I Live imaging of GFP - RAB6A in control , dynarrestin - treated and CC1 - p150 - expressing aRG cells at E15 . 5 . Scale bars = 5µm . Distance = 5µm , time = 30 seconds . J . Number of RAB6A + vesicles in the apical process of DMSO and dynarrestin - treated mouse aRG cells . K . Relative time spent by RAB6A + vesicles in apical movement phase , in DMSO and dynarrestin - treated mouse aRG cells . L . Number of RAB6A + vesicles in the apical process of mCherry and CC1 - p150 - expressing aRG cells . M . Relative time spent by RAB6A + vesicles in apical movement phase , in mCherry and CC1 - p150 - expressing aRG cells . DMSO treatment slightly affected RAB6A dynamics , as compared to mCherry control . Data information : ( J , K , L , M ) 216 vesicles from N = 11 cells for DMSO , 145 vesicles from N = 25 cells for dynarrestin , 173 vesicles from N = 17 cells for mCherry control , 71 vesicles from N = 15 cells for CC1 - p150 . Mann - Whitney U test * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . All boxplots : whiskers indicate min and max , boxes indicate 25th and 75th percentile , and central band indicates the median .

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 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 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 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

I Live imaging of GFP-RAB6A in control, dynarrestin-treated and CC1-p150-expressing aRG cells at E15.5. Scale bars = 5µm. Distance = 5µm, time = 30 seconds. J. Number of RAB6A+ vesicles in the apical process of DMSO and dynarrestin-treated mouse aRG cells. K. Relative time spent by RAB6A+ vesicles in apical movement phase, in DMSO and dynarrestin-treated mouse aRG cells. L. Number of RAB6A+ vesicles in the apical process of mCherry and CC1-p150-expressing aRG cells. M. Relative time spent by RAB6A+ vesicles in apical movement phase, in mCherry and CC1-p150-expressing aRG cells. DMSO treatment slightly affected RAB6A dynamics, as compared to mCherry control. Data information: (J, K, L, M) 216 vesicles from N=11 cells for DMSO, 145 vesicles from N=25 cells for dynarrestin, 173 vesicles from N=17 cells for mCherry control, 71 vesicles from N=15 cells for CC1-p150. Mann-Whitney U test *p ≤ 0.05, **p ≤ 0.01, *** p ≤ 0.001. All boxplots: whiskers indicate min and max, boxes indicate 25th and 75th percentile, and central band indicates the median.


C . RUSH assay for CRB3 - GFP in control ( mcherry ) and dynactin - inhibited radial glial cells ( CC1 - p150 - dsRed ) , electroporated at E . 15 . 5 and imaged at E16 . 5 . Scale bar = 5µm . D . CRB3 localization at the apical surface upon release . E . Percentage of cells with 100 % of CRB3 signal at the apical surface upon release . Data information : ( D , E ) mCherry ( control ) : N = 3 brains per timepoint ( 361 cells total ) . CC1 - p150 : N = 3 brains per timepoint ( 2 brains for T0 ) ( 268 cells total ) . Fisher ' s exact test and Benjamini - Hochberg procedure , * * * p ≤ 0 . 001 . All error bars indicate SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. RUSH assay for CRB3-GFP in control (mcherry) and dynactin-inhibited radial glial cells (CC1-p150-dsRed), electroporated at E.15.5 and imaged at E16.5. Scale bar = 5µm. D. CRB3 localization at the apical surface upon release. E. Percentage of cells with 100% of CRB3 signal at the apical surface upon release. Data information: (D, E) mCherry (control): N= 3 brains per timepoint (361 cells total). CC1-p150: N=3 brains per timepoint (2 brains for T0) (268 cells total). Fisher's exact test and Benjamini-Hochberg procedure, *** p ≤ 0.001. All error bars indicate SD.


A . PAX6 and phospho - Histone 3 ( p - H3 ) staining in WT and LIS1 KO E12 . 5 brains . Cortices were subdivided into 5 bins of equal size along the radial axis . Scale bar = 25µm . B . Percentage of PH3 + / PAX6 + cells located above the ventricular surface of WT and LIS1 KO E12 . 5 brains . WT : 1192 cells from N = 6 brains . LIS1 KO : 589 cells from N = 3 brains . Data information : ( B , Mann - Whitney U test , * p ≤ 0 . 05 . All error bars indicate SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. PAX6 and phospho-Histone 3 (p-H3) staining in WT and LIS1 KO E12.5 brains. Cortices were subdivided into 5 bins of equal size along the radial axis. Scale bar = 25µm. B. Percentage of PH3+/PAX6+ cells located above the ventricular surface of WT and LIS1 KO E12.5 brains. WT: 1192 cells from N=6 brains. LIS1 KO: 589 cells from N=3 brains. Data information: (B, Mann-Whitney U test, *p ≤ 0.05. All error bars indicate SD.


C . Phospho - Vimentin ( p - VIM ) staining in WT and LIS1 KO E12 . 5 brains . Scale bar = 25µm . D . Percentage p - VIM + cells dividing ectopically , away from the ventricular surface of WT and LIS1 KO E12 . 5 brains . WT : 1056 cells from N = 6 brains . LIS1 KO : 879 cells from N = 3 brains . Data information : D ) Mann - Whitney U test , * p ≤ 0 . 05 . All error bars indicate SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Phospho-Vimentin (p-VIM) staining in WT and LIS1 KO E12.5 brains. Scale bar = 25µm. D. Percentage p-VIM+ cells dividing ectopically, away from the ventricular surface of WT and LIS1 KO E12.5 brains. WT: 1056 cells from N=6 brains. LIS1 KO: 879 cells from N=3 brains. Data information: D) Mann-Whitney U test, *p ≤ 0.05. All error bars indicate SD.


A . CRB3 and PALS1 staining in WT , RAB6A / B dKO ( E15 . 5 ) and LIS1 KO ( E12 . 5 ) brains . Scale bar = 25µm .

O O O O O O O O O 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. CRB3 and PALS1 staining in WT, RAB6A/B dKO (E15.5) and LIS1 KO (E12.5) brains. Scale bar = 25µm.


B . Quantification of CRB3 staining interruptions along the ventricular boundary of WT ( E12 . 5 and E15 . 5 ; N = 13 brains ) , LIS1 KO ( E12 . 5 , N = 4 brains ) ) and RAB6A / B dKO ( E15 . 5 , N = 4 brains ) . Data information : ( B , Kruskal - Wallis test with a Dunn post - hoc test and Benjamini - Hochberg procedure , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . All error bars indicate SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 CRB3 staining interruptions along the ventricular boundary of WT (E12.5 and E15.5; N=13 brains), LIS1 KO (E12.5, N=4 brains)) and RAB6A/B dKO (E15.5, N=4 brains). Data information: (B, Kruskal-Wallis test with a Dunn post-hoc test and Benjamini-Hochberg procedure, *p ≤ 0.05, *** p ≤ 0.001. All error bars indicate SD.


C . N - Cadherin staining in WT , RAB6A / B dKO ( E15 . 5 ) and LIS1 KO ( E12 . 5 ) brains . Scale bar = 25µm .

C. N-Cadherin staining in WT, RAB6A/B dKO (E15.5) and LIS1 KO (E12.5) brains. Scale bar = 25µm.


D . Quantification of N - Cadh staining interruptions along the ventricular boundary of WT ( E12 . 5 and E15 . 5 ; N = 6 brains ) , LIS1 KO ( E12 . 5 , N = 3 brains ) and RAB6A / B dKO ( E15 . 5 , N = 3 brains ) . Data information : D ) Kruskal - Wallis test with a Dunn post - hoc test and Benjamini - Hochberg procedure , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 . All error bars indicate SD .

D. Quantification of N-Cadh staining interruptions along the ventricular boundary of WT (E12.5 and E15.5; N=6 brains), LIS1 KO (E12.5, N=3 brains) and RAB6A/B dKO (E15.5, N=3 brains). Data information: D) Kruskal-Wallis test with a Dunn post-hoc test and Benjamini-Hochberg procedure, *p ≤ 0.05, *** p ≤ 0.001. All error bars indicate SD.


A ) SH - SY5Y neuroblastoma cells were either cultivated in RPMI + 10 % FCS or starved in HBSS with or without addition of LY294002 or rapamycin . After 16 h PINK1 mRNA expression was determined by RT - qPCR and the PINK1 mRNA content of cells kept in RPMI + 10 % FCS was set as 1 . Induction of autophagy by starvation or rapamycin resulted in an induction of PINK1 expression , comparable to the positive control LY294002 ; n = 4 ; * expression changes compared to the PINK1 expression in RPMI + 10 % FCS : LY294002 : p < 0 . 01 ; rapamycin : p < 1 × 10 − 4 ; HBSS : p < 0 . 005 ; HBSS + LY294002 : p < 5 × 10 − 6 ; HBSS + rapamycin : p < 0 . 0005 ; # expression changes compared to the PINK1 expression in RPMI + 10 % FCS + LY294002 : p < 0 . 05 ; + expression changes compared to the PINK1 expression in RPMI + 10 % FCS + rapamycin : p < 0 . 0005 .

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

A) SH-SY5Y neuroblastoma cells were either cultivated in RPMI+10% FCS or starved in HBSS with or without addition of LY294002 or rapamycin. After 16 h PINK1 mRNA expression was determined by RT-qPCR and the PINK1 mRNA content of cells kept in RPMI+10% FCS was set as 1. Induction of autophagy by starvation or rapamycin resulted in an induction of PINK1 expression, comparable to the positive control LY294002; n = 4; * expression changes compared to the PINK1 expression in RPMI+10% FCS: LY294002: p<0.01; rapamycin: p<1×10−4; HBSS: p<0.005; HBSS+LY294002: p<5×10−6; HBSS+ rapamycin: p<0.0005; # expression changes compared to the PINK1 expression in RPMI+10% FCS+LY294002: p<0.05; + expression changes compared to the PINK1 expression in RPMI+10% FCS+rapamycin: p<0.0005.


B ) SH - SY5Y cells were either stably transduced with a control ( nt ) shRNA or a shRNA directed against PINK1 and cultivated in RPMI medium containing 5 % or 10 % FCS . Their PINK1 mRNA content was determined by RT - qPCR and PINK1 mRNA content of nt cells kept in medium with 10 % FCS was set as 1 . Serum reduction increased PINK1 mRNA in nt cells in accordance with the data shown in Fig . 1A , while stable PINK1 knockdown ( kd ) reduced PINK1 content under both conditions ; n = 3 ; * expression changes compared to the PINK1 expression in RPMI + 10 % FCS : PINK1 kd 10 % FCS p < 0 . 0005 ; PINK1 induction by 5 % FCS : p < 0 . 05 ; # expression changes compared to the PINK1 expression in RPMI + 5 % FCS : PINK1 kd 5 % FCS : p < 0 . 005 .

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

B) SH-SY5Y cells were either stably transduced with a control (nt) shRNA or a shRNA directed against PINK1 and cultivated in RPMI medium containing 5% or 10% FCS. Their PINK1 mRNA content was determined by RT-qPCR and PINK1 mRNA content of nt cells kept in medium with 10% FCS was set as 1. Serum reduction increased PINK1 mRNA in nt cells in accordance with the data shown in Fig. 1A, while stable PINK1 knockdown (kd) reduced PINK1 content under both conditions; n = 3; * expression changes compared to the PINK1 expression in RPMI+10% FCS: PINK1 kd 10% FCS p<0.0005; PINK1 induction by 5% FCS: p<0.05; # expression changes compared to the PINK1 expression in RPMI+5% FCS: PINK1 kd 5% FCS: p<0.005.


C ) SH - SY5Y cells without ( nt ) or with stable PINK1 knockdown ( kd ) were kept in medium with 5 % FCS and either untreated or treated for 2 h with CCCP to stabilize PINK1 . Afterwards the PINK1 63 kDa protein ( arrowhead ) and actin protein levels were determined by western blotting ( see representative gel on the right ) . The quantification revealed a reduction of PINK1 protein under both conditions in PINK1 kd cells .

O O O O O O O O O O O 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

C) SH-SY5Y cells without (nt) or with stable PINK1 knockdown (kd) were kept in medium with 5% FCS and either untreated or treated for 2 h with CCCP to stabilize PINK1. Afterwards the PINK1 63 kDa protein (arrowhead) and actin protein levels were determined by western blotting (see representative gel on the right). The quantification revealed a reduction of PINK1 protein under both conditions in PINK1 kd cells.


2A ) SH - SY5Y cells were starved for 2 h in HBSS with Bafilomycin ( + Baf ) or left untreated in RPMI + 5 % FCS ( - Baf ) . The LC3 - II and actin content were determined by western blotting . A representative blot is shown on the right , showing only LC3 - II bands in the Bafilomycin - treated samples . The LC3 - II bands of Bafilomycin treated samples were normalized to actin and the relative LC3 - II content of nt cells was set as 1 . Cells with stable PINK1 knockdown exhibited a reduced LC3 - II / actin ratio compared to the control ( nt ) cells ; n = 4 , p < 0 . 005 .

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 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

2A) SH-SY5Y cells were starved for 2 h in HBSS with Bafilomycin (+Baf) or left untreated in RPMI+5% FCS (- Baf). The LC3-II and actin content were determined by western blotting. A representative blot is shown on the right, showing only LC3-II bands in the Bafilomycin-treated samples. The LC3-II bands of Bafilomycin treated samples were normalized to actin and the relative LC3-II content of nt cells was set as 1. Cells with stable PINK1 knockdown exhibited a reduced LC3-II/actin ratio compared to the control (nt) cells; n = 4, p<0.005.


2B ) Cortical neurons from 3 different isolations ( 10 - 20 DIV ) of WT and PINK1 KO mice were either non - starved or starved for 2 h in HBSS and the LC3 - II and actin content was determined by western blotting . A representative blot is shown on the right . The relative LC3 - II content of WT and PINK1 KO mice , respectively , was set as 1 . Primary neurons showed a strong upregulation of autophagy in reaction to starvation but cells derived from PINK1 KO mice exhibited a reduced LC3 - II / actin ratio compared to the WT cells ; n = 5 , p < 0 . 001 .

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

2B) Cortical neurons from 3 different isolations (10-20 DIV) of WT and PINK1 KO mice were either non-starved or starved for 2 h in HBSS and the LC3-II and actin content was determined by western blotting. A representative blot is shown on the right. The relative LC3-II content of WT and PINK1 KO mice, respectively, was set as 1. Primary neurons showed a strong upregulation of autophagy in reaction to starvation but cells derived from PINK1 KO mice exhibited a reduced LC3-II/actin ratio compared to the WT cells; n = 5, p<0.001.


2C ) HeLa cells were transiently transfected with scrambled siRNA or PINK1 siRNA . After 48 h the LC3 - II and actin content was determined by western blotting . A representative blot is shown on the right . The relative LC3 - II content of cells transfected with scrambled siRNA was set as 1 . Transient PINK1 knockdown resulted in a reduced LC3 - II / actin ratio compared to the cells transfected with scrambled siRNA ; n = 6 ; p < 0 . 005 .

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

2C) HeLa cells were transiently transfected with scrambled siRNA or PINK1 siRNA. After 48 h the LC3-II and actin content was determined by western blotting. A representative blot is shown on the right. The relative LC3-II content of cells transfected with scrambled siRNA was set as 1. Transient PINK1 knockdown resulted in a reduced LC3-II/actin ratio compared to the cells transfected with scrambled siRNA; n = 6; p<0.005.


2D ) HeLa cells were transiently transfected with PINK1 - GFP , PINK1G309D - GFP or GFP . After 24 h cells were starved for 2 h in HBSS . Afterwards the LC3 - II and actin content were determined by western blotting . A representative blot is shown on the right . The relative LC3 - II content of cells transfected with GFP was set as 1 . Transient PINK1 or PINK1G309D overexpression resulted in an increased LC3 - II / actin ratio compared to cells transfected with GFP ; n = 3 , PINK1 : p < 0 . 01 ; PINK1G309D : p < 0 . 05 .

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

2D) HeLa cells were transiently transfected with PINK1-GFP, PINK1G309D-GFP or GFP. After 24 h cells were starved for 2 h in HBSS. Afterwards the LC3-II and actin content were determined by western blotting. A representative blot is shown on the right. The relative LC3-II content of cells transfected with GFP was set as 1. Transient PINK1 or PINK1G309D overexpression resulted in an increased LC3-II/actin ratio compared to cells transfected with GFP; n = 3, PINK1: p<0.01; PINK1G309D: p<0.05.


3A ) Regular , non - transduced SH - SY5Y cells were cultivated either in RPMI + 10 % FCS or starved in HBSS medium for 24 and 40 h . The LAMP - 2 and actin content were determined by western blotting and the relative LAMP - 2 content of untreated cells was set as 1 . Starvation enhanced LAMP - 2protein levels ; n = 3 , p < 0 . 05 .

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

3A) Regular, non-transduced SH-SY5Y cells were cultivated either in RPMI+10% FCS or starved in HBSS medium for 24 and 40 h. The LAMP-2 and actin content were determined by western blotting and the relative LAMP-2 content of untreated cells was set as 1. Starvation enhanced LAMP-2protein levels; n = 3, p<0.05.


3B ) Transduced nt and PINK1 knockdown ( kd ) SH - SY5Y cells were cultivated either in RPMI + 5 % FCS or starved for 24 h and 40 h in HBSS . The LAMP - 2 and actin content were determined by western blotting . A representative blot after 40 h starvation in HBSS is shown on the right . The relative LAMP - 2 content of untreated cells was set as 1 . Cells with stable PINK1 knockdown exhibited after 40 h a reduced LAMP - 2 / actin ratio compared to the nt cells ; n = 4 , p < 0 . 05 .

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

3B) Transduced nt and PINK1 knockdown (kd) SH-SY5Y cells were cultivated either in RPMI+5% FCS or starved for 24 h and 40 h in HBSS. The LAMP-2 and actin content were determined by western blotting. A representative blot after 40 h starvation in HBSS is shown on the right. The relative LAMP-2 content of untreated cells was set as 1. Cells with stable PINK1 knockdown exhibited after 40 h a reduced LAMP-2/actin ratio compared to the nt cells; n = 4, p<0.05.


3C ) nt and PINK1 knockdown ( kd ) SH - SY5Y cells were starved for the indicated times with HBSS and afterwards stained for Lamp - 2 . Micrographs were taken with constant microscopical settings . LAMP - 2 positive signals in each cell were quantified with ImageJ as described in Material and Methods . Knockdown of PINK1 resulted in a decreased amount of LAMP - 2 positive stainings / cell after starvation ; n = 2 , at least 8 fields of view / condition , 89 - 124 cells / condition ; 24 h : p < 0 . 001 ; 40 h : p < 0 . 05 .

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

3C) nt and PINK1 knockdown (kd) SH-SY5Y cells were starved for the indicated times with HBSS and afterwards stained for Lamp-2. Micrographs were taken with constant microscopical settings. LAMP-2 positive signals in each cell were quantified with ImageJ as described in Material and Methods. Knockdown of PINK1 resulted in a decreased amount of LAMP-2 positive stainings/cell after starvation; n = 2, at least 8 fields of view/condition, 89-124 cells/condition; 24 h: p<0.001; 40 h: p<0.05.


3D ) nt and PINK1 knockdown ( kd ) SH - SY5Y cells were stained with the photo - reactive dye MTR and either irradiated for 45 min or left untreated . 24 h and 48 h after irradiation the LAMP - 2 and actin content were determined by western blotting . A representative blot 48 h after irradiation is shown on the right . The relative LAMP - 2 content of untreated cells was set as 1 . Cells with stable PINK1 knockdown exhibited after 48 h a reduced Lamp - 2 / actin ratio compared to the control ( nt ) cells ; n = 5 , p < 0 . 001 .

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

3D) nt and PINK1 knockdown (kd) SH-SY5Y cells were stained with the photo-reactive dye MTR and either irradiated for 45 min or left untreated. 24 h and 48 h after irradiation the LAMP-2 and actin content were determined by western blotting. A representative blot 48 h after irradiation is shown on the right. The relative LAMP-2 content of untreated cells was set as 1. Cells with stable PINK1 knockdown exhibited after 48 h a reduced Lamp-2/actin ratio compared to the control (nt) cells; n = 5, p<0.001.


3E ) Acid phosphatase activity as parameter for lysosomal activity was quantified in nt and PINK1 knockdown ( kd ) SH - SY5Y cells cultivated in RPMI medium with 5 % FCS . The phosphatase activity was measured and normalized to the protein content . The phosphatase activity of nt cells was set as 1 . PINK1 knockdown mediated a reduction of lysosomal activity ; n = 3 ; p < 0 . 05 .

3E) Acid phosphatase activity as parameter for lysosomal activity was quantified in nt and PINK1 knockdown (kd) SH-SY5Y cells cultivated in RPMI medium with 5% FCS. The phosphatase activity was measured and normalized to the protein content. The phosphatase activity of nt cells was set as 1. PINK1 knockdown mediated a reduction of lysosomal activity; n = 3; p<0.05.


4A ) Oxygen consumption rate ( OCR ) was measured in nt and PINK1 knockdown ( kd ) SH - SY5Y cells without ( nt ) or with PINK1 knockdown ( kd ) cultivated in RPMI medium with 5 % FCS . Although oxygen consumption appeared to be slightly impaired in cells with PINK1 kd , no significant changes were observed ; n = 1 ( quadruplicates ) .

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

4A) Oxygen consumption rate (OCR) was measured in nt and PINK1 knockdown (kd) SH-SY5Y cells without (nt) or with PINK1 knockdown (kd) cultivated in RPMI medium with 5% FCS. Although oxygen consumption appeared to be slightly impaired in cells with PINK1 kd, no significant changes were observed; n = 1 (quadruplicates).


4B ) Energy charge ( EC ) of nt and PINK1 knockdown ( kd ) SH - SY5Y cells grown in RPMI + 5 % FCS was determined as detailed in Material Methods . PINK1 knockdown resulted in a lower EC compared to nt cells ; n = 5 , p0 . 05 .

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4B) Energy charge (EC) of nt and PINK1 knockdown (kd) SH-SY5Y cells grown in RPMI+5% FCS was determined as detailed in Material Methods. PINK1 knockdown resulted in a lower EC compared to nt cells; n = 5, p0.05.


4C ) nt and PINK1 knockdown ( kd ) SH - SY5Y cells were grown for 2 months in RPMI + 5 % FCS and their population doublings were determined . Starting from day 12 cells with PINK1 kd exhibited impaired cell population growth ; n = 1 ( triplicates ) ; * p < 0 . 05 ( day 12 ) - p < 1 × 10 − 6 ( day 61 ) .

4C) nt and PINK1 knockdown (kd) SH-SY5Y cells were grown for 2 months in RPMI+5% FCS and their population doublings were determined. Starting from day 12 cells with PINK1 kd exhibited impaired cell population growth; n = 1 (triplicates); * p<0.05 (day 12) - p<1×10−6 (day 61).


4D ) SH - SY5Y cells were grown in RPMI + 5 % FCS and the amount of nt and PINK1 knockdown ( kd ) cells in S - phase was determined by BrdU incorporation . No differences in cell proliferation were visible between cells without ( nt ) and with PINK1 kd ; n = 3 .

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4D) SH-SY5Y cells were grown in RPMI+5% FCS and the amount of nt and PINK1 knockdown (kd) cells in S-phase was determined by BrdU incorporation. No differences in cell proliferation were visible between cells without (nt) and with PINK1 kd; n = 3.


5A ) nt and PINK1 knockdown ( kd ) SH - SY5Y cells were either kept in RPMI medium with 5 % FCS or starved with HBSS for 12 h . DEVD cleavage as parameter for caspase - 3 activity was analyzed and normalized to the protein content . Cells with PINK1 knockdown demonstrated a significantly increased caspase - 3 activity under both conditions ; n = 4 ; RPMI + 5 % FCS : p < 0 . 05 ; HBSS : p < 0 . 01 .

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5A) nt and PINK1 knockdown (kd) SH-SY5Y cells were either kept in RPMI medium with 5% FCS or starved with HBSS for 12 h. DEVD cleavage as parameter for caspase-3 activity was analyzed and normalized to the protein content. Cells with PINK1 knockdown demonstrated a significantly increased caspase-3 activity under both conditions; n = 4; RPMI+5% FCS: p<0.05; HBSS: p<0.01.


5B ) nt and PINK1 knockdown ( kd ) SH - SY5Y cells were either kept in RPMI medium with 5 % FCS for 48 h or starved with HBSS for the indicated time points . In the representative western blot the appearance of the PARP 89 kDa cleaved product is depicted as well as GAPDH for normalizing .

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5B) nt and PINK1 knockdown (kd) SH-SY5Y cells were either kept in RPMI medium with 5% FCS for 48 h or starved with HBSS for the indicated time points. In the representative western blot the appearance of the PARP 89 kDa cleaved product is depicted as well as GAPDH for normalizing.


5C ) nt and PINK1 knockdown ( kd ) SH - SY5Y cells were either kept in RPMI medium with 5 % FCS for 48 h or starved with HBSS for the indicated time points . For quantification the cleaved PARP 89 kDa product was normalized to the full length 113 kDa PARP and the resulting value normalized to GAPDH . Cells with PINK1 knockdown demonstrated increased PARP cleavage that was significant after 24 h starvation ; n = 4 ; 24 h : p < 0 . 005 .

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5C) nt and PINK1 knockdown (kd) SH-SY5Y cells were either kept in RPMI medium with 5% FCS for 48 h or starved with HBSS for the indicated time points. For quantification the cleaved PARP 89 kDa product was normalized to the full length 113 kDa PARP and the resulting value normalized to GAPDH. Cells with PINK1 knockdown demonstrated increased PARP cleavage that was significant after 24 h starvation; n = 4; 24 h: p<0.005.


5D ) HeLa cells were transfected with scrambled siRNA or PINK1 siRNA and GFP or GFP - Parkin or PINK1 - GFP or PINK1G309D - GFP . After transfection cells were starved for additional 24 h with HBSS and afterwards the amount of adherent cells was determined . The number of adherent cells transfected with scrambled siRNA and the indicated plasmid was set as 1 . Starvation - mediated increased cell loss after PINK1 knockdown could be rescued by PINK1 or PINK1G309D - GFP but not by GFP or Parkin ; GFP : n = 5 , p < 0 . 005 ; Parkin : n = 3 , p < 0 . 05 ; PINK1 : n = 5 , ns ; PINK1G309D - GFP : n = 3 , ns .

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5D) HeLa cells were transfected with scrambled siRNA or PINK1 siRNA and GFP or GFP-Parkin or PINK1-GFP or PINK1G309D-GFP. After transfection cells were starved for additional 24 h with HBSS and afterwards the amount of adherent cells was determined. The number of adherent cells transfected with scrambled siRNA and the indicated plasmid was set as 1. Starvation-mediated increased cell loss after PINK1 knockdown could be rescued by PINK1 or PINK1G309D-GFP but not by GFP or Parkin; GFP: n = 5, p<0.005; Parkin: n = 3, p<0.05; PINK1: n = 5, ns; PINK1G309D-GFP: n = 3, ns.


5E ) HeLa cells and HeLa cells stably overexpressing LC3 were transfected with scrambled siRNA or PINK1 siRNA . After 48 h cells were starved in HBSS for additional 24 h and afterwards DEVD cleavage as parameter for caspase - 3 activity was analyzed and normalized to 1 , 000 , 000 cells . PINK1 knockdown increased caspase - 3 activity significantly , which was prevented by LC3 overexpression ; n = 3 ; p < 0 . 05 .

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5E) HeLa cells and HeLa cells stably overexpressing LC3 were transfected with scrambled siRNA or PINK1 siRNA. After 48 h cells were starved in HBSS for additional 24 h and afterwards DEVD cleavage as parameter for caspase-3 activity was analyzed and normalized to 1,000,000 cells. PINK1 knockdown increased caspase-3 activity significantly, which was prevented by LC3 overexpression; n = 3; p<0.05.


5F ) HeLa cells and HeLa cells stably expressing LC3 were transfected with scrambled or PINK1 siRNA . 48 h post transfection cells were either left untreated ( + medium ) or starved ( + HBSS ) for additional 24 h and afterwards the amount of adherent cells was determined . The amount of non - starved cells was set as 1 . PINK1 knockdown resulted in elevated cell loss , which was prevented by LC3 overexpression ; HeLa : n = 6 , p < 0 . 05 ; HeLa LC3 : n = 5 .

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5F) HeLa cells and HeLa cells stably expressing LC3 were transfected with scrambled or PINK1 siRNA. 48 h post transfection cells were either left untreated (+ medium) or starved (+HBSS) for additional 24 h and afterwards the amount of adherent cells was determined. The amount of non-starved cells was set as 1. PINK1 knockdown resulted in elevated cell loss, which was prevented by LC3 overexpression; HeLa: n = 6, p<0.05; HeLa LC3: n = 5.


A . Confocal images of mitochondrial morphology in wild - type ( WT ) and Drp1 - / - 293T cells transfected with empty vector ( left panel ) and Myc - hFis1 ( right panel ) , stained with MitoTracker ( red ) followed by immunostaining with anti - Myc antibody ( green ) .

A. Confocal images of mitochondrial morphology in wild-type (WT) and Drp1-/- 293T cells transfected with empty vector (left panel) and Myc-hFis1 (right panel), stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green).


B . Quantitative analyses of fragmented mitochondria size ( mean area ( μm2 ) per mitochondrion ) after Myc - hFis1 overexpression in WT and Drp1 - / - 293T cells using Image J software ( Particle analysis ) in three independent experiments . In each cell , only dispersed individual mitochondria were analyzed . The total number of mitochondria ( mito ) analyzed is indicated for each condition .

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B. Quantitative analyses of fragmented mitochondria size (mean area (μm2) per mitochondrion) after Myc-hFis1 overexpression in WT and Drp1-/- 293T cells using Image J software (Particle analysis) in three independent experiments. In each cell, only dispersed individual mitochondria were analyzed. The total number of mitochondria (mito) analyzed is indicated for each condition.


C . Quantitative analysis of mean mitochondria number per cell in WT and Drp1 - / - 293T cells overexpressing Myc - hFis1 using Image J software ( Particle analysis ) in three independent experiments .

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C. Quantitative analysis of mean mitochondria number per cell in WT and Drp1-/- 293T cells overexpressing Myc-hFis1 using Image J software (Particle analysis) in three independent experiments.


E . Percentages of cells with indicated mitochondrial morphologies in WT and Drp1 - / - 293T cells transfected with empty vector ( control ) , Myc - hFis1 and either Myc - or GFP - tagged mutants as indicated in three independent experiments .

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E. Percentages of cells with indicated mitochondrial morphologies in WT and Drp1-/- 293T cells transfected with empty vector (control), Myc-hFis1 and either Myc- or GFP-tagged mutants as indicated in three independent experiments.


A . Confocal images of mitochondrial morphology in WT 293T cells treated with scrambled siRNA ( control ) and Dyn2 siRNA as indicated , followed by transfection with empty vector ( left panel ) and Myc - hFis1 ( right three panels ) as indicated . Cells were stained with MitoTracker ( red ) followed by immunostaining with anti - Myc antibody ( green ) .

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A. Confocal images of mitochondrial morphology in WT 293T cells treated with scrambled siRNA (control) and Dyn2 siRNA as indicated, followed by transfection with empty vector (left panel) and Myc-hFis1 (right three panels) as indicated. Cells were stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green).


B . Western blot of Dyn2 , Myc - hFis1 , Drp1 and GAPDH in WT 293T cells collected from the experiments in ( A ) .

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B. Western blot of Dyn2, Myc-hFis1, Drp1 and GAPDH in WT 293T cells collected from the experiments in (A).


C . Percentages of cells with indicated mitochondrial morphologies in WT 293T cells treated with scrambled siRNA and Dyn2 siRNA as indicated , followed by transfection with empty vector or Myc - hFis1 in three independent experiments .

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C. Percentages of cells with indicated mitochondrial morphologies in WT 293T cells treated with scrambled siRNA and Dyn2 siRNA as indicated, followed by transfection with empty vector or Myc-hFis1 in three independent experiments.


D . Confocal images of mitochondrial morphology in Drp1 - / - 293T cells treated with scrambled siRNA and Dyn2 siRNA as indicated , followed by transfection with empty vector ( left panel ) and Myc - hFis1 ( right three panels ) . Cells were stained with MitoTracker ( red ) followed by immunostaining with anti - Myc antibody ( green ) . Representative examples of tubular , fragmented and tubular cluster phenotypes are indicated .

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D. Confocal images of mitochondrial morphology in Drp1-/- 293T cells treated with scrambled siRNA and Dyn2 siRNA as indicated, followed by transfection with empty vector (left panel) and Myc-hFis1 (right three panels). Cells were stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green). Representative examples of tubular, fragmented and tubular cluster phenotypes are indicated.


E . Western blot of Dyn2 , Myc - hFis1 and GAPDH in Drp1 - / - 293T cells collected from the experiments in ( D ) .

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E. Western blot of Dyn2, Myc-hFis1 and GAPDH in Drp1-/- 293T cells collected from the experiments in (D).


F . Percentages of cells with indicated mitochondrial morphologies in Drp1 - / - 293T cells treated with scrambled siRNA or Dyn2 siRNA , followed by transfection with empty vector or Myc - hFis1 as indicated in three independent experiments .

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F. Percentages of cells with indicated mitochondrial morphologies in Drp1-/- 293T cells treated with scrambled siRNA or Dyn2 siRNA, followed by transfection with empty vector or Myc-hFis1 as indicated in three independent experiments.


A . Confocal images of mitochondrial morphology in Drp1 - / - 293T cells transfected with scrambled siRNA ( control ) or with hFis1 - siRNA and then stained with MitoTracker ( red ) .

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A. Confocal images of mitochondrial morphology in Drp1-/- 293T cells transfected with scrambled siRNA (control) or with hFis1-siRNA and then stained with MitoTracker (red).


B . Confocal images of mitochondrial morphology in Drp1 - / - 293T cells transfected with either Mfn1 - Myc , Mfn2 - Myc or OPA1 - Myc , stained with MitoTracker ( red ) followed by immunostaining with anti - Myc antibody ( green ) .

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B. Confocal images of mitochondrial morphology in Drp1-/- 293T cells transfected with either Mfn1-Myc, Mfn2-Myc or OPA1-Myc, stained with MitoTracker (red) followed by immunostaining with anti-Myc antibody (green).


C . Percentages ( mean ± s . e . m . ) of cells with indicated mitochondrial morphologies in Drp1 - / - 293T cells transfected with either scrambled siRNA ( Ctr ) , hFis1 siRNA , Mfn1 - Myc , Mfn2 - Myc or OPA1 - Myc in three independent experiments for each condition ( n represents the number of cells analyzed ) .

C. Percentages (mean ± s.e.m.) of cells with indicated mitochondrial morphologies in Drp1-/- 293T cells transfected with either scrambled siRNA (Ctr), hFis1 siRNA, Mfn1-Myc, Mfn2-Myc or OPA1-Myc in three independent experiments for each condition (n represents the number of cells analyzed).


D . hFis1 interacts with Mfn1 , Mfn2 and OPA1 as well as Drp1 , but not with Dyn2 at endogenous levels following chemical crosslinking . Wild - type ( WT ) and Drp1 - / - 293T cells were in vivo crosslinked with 1 % formaldehyde ( FA ) and cell lysates were used for co - immunoprecipitation ( IP ) with Protein G beads bound to rabbit normal IgG ( negative control ) or rabbit anti - hFis1 antibody as indicated , followed by immunoblotting with indicated antibodies .

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D. hFis1 interacts with Mfn1, Mfn2 and OPA1 as well as Drp1, but not with Dyn2 at endogenous levels following chemical crosslinking. Wild-type (WT) and Drp1-/- 293T cells were in vivo crosslinked with 1% formaldehyde (FA) and cell lysates were used for co-immunoprecipitation (IP) with Protein G beads bound to rabbit normal IgG (negative control) or rabbit anti-hFis1 antibody as indicated, followed by immunoblotting with indicated antibodies.


hFis1 binds to Mfn1 , Mfn2 and OPA1 at endogenous levels also in the absence of chemical crosslinking . Cell lysates prepared from WT 293T ( E ) cells without chemical crosslinking were used for co - immunoprecipitation ( IP ) with Protein G beads bound to rabbit normal IgG ( negative control ) or rabbit anti - hFis1 antibody as indicated , followed by Western blotting with indicated antibodies .

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hFis1 binds to Mfn1, Mfn2 and OPA1 at endogenous levels also in the absence of chemical crosslinking. Cell lysates prepared from WT 293T (E) cells without chemical crosslinking were used for co-immunoprecipitation (IP) with Protein G beads bound to rabbit normal IgG (negative control) or rabbit anti-hFis1 antibody as indicated, followed by Western blotting with indicated antibodies.


F . hFis1 binds to Mfn1 , Mfn2 and OPA1 at endogenous levels also in the absence of chemical crosslinking . Cell lysates prepared from HeLa ( F ) cells without chemical crosslinking were used for co - immunoprecipitation ( IP ) with Protein G beads bound to rabbit normal IgG ( negative control ) or rabbit anti - hFis1 antibody as indicated , followed by Western blotting with indicated antibodies .

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F. hFis1 binds to Mfn1, Mfn2 and OPA1 at endogenous levels also in the absence of chemical crosslinking. Cell lysates prepared from HeLa (F) cells without chemical crosslinking were used for co-immunoprecipitation (IP) with Protein G beads bound to rabbit normal IgG (negative control) or rabbit anti-hFis1 antibody as indicated, followed by Western blotting with indicated antibodies.


G , H . Interaction of hFis1 with Mfn1 / 2 and with OPA1 are independent events . WT 293T cells were treated with control , OPA1 ( G ) or Mfn1 plus Mfn2 ( H ) siRNA , followed by in vivo crosslinking with 1 % FA . Cell lysates were used for co - IP with Protein G beads bound to rabbit normal IgG ( negative control ) or rabbit anti - hFis1 antibody as indicated , followed by immunoblotting with indicated antibodies .

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G, H. Interaction of hFis1 with Mfn1/2 and with OPA1 are independent events. WT 293T cells were treated with control, OPA1 (G) or Mfn1 plus Mfn2 (H) siRNA, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with Protein G beads bound to rabbit normal IgG (negative control) or rabbit anti-hFis1 antibody as indicated, followed by immunoblotting with indicated antibodies.


I . Interactions between Mfn1 / 2 and OPA1 occur independent of hFis1 . WT 293T cells were treated with control or hFis1 siRNA , followed by in vivo crosslinking with 1 % FA . Cell lysates were used for co - IP with Protein G beads bound to mouse normal IgG ( negative control ) or mouse anti - OPA1 antibody as indicated , followed by immunoblotting with indicated antibodies .

I. Interactions between Mfn1/2 and OPA1 occur independent of hFis1. WT 293T cells were treated with control or hFis1 siRNA, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with Protein G beads bound to mouse normal IgG (negative control) or mouse anti-OPA1 antibody as indicated, followed by immunoblotting with indicated antibodies.


J . Interaction between Mfn1 and Mfn2 is not affected by hFis1 overexpression . 293T cells were transfected with empty vector or Myc - hFis1 , followed by in vivo crosslinking with 1 % FA . Cell lysates were used for co - IP with Protein G beads bound to mouse normal IgG or mouse anti - Mfn1 antibody , followed by immunoblotting with indicated antibodies .

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J. Interaction between Mfn1 and Mfn2 is not affected by hFis1 overexpression. 293T cells were transfected with empty vector or Myc-hFis1, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with Protein G beads bound to mouse normal IgG or mouse anti-Mfn1 antibody, followed by immunoblotting with indicated antibodies.


B . Interaction of different hFis1 mutants with Mfns and OPA1 . hFis1 - / - 293T cells were transfected with the full - length and the truncated hFis1 mutants as indicated , followed by in vivo crosslinking with 1 % FA . Cell lysates were used for co - IP with anti - Myc or anti - GFP agarose beads and the immunoprecipitates were analyzed by Western blotting with indicated antibodies .

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B. Interaction of different hFis1 mutants with Mfns and OPA1. hFis1-/- 293T cells were transfected with the full-length and the truncated hFis1 mutants as indicated, followed by in vivo crosslinking with 1% FA. Cell lysates were used for co-IP with anti-Myc or anti-GFP agarose beads and the immunoprecipitates were analyzed by Western blotting with indicated antibodies.


A , B . Representative confocal images of mitochondrial fusion in WT 293T polykaryons in cultures subjected to the PEG - based fusion assay in the presence or absence of exogenous hFis1 . WT 293T cells stably expressing mitoGFP or mitoRFP were co - cultured , subsequently transfected with empty vector ( control ) or with Myc - hFis1 as indicated and then treated with PEG for cell fusion . Mitochondrial fusion is indicated by co - localization of mitoGFP and mitoRFP ( i . e . yellow mitochondria ) . Left panel : empty vector transfected ; right panel : Myc - hFis1transfected . Insets represent magnification of the boxed areas in upper panel ( A ) . Quantitative analysis of the extent of mitochondrial fusion in individual hybrid cells was performed by the Pearson ' s correlation coefficient ( PCC ) in three independent experiments for each condition and data summarized in ( B ) .

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A, B. Representative confocal images of mitochondrial fusion in WT 293T polykaryons in cultures subjected to the PEG-based fusion assay in the presence or absence of exogenous hFis1. WT 293T cells stably expressing mitoGFP or mitoRFP were co-cultured, subsequently transfected with empty vector (control) or with Myc-hFis1 as indicated and then treated with PEG for cell fusion. Mitochondrial fusion is indicated by co-localization of mitoGFP and mitoRFP (i.e. yellow mitochondria). Left panel: empty vector transfected; right panel: Myc-hFis1transfected. Insets represent magnification of the boxed areas in upper panel (A). Quantitative analysis of the extent of mitochondrial fusion in individual hybrid cells was performed by the Pearson's correlation coefficient (PCC) in three independent experiments for each condition and data summarized in (B).


C , D . Representative confocal images of mitochondrial fusion in WT 293T cells assessed by the mito - PAGFP - based fusion assay in the presence or absence of exogenous hFis1 . WT 293T cells co - transfected with mito - PAGFP ( 0 . 5 µg ) , mito - DsRed ( 0 . 2 µg ) , and either empty vector ( 0 . 5 µg , the upper panel ) or hFis1 ( 0 . 5 µg , the lower panel ) were photoactivated in a small region of interest ( ROI ) ( white circle , 3 µm diameter ) in preactivation images of mitochondria seen by mito - DsRed ( red ) . After photoactivation , sequential Z - stack images with photoactivated GFP ( green ) and mitochondrial marker ( mito - DsRed ) were collected at indicated time points using series of Z - sections from the top to the cell bottom with intervals between sections set to 0 . 5 - 0 . 75 μm ( C ) . Mitochondrial fusion was quantified by analyzing changes in fluorescence intensity of photoactivated mito - PAGFP in ROIs at 40 sec , 15 , 30 , 45 min after photoactivation . The dilution rates ( percentage ) of the GFP fluorescence intensity at different time points were normalized by the fluorescence intensity at 40 sec after photoactivation ( D ) .

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C, D. Representative confocal images of mitochondrial fusion in WT 293T cells assessed by the mito-PAGFP-based fusion assay in the presence or absence of exogenous hFis1. WT 293T cells co-transfected with mito-PAGFP (0.5 µg), mito-DsRed (0.2 µg), and either empty vector (0.5 µg, the upper panel) or hFis1 (0.5 µg, the lower panel) were photoactivated in a small region of interest (ROI) (white circle, 3 µm diameter) in preactivation images of mitochondria seen by mito-DsRed (red). After photoactivation, sequential Z-stack images with photoactivated GFP (green) and mitochondrial marker (mito-DsRed) were collected at indicated time points using series of Z-sections from the top to the cell bottom with intervals between sections set to 0.5-0.75 μm (C). Mitochondrial fusion was quantified by analyzing changes in fluorescence intensity of photoactivated mito-PAGFP in ROIs at 40 sec, 15, 30, 45 min after photoactivation. The dilution rates (percentage) of the GFP fluorescence intensity at different time points were normalized by the fluorescence intensity at 40 sec after photoactivation (D).


E , F . Representative confocal images of mitochondrial fusion in WT 293T polykaryons in cultures subjected to the PEG - based fusion assay in the presence or absence of endogenous hFis1 . Both WT 293T cells stably expressing mitoGFP or mitoRFP were transfected with scrambled siRNA ( control siRNA ) or hFis1 - siRNA as indicated and then co - cultured and fused using PEG treatment . Left panel : control siRNA transfected ; right panel : hFis1 - siRNA transfected ( E ) . Quantitative analysis of the mitochondrial fusion in individual hybrid cells was performed by the Pearson ' s correlation coefficient in three independent experiments and data summarized in ( F ) .

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E, F. Representative confocal images of mitochondrial fusion in WT 293T polykaryons in cultures subjected to the PEG-based fusion assay in the presence or absence of endogenous hFis1. Both WT 293T cells stably expressing mitoGFP or mitoRFP were transfected with scrambled siRNA (control siRNA) or hFis1-siRNA as indicated and then co-cultured and fused using PEG treatment. Left panel: control siRNA transfected; right panel: hFis1-siRNA transfected (E). Quantitative analysis of the mitochondrial fusion in individual hybrid cells was performed by the Pearson's correlation coefficient in three independent experiments and data summarized in (F).


G , H . Representative confocal images of mitochondrial fusion in WT 293T cells assessed by the mito - PAGFP - based fusion assay in the presence or absence of endogenous hFis1 . Scrambled siRNA or hFis1 siRNA treated cells were co - transfected with mito - PAGFP and mito - DsRed and were subsequently photoactivated ( G ) as described in ( C ) . Mitochondrial fusion was quantified ( H ) as described in ( D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative confocal images of mitochondrial fusion in WT 293T cells assessed by the mito-PAGFP-based fusion assay in the presence or absence of endogenous hFis1. Scrambled siRNA or hFis1 siRNA treated cells were co-transfected with mito-PAGFP and mito-DsRed and were subsequently photoactivated (G) as described in (C). Mitochondrial fusion was quantified (H) as described in (D).


A , B . Representative confocal images of mitochondrial fusion in Drp1 - / - 293T polykaryons in cultures subjected to the PEG - based fusion assay in the presence or absence of exogenous hFis1 . Drp1 - / - cells stably expressing mitoGFP or mitoRFP were co - cultured , subsequently transfected with empty vector ( control ) or Myc - hFis1 as indicated and then stimulated with PEG treatment for cell fusion , followed by immunostaining with anti - Myc antibody ( green ) . Mitochondrial fusion was indicated by co - localization of mitoGFP and mitoRFP ( i . e . yellow mitochondria ) . Left panel : empty vector transfected ; right panel : Myc - hFis1 transfected . Insets represent magnification of the boxed areas in upper panel ( A ) . Quantitative analyses for measuring the extent of mitochondrial fusion in individual hybrid cells were performed by the Pearson ' s correlation coefficient ( PCC ) in three independent experiments and summarized in ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 confocal images of mitochondrial fusion in Drp1-/- 293T polykaryons in cultures subjected to the PEG-based fusion assay in the presence or absence of exogenous hFis1. Drp1-/- cells stably expressing mitoGFP or mitoRFP were co-cultured, subsequently transfected with empty vector (control) or Myc-hFis1 as indicated and then stimulated with PEG treatment for cell fusion, followed by immunostaining with anti-Myc antibody (green). Mitochondrial fusion was indicated by co-localization of mitoGFP and mitoRFP (i.e. yellow mitochondria). Left panel: empty vector transfected; right panel: Myc-hFis1 transfected. Insets represent magnification of the boxed areas in upper panel (A). Quantitative analyses for measuring the extent of mitochondrial fusion in individual hybrid cells were performed by the Pearson's correlation coefficient (PCC) in three independent experiments and summarized in (B).


C , D . Representative confocal images of mitochondrial fusion in Drp1 - / - 293T cells assessed by the mito - PAGFP - based fusion assay in the presence or absence of exogenous hFis1 . Drp1 - / - cells co - transfected with mito - PAGFP , mito - DsRed , and either empty vector ( the upper panel ) or hFis1 ( the lower panel ) were photoactivated in a small region of interest ( ROI ) ( white circle , 3 µm diameter ) as indicated in preactivation images of mitochondria seen by mito - DsRed ( red ) . After photoactivation , Z - stack images with photoactivated GFP ( green ) and mitochondrial marker ( mito - DsRed ) were collected at times 40 sec , 15 , 30 , 45 min as indicated ( C ) . Mitochondrial fusion was quantified by analysis of changes in the fluorescence intensity of photoactivated mito - PAGFP in ROIs at 40 sec , 15 , 30 , 45 min after photoactivation . The dilution rates ( percentage ) of the GFP fluorescence intensity at different time points were normalized by the fluorescence intensity at 40 sec after photoactivation ( D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 confocal images of mitochondrial fusion in Drp1-/- 293T cells assessed by the mito-PAGFP-based fusion assay in the presence or absence of exogenous hFis1. Drp1-/- cells co-transfected with mito-PAGFP, mito-DsRed, and either empty vector (the upper panel) or hFis1 (the lower panel) were photoactivated in a small region of interest (ROI) (white circle, 3 µm diameter) as indicated in preactivation images of mitochondria seen by mito-DsRed (red). After photoactivation, Z-stack images with photoactivated GFP (green) and mitochondrial marker (mito-DsRed) were collected at times 40 sec, 15, 30, 45 min as indicated (C). Mitochondrial fusion was quantified by analysis of changes in the fluorescence intensity of photoactivated mito-PAGFP in ROIs at 40 sec, 15, 30, 45 min after photoactivation. The dilution rates (percentage) of the GFP fluorescence intensity at different time points were normalized by the fluorescence intensity at 40 sec after photoactivation (D).


E , F . Representative confocal images of mitochondrial fusion in Drp1 - / - 293T polykaryons in cultures subjected to the PEG - based fusion assay in the presence or absence of endogenous hFis1 . Drp1 - / - cells stably expressing mitoGFP or mitoRFP were transfected with scrambled siRNA ( control siRNA ) or with hFis1 - siRNA as indicated and then co - cultured and fused by PEG treatment . Left panel : control siRNA transfected ; right panel : hFis1 - siRNA transfected ( E ) . Quantitative analysis of extent of mitochondrial fusion in individual hybrid cells was performed by the Pearson ' s correlation coefficient ( PCC ) in three independent experiments and summarized in ( 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 O O O

E, F. Representative confocal images of mitochondrial fusion in Drp1-/- 293T polykaryons in cultures subjected to the PEG-based fusion assay in the presence or absence of endogenous hFis1. Drp1-/- cells stably expressing mitoGFP or mitoRFP were transfected with scrambled siRNA (control siRNA) or with hFis1-siRNA as indicated and then co-cultured and fused by PEG treatment. Left panel: control siRNA transfected; right panel: hFis1-siRNA transfected (E). Quantitative analysis of extent of mitochondrial fusion in individual hybrid cells was performed by the Pearson's correlation coefficient (PCC) in three independent experiments and summarized in (F).


G , H . Representative confocal images of mitochondrial fusion in Drp1 - / - 293T cells monitored by the mito - PAGFP - based fusion assay in the presence or absence of endogenous hFis1 . Scrambled siRNA or hFis1 siRNA treated cells were co - transfected with mito - PAGFP and mito - DsRed and were subsequently photoactivated ( G ) as described in ( C ) . Mitochondrial fusion was quantified by analysis of changes in the fluorescence intensity of photoactivated mito - PAGFP ( H ) as described in ( D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative confocal images of mitochondrial fusion in Drp1-/- 293T cells monitored by the mito-PAGFP-based fusion assay in the presence or absence of endogenous hFis1. Scrambled siRNA or hFis1 siRNA treated cells were co-transfected with mito-PAGFP and mito-DsRed and were subsequently photoactivated (G) as described in (C). Mitochondrial fusion was quantified by analysis of changes in the fluorescence intensity of photoactivated mito-PAGFP (H) as described in (D).


A , B . GTP hydrolysis activities of Mfn1 , Mfn2 , Mfn2K109A and OPA1 in the presence or absence of recombinant GST - hFis1 or GST ( negative control ) were determined by measuring the concentration of free phosphate ( Pi ) released from GTP . Myc - tagged Mfn1 , Mfn2 , Mfn2K109A and OPA1 were transiently expressed separately in 293T cells and immunopurified by anti - Myc agarose beads . GTPase activities of immunopurified Myc - tagged Mfn1 , Mfn2 , Mfn2K109A and OPA1 were determined after treatment with or without recombinant GST - hFis1 or GST protein ( A ) . The input levels of immunopurified Mfn1 - Myc , Mfn2 - Myc and OPA1 - Myc ( left panel ) as well as recombinant GST - hFis1 ( ~ 42 kDa ) and GST ( ~ 26 kDa ) ( right panel ) were assessed by immunoblotting with indicated antibodies ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. GTP hydrolysis activities of Mfn1, Mfn2, Mfn2K109A and OPA1 in the presence or absence of recombinant GST-hFis1 or GST (negative control) were determined by measuring the concentration of free phosphate (Pi) released from GTP. Myc-tagged Mfn1, Mfn2, Mfn2K109A and OPA1 were transiently expressed separately in 293T cells and immunopurified by anti-Myc agarose beads. GTPase activities of immunopurified Myc-tagged Mfn1, Mfn2, Mfn2K109A and OPA1 were determined after treatment with or without recombinant GST-hFis1 or GST protein (A). The input levels of immunopurified Mfn1-Myc, Mfn2-Myc and OPA1-Myc (left panel) as well as recombinant GST-hFis1 (~42 kDa) and GST (~26 kDa) (right panel) were assessed by immunoblotting with indicated antibodies (B).


C , D . GTP hydrolysis activities of Drp1 in the presence or absence of recombinant GST - hFis1 or GST ( negative control ) were determined as described in ( A ) . Untagged WT Drp1 and Drp1Q34A were transiently expressed in Drp1 - / - 293T cells and immunopurified by protein G beads pre - incubated with Drp1 antibody . GTPase activities of immunopurified Drp1 and Drp1Q34A were determined after treatment with or without recombinant hFis1 or GST protein ( C ) . The input levels of immunopurified Drp1 and Drp1Q34A were assessed by immunoblotting with anti - Drp1 antibody ( D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. GTP hydrolysis activities of Drp1 in the presence or absence of recombinant GST-hFis1 or GST (negative control) were determined as described in (A). Untagged WT Drp1 and Drp1Q34A were transiently expressed in Drp1-/- 293T cells and immunopurified by protein G beads pre-incubated with Drp1 antibody. GTPase activities of immunopurified Drp1 and Drp1Q34A were determined after treatment with or without recombinant hFis1 or GST protein (C). The input levels of immunopurified Drp1 and Drp1Q34A were assessed by immunoblotting with anti-Drp1 antibody (D).


E , F . GTP hydrolysis activities of Dyn2 in the presence or absence of recombinant GST - hFis1 or GST ( negative control ) were determined as described in ( A ) . Endogenous Dyn2 in Drp1 - / - 293T cells was immunopurified by protein G beads pre - incubated with anti - Dyn2 antibody . GTPase activity of immunopurified Dyn2 was determined after treatment with or without recombinant GST - hFis1 or GST protein ( E ) . The input levels of immunopurified Dyn2 were assessed by immunoblotting with anti - Dyn2 antibody ( 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

E, F. GTP hydrolysis activities of Dyn2 in the presence or absence of recombinant GST-hFis1 or GST (negative control) were determined as described in (A). Endogenous Dyn2 in Drp1-/- 293T cells was immunopurified by protein G beads pre-incubated with anti-Dyn2 antibody. GTPase activity of immunopurified Dyn2 was determined after treatment with or without recombinant GST-hFis1 or GST protein (E). The input levels of immunopurified Dyn2 were assessed by immunoblotting with anti-Dyn2 antibody (F).


G . Summary of the effects of pro - fusion GTPases ( Mfns and OPA1 ) on hFis1 - induced mitochondrial fragmentation in Drp1 - / - cells . Percentages ( mean ± s . e . m . ) of cells with indicated mitochondrial morphologies in Drp1 - / - 293T cells co - expressing Myc - hFis1 and either empty vector , Mfn1 - Myc , Mfn2 - Myc or OPA1 - Myc in three independent experiments . n represents the number of cells analyzed . Corresponding confocal images shown in Fig EV4 .

G. Summary of the effects of pro-fusion GTPases (Mfns and OPA1) on hFis1-induced mitochondrial fragmentation in Drp1-/- cells. Percentages (mean ± s.e.m.) of cells with indicated mitochondrial morphologies in Drp1-/- 293T cells co-expressing Myc-hFis1 and either empty vector, Mfn1-Myc, Mfn2-Myc or OPA1-Myc in three independent experiments. n represents the number of cells analyzed. Corresponding confocal images shown in Fig EV4.


A . Ablation of OPA1 using CRISPR - Cas9 gene editing in Drp1 - / - 293T cells was confirmed by Western blotting analysis .

O 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. Ablation of OPA1 using CRISPR-Cas9 gene editing in Drp1-/- 293T cells was confirmed by Western blotting analysis.


B . Confocal images of mitochondrial morphology in Drp1 - / - ( left panel ) and Drp1 / OPA1DKO 293T ( right panel ) cells . ( Aggr / frag : aggregation / fragmentation of mitochondria ; Bulb / tubular : bulb - like / tubular mitochondria ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Confocal images of mitochondrial morphology in Drp1-/- (left panel) and Drp1/OPA1DKO 293T (right panel) cells. (Aggr/frag: aggregation/fragmentation of mitochondria; Bulb/tubular: bulb-like/tubular mitochondria).


C . Confocal images of mitochondrial morphology in Drp1 / OPA1DKO 293T cells treated with the indicated siRNAs , combinations of siRNAs or with Myc - hFis1 plasmid ( lower three panels ) compared to mitochondrial morphology in Drp1 - / - 293T cells overexpressing Myc - hFis1 or not ( upper panel ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Confocal images of mitochondrial morphology in Drp1/OPA1DKO 293T cells treated with the indicated siRNAs, combinations of siRNAs or with Myc-hFis1 plasmid (lower three panels) compared to mitochondrial morphology in Drp1-/- 293T cells overexpressing Myc-hFis1 or not (upper panel).


D . Percentages ( mean ± s . e . m . ) of cells with indicated mitochondrial morphologies in Drp1 - / - 293T cells transfected with empty vector or Myc - hFis1 plasmid , and in Drp1 / OPA1DKO 293T cells treated with the indicated siRNA , combinations of siRNA or Myc - hFis1 plasmid . The data were collected from three independent experiments . Total cell numbers ( n ) used for statistical analysis are indicated for each 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 O O 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. Percentages (mean ± s.e.m.) of cells with indicated mitochondrial morphologies in Drp1-/- 293T cells transfected with empty vector or Myc-hFis1 plasmid, and in Drp1/OPA1DKO 293T cells treated with the indicated siRNA, combinations of siRNA or Myc-hFis1 plasmid. The data were collected from three independent experiments. Total cell numbers (n) used for statistical analysis are indicated for each condition.


E . Knockdown of the indicated proteins by siRNA in Drp1 / OPA1DKO 293T cells was confirmed by Western blotting analysis .

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

E. Knockdown of the indicated proteins by siRNA in Drp1/OPA1DKO 293T cells was confirmed by Western blotting analysis.


Schematic diagram of ATXN8OS ( top strand ) and ATXN8 ( bottom strand ) . RNA - seq read coverage visualized by Integrative Genomic Viewer across the ATXN8 / ATXN8OS locus is depicted .

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Schematic diagram of ATXN8OS (top strand) and ATXN8 (bottom strand). RNA-seq read coverage visualized by Integrative Genomic Viewer across the ATXN8/ATXN8OS locus is depicted.


B Bar graph showing relative mRNA levels of ATXN8 and ATXN8OS . FPKM values for each transcript are normalized to FPKM value for ATXN8OS . ( * * * * ; p < 0 . 0001 ; mean ± SEM ; unpaired t test ) FPKM = fragments per kilo base of transcript per million mapped reads .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Bar graph showing relative mRNA levels of ATXN8 and ATXN8OS. FPKM values for each transcript are normalized to FPKM value for ATXN8OS. (****; p<0.0001; mean ± SEM; unpaired t test) FPKM= fragments per kilo base of transcript per million mapped reads.


D Immunohistochemistry of 10 - 12 month old end - stage SCA8 BAC mice brain tissue shows accumulation of novel polySer RAN protein ( detected by unique antibody to the polySer protein C - terminus , α - SerCT ) in cerebellar white matter , brainstem , hippocampus and layer II of the frontal cortex . Representative polySer aggregates are indicated by black arrows . No aggregates are found in age matched non - transgenic ( NT ) littermates ( n = 6 for each cohort ) .

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

D Immunohistochemistry of 10-12 month old end-stage SCA8 BAC mice brain tissue shows accumulation of novel polySer RAN protein (detected by unique antibody to the polySer protein C-terminus, α-SerCT) in cerebellar white matter, brainstem, hippocampus and layer II of the frontal cortex. Representative polySer aggregates are indicated by black arrows. No aggregates are found in age matched non-transgenic (NT) littermates (n=6 for each cohort).


F Immunohistochemistry of human SCA8 autopsy tissue ( n = 4 - 7 ) shows accumulation of novel polySer RAN protein in cerebellar white matter , brainstem and frontal cortex .

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

F Immunohistochemistry of human SCA8 autopsy tissue (n=4-7) shows accumulation of novel polySer RAN protein in cerebellar white matter, brainstem and frontal cortex.


A Immunohistochemistry ( IHC ) of end - stage SCA8 BAC mouse cerebellum shows that polySer but not polyGln is found in the molecular layer and cerebellar white matter and that polyGln but not polySer accumulates in Purkinje cells . Representative polySer aggregates are indicated by black arrows . Inset : higher magnification of molecular layer and white matter .

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

A Immunohistochemistry (IHC) of end-stage SCA8 BAC mouse cerebellum shows that polySer but not polyGln is found in the molecular layer and cerebellar white matter and that polyGln but not polySer accumulates in Purkinje cells. Representative polySer aggregates are indicated by black arrows. Inset: higher magnification of molecular layer and white matter.


B IF double staining shows no co - localization of polyGln and polySer in frontal cortex , pons or hippocampus of end - stage SCA8 BAC mice .

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B IF double staining shows no co-localization of polyGln and polySer in frontal cortex, pons or hippocampus of end-stage SCA8 BAC mice.


C IF double staining of end - stage SCA8 BAC frontal cortex shows exclusive localization of polyGln ( mouse α - Gln , red , bottom panel ) in neurons ( rabbit α - NeuN , green , bottom panel ) . In contrast , polySer ( Rabbit α - SerCT , red , top panel ) shows widespread accumulation in the frontal cortex including within neurons ( mouse α - NeuN , green , top panel ) .

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

C IF double staining of end-stage SCA8 BAC frontal cortex shows exclusive localization of polyGln (mouse α-Gln, red, bottom panel) in neurons (rabbit α-NeuN, green, bottom panel). In contrast, polySer (Rabbit α-SerCT, red, top panel) shows widespread accumulation in the frontal cortex including within neurons (mouse α-NeuN, green, top panel).


D IHC of SCA8 human cerebellum shows that PolySer accumulates in the white matter but not in the Purkinje cells ( left panels ) while polyGln accumulates in Purkinje cells but not cerebellar white matter ( right panels ) .

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

D IHC of SCA8 human cerebellum shows that PolySer accumulates in the white matter but not in the Purkinje cells (left panels) while polyGln accumulates in Purkinje cells but not cerebellar white matter (right panels).


A Representative images of the vestibular nucleus ( upper panels ) , cuneate nucleus ( middle panels ) and motor cortex layers II / III ( lower panels ) of SCA8 BAC mice at 2 months ( left panels ) , 6 months ( middle panels ) and end - stage ( 10 months , right panels ) stained with α - SerCT . Representative aggregates are indicated by black arrows . Corresponding quantifications of polySer aggregates for each region on the right ( n = 3 ; mean ± SEM ; One - way ANOVA with Tukey ' s post - hoc test ; * p < 0 . 025 , * * p < 0 . 005 , * * * p < 0 . 0005 , * * * * 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 the vestibular nucleus (upper panels), cuneate nucleus (middle panels) and motor cortex layers II/III (lower panels) of SCA8 BAC mice at 2 months (left panels), 6 months (middle panels) and end-stage (10 months, right panels) stained with α-SerCT. Representative aggregates are indicated by black arrows. Corresponding quantifications of polySer aggregates for each region on the right (n=3; mean ± SEM; One-way ANOVA with Tukey's post-hoc test; * p<0.025, ** p<0.005, *** p<0.0005, **** p<0.0001)


B Open field analysis of 2 month old animals show a significant decrease in ambulatory distance ( cm ) and ambulatory time ( s ) and a significant increase in resting time ( s ) ( NT n = 19 , SCA8 BAC n = 23 , * * p < 0 . 01 , * * * p < 0 . 001 ; mean ± SEM ; unpaired t test ) .

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B Open field analysis of 2 month old animals show a significant decrease in ambulatory distance (cm) and ambulatory time (s) and a significant increase in resting time (s) (NT n=19, SCA8 BAC n=23, ** p<0.01, *** p<0.001; mean ± SEM; unpaired t test).


A Disease - specific sensitivity to vacuolization after prolonged storage in ethanol shown by H & E in cerebellar white matter and brainstem of 10 - 12 month old end - stage SCA8 BAC mice but not in age matched NT ( n = 3 ) ( panels 1 & 2 ) . Demyelination shown by luxol fast blue staining ( LFB ) ( panel 3 ) and axonal degeneration shown by α - SMI - 32 ( panel 4 ; degenerated axons are indicated by black arrows ) observed in sites of polySer accumulation shown by α - SerCT ( panel 5 ; representative aggregates are indicated by black arrows ) in deep cerebellar white matter and brainstem in SCA8 BAC mice ( lower panels ) but not in NT mice ( upper panels ) ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Disease-specific sensitivity to vacuolization after prolonged storage in ethanol shown by H&E in cerebellar white matter and brainstem of 10-12 month old end-stage SCA8 BAC mice but not in age matched NT (n=3) (panels 1&2). Demyelination shown by luxol fast blue staining (LFB) (panel 3) and axonal degeneration shown by α-SMI-32 (panel 4; degenerated axons are indicated by black arrows) observed in sites of polySer accumulation shown by α-SerCT (panel 5; representative aggregates are indicated by black arrows) in deep cerebellar white matter and brainstem in SCA8 BAC mice (lower panels) but not in NT mice (upper panels) (n=3).


Demyelination shown by luxol fast blue staining ( LFB ) ( panel 1 ) and axonal degeneration shown by α - SMI - 32 ( panel 2 ) observed sites of polySer accumulation shown by a - SerCT2 ( panel 3 ) in deep cerebellar white matter was found in SCA8 autopsy tissue but not in control brains ( 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 O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Demyelination shown by luxol fast blue staining (LFB) (panel 1) and axonal degeneration shown by α-SMI-32 (panel 2) observed sites of polySer accumulation shown by a-SerCT2 (panel 3) in deep cerebellar white matter was found in SCA8 autopsy tissue but not in control brains (n=3).


C Immunohistochemistry using CC1 ( α - APC ) antibody shows significantly lower numbers of mature oligodendrocytes in SCA8 BAC mice compared to NT mice ( NT n = 5 , SCA8 BAC n = 5 ; * * * * p < 0 . 0001 ; mean ± SEM ; unpaired t test ) .

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C Immunohistochemistry using CC1 (α- APC) antibody shows significantly lower numbers of mature oligodendrocytes in SCA8 BAC mice compared to NT mice (NT n=5, SCA8 BAC n=5; **** p<0.0001; mean ± SEM; unpaired t test).


D Immunofluorescence using α - GFAP antibody shows significant increase in astrogliosis in SCA8 BAC mice compared to NT mice ( NT n = 3 , SCA8 BAC n = 3 , * * p < 0 . 01 ; mean ± SEM ; unpaired t test ) .

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D Immunofluorescence using α-GFAP antibody shows significant increase in astrogliosis in SCA8 BAC mice compared to NT mice (NT n=3, SCA8 BAC n=3, ** p<0.01; mean ± SEM; unpaired t test).


E Cell death measured by lactase dehydrogenase ( LDH ) assay in T98 cells transfected with codon replacement constructs expressing polyGln ( light blue ) or polySer ( dark blue ) . NT = nontransfected , EV = empty vector ( n = 5 , * * p < 0 . 003 ; * * * p < 0 . 0003 ; mean ± SEM ; unpaired t test ) .

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E Cell death measured by lactase dehydrogenase (LDH) assay in T98 cells transfected with codon replacement constructs expressing polyGln (light blue) or polySer (dark blue). NT=nontransfected, EV=empty vector (n=5, ** p<0.003; *** p< 0.0003; mean ± SEM; unpaired t test).


qRT - PCR showing Eif3f expression levels are increased 2 - fold in SCA8 cerebellar white matter compared to SCA8 cerebellar grey matter ( n = 3 , * * p < 0 . 01 ; mean ± SEM ; unpaired t test ) .

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qRT-PCR showing Eif3f expression levels are increased 2-fold in SCA8 cerebellar white matter compared to SCA8 cerebellar grey matter (n=3, **p<0.01; mean ± SEM; unpaired t test).


C Dot blot detection of polySer expressionin using α - FLAG antibody showing decrease in RAN polySer but not ATG - polySer when HEK293 cells are co - transfected with eIF3F siRNA . D Quantification of polySer detection ( n = 5 , * p < 0 . 01 ; n . s . no significance ; mean ± SEM ; unpaired t test ) .

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C Dot blot detection of polySer expressionin using α-FLAG antibody showing decrease in RAN polySer but not ATG-polySer when HEK293 cells are co-transfected with eIF3F siRNA. D Quantification of polySer detection (n=5, * p<0.01; n.s. no significance; mean ± SEM; unpaired t test).


E Detection of polyAla expression using α - HA antibody showing decrease in RAN polyAla but not ATG - polyAla when HEK293 cells are co - transfected with eIF3F siRNA . F Detection of polyGP expression using α - FLAG antibody showing a decrease in RAN polyGP when HEK293 cells are co - transfected with eIF3F siRNA . . G Quantification of polyAla and polyGP detection ( n = 5 , * p < 0 . 01 ; n . s . no significance ; mean ± SEM ; unpaired t test ) .

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E Detection of polyAla expression using α-HA antibody showing decrease in RAN polyAla but not ATG-polyAla when HEK293 cells are co-transfected with eIF3F siRNA. F Detection of polyGP expression using α-FLAG antibody showing a decrease in RAN polyGP when HEK293 cells are co-transfected with eIF3F siRNA.. G Quantification of polyAla and polyGP detection (n=5, * p<0.01; n.s. no significance; mean ± SEM; unpaired t test).


( A and B ) FACS - sorted T cell populations and Monocyte - derived Macrophages were treated with the indicated stimuli : Nigericin ( Nig . 4h ) , NeedleTox ( 4h ) , Val - boroPro ( VbP 22h ) , ABT737 / S63845 ( A / S 22h ) . When indicated , cells were primed with LPS for 2 hours prior to stimulation . When indicated , MCC950 was added to the media 30 minutes prior to the addition of Nigericin . LDH - activity ( A ) and IL - 1β and IL - 18 concentration ( B and C , respectively ) in the supernatant were determined by LDH cytotoxicity assay and ELISA respectively . Individual data points ± SEM from three independent donors are shown . Statistics indicate significance by two - way ANOVA : * * * p ≤ 0 . 001 ; * * p ≤ 0 . 01 ; * p ≤ 0 . 05 ; ns , not significant . P - values were corrected for multiple comparisons ( Dunnett ) .

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(A and B) FACS-sorted T cell populations and Monocyte-derived Macrophages were treated with the indicated stimuli: Nigericin (Nig. 4h), NeedleTox (4h), Val-boroPro (VbP 22h), ABT737/S63845 (A/S 22h). When indicated, cells were primed with LPS for 2 hours prior to stimulation. When indicated, MCC950 was added to the media 30 minutes prior to the addition of Nigericin. LDH-activity (A) and IL-1β and IL-18 concentration (B and C, respectively) in the supernatant were determined by LDH cytotoxicity assay and ELISA respectively. Individual data points ± SEM from three independent donors are shown. Statistics indicate significance by two-way ANOVA: ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; ns, not significant. P-values were corrected for multiple comparisons (Dunnett).


A ) Macrophages and CD4 T cells from the same donor were subjected to the indicated treatments and morphologic changes as well as PI - uptake were monitored by live - cell imaging microscopy using a 25x objective . Representative images from indicated time points are shown . Cyan color coding is used for the fluorescent PI - signal . One donor out of two is shown . ( B ) Representative images were acquired with a 63x objective at the end of the experiment shown in ( A ) at 16 hours . One donor out of two is shown . Scale bars : 25 µm .

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A) Macrophages and CD4 T cells from the same donor were subjected to the indicated treatments and morphologic changes as well as PI-uptake were monitored by live-cell imaging microscopy using a 25x objective. Representative images from indicated time points are shown. Cyan color coding is used for the fluorescent PI-signal. One donor out of two is shown. (B) Representative images were acquired with a 63x objective at the end of the experiment shown in (A) at 16 hours. One donor out of two is shown. Scale bars: 25 µm.


( C ) CD4 T cells were treated with VbP or ABT737 / S63845 . Lysate and supernatant samples were collected at the indicated time points . Samples were analyzed by immunoblotting . αSS and αLS indicate the use of a small subunit or large subunit - specific caspase - 1 antibody respectively . ( Lys = lysate , SN = supernatant , FL = full length ) . One representative experiment out of three is shown .

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(C) CD4 T cells were treated with VbP or ABT737/S63845. Lysate and supernatant samples were collected at the indicated time points. Samples were analyzed by immunoblotting. αSS and αLS indicate the use of a small subunit or large subunit-specific caspase-1 antibody respectively. (Lys = lysate, SN = supernatant, FL = full length). One representative experiment out of three is shown.


( A ) Gene expression profile of 15 inflammasome - related genes in human T cell and monocyte subsets . TPM values were retrieved from a public RNASeq dataset and log2 transformed . Both , rows and columns are clustered using euclidean distance and complete linkage . ( Tfh , T follicular helper cells ; Tregs , T regulatory cells ; Th , T helper cells ; CM , central memory T cells ; EM , effector memory T cells ; TE , terminal effector T cells ; CD14 + C16 - , classical monocytes ; CD14 + CD16 + , intermediate monocytes ; CD14 - CD16 + , non - classical monocytes ) .

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(A) Gene expression profile of 15 inflammasome-related genes in human T cell and monocyte subsets. TPM values were retrieved from a public RNASeq dataset and log2 transformed. Both, rows and columns are clustered using euclidean distance and complete linkage. (Tfh, T follicular helper cells; Tregs, T regulatory cells; Th, T helper cells; CM, central memory T cells; EM, effector memory T cells; TE, terminal effector T cells; CD14+ C16-, classical monocytes; CD14+ CD16+, intermediate monocytes; CD14- CD16+, non-classical monocytes).


( B ) Immunoblotting of CD4 T cells and MDMs treated with Pam3CSK4 for 6 hours . * indicates an unspecific band . One representative experiment out of three is shown .

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(B) Immunoblotting of CD4 T cells and MDMs treated with Pam3CSK4 for 6 hours. * indicates an unspecific band. One representative experiment out of three is shown.


( C ) Immunoblotting of polyclonal CD4 T cells targeted with two gRNAs for each indicated target gene . One representative experiment out of three is shown .

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(C) Immunoblotting of polyclonal CD4 T cells targeted with two gRNAs for each indicated target gene. One representative experiment out of three is shown.


( D ) CD4 T cells from the indicated polyclonal gene targeting approach were treated with the indicated stimuli for 18 hours . Individual data points ± SEM from three independent donors are shown . Statistics indicate significance by two - way ANOVA : * * * p ≤ 0 . 001 ; ns , not significant . P - values were corrected for multiple comparisons ( Dunnett ) .

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(D) CD4 T cells from the indicated polyclonal gene targeting approach were treated with the indicated stimuli for 18 hours. Individual data points ± SEM from three independent donors are shown. Statistics indicate significance by two-way ANOVA: ***p ≤ 0.001; ns, not significant. P-values were corrected for multiple comparisons (Dunnett).


( B ) MiSeq analysis of the indicated gene targeting approaches from samples collected at the indicated time points post editing . DPP9 and DPP8 were targeted with one gRNA / target gene , CARD8 was targeted with two gRNAs . On the day of nucleofection , T cells were activated with human T activator CD3 / CD28 beads and expanded in the presence of IL - 2 . On day 10 , cells were switched to IL - 7 / IL - 15 containing medium . ( C ) Fraction of wildtype and in - frame reads are depicted . One representative experiment out of three is shown .

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(B) MiSeq analysis of the indicated gene targeting approaches from samples collected at the indicated time points post editing. DPP9 and DPP8 were targeted with one gRNA/target gene, CARD8 was targeted with two gRNAs. On the day of nucleofection, T cells were activated with human T activator CD3/CD28 beads and expanded in the presence of IL-2. On day 10, cells were switched to IL-7/IL-15 containing medium. (C) Fraction of wildtype and in-frame reads are depicted. One representative experiment out of three is shown.


( A ) CD4 T cells were activated with human T activator CD3 / CD28 beads ( bead : cell ratio 1 : 5 ) for 4 days . Controls were kept resting in the presence of IL - 7 and IL - 15 . On day 4 cells were stimulated as indicated for 18 hours and cytotoxicity was assessed by LDH - activity in the supernatant . Individual data points ± SEM from four independent donors . Statistics indicate significance by two - way ANOVA : * * * p ≤ 0 . 001 ; ns , not significant . P - values were corrected for multiple comparisons ( Sidak ) .

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(A) CD4 T cells were activated with human T activator CD3/CD28 beads (bead:cell ratio 1:5) for 4 days. Controls were kept resting in the presence of IL-7 and IL-15. On day 4 cells were stimulated as indicated for 18 hours and cytotoxicity was assessed by LDH-activity in the supernatant. Individual data points ± SEM from four independent donors. Statistics indicate significance by two-way ANOVA: ***p ≤ 0.001; ns, not significant. P-values were corrected for multiple comparisons (Sidak).


( B ) Immunoblotting of CD4 T cell lysates treated as in ( A ) for 3 to 4 days . * indicates an unspecific band . ( C ) Quantification of immunoblots shown in ( B ) and additional donors ( n = 9 for all but GSDMD , GSDMD n = 8 , for one donor quantification of the GSDMD band was considered unreliable due to uneven substrate distribution ) . Individual data points ± SEM are shown . Statistics indicate significance by one - way ANOVA of log2 - transformed fold change values of band intensities ( activated / non - activated T cells ) normalized to β - Actin : * * * p ≤ 0 . 001 ; * p ≤ 0 . 05 ; ns , not significant . P - values were corrected for multiple comparisons ( Sidak ) . All band intensity ratios were tested against β - Actin . CARD8 full length was additionally tested against CARD8 cleaved .

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(B) Immunoblotting of CD4 T cell lysates treated as in (A) for 3 to 4 days. * indicates an unspecific band. (C) Quantification of immunoblots shown in (B) and additional donors (n= 9 for all but GSDMD, GSDMD n=8, for one donor quantification of the GSDMD band was considered unreliable due to uneven substrate distribution). Individual data points ± SEM are shown. Statistics indicate significance by one-way ANOVA of log2-transformed fold change values of band intensities (activated/non-activated T cells) normalized to β-Actin: ***p ≤ 0.001; *p ≤ 0.05; ns, not significant. P-values were corrected for multiple comparisons (Sidak). All band intensity ratios were tested against β-Actin. CARD8 full length was additionally tested against CARD8 cleaved.


( E ) Representative western blot of scr or miR - 30c transfected TREx cells analysed for ORF65 expression with GAPDH as a loading control . Densitometry analysis performed on n = 3 . Data information : data are presented as mean ± SD . * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

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(E) Representative western blot of scr or miR-30c transfected TREx cells analysed for ORF65 expression with GAPDH as a loading control. Densitometry analysis performed on n=3. Data information: data are presented as mean ± SD. **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( F ) Representative western blot of scr or miR - 29b transfected TREx cells analysed for ORF65 expression with GAPDH as a loading control . Densitometry analysis performed on n = 3 . Data information : data are presented as mean ± SD . * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

(F) Representative western blot of scr or miR-29b transfected TREx cells analysed for ORF65 expression with GAPDH as a loading control. Densitometry analysis performed on n=3. Data information: data are presented as mean ± SD. **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( C ) Representative western blot of ORF65 levels in scr and circHIPK3 KD cell lines with GAPDH as a loading control . Densitometry analysis of n = 3 western blots . Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

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(C) Representative western blot of ORF65 levels in scr and circHIPK3 KD cell lines with GAPDH as a loading control. Densitometry analysis of n=3 western blots. Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( B ) Representative western blot of DLL4 expression in TREx cells 0 and 24 hours post induction . GAPDH was used a loading control ( n = 3 ) . Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

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(B) Representative western blot of DLL4 expression in TREx cells 0 and 24 hours post induction. GAPDH was used a loading control (n=3). Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.


( D ) Representative western blot of DLL4 levels in scr and circHIPK3 stable TREx cells at 0 and 24 hours post induction with GAPDH as a loading control . Densitometry analysis of n = 3 . Data information : data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( Unpaired Student ' s t - test ) . All repeats are biological .

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(D) Representative western blot of DLL4 levels in scr and circHIPK3 stable TREx cells at 0 and 24 hours post induction with GAPDH as a loading control. Densitometry analysis of n=3. Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.