-352 autophosphorylation is shown. WT CDKL5 uses ATPγS, KD is inactive, F89A uses benzyl-ATPγS with reduced efficiency, F89A/ C152A mutant has rescued activity. Quantification of Thio-P signal is normalized for CDKL5 levels (HA) and KD background is subtracted. Fisher's LSD: n = 4 replicates. bn = benzyl


E - H In vitro kinase assays showing efficient MAP1S phosphorylation by CDKL5 . 50 ng ( 40 nM ) AS - CDKL5 phosphorylates 150 ng ( 50 nM ) MAP1S very rapidly ( E , F ) of phosphorylated MAP1S is normalized to maximum . n = 2

O O O O O O O O O O O O O O O O O O O O O O O O O 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 - H In vitro kinase assays showing efficient MAP1S phosphorylation by CDKL5. 50 ng (40 nM) AS-CDKL5 phosphorylates 150 ng (50 nM) MAP1S very rapidly (E, F) Quantification of phosphorylated MAP1S is normalized to maximum intensity. n = 2 replicates


E - H In vitro kinase assays showing efficient MAP1S phosphorylation by CDKL5 In 30 minutes of incubation , 150 ng MAP1S is phosphorylated by small amounts of CDKL5 ( G , H ) . of phosphorylated MAP1S is normalized to maximum . n = 2

O O O O O O O O O O O O O O O O O O O O O O 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 - H In vitro kinase assays showing efficient MAP1S phosphorylation by CDKL5 In 30 minutes of incubation, 150 ng MAP1S is phosphorylated by small amounts of CDKL5 (G, H). Quantification of phosphorylated MAP1S is normalized to maximum intensity. n = 2 replicates


I - L Western blots of in vitro kinase assays showing loss of phosphorylation in phosphomutants ARHGEF2 S122A ( I Substrate are shown by coommassie

O 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 - L Western blots of in vitro kinase assays showing loss of phosphorylation in phosphomutants ARHGEF2 S122A (I Substrate levels are shown by coommassie staining underneath


I - L Western blots of in vitro kinase assays showing loss of phosphorylation in phosphomutant EB2 S222A ( J Substrate are shown by coommassie

I - L Western blots of in vitro kinase assays showing loss of phosphorylation in phosphomutant EB2 S222A (J Substrate levels are shown by coommassie staining underneath


I - L Western blots of in vitro kinase assays showing loss of phosphorylation in phosphomutant MAP1S S786 / 812A ( K ) . Substrate are shown by coommassie underneath . MAP1S phosphorylation is quantified in ( L ) . Dunnett ' s multiple comparison : n = 3

I - L Western blots of in vitro kinase assays showing loss of phosphorylation in phosphomutant MAP1S S786/812A (K). Substrate levels are shown by coommassie staining underneath. MAP1S phosphorylation is quantified in (L). Dunnett's multiple comparison: n = 3 replicates


A , B In vivo substrate phosphorylations are assessed by Western blotting of 5 WT ( CDKL5 + / Y ) 5 CDKL5 KO ( CDKL5 - / Y ) mouse cortical lysates using phosphospecific antibodies . EB2 pS222 MAP1S pS812 are dramatically reduced in KOs . of substrate phosphorylation is normalized for total level . Student ' s t - : n = 5 per

A, B In vivo substrate phosphorylations are assessed by Western blotting of 5 WT (CDKL5 +/Y) and 5 CDKL5 KO (CDKL5 -/Y) mouse cortical lysates using phosphospecific antibodies. EB2 pS222 and MAP1S pS812 are dramatically reduced in KOs. Quantification of substrate phosphorylation is normalized for total protein level. Student's t-test: n = 5 animals per genotype


C , D Developmental expression of CDKL5 of its substrate phosphorylations in P4 - P50 cortex . CDKL5 - dependent substrate phosphorylation is quantified by subtracting KO from WT for each . n = 3 per per genotype

O O O O O O O O O O O O O O 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, D Developmental expression of CDKL5 and levels of its substrate phosphorylations in P4 - P50 cortex. CDKL5-dependent substrate phosphorylation is quantified by subtracting KO from WT levels for each age. n = 3 animals per age per genotype


E , F Expression of CDKL5 of substrate phosphorylation in P8 Nex - Cre conditional KO cortex . CDKL5 expression is almost completely lost . of substrate phosphorylation is normalized for total level . Student ' s t - : n = 3 per genotype

O O O O O O O O O O O O O O O O O O O O O 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 Expression of CDKL5 and levels of substrate phosphorylation in P8 Nex-Cre conditional KO cortex. CDKL5 protein expression is almost completely lost. Quantification of substrate phosphorylation is normalized for total protein level. Student's t-test: n = 3 animals per genotype


G CDKL5 WT CDKL5 KO neurons in culture are co - stained with neuronal dendrite marker MAP2 , EB2 EB2 pS222 . EB2 pS222 is lost in CDKL5 KO dendrites . Scale bar is 20 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

G CDKL5 WT and CDKL5 KO neurons in culture are co-stained with neuronal dendrite marker MAP2, EB2 and EB2 pS222. EB2 pS222 is lost in CDKL5 KO dendrites. Scale bar is 20 μm


A - C lysates were obtained from cortical neuronal cultures treated with 50 mM KCl applied directly to culture media for indicated durations . 100 µm AP5 was used to block NMDAR activity during KCl treatment . of phosphorylated EB2 ( B ) total CDKL5 ( C ) are normalized to 0 minutes time point . EB2 phosphorylation is significantly decreased after 10 minutes of KCl treatment compared to 0 minutes . CDKL5 are slightly reduced after 40 minutes compared to 0 minutes . Dunnett ' s multiple comparison : n = 3

O O 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

A - C Protein lysates were obtained from cortical neuronal cultures treated with 50 mM KCl applied directly to culture media for indicated durations. 100 µm AP5 was used to block NMDAR activity during KCl treatment. Quantification of phosphorylated EB2 (B) and total CDKL5 (C) are normalized to 0 minutes time point. EB2 phosphorylation is significantly decreased after 10 minutes of KCl treatment compared to 0 minutes. CDKL5 protein levels are slightly reduced after 40 minutes compared to 0 minutes. Dunnett's multiple comparison: n = 3 replicates


D , E lysates were obtained from cortical neuronal cultures treated with 50 μM NMDA applied directly to culture media for indicated durations . of phosphorylated EB2 total CDKL5 is normalized to 0 minutes time point . EB2 S222 phosphorylation is significantly reduced after 10 minutes compared to 0 minutes . Dunnett ' s multiple comparison : n = 3 replicates

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

D, E Protein lysates were obtained from cortical neuronal cultures treated with 50 μM NMDA applied directly to culture media for indicated durations. Quantification of phosphorylated EB2 and total CDKL5 is normalized to 0 minutes time point. EB2 S222 phosphorylation is significantly reduced after 10 minutes compared to 0 minutes. Dunnett's multiple comparison: n = 3 replicates


A Representative kymographs of EB3 - tdTomato in cortical neuron dendrites at DIV14

A Representative kymographs of EB3-tdTomato in cortical neuron dendrites at DIV14


B - D In CDKL5 KOs ( CDKL5 - / Y ) , comet ( B ) lifetime ( C ) is increased when compared to WT ( CDKL5 + / Y ) . Comet velocity ( D ) is unchanged . Student ' s t - : n = 16 / 615 15 / 381 neurons / comets in WT KO , respectively

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - D In CDKL5 KOs (CDKL5 -/Y), comet distance (B) and lifetime (C) is increased when compared to WT (CDKL5 +/Y). Comet velocity (D) is unchanged. Student's t-test: n = 16/615 and 15/381 neurons/comets in WT and KO, respectively


E Representative kymographs of CDKL5 WT KO neurons transfected with control shRNA MAP1S shRNA

E Representative kymographs of CDKL5 WT and KO neurons transfected with control shRNA and MAP1S shRNA


F Comet lifetime is higher in control shRNA expressing KOs than WTs . MAP1S shRNA expression reduces comet lifetime in KO neurons . EB2 shRNA - mediated knockdown does not alter microtubule dynamics in WT or KO . Tukey ' s multiple comparison : n = 12 - 15 neurons , 388 - 741 comets per condition

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Comet lifetime is higher in control shRNA expressing KOs than WTs. MAP1S shRNA expression reduces comet lifetime in KO neurons. EB2 shRNA-mediated knockdown does not alter microtubule dynamics in WT or KO. Tukey's multiple comparison: n = 12-15 neurons, 388-741 comets per condition


A , B Representative stills ( A ) kymographs ( B ) of TrkB - RFP overexpressed in dendrites of WT CDKL5 KO DIV14 cortical neurons . Scale bar is 5 μm . C , D No significant change is observed in of dynamic vs stationary vesicles ( C ) or anterograde vs retrograde runs ( D ) . S = stationary , D = dynamic , A = anterograde , R = retrograde . Vesicles with a net > 5 μm were considered dynamic . E , F Both anterograde retrograde runs show a trend towards reduced velocity in CDKL5 KO neurons ( E ) . Anterograde run lengths are significantly reduced in KO compared to WT neurons ( F )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B Representative stills (A) and kymographs (B) of TrkB-RFP overexpressed in dendrites of WT and CDKL5 KO DIV14 cortical neurons. Scale bar is 5 μm. C, D No significant change is observed in the ratio of dynamic vs stationary vesicles (C) or anterograde vs retrograde runs (D). S = stationary, D = dynamic, A = anterograde, R = retrograde. Vesicles with a net distance > 5 μm were considered dynamic. E, F Both anterograde and retrograde runs show a trend towards reduced velocity in CDKL5 KO neurons (E). Anterograde run lengths are significantly reduced in KO compared to WT neurons (F)


B COS - 7 expressing HA - MAP1S LC , Flag - CDKL5 WT / kinase dead ( KD ) is shown with endogenous α - tubulin . MAP1S does not localize to microtubules when phosphorylated by CDKL5 . Scale bar is 20 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

B COS-7 cells expressing HA-MAP1S LC, Flag-CDKL5 WT / kinase dead (KD) is shown with endogenous α-tubulin staining. MAP1S does not localize to microtubules when phosphorylated by CDKL5. Scale bar is 20 μm


C , D of where α - tubulin MAP1S LC are colocalised ( C ) of MAP1S LC α - tubulin colocalisation by Pearson correlation coefficient ( D ) . Tukey ' s multiple comparison : n = 22 - 24 per condition

O O O O O O O O O O O O O O O O O O O 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, D The percentage of cells where α-tubulin and MAP1S LC are colocalised (C) and the quantification of MAP1S LC and α-tubulin colocalisation by Pearson correlation coefficient (D). Tukey's multiple comparison: n = 22-24 cells per condition


E , F Taxol - stabilised microtubule co - sedimentation shows direct binding of MAP1S LC to microtubules is reduced upon prior phosphorylation by CDKL5 . of soluble MAP1S in supernatant ( s / n ) fraction is normalized to MT - bound pellet fraction . 3 : 1 MT : MAP1S LC used here . Tukey ' s multiple comparison : n = 3 technical repeats

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Taxol-stabilised microtubule co-sedimentation assay shows direct binding of MAP1S LC to microtubules is reduced upon prior phosphorylation by CDKL5. Quantification of soluble MAP1S in the supernatant (s/n) fraction is normalized to the MT-bound pellet fraction. 3:1 MT:MAP1S LC ratio used here. Tukey's multiple comparison: n = 3 technical repeats


G HA - MAP1S LC is overexpressed stained with anti - HA antibody in cultured cortical neurons ( red ) . Numerous loops at dendrites ' tips are observed ( arrowheads ) . Scale bar is 5 μm

G HA-MAP1S LC is overexpressed and stained with anti-HA antibody in cultured cortical neurons (red). Numerous loops at the dendrites' tips are observed (arrowheads). Scale bar is 5 μm


A , B Western blots for total EB2 , pS223 tubulin using lysates obtained from neurons derived from three with CDD their related control . 1 = p . D135 _ F154del , ; 2 = p . R59X , ; 3 = p . R59X , . of EB2 phosphorylation is normalized for total level . Student ' s t - : n = 3 / controls . * p < 0 . 05 , error bars are

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B Western blots for total EB2, pS223 and tubulin using protein lysates obtained from neurons derived from three patients with CDD and their related control. 1 = p.D135_F154del, male; 2 = p.R59X, male; 3 = p.R59X, female. Quantification of EB2 phosphorylation is normalized for total protein level. Student's t-test: n = 3 patients/controls. * p < 0.05, error bars are SEM


C Representative of human iPSC - derived neurons co - stained with neuronal dendrite marker MAP2 , EB2 EB2 pS223 . EB2 phosphorylation is present in dendrites of control neurons but not in CDD neurons . Enlarged of boxed areas are shown on right panel . Scale bar is 20 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

C Representative images of human iPSC-derived neurons co-stained with the neuronal dendrite marker MAP2, EB2 and EB2 pS223. EB2 phosphorylation is present in dendrites of control neurons but not in CDD neurons. Enlarged images of the boxed areas are shown on the right panel. Scale bar is 20 μm


A . Quantitative of developmental expression of Lrig1 mRNA in rat hippocampus by real - time RT - PCR . results are shown as mean of n = 3 independent assays . of Lrig1 mRNA were normalized using expression of housekeeping gene Tbp ( TATA binding ) . insert shows expression of Lrig1 in embryonic E17 . 5 rat hippocampus examined by RT - PCR . Control sample without reverse transcriptase ( - RT ) is also shown .

A. Quantitative analysis of developmental expression of Lrig1 mRNA in rat hippocampus by real-time RT-PCR. The results are shown as mean SEM of n=3 independent assays. The levels of Lrig1 mRNA were normalized using the expression of the housekeeping gene Tbp (TATA binding protein). The insert shows the expression of Lrig1 in embryonic E17.5 rat hippocampus examined by RT-PCR. Control sample without reverse transcriptase (-RT) is also shown.


B . Localization of Lrig1 ( red ) in coronal sections from P15 ratbrain by immunofluorescence . Anti - Lrig1ECD antibodies label dentate gyrus , CA1 - CA3 hippocampalneurons pyramidal corticalneurons ( * ) . Scale bar , 400 m .

O O O O O O O O O O O O O O O O O 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. Localization of Lrig1 (red) in coronal sections from P15 ratbrain by immunofluorescence. Anti-Lrig1ECD antibodies label dentate gyrus cells, CA1-CA3 hippocampalneurons and pyramidal corticalneurons (*). Scale bar, 400 m.


C - E . Confocal image of cortical ( C ) hippocampal ( D - E ) pyramidal neurons stained with rabbit anti - Lrig1ECD antibody . Arrows indicate Lrig1 in proximal segments of apical dendrites of CA1 - CA3hippocampal pyramidal cortical neurons ( layer V ) . Scale bars , 20 m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E. Confocal image of cortical (C) and hippocampal (D-E) pyramidal neurons stained with rabbit anti-Lrig1ECD antibody. Arrows indicate Lrig1 staining in proximal segments of apical dendrites of CA1-CA3hippocampal and pyramidal cortical neurons (layer V). Scale bars, 20 m.


F . Immunofluorescence of Lrig1 ( red ) with anti - Lrig1ECD antibody neuronal marker ßIII tubulin ( green ) in dissociated hippocampal cultured for 7 DIV . Yellow indicates neuronal expression of Lrig1 . Scale bar , 15 m .

F. Immunofluorescence staining of Lrig1 (red) with anti-Lrig1ECD antibody and the neuronal marker ßIII tubulin (green) in dissociated hippocampal cells cultured for 7 DIV. Yellow indicates neuronal expression of Lrig1. Scale bar, 15 m.


G . Immunofluorescence of Lrig1 ( red ) with anti - Lrig1ECD antibody astrocytic marker S100 ( green ) in dissociated hippocampal cultured for 7 DIV . Scale bar , 20 m .

O O O O O O O O O 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. Immunofluorescence staining of Lrig1 (red) with anti-Lrig1ECD antibody and the astrocytic marker S100 (green) in dissociated hippocampal cells cultured for 7 DIV. Scale bar, 20 m.


H . Localization of Lrig1 ( red ) with anti - Lrig1ECD antibody somatodendritic marker MAP - 2 ( blue ) by immunocytochemistry in dissociated rat hippocampalneurons after 12 DIV . Scale bar , 20 m .

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

H. Localization of Lrig1 (red) with anti-Lrig1ECD antibody and the somatodendritic marker MAP-2 (blue) by immunocytochemistry in dissociated rat hippocampalneurons after 12 DIV. Scale bar, 20 m.


A . Representative of mouse hippocampal neurons transfected with either GFP - expressing control or Lrig1 shRNA vector at 9 DIV maintained for 3 additional days in vitro ( 9 + 3 DIV ) . Scale bar , 15 m . Boxed represents a higher - magnification image showing profuse proximal dendritic arborization of Lrig1 - shRNA transfected neurons . B . Sholl of dendritic arbor from hippocampal neurons transfected with either control or Lrig1 shRNA - GFP vector at 9 DIV maintained for 3 additional days in vitro ( 9 + 3 DIV ) . Data are shown as mean of n = 3 independent experiments . * p < 0 . 05 * * p < 0 . 01 by two - way ANOVA followed by Bonferroni multiple comparisons . C - G . of ( C ) , secondary ( D ) dendrites as well as total dendritic branching ( E ) , terminal dendritic points ( F ) , total dendritic ( G ) of hippocampal neurons transfected with either control or Lrig1 shRNA - GFP vector . results are shown as mean of n = 3 independent experiments . * p < 0 . 05 by Students t .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of mouse hippocampal neurons transfected with either GFP-expressing control or Lrig1 shRNA vector at 9 DIV and maintained for 3 additional days in vitro (9+3 DIV). Scale bar, 15 m. Boxed area represents a higher-magnification image showing the profuse proximal dendritic arborization of Lrig1-shRNA transfected neurons.B. Sholl analysis of the dendritic arbor from hippocampal neurons transfected with either control or Lrig1 shRNA-GFP vector at 9 DIV and maintained for 3 additional days in vitro (9+3 DIV). Data are shown as mean SEM of n=3 independent experiments. *p<0.05 and **p<0.01 by two-way ANOVA followed by Bonferroni multiple comparisons test.C-G. Quantification of primary (C), and secondary (D) dendrites as well as total dendritic branching (E), terminal dendritic points (F), and total dendritic length (G) of hippocampal neurons transfected with either control or Lrig1 shRNA-GFP vector. The results are shown as mean SEM of n=3 independent experiments. *p<0.05 by Students t test.


H . Knockdown efficiency was analyzed by real - time RT - PCR in MN1 transfected with control or Lrig1 shRNA vectors . Transfected were enriched by puromycin treatment in order to increase population of expressing control or Lrig1 shRNA constructs . Data are shown as values of a representative measured in triplicates . n = 2 independent experiments were performed .

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

H. Knockdown efficiency was analyzed by real-time RT-PCR in MN1 cells transfected with control or Lrig1 shRNA vectors. Transfected cells were enriched by puromycin treatment in order to increase the population of cells expressing control or Lrig1 shRNA constructs. Data are shown as individual values of a representative assay measured in triplicates. n=2 independent experiments were performed.