Figure 5In vitro HMTase assays . Recombinant H3N - GST ( WT or K9R ) pre - ubiquitylated by the CLRC was purified and used in the HMTase assay with 6 × His - tagged recombinant Clr4 ( His - Clr4 ) . Proteins were analyzed by SDS - PAGE and Coomassie Brilliant Blue ( CBB ) staining ( left ) and autoradiography ( right ) . C , D In vitro HMTase assay using disulfide - linked ubiquitylated histone H3 . H3K14CUb and control histone H3 ( H3C110A ) were used as substrates in the HMTase assay , and analyzed The 3H - methyl group incorporation was measured using a liquid scintillation counter ( D ) . Error bars indicate the standard deviations calculated from three independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5In vitro HMTase assays. Recombinant H3N-GST (WT or K9R) pre-ubiquitylated by the CLRC was purified and used in the HMTase assay with 6×His-tagged recombinant Clr4 (His-Clr4). Proteins were analyzed by SDS-PAGE and Coomassie Brilliant Blue (CBB) staining (left) and autoradiography (right).C,D In vitro HMTase assay using disulfide-linked ubiquitylated histone H3. H3K14CUb and control histone H3 (H3C110A) were used as substrates in the HMTase assay, and analyzed The 3H-methyl group incorporation was measured using a liquid scintillation counter (D). Error bars indicate the standard deviations calculated from three independent experiments.


Figure 6B Recombinant proteins were resolved by 10 % SDS - PAGE and visualized by CBB staining . C In vitro HMTase assay using disulfide - linked ubiquitylated histone H3 . H3K14CUb and control histone H3 ( H3C110A ) were used as substrates in the HMTase assay , and analyzedD In vitro pulldown assays of GST - Clr4 proteins with biotinylated ubiquitin . Bound ubiquitin was analyzed by Western blotting with HRP - conjugated streptavidin ( left ) . Precipitated GST proteins were analyzed by SDS - PAGE and CBB staining ( right ) . F Whole cell lysates prepared from control cells , cells expressing FLAG - tagged wild - type Clr4 ( F - clr4 ) or mutant Clr4 ( F - clr4 ∆ 63 - 127 ) , or clr4 ∆ cells were subjected to immunoblotting using the indicated antibodies . G Heterochromatic silencing assays of wild - type and clr4 mutant strains . Silencing at the mating - type Kint2 : : ura4 + was evaluated . H ChIP analysis of H3K9me2 levels associated with the silencing marker gene inserted in the mating - type loci ( Kint2 : : ura4 + ) and the mating - type cenH and centromeric dg loci , relative to the control act1 + locus . Error bars indicate standard errors from three technical replicates . I The levels of Kint2 : : ura4 + , mating - type cenH , and centromeric dg transcripts were quantified by RT - qPCR and calculated as fold change to the wild - type expression . Error bars indicate standard errors from three technical replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6B Recombinant proteins were resolved by 10% SDS-PAGE and visualized by CBB staining.C In vitro HMTase assay using disulfide-linked ubiquitylated histone H3. H3K14CUb and control histone H3 (H3C110A) were used as substrates in the HMTase assay, and analyzedD In vitro pulldown assays of GST-Clr4 proteins with biotinylated ubiquitin. Bound ubiquitin was analyzed by Western blotting with HRP-conjugated streptavidin (left). Precipitated GST proteins were analyzed by SDS-PAGE and CBB staining (right).F Whole cell lysates prepared from control cells, cells expressing FLAG-tagged wild-type Clr4 (F-clr4) or mutant Clr4 (F-clr4∆63-127), or clr4∆ cells were subjected to immunoblotting using the indicated antibodies.G Heterochromatic silencing assays of wild-type and clr4 mutant strains. Silencing at the mating-type Kint2::ura4+ was evaluated.H ChIP analysis of H3K9me2 levels associated with the silencing marker gene inserted in the mating-type loci (Kint2::ura4+) and the mating-type cenH and centromeric dg loci, relative to the control act1+ locus. Error bars indicate standard errors from three technical replicates.I The levels of Kint2::ura4+, mating-type cenH, and centromeric dg transcripts were quantified by RT-qPCR and calculated as fold change to the wild-type expression. Error bars indicate standard errors from three technical replicates.


Figure 1A Representative images of Bem1 - GFP and the PS marker LactC2 - GFP in wild type and cho1 ∆ cells . Images show average intensity projections of deconvolved z - stacks . Black and white pixels were inverted to improve contrast for display . B Frequency of cells displaying polarized Bem1 - GFP signal in wild type and cho1 ∆ cells . Values are means ± SD , n > 100 cells in each of 3 independent experiments . Paired Student ' s t - tests were performed . C Imaging of the PI4P probe GFP - 2xPHOsh2 in wild type and cho1 ∆ cells . Images show average intensity projections of deconvolved z - stacks in which black and white pixels were inverted . D Scatter dot plot showing PI4P levels at the plasma membrane ( see experimental procedure for details of the quantification ) in wild type and cho1 ∆ cells . Values are means ± SD , n > 100 cells , observed over 3 experiments . Mann - Whitney tests were performed . E Ratio of PI4P average fluorescence intensity in the bud versus mother cell in the cho1 ∆ mutant . Values are means ± SD , n > 100 cells , observed over 3 experiments . A value > 1 indicates enrichment of the fluorescence in the bud . F Images of Bem1 - GFP in cho1 ∆ 9xMyc - AID - stt4 cells after 30 min treatment with or without 0 . 5 mM auxin . Images are average intensity projections of deconvolved z - stacks . G Frequency of cells with polarized Bem1 - GFP signal or Bem1 - GFP in puncta in cho1 ∆ 9xMyc - AID - stt4 cells treated with or without auxin as shown in F . Values are means ± SD , n > 100 cells in each of 6 independent experiments . Paired Student ' s t - tests were performed . H Images of Bem1 - GFP ( cyan ) and Cdc24 - mCherry ( red ) signals in cho1 ∆ 9xMyc - AID - stt4 cells with or without auxin . Images display maximum intensity projections of z - stacks . I Maximum intensity projections of z - stacks displaying Bem1 - GFP in the mutants indicated . Bem1 - GFP was imaged in mss4 - 102 cho1 ∆ cells after shifting to the restrictive temperature for 30 minutes . J Frequency of cells with polarized Bem1 - GFP signal after incubation at the time and temperature shown . Values are means ± SD , n > 200 cells in each of 3 independent experiments . Paired Student ' s t - tests were performed .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A Representative images of Bem1-GFP and the PS marker LactC2-GFP in wild type and cho1∆ cells. Images show average intensity projections of deconvolved z-stacks. Black and white pixels were inverted to improve contrast for display. B Frequency of cells displaying polarized Bem1-GFP signal in wild type and cho1∆ cells. Values are means ± SD, n > 100 cells in each of 3 independent experiments. Paired Student's t-tests were performed. C Imaging of the PI4P probe GFP-2xPHOsh2 in wild type and cho1∆ cells. Images show average intensity projections of deconvolved z-stacks in which black and white pixels were inverted.D Scatter dot plot showing PI4P levels at the plasma membrane (see experimental procedure for details of the quantification) in wild type and cho1∆ cells. Values are means ± SD, n > 100 cells, observed over 3 experiments. Mann-Whitney tests were performed.E Ratio of PI4P average fluorescence intensity in the bud versus mother cell in the cho1∆ mutant. Values are means ± SD, n > 100 cells, observed over 3 experiments. A value > 1 indicates enrichment of the fluorescence in the bud.F Images of Bem1-GFP in cho1∆ 9xMyc-AID-stt4 cells after 30 min treatment with or without 0.5 mM auxin. Images are average intensity projections of deconvolved z-stacks.G Frequency of cells with polarized Bem1-GFP signal or Bem1-GFP in puncta in cho1∆ 9xMyc-AID-stt4 cells treated with or without auxin as shown in F. Values are means ± SD, n > 100 cells in each of 6 independent experiments. Paired Student's t-tests were performed.H Images of Bem1-GFP (cyan) and Cdc24-mCherry (red) signals in cho1∆ 9xMyc-AID-stt4 cells with or without auxin. Images display maximum intensity projections of z-stacks.I Maximum intensity projections of z-stacks displaying Bem1-GFP in the mutants indicated. Bem1-GFP was imaged in mss4-102 cho1∆ cells after shifting to the restrictive temperature for 30 minutes.J Frequency of cells with polarized Bem1-GFP signal after incubation at the time and temperature shown. Values are means ± SD, n > 200 cells in each of 3 independent experiments. Paired Student's t-tests were performed.


Figure 2B Upper panel . SDS - PAGE stained with Coomassie blue in which Bem1 is indicated . Lower panel . The liposomes were composed of 100 % phosphatidylcholine ( PC ) , 80 % PC and 20 % phosphatidylethanolamine ( PE ) , 95 % PC and 5 % phosphatidic acid ( PA ) , 95 % PC and 5 % phosphoatidylinositol ( PI ) , 95 % PC and 5 % PI ( 4 , 5 ) P2 ( PI ( 4 , 5 ) P2 ) , 95 % PC and 5 % PI4P ( PI4P ) , 80 % PC and 20 % phosphatidylserine ( PS ) or 75 % PC 20 % PS 5 % PI4P ( PS + PI4P ) . ( S ) supernatant , ( P ) pellet . C Liposomes composed of either 100 % PC ± 40 % ergosterol or 75 % PC , 20 % PS , 5 % Bem1 ± 40 % ergosterol replacing PC were incubated with Bem1 and treated as in panel ( B ) . E Percentage of the indicated bem1 constructs associated with liposomes containing 75 % PC , 20 % PS and 5 % PI4P . F Lipid ordering determined by 2H solid - state NMR analysis of liposomes containing POPC - d31 / POPS ( 4 : 1 molar ratio ) in the presence or absence of Bem1 BC motifs . Calculation of oriented - like spectra from Pake patterns ( de - Pake - ing ) and simulation of 2H solid - state NMR spectra were applied to measure individual quadrupolar splittings for POPC - d31 and determine order parameter accurately . Lipid ordering determined by 2H solid - state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs . Calculation of oriented - like spectra from Pake patterns ( de - Pake - ing ) and simulation of 2H solid - state NMR spectra were applied to measure individual quadrupolar splittings for POPC - d31 and determine order parameter accurately . containing POPC - d31 / PI4P ( 19 : 1 molar ratio ) . Lipid ordering determined by 2H solid - state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs . Calculation of oriented - like spectra from Pake patterns ( de - Pake - ing ) and simulation of 2H solid - state NMR spectra were applied to measure individual quadrupolar splittings for POPC - d31 and determine order parameter accurately . containing POPC - d31 / POPC / PI4P ( 15 : 4 : 1 molar ratio ) . Lipid ordering determined by 2H solid - state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs . Calculation of oriented - like spectra from Pake patterns ( de - Pake - ing ) and simulation of 2H solid - state NMR spectra were applied to measure individual quadrupolar splittings for POPC - d31 and determine order parameter accurately . containing POPC - d31 / POPS / Yeast lipid extract / ergosterol ( 1 : 1 : 1 : 2 molar ratio ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B Upper panel. SDS-PAGE stained with Coomassie blue in which Bem1 is indicated. Lower panel. The liposomes were composed of 100% phosphatidylcholine (PC), 80% PC and 20% phosphatidylethanolamine (PE), 95% PC and 5% phosphatidic acid (PA), 95% PC and 5% phosphoatidylinositol (PI), 95% PC and 5% PI(4,5)P2 (PI(4,5)P2), 95% PC and 5% PI4P (PI4P), 80% PC and 20% phosphatidylserine (PS) or 75% PC 20% PS 5% PI4P (PS+PI4P). (S) supernatant, (P) pellet.C Liposomes composed of either 100% PC ± 40% ergosterol or 75% PC, 20% PS, 5% Bem1 ± 40% ergosterol replacing PC were incubated with Bem1 and treated as in panel (B).E Percentage of the indicated bem1 constructs associated with liposomes containing 75% PC, 20% PS and 5% PI4P.F Lipid ordering determined by 2H solid-state NMR analysis of liposomes containing POPC-d31/POPS (4:1 molar ratio) in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately.Lipid ordering determined by 2H solid-state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately. containing POPC-d31/PI4P (19:1 molar ratio).Lipid ordering determined by 2H solid-state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately. containing POPC-d31/POPC/PI4P (15:4:1 molar ratio).Lipid ordering determined by 2H solid-state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately. containing POPC-d31/POPS/Yeast lipid extract/ergosterol (1:1:1:2 molar ratio).


Figure 3B Percentage of the different full - length bem1 bc mutants associated with liposomes containing 75 % PC 20 % PS and 5 % PI4P . C Fluorescence intensity change associated with the nucleotide exchange of GDP - Cdc42 for mant - GTP Cdc42 . Fluorescence was measured after the addition of GDP - Cdc42 to reactions containing Mant - GTP ( 100 nM ) , GMP - PNP ( 100 µM ) and the proteins indicated . D Ten - fold serial dilutions of cells and subsequent colony formation on the indicated plates , where expression of wild type GALp - CDC42 is either induced in the presence of Gal or repressed in Dex . Note how mutation of the wild type Cdc42 ( KKSKK ) to MMSMM is lethal ( see blue box ) , whereas appending the Bem1 BC - 1 motif to this cdc42 mutant restores viability ( red box ) . E Representative images of the mEOS - cdc42 mutants signal ( cyan ) after inducing the expression of GAL1p - CDC42 in the presence of Gal or repressing it in the presence of Dex . The cells in the blue and red boxes correspond to the cells in the blue and red box in panel ( D ) . Images are average fluorescence intensity projections in which black and white pixels have been inverted .

O B-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B Percentage of the different full-length bem1 bc mutants associated with liposomes containing 75% PC 20% PS and 5% PI4P.C Fluorescence intensity change associated with the nucleotide exchange of GDP-Cdc42 for mant-GTP Cdc42. Fluorescence was measured after the addition of GDP-Cdc42 to reactions containing Mant-GTP (100 nM), GMP-PNP (100 µM) and the proteins indicated.D Ten-fold serial dilutions of cells and subsequent colony formation on the indicated plates, where expression of wild type GALp-CDC42 is either induced in the presence of Gal or repressed in Dex. Note how mutation of the wild type Cdc42 (KKSKK) to MMSMM is lethal (see blue box), whereas appending the Bem1 BC-1 motif to this cdc42 mutant restores viability (red box).E Representative images of the mEOS-cdc42 mutants signal (cyan) after inducing the expression of GAL1p-CDC42 in the presence of Gal or repressing it in the presence of Dex. The cells in the blue and red boxes correspond to the cells in the blue and red box in panel (D). Images are average fluorescence intensity projections in which black and white pixels have been inverted.


Figure 4A Ten - fold serial dilutions of the indicated mutant cells and subsequent colony formation at the temperatures indicated . Note how mutations on both the BC motifs and PX in bem1 compromise growth at the restrictive temperature . B Western blots probed with anti - HA antibody to detect the indicated bem1 constructs tagged with 3XHA ( top panel ) . Detection of Ade13 was used as loading control ( bottom panel ) . C Representative images of the indicated bem1 mutants tagged with GFP at the BEM1 locus . The inverted fluorescence images are average intensity projections . D Frequency of cells with polarized Bem1 - GFP . E Scatter dot plot showing the fluorescence levels of the indicated bem1 strains at the cell pole . The measurements were made on average intensity projections .

O B-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A Ten-fold serial dilutions of the indicated mutant cells and subsequent colony formation at the temperatures indicated. Note how mutations on both the BC motifs and PX in bem1 compromise growth at the restrictive temperature.B Western blots probed with anti-HA antibody to detect the indicated bem1 constructs tagged with 3XHA (top panel). Detection of Ade13 was used as loading control (bottom panel).C Representative images of the indicated bem1 mutants tagged with GFP at the BEM1 locus. The inverted fluorescence images are average intensity projections.D Frequency of cells with polarized Bem1-GFP. E Scatter dot plot showing the fluorescence levels of the indicated bem1 strains at the cell pole. The measurements were made on average intensity projections.


Figure 5B Percentage of full - length Cdc24 and cdc24 ph domain mutant ( cdc24 ph ) associated with liposomes containing 75 % PC 20 % PS and 5 % PI4P . C Percentage of Cdc24 and cdc24 ph mutant associated with liposomes of the composition shown in ( B ) in the presence of the indicated bem1 protein . Note how additive mutations in the Bem1 lipid binding sequences reduce the percentage of Cdc24 associated with the liposomes . D Percentage of Cdc24 and cdc24 ph associated with liposomes of the composition shown in ( B ) as a function of the indicated bem1 protein concentration . The curves denote the regression fit of the data to equation 2 in the materials and methods . E Ten - fold serial dilutions and subsequent colony formation of the indicated mutant cells at the indicated temperatures . F DIC images of the indicated bem1 and cdc24 mutants showing the increased morphological defects ensuing from loss of lipid tethering in the bem1 and cdc24 mutants . G Western blots probed with anti - HA antibody to detect Cdc24 or cdc24 ph tagged with 3XHA in the indicated mutant strains ( top panel ) . Detection of Ade13 was used as a loading control ( bottom panel ) .

O B-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5B Percentage of full-length Cdc24 and cdc24 ph domain mutant (cdc24 ph) associated with liposomes containing 75% PC 20% PS and 5% PI4P.C Percentage of Cdc24 and cdc24 ph mutant associated with liposomes of the composition shown in (B) in the presence of the indicated bem1 protein. Note how additive mutations in the Bem1 lipid binding sequences reduce the percentage of Cdc24 associated with the liposomes.D Percentage of Cdc24 and cdc24 ph associated with liposomes of the composition shown in (B) as a function of the indicated bem1 protein concentration. The curves denote the regression fit of the data to equation 2 in the materials and methods.E Ten-fold serial dilutions and subsequent colony formation of the indicated mutant cells at the indicated temperatures.F DIC images of the indicated bem1 and cdc24 mutants showing the increased morphological defects ensuing from loss of lipid tethering in the bem1 and cdc24 mutants.G Western blots probed with anti-HA antibody to detect Cdc24 or cdc24 ph tagged with 3XHA in the indicated mutant strains (top panel). Detection of Ade13 was used as a loading control (bottom panel).


Figure 6B Global average MSD curves of mEOS - Cdc42 in the strains indicated at the Pole ( P ) and Non - Pole ( NP ) of cells . Trajectories longer than 6 frames were analyzed . Number of trajectories analyzed : BEM1 ( n = 11 cells : Non Pole : 1814 tracks ; Pole : 706 ) ; bem1 bc - 14E px ( n = 10 cells : Non Pole : 1793 tracks ; Pole : 908 ) ; bem1 bc - 14E ( n = 13 cells : Non Pole : 1714 tracks ; Pole : 975 ) . Error bars display s . e . m . C D coefficients of mEOS - Cdc42 in the strains indicated ( in box - plots displaying the median ( line ) , the 25 - 75 percentiles ( box ) and the mean ( cross ) ) , which were compared using a non - parametric , two - tailed Mann - Whitney rank sum test . D SR - Tesseler images of mEOS - Cdc42 nanocluster organization in BEM1 ( 5312 localizations shown in image ) and bem1 bc - 14E px cells ( 3739 localizations shown ) . Insets show mEOS - Cdc42 after 491 nm widelfield laser excitation to identify the cell pole . Scale bars : 2 μm . A zoom of the pole region shows the organization of the detected nanoclusters , circled in light blue , in the strains indicated . Scale bar in the zoom : 60 nm . E Distribution of nanocluster area at the pole ( P ) and non - pole ( NP ) regions of BEM1 ( diameter NP : 59 nm ± 1 nm ( s . e . m ) ; P : 74 nm ± 3 . 2 nm ) ( n = 16 cells . NP : 570 clusters ; P : 162 clusters ) and bem1 bc - 14E px cells ( diameter NP : 59 nm ± 1 . 5 nm ( s . e . m ) ; P : 57 nm ± 2 . 4 nm ) ( n = 15 cells . NP : 421 clusters ; P : 141 clusters ) . Data are presented as scatter dot - plots displaying the median as a line and the 25 - 75 percentiles . Data were compared using non - parametric , two - tailed Mann - Whitney rank sum test . F Active Cdc42 - GTP levels were quantified in the strains indicated using a gic2 ( 1 - 208 ) - yeGFP probe . Values are mean ± SD for n = 30 cells observed over 2 experiments . Data were compared using a Mann - Whitney test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6B Global average MSD curves of mEOS-Cdc42 in the strains indicated at the Pole (P) and Non-Pole (NP) of cells. Trajectories longer than 6 frames were analyzed. Number of trajectories analyzed: BEM1 (n = 11 cells: Non Pole: 1814 tracks; Pole: 706); bem1 bc-14E px (n = 10 cells: Non Pole: 1793 tracks; Pole: 908); bem1 bc-14E (n = 13 cells: Non Pole: 1714 tracks; Pole: 975). Error bars display s.e.m. C D coefficients of mEOS-Cdc42 in the strains indicated (in box-plots displaying the median (line), the 25-75 percentiles (box) and the mean (cross)), which were compared using a non-parametric, two-tailed Mann-Whitney rank sum test. D SR-Tesseler images of mEOS-Cdc42 nanocluster organization in BEM1 (5312 localizations shown in image) and bem1 bc-14E px cells (3739 localizations shown). Insets show mEOS-Cdc42 after 491 nm widelfield laser excitation to identify the cell pole. Scale bars: 2 μm. A zoom of the pole region shows the organization of the detected nanoclusters, circled in light blue, in the strains indicated. Scale bar in the zoom: 60 nm.E Distribution of nanocluster area at the pole (P) and non-pole (NP) regions of BEM1 (diameter NP: 59 nm ± 1 nm (s.e.m); P: 74 nm ± 3.2 nm) (n = 16 cells. NP: 570 clusters; P: 162 clusters) and bem1 bc-14E px cells (diameter NP: 59 nm ± 1.5 nm (s.e.m); P: 57 nm ± 2.4 nm) (n = 15 cells. NP: 421 clusters; P: 141 clusters). Data are presented as scatter dot-plots displaying the median as a line and the 25-75 percentiles. Data were compared using non-parametric, two-tailed Mann-Whitney rank sum test.F Active Cdc42-GTP levels were quantified in the strains indicated using a gic2(1-208)-yeGFP probe. Values are mean ± SD for n = 30 cells observed over 2 experiments. Data were compared using a Mann-Whitney test.


Figure 1Mitochondria - enriched fractions were isolated from HdhQ7 or HdhQ111 cells . mtHSF1 was examined by WB analysis . VDAC was analyzed as a loading control . ( n = 6 biological replicates ) . HdhQ7 WT striatal cells were exposed to 5 mM 3 - NP for 2 h . mtHSF1 levels were determined by immunoblotting . VDAC was used as a loading control . ( n = 5 biological replicates ) . HSF1 was measured in the mitochondria - enriched fractions of the HD transgenic YAC128 mice ( 9 months old ) or age - matched littermates . ( n = 5 biological replicates ) . mtHSF1 was analyzed in HD patient fibroblasts ( GM04208 , GM04222 , and GM21756 ) and fibroblasts from healthy individuals . ( n = 5 for Con1 / GM04208 and Con2 / GM04222 groups ; n = 3 for Con3 / GM21756 group ) . Representative immunoblot of HSF1 levels in mitochondria isolated from patient - derived iPSCs and control iPSCs . ( n = 3 biological replicates ) . Representative images of striatal organoids . Left , immunostaining for the MSN marker DARPP32 ( green ) and the radial glial marker SOX2 ( red ) in striatal organoids at D60 . Middle , immunostaining for the LGE progenitor markers Ctip2 ( green ) and GSH2 ( red ) at D30 . Right panel , immunostaining for the neuronal marker NeuN ( green ) and the proliferation marker Ki67 ( red ) at D30 . The images are representative of 12 independent differentiation experiments of H9 and IMR90 - 4 cells . HO = Hoechst ( blue ) . The scale bar represents 100 µm . Representative images and scatterplot confirming the increased HSF1 ( green ) translocation to mitochondria ( Tom20 , red ) . The data were obtained from 3 independent biological experiments . Con : n = 15 organoids ; HD : n = 14 organoids . Two - tailed unpaired t - test ; Means ± SEMs . Images were taken using Structured Illumination Microscopy ( SIM ) . The subcellular localization of HSF1 in HD striatal organoids was observed by immunoelectron microscopy . The arrows ( red ) mark HSF1 in mitochondria . The negative control lacked the HSF1 antibody . The scale bar represents 200 nm . HTT was knocked down in HdhQ7 or HdhQ111 cells by lentiviral infection . The protein levels of HSF1 were detected in mitochondrial fractions . ( n = 3 biological replicates ) .

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Figure 1Mitochondria-enriched fractions were isolated from HdhQ7 or HdhQ111 cells. mtHSF1 was examined by WB analysis. VDAC was analyzed as a loading control. (n=6 biological replicates).HdhQ7 WT striatal cells were exposed to 5 mM 3-NP for 2 h. mtHSF1 levels were determined by immunoblotting. VDAC was used as a loading control. (n=5 biological replicates).HSF1 was measured in the mitochondria-enriched fractions of the HD transgenic YAC128 mice (9 months old) or age-matched littermates. (n=5 biological replicates).mtHSF1 was analyzed in HD patient fibroblasts (GM04208, GM04222, and GM21756) and fibroblasts from healthy individuals. (n = 5 for Con1/ GM04208 and Con2/ GM04222 groups; n = 3 for Con3/ GM21756 group).Representative immunoblot of HSF1 levels in mitochondria isolated from patient-derived iPSCs and control iPSCs. (n=3 biological replicates).Representative images of striatal organoids. Left, immunostaining for the MSN marker DARPP32 (green) and the radial glial marker SOX2 (red) in striatal organoids at D60. Middle, immunostaining for the LGE progenitor markers Ctip2 (green) and GSH2 (red) at D30. Right panel, immunostaining for the neuronal marker NeuN (green) and the proliferation marker Ki67 (red) at D30. The images are representative of 12 independent differentiation experiments of H9 and IMR90-4 cells. HO= Hoechst (blue). The scale bar represents 100 µm.Representative images and scatterplot confirming the increased HSF1 (green) translocation to mitochondria (Tom20, red). The data were obtained from 3 independent biological experiments. Con: n=15 organoids; HD: n=14 organoids. Two-tailed unpaired t-test; Means ± SEMs. Images were taken using Structured Illumination Microscopy (SIM).The subcellular localization of HSF1 in HD striatal organoids was observed by immunoelectron microscopy. The arrows (red) mark HSF1 in mitochondria. The negative control lacked the HSF1 antibody. The scale bar represents 200 nm.HTT was knocked down in HdhQ7 or HdhQ111 cells by lentiviral infection. The protein levels of HSF1 were detected in mitochondrial fractions. (n=3 biological replicates).


Figure 2HdhQ7 cells were transfected with Flag - mtHSF1 and stained with anti - Tom20 ( green ) and anti - Flag ( red ) antibodies . Mitochondrial morphology was examined by confocal microscopy . Scatterplot with bar showed the percentage of cells with mitochondrial fragmentation . The scale bar represents 40 µm . ( n = 3 biological replicates , at least 100 cells per group were counted ) . The total protein levels of p - Drp1 S616 , p - Drp1S637 , Drp1 , OPA1 , MFN2 , Flag - mtHSF1 and actin ( loading control ) were determined by WB analysis . ( n = 3 biological replicates ) . Brain sections were stained with an anti - Tom20 antibody ( red ) , an anti - Flag antibody ( green ) and DAPI ( blue ) . Mitochondrial morphology was analyzed in Flag + or Flag - regions by confocal microscopy . The scale bar presents 2 mm , and the enlarged image scale bar is 40 µm . ( n = 6 mice / group ) . Striata injected with AAVs were lysed . p - Drp1 S616 was tested by immunoblotting . ( n = 5 mice / group ) . Mitochondrial morphology was analyzed by electron microscopy ( EM ) in mice injected with AAV - Con or AAV - mtHSF1 . Scatterplot showed the length of mitochondria . The scale bar presents 4 µm , and the enlarged image scale bar is 400 nm . ( n = 3 mice / group ) . AAV - Con group , n = 268 mitochondria were measured ; AAV - Flag - mtHSF1 group , n = 179 mitochondria were measured . Representative images and scatterplot of mitochondrial length ( Tom20 , red ) in Flag + or Flag - cells . The data were obtained from 3 independent biological experiments . Flag - : n = 154 mitochondria ; Flag + : n = 132 mitochondria . Representative images and scatterplot showing that the number of p - Drp1 S616 puncta in Flag + cells was greater than that in Flag - cells . The data were obtained from 3 independent biological experiments . Flag - : n = 70 cells from 10 organoids ; Flag + : n = 55 cells from 10 organoids . The images were obtained using structured illumination microscopy ( SIM ) .

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Figure 2HdhQ7 cells were transfected with Flag-mtHSF1 and stained with anti-Tom20 (green) and anti-Flag (red) antibodies. Mitochondrial morphology was examined by confocal microscopy. Scatterplot with bar showed the percentage of cells with mitochondrial fragmentation. The scale bar represents 40 µm. (n=3 biological replicates, at least 100 cells per group were counted).The total protein levels of p-Drp1 S616, p-Drp1S637, Drp1, OPA1, MFN2, Flag-mtHSF1 and actin (loading control) were determined by WB analysis. (n=3 biological replicates).Brain sections were stained with an anti-Tom20 antibody (red), an anti-Flag antibody (green) and DAPI (blue). Mitochondrial morphology was analyzed in Flag+ or Flag- regions by confocal microscopy. The scale bar presents 2 mm, and the enlarged image scale bar is 40 µm. (n=6 mice/group).Striata injected with AAVs were lysed. p-Drp1 S616 was tested by immunoblotting. (n=5 mice/group).Mitochondrial morphology was analyzed by electron microscopy (EM) in mice injected with AAV-Con or AAV-mtHSF1. Scatterplot showed the length of mitochondria. The scale bar presents 4 µm, and the enlarged image scale bar is 400 nm. (n=3 mice/group). AAV-Con group, n=268 mitochondria were measured; AAV-Flag-mtHSF1 group, n=179 mitochondria were measured.Representative images and scatterplot of mitochondrial length (Tom20, red) in Flag+ or Flag- cells. The data were obtained from 3 independent biological experiments. Flag-: n=154 mitochondria; Flag+: n=132 mitochondria.Representative images and scatterplot showing that the number of p-Drp1 S616 puncta in Flag+ cells was greater than that in Flag- cells. The data were obtained from 3 independent biological experiments. Flag-: n=70 cells from 10 organoids; Flag+: n=55 cells from 10 organoids. The images were obtained using structured illumination microscopy (SIM).


Figure 3The mtDNA content was examined with primers of D - loop , mtCO1 , mtCO2 and mtND2 by qPCR in control or Flag - mtHSF1 - expressing striatal cells . Scatterplot with bar showed the relative percentage of mtDNA content . ( n = 3 biological replicates ) . Measurement of mitochondrial respiratory activity by Seahorse analysis . ( n = 9 - 10 biological replicates ) Cells were treated with EtBr for six days to delete mtDNA . mtDNA repopulation efficiency was measured by qPCR after withdrawal of EtBr . ( n = 3 biological replicates ) . D - E . Cell death was determined by detection of LDH release into the medium and cleavage of PARP . Scatterplot with bar showed the relative percentage of LDH release . ( n = 3 biological replicates ) . qPCR analysis of the mtDNA content of AAV - Con - injected mice or AAV - mtHSF1 - injected mice . Scatterplot with bar showed the relative percentage of mtDNA content ( n = 3 mice / group ) . Cleaved - Caspase3 in AAV - Con - or AAV - mtHSF1 - injected mice was detected by WB analysis . ( n = 5 mice / group ) . Representative images and scatterplot showing the number of mtDNA ( purple ) colocalized with mitochondria ( Tom20 , green ) in striatal organoids injected with AAV - Con or AAV - mtHSF1 . The data were obtained from 3 independent biological experiments . Con : n = 65 cells from 20 organoids ; Flag - mtHSF1 : n = 70 cells from 20 organoids . Two - tailed unpaired t - test was used . Images were captured by Structured Illumination Microscopy ( SIM ) . The typical nuclear fragmentation morphology of AAV - Con - ( GFP ) or AAV - mtHSF1 - injected striatal organoids was measured . Scatterplot showed the quantitative result of nuclear fragmentation . The arrows ( yellow ) mark fragmented nuclei . Two - tailed unpaired t - test was used . The data were obtained from 3 independent biological experiments . Con : n = 14 organoids ; Flag - mtHSF1 : n = 15 organoids .

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Figure 3The mtDNA content was examined with primers of D-loop, mtCO1, mtCO2 and mtND2 by qPCR in control or Flag-mtHSF1-expressing striatal cells. Scatterplot with bar showed the relative percentage of mtDNA content. (n=3 biological replicates).Measurement of mitochondrial respiratory activity by Seahorse analysis. (n=9-10 biological replicates)Cells were treated with EtBr for six days to delete mtDNA. mtDNA repopulation efficiency was measured by qPCR after withdrawal of EtBr. (n=3 biological replicates).D-E. Cell death was determined by detection of LDH release into the medium and cleavage of PARP. Scatterplot with bar showed the relative percentage of LDH release. (n=3 biological replicates).qPCR analysis of the mtDNA content of AAV-Con-injected mice or AAV-mtHSF1-injected mice. Scatterplot with bar showed the relative percentage of mtDNA content (n=3 mice/group).Cleaved-Caspase3 in AAV-Con- or AAV-mtHSF1-injected mice was detected by WB analysis. (n=5 mice/group).Representative images and scatterplot showing the number of mtDNA (purple) colocalized with mitochondria (Tom20, green) in striatal organoids injected with AAV-Con or AAV-mtHSF1. The data were obtained from 3 independent biological experiments. Con: n=65 cells from 20 organoids; Flag-mtHSF1: n=70 cells from 20 organoids. Two-tailed unpaired t-test was used. Images were captured by Structured Illumination Microscopy (SIM).The typical nuclear fragmentation morphology of AAV-Con- (GFP) or AAV-mtHSF1-injected striatal organoids was measured. Scatterplot showed the quantitative result of nuclear fragmentation. The arrows (yellow) mark fragmented nuclei. Two-tailed unpaired t-test was used. The data were obtained from 3 independent biological experiments. Con: n=14 organoids; Flag-mtHSF1: n=15 organoids.


Figure 4Total cell lysates were subjected to immunoprecipitation with an anti - HSF1 antibody followed by WB analysis . ( n = 3 biological replicates ) . HdhQ7 or HdhQ111 cell extracts were incubated with DSS ( 1 mM ) for 30 min at RT . SSBP1 oligomers were detected by immunoblotting with an anti - SSBP1 antibody . ( n = 3 biological replicates ) . SSBP1 oligomers in control and patient fibroblasts ( GM04208 and GM21756 ) were analyzed . ( n = 3 biological replicates ) . SSBP1 oligomers were measured in WT mice or YAC128 mice . ( n = 3 mice / group ) . Cells were transfected with an empty vector or Flag - mtHSF1 for 48 h . The total cell lysates were harvested and crosslinked with DSS . SSBP1 oligomers were tested by immunoblotting . ( n = 4 biological replicates ) . Flag - mtHSF1 was expressed in the striata of WT mice for three weeks . SSBP1 oligomers were determined by immunoblotting . ( n = 5 mice / group ) . Flag - mtHSF1 or sh - SSBP1 was delivered into cells via lentiviral infection . The expression of SSBP1 and mtHSF1 was detected by WB analysis . The mtDNA content was examined by qPCR analysis . ( n = 3 biological replicates ) .

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Figure 4Total cell lysates were subjected to immunoprecipitation with an anti-HSF1 antibody followed by WB analysis. (n=3 biological replicates).HdhQ7 or HdhQ111 cell extracts were incubated with DSS (1 mM) for 30 min at RT. SSBP1 oligomers were detected by immunoblotting with an anti-SSBP1 antibody. (n=3 biological replicates).SSBP1 oligomers in control and patient fibroblasts (GM04208 and GM21756) were analyzed. (n=3 biological replicates).SSBP1 oligomers were measured in WT mice or YAC128 mice. (n=3 mice/group).Cells were transfected with an empty vector or Flag-mtHSF1 for 48 h. The total cell lysates were harvested and crosslinked with DSS. SSBP1 oligomers were tested by immunoblotting. (n=4 biological replicates).Flag-mtHSF1 was expressed in the striata of WT mice for three weeks. SSBP1 oligomers were determined by immunoblotting. (n=5 mice/group).Flag-mtHSF1 or sh-SSBP1 was delivered into cells via lentiviral infection. The expression of SSBP1 and mtHSF1 was detected by WB analysis. The mtDNA content was examined by qPCR analysis. (n=3 biological replicates).


Figure 5Primary neurons expressing Flag - mtHSF1 were stained with anti - DARPP32 ( green ) and anti - Flag ( red ) antibodies . The arrows ( white ) mark Flag + or Flag - primary neurons . The neurite length of Flag + or Flag - neurons was measured by Image J and showed by scatterplot . The scale bar represents 50 µm . ( n = 155 for Flag - group and n = 154 for Flag + group ) . Human striatal organoids were injected with AAV - Con ( GFP ) or AAV - mtHSF1 at D42 . Scatterplot showed the percentage of NeuN + cells ( red ) among Flag + or GFP + cells ( green ) after injection for 2 weeks . The data were obtained from 3 independent biological experiments . Con : n = 11 organoids ; Flag - mtHSF1 : n = 13 organoids . The scale bar represents 100 µm . Representative images of immunostaining with DARPP32 ( red ) and Flag ( green ) . The DARPP32 fluorescence density was analyzed in Flag + or Flag - sections by confocal microscopy and showed by scatterplot . Thickness : 30 µm . The scale bar is 2 mm , and the enlarged image scale bar is 40 µm . ( n = 12 mice / group ) . The striatum of AAV - Con - or AAV - mtHSF1 - injected mice were harvested . The DARPP32 protein levels were tested by WB analysis . ( n = 6 mice / group ) . The percentage of NeuN + cells in Flag + or Flag - sections was determined by confocal microscopy and showed by scatterplot . Thickness : 10 µm . ( n = 9 mice / group ) . Mouse weight and motor activity were recorded post AAV injection . ( n = 7 - 13 mice / group ) .

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Figure 5Primary neurons expressing Flag-mtHSF1 were stained with anti-DARPP32 (green) and anti-Flag (red) antibodies. The arrows (white) mark Flag+ or Flag- primary neurons. The neurite length of Flag+ or Flag- neurons was measured by Image J and showed by scatterplot. The scale bar represents 50 µm. (n = 155 for Flag- group and n = 154 for Flag+ group).Human striatal organoids were injected with AAV-Con (GFP) or AAV-mtHSF1 at D42. Scatterplot showed the percentage of NeuN+ cells (red) among Flag+ or GFP+ cells (green) after injection for 2 weeks. The data were obtained from 3 independent biological experiments. Con: n=11 organoids; Flag-mtHSF1: n=13 organoids. The scale bar represents 100 µm.Representative images of immunostaining with DARPP32 (red) and Flag (green). The DARPP32 fluorescence density was analyzed in Flag+ or Flag- sections by confocal microscopy and showed by scatterplot. Thickness: 30 µm. The scale bar is 2 mm, and the enlarged image scale bar is 40 µm. (n=12 mice/group).The striatum of AAV-Con- or AAV-mtHSF1-injected mice were harvested. The DARPP32 protein levels were tested by WB analysis. (n=6 mice/group).The percentage of NeuN+ cells in Flag+ or Flag- sections was determined by confocal microscopy and showed by scatterplot. Thickness: 10 µm. (n=9 mice/group).Mouse weight and motor activity were recorded post AAV injection. (n=7-13 mice/group).


Figure 6Total lysates of HdhQ7 or HdhQ111 cells were subjected to immunoprecipitation with an anti - HSF1 antibody followed by immunoblotting . ( n = 3 biological replicates ) . Drp1 and HSF1 binding in vitro was analyzed by WB analysis . ( n = 3 biological replicates ) . Mitochondrial fractions were isolated from WT or Drp1 - KO MEFs . The protein levels of Drp1 and HSF1 were tested by immunoblotting . ( n = 3 biological replicates ) . GST pulldown assay of Drp1 and HSF1 in the presence or absence of DH1 . ( n = 3 biological replicates ) . HdhQ7 or HdhQ111 cells ( 1 μM / day , 3 days ) , were treated with DH1 or TAT . mtHSF1 was measured by immunoblotting . ( n = 3 - 5 biological replicates ) . H . YAC128 or age - matched littermates ( 3 mg / kg / day , 3 months ) , were treated with DH1 or TAT . mtHSF1 was measured by immunoblotting . ( n = 3 - 5 biological replicates ) . I . striatal organoids differentiated from patient iPSCs or control iPSCs ( 1 μM / day , 7 days ) were treated with DH1 or TAT . mtHSF1 was measured by immunoblotting . ( n = 3 - 5 biological replicates ) . J . patient fibroblasts ( 1 μM / day , 5 days ) were treated with DH1 or TAT . mtHSF1 was measured by immunoblotting . ( n = 3 - 5 biological replicates ) . HdhQ7 or HdhQ111 were treated with DH1 or TAT ( 1 μM / day , 3 days ) . SSBP1 oligomers were analyzed by immunoblotting . ( n = 3 - 6 biological replicates ) . L . patient fibroblasts were treated with DH1 or TAT ( 1 μM / day , 3 days ) . SSBP1 oligomers were analyzed by immunoblotting . ( n = 3 - 6 biological replicates ) .

O B-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Total lysates of HdhQ7 or HdhQ111 cells were subjected to immunoprecipitation with an anti-HSF1 antibody followed by immunoblotting. (n=3 biological replicates).Drp1 and HSF1 binding in vitro was analyzed by WB analysis. (n=3 biological replicates).Mitochondrial fractions were isolated from WT or Drp1-KO MEFs. The protein levels of Drp1 and HSF1 were tested by immunoblotting. (n=3 biological replicates).GST pulldown assay of Drp1 and HSF1 in the presence or absence of DH1. (n=3 biological replicates).HdhQ7 or HdhQ111 cells (1 μM/day, 3 days), were treated with DH1 or TAT. mtHSF1 was measured by immunoblotting. (n=3-5 biological replicates).H. YAC128 or age-matched littermates (3 mg/kg/day, 3 months), were treated with DH1 or TAT. mtHSF1 was measured by immunoblotting. (n=3-5 biological replicates).I. striatal organoids differentiated from patient iPSCs or control iPSCs (1 μM/day, 7 days) were treated with DH1 or TAT. mtHSF1 was measured by immunoblotting. (n=3-5 biological replicates).J. patient fibroblasts (1 μM/day, 5 days) were treated with DH1 or TAT. mtHSF1 was measured by immunoblotting. (n=3-5 biological replicates).HdhQ7 or HdhQ111 were treated with DH1 or TAT (1 μM/day, 3 days). SSBP1 oligomers were analyzed by immunoblotting. (n=3-6 biological replicates).L. patient fibroblasts were treated with DH1 or TAT (1 μM/day, 3 days). SSBP1 oligomers were analyzed by immunoblotting. (n=3-6 biological replicates).


Figure 7Immunostaining for mitochondria ( Tom20 , green ) and mtDNA ( anti - DNA , red ) in striatal organoids treated with DH1 or TAT at D60 . Scatterplots with bar showed the length of mitochondria and the number of mtDNA colocalized with mitochondria . The data were obtained from 3 independent biological experiments . Left , n = 222 - 311 mitochondria per group ; Right , n = 14 - 20 organoids per group . Images were taken using Structured Illumination Microscopy ( SIM ) . The scale bar represents 10 μm . Immunostaining for mitochondria ( Tom20 , red ) and p - Drp1 S616 ( green ) in striatal treated with DH1 or TAT at D60 . Scatterplot with bar showed the number of p - Drp1 S616 per cell in organoids . The data were obtained from 3 independent biological experiments . n = 29 - 34 cells from at least 10 organoids . The scale bar represents 5 μm . Scatterplot with bar showed the ratio of MSNs ( DARPP32 , green ) in striatal organoids in the presence or absence of DH1 . The data were obtained from 3 independent biological experiments . n = 30 organoids per group . Representative tracing images of DARPP32 + neurons treated with DH1 or TAT at D45 . Scatterplot with bar showed the results of longest neurites length . The data were obtained from 3 independent biological experiments . n = 45 - 92 neurons per group . Representative images and scatterplot with bar of nuclear fragmentation in striatal organoids treated with DH1 . The arrows ( white ) mark fragmented nuclei . The data were obtained from 3 independent biological experiments . n = 7 - 10 organoids per group . The scale bar represents 20 μm . H . PGC1α protein levels were determined by immunoblotting . ( n = 3 biological replicates ) . I . DARPP32 protein levels were determined by immunoblotting . ( n = 3 biological replicates ) . Mouse behavior was tested after DH1 treatment . ( n = 9 - 12 mice / group ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O

Figure 7Immunostaining for mitochondria (Tom20, green) and mtDNA (anti-DNA, red) in striatal organoids treated with DH1 or TAT at D60. Scatterplots with bar showed the length of mitochondria and the number of mtDNA colocalized with mitochondria. The data were obtained from 3 independent biological experiments. Left, n=222-311 mitochondria per group; Right, n=14-20 organoids per group. Images were taken using Structured Illumination Microscopy (SIM). The scale bar represents 10 μm.Immunostaining for mitochondria (Tom20, red) and p-Drp1 S616 (green) in striatal  treated with DH1 or TAT at D60. Scatterplot with bar showed the number of p-Drp1 S616 per cell in organoids. The data were obtained from 3 independent biological experiments. n=29-34 cells from at least 10 organoids. The scale bar represents 5 μm.Scatterplot with bar showed the ratio of MSNs (DARPP32, green) in striatal organoids in the presence or absence of DH1. The data were obtained from 3 independent biological experiments. n=30 organoids per group.Representative tracing images of DARPP32+ neurons treated with DH1 or TAT at D45. Scatterplot with bar showed the results of longest neurites length. The data were obtained from 3 independent biological experiments. n=45-92 neurons per group.Representative images and scatterplot with bar of nuclear fragmentation in striatal organoids treated with DH1. The arrows (white) mark fragmented nuclei. The data were obtained from 3 independent biological experiments. n=7-10 organoids per group. The scale bar represents 20 μm.H. PGC1α protein levels were determined by immunoblotting. (n=3 biological replicates).I. DARPP32 protein levels were determined by immunoblotting. (n=3 biological replicates).Mouse behavior was tested after DH1 treatment. (n=9-12 mice/group).


Fig . 1 b In HEK293 cells , recombinantly expressed GluN1 / GluN2A and GluN1 / GluN2A ( N615S ) channels were activated by fast glutamate application ( 1 mM ; in the continuous presence of the co - agonist glycine , 10 µM ) at holding potentials from - 100 to + 40 mV in different extracellular Mg2 + concentrations . NMDAR - mediated peak currents were normalized to those obtained at + 40 mV . Data points represent mean ± SEM for n = 4 - 7 different HEK293 cells . Representative current traces evoked in 0 mM Mg2 + at - 60 mV , with 20 and 600 ms applications , are shown below the IV plots and were used to determine the current kinetics ( Supplementary Table 1 ) . d Reverse transcription PCR ( RT - PCR ) - sequence analyses of total brain mRNA show the A - to - G mutation in pos . 0 and two diagnostic silent mutations at pos . - 11 and - 14 in the pore loop encoding gene segment in Grin2a + / + , Grin2aS / S , Grin2a + / S mice . In Grin2a + / S mice , the overlay of two different colored " nucleotide " peaks , at position 0 , - 11 , and - 14 indicate equimolar amounts of mRNA from the Grin2a + and the targeted Grin2aS alleles . e Immunoblots of forebrain protein lysates of 4 - week - old mice ( Supplementary Fig . 2 ) indicate no genotype - specific differences of GluN1 , GluN2A , and the AMPAR subunit GluA1 expression relative to the β - actin levels ( p > 0 . 05 ) . The levels of PSD95 and αCaMKII ( in its phosphorylated state , pCaMKII ) are also comparable between genotypes relative to the GAPDH expression . The GluN2B expression level in the membrane fraction was significantly increased in Grin2aS / S mice when compared to Grin2a + / + and Grin2a + / S mice but not in the levels of total protein lysates ( for statistics : Supplementary Statistics to Fig . 1 ) . The number of mice is given in brackets . Error bars represent mean ± SEM .

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

Fig. 1 b In HEK293 cells, recombinantly expressed GluN1/GluN2A and GluN1/GluN2A(N615S) channels were activated by fast glutamate application (1 mM; in the continuous presence of the co-agonist glycine, 10 µM) at holding potentials from -100 to +40 mV in different extracellular Mg2+ concentrations. NMDAR-mediated peak currents were normalized to those obtained at +40 mV. Data points represent mean ± SEM for n = 4-7 different HEK293 cells. Representative current traces evoked in 0 mM Mg2+ at - 60 mV, with 20 and 600 ms applications, are shown below the IV plots and were used to determine the current kinetics (Supplementary Table 1).  d Reverse transcription PCR (RT-PCR)-sequence analyses of total brain mRNA show the A-to-G mutation in pos. 0 and two diagnostic silent mutations at pos. -11 and -14 in the pore loop encoding gene segment in Grin2a+/+, Grin2aS/S, Grin2a+/S mice. In Grin2a+/S mice, the overlay of two different colored "nucleotide" peaks, at position 0, -11, and -14 indicate equimolar amounts of mRNA from the Grin2a+ and the targeted Grin2aS alleles.  e Immunoblots of forebrain protein lysates of 4-week-old mice (Supplementary Fig. 2) indicate no genotype-specific differences of GluN1, GluN2A, and the AMPAR subunit GluA1 expression relative to the β-actin levels (p > 0.05). The levels of PSD95 and αCaMKII (in its phosphorylated state, pCaMKII) are also comparable between genotypes relative to the GAPDH expression. The GluN2B expression level in the membrane fraction was significantly increased in Grin2aS/S mice when compared to Grin2a+/+ and Grin2a+/S mice but not in the levels of total protein lysates (for statistics: Supplementary Statistics to Fig. 1). The number of mice is given in brackets. Error bars represent mean ± SEM.


Fig . 2 a ( left ) In the absence of extracellular Mg2 + the synaptic AMPA / NMDA ratio of CA1 pyramidal cells in acute hippocampal slices is not altered in Grin2a + / S and Grin2aS / S mice compared to control littermates . ( right ) In the presence of extracellular Mg2 + the strong reduction of the AMPA / NMDA ratio in Grin2aS / S mice relates to the increased NMDA currents at CA1 synapses ( Tukey ' s test ) . Example traces are depicted to the right of each bar graph . Data from the same experimental group were pooled across animals and are presented as mean ± SEM ( see also ref . 35 ) with p < 0 . 05 being designated as statistically significant . Numbers in bar graphs indicate the number of slices . b Paired - pulse facilitation at CA3 - to - CA1 synapses excluded strong alterations of presynaptic function in mutant mice . The stimulation strengths ( in nC ) necessary to elicit a pre - volley of 1 . 0 and 1 . 5 mV and the resulting fEPSP amplitudes were comparable in all genotypes but showed only a trend towards lower fEPSP amplitudes recorded at 1 . 5 mV pre - volley amplitudes in Grin2aS / S mice . The fEPSP amplitudes necessary to elicit a just detectable population spike ( 1 ) and a population spike of 2 mV amplitude ( 2 ) and the paired - pulse facilitation ratio ( PPF ) at an interstimulus interval of 50 ms did not indicate any synaptic impairments in Grin2aS / S and Grin2a + / S mice compared to WT littermates . The number of slices is indicated in the bar graphs or in brackets . c Field LTP ( fLTP ) at CA3 - to - CA1 synapses , induced by tetanic stimulation in slices ( 1 s ; 100 Hz ; arrow ) , was comparable in all three genotypes . The inset in c gives a schematic view of the stimulating ( white arrow ) and the recording ( green arrow ) electrode positions in str . radiatum ( r ) and str . oriens ( o ) . d Similarly , CA1 - to - CA3 fLTP ( induction : 2 × 1 s ; 100 Hz ; arrow ) in freely moving mice was comparable between Grin2aS / S and Grin2a + / + mice . The inset shows a Nissl - stained slice of one recorded mouse post mortem . e In mutant mice , but not in control littermates , the GluN2B - containing NMDAR contributes significantly to the magnitude of LTP , since LTP was significantly reduced by the GluN2B - specific antagonist CP101 , 106 ( CP ) . Recordings of the non - tetanized control pathway in c and e are given as dashed lines . Error bars represent mean ± SEM ( for statistics : Supplementary Statistics to Fig . 2 ) .

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

Fig. 2 a (left) In the absence of extracellular Mg2+ the synaptic AMPA/NMDA ratio of CA1 pyramidal cells in acute hippocampal slices is not altered in Grin2a+/S and Grin2aS/S mice compared to control littermates. (right) In the presence of extracellular Mg2+ the strong reduction of the AMPA/NMDA ratio in Grin2aS/S mice relates to the increased NMDA currents at CA1 synapses (Tukey's test). Example traces are depicted to the right of each bar graph. Data from the same experimental group were pooled across animals and are presented as mean ± SEM (see also ref. 35) with p < 0.05 being designated as statistically significant. Numbers in bar graphs indicate the number of slices.  b Paired-pulse facilitation at CA3-to-CA1 synapses excluded strong alterations of presynaptic function in mutant mice. The stimulation strengths (in nC) necessary to elicit a pre-volley of 1.0 and 1.5 mV and the resulting fEPSP amplitudes were comparable in all genotypes but showed only a trend towards lower fEPSP amplitudes recorded at 1.5 mV pre-volley amplitudes in Grin2aS/S mice. The fEPSP amplitudes necessary to elicit a just detectable population spike (1) and a population spike of 2 mV amplitude (2) and the paired-pulse facilitation ratio (PPF) at an interstimulus interval of 50 ms did not indicate any synaptic impairments in Grin2aS/S and Grin2a+/S mice compared to WT littermates. The number of slices is indicated in the bar graphs or in brackets.  c Field LTP (fLTP) at CA3-to-CA1 synapses, induced by tetanic stimulation in slices (1 s; 100 Hz; arrow), was comparable in all three genotypes. The inset in c gives a schematic view of the stimulating (white arrow) and the recording (green arrow) electrode positions in str. radiatum (r) and str. oriens (o). d Similarly, CA1-to-CA3 fLTP (induction: 2 × 1 s; 100 Hz; arrow) in freely moving mice was comparable between Grin2aS/S and Grin2a+/+ mice. The inset shows a Nissl-stained slice of one recorded mouse post mortem. e In mutant mice, but not in control littermates, the GluN2B-containing NMDAR contributes significantly to the magnitude of LTP, since LTP was significantly reduced by the GluN2B-specific antagonist CP101,106 (CP). Recordings of the non-tetanized control pathway in c and e are given as dashed lines. Error bars represent mean ± SEM (for statistics: Supplementary Statistics to Fig. 2).


Fig . 3 aGrin2aS / S mice had a reduced body weight ( by 18 % in adults ) , significantly lower score in nesting and in overnight burrowing activity ( filled circles : 30 - week - old mice , white dots : 18 - week - old mice ) . b Grin2aS / S mice showed the clasping reflex . c During the 12 h dark phase on the Lafayette running wheel the running distance is reduced in Grin2aS / S mice . d Nissl staining revealed unaltered cell density throughout the brain ( top ) and layering of hippocampal subfields , including CA1 ( bottom ) , in Grin2aS / S mice compared to Grin2a + / + . Error bars represent mean ± SEM . e In the TUNEL assay no apoptotic cells could be detected in the hippocampus of Grin2aS / S mice , compared to the staining of the nuclease - treated positive control slice . f No aberrant mossy fiber sprouting in either genotype in the dentate gyrus inner ( IML ) and outer molecular layer ( OML ) can be found by Timm staining of coronal sections . g The distribution of NeuN - positive neurons and of GFAP - positive glial cells are indistinguishable between Grin2aS / S and Grin2a + / + mice . Mossy fiber projections visualized by anti - calbindin ( CB ) staining , as well as numbers of parvalbumin ( PV ) - positive interneurons are similar between both genotypes . Anti - GluA1 immunosignal in all hippocampal layers is comparable between brain sections of Grin2aS / S mice and control littermates . CA1 cornu ammonis region 1 , CA3 cornu ammonis region 3 , DG dentate gyrus , mol stratum moleculare , ori stratum oriens , rad , stratum radiatum . Scale bars in d - g are in mm . The number of animals is given below the bars . For the Nissl stain , Tunnel test and Timm stain 3 mice were used per genotype . For the immunohistological analysis of glial fibrillary acidic protein ( GFAP ) , neuronal nuclear antigen ( NeuN ) , Calbindin ( CB ) , and Parvalbumin ( PV ) five mice and for the GluA1 immunofluorescence stain , three mice were used ( for statistics : Supplementary Statistics to Fig . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fig. 3 aGrin2aS/S mice had a reduced body weight (by 18% in adults), significantly lower score in nesting and in overnight burrowing activity (filled circles: 30-week-old mice, white dots: 18-week-old mice).  b Grin2aS/S mice showed the clasping reflex. c During the 12 h dark phase on the Lafayette running wheel the running distance is reduced in Grin2aS/S mice.  d Nissl staining revealed unaltered cell density throughout the brain (top) and layering of hippocampal subfields, including CA1 (bottom), in Grin2aS/S mice compared to Grin2a+/+. Error bars represent mean ± SEM. e In the TUNEL assay no apoptotic cells could be detected in the hippocampus of Grin2aS/S mice, compared to the staining of the nuclease-treated positive control slice. f No aberrant mossy fiber sprouting in either genotype in the dentate gyrus inner (IML) and outer molecular layer (OML) can be found by Timm staining of coronal sections.  g The distribution of NeuN-positive neurons and of GFAP-positive glial cells are indistinguishable between Grin2aS/S and Grin2a+/+ mice. Mossy fiber projections visualized by anti-calbindin (CB) staining, as well as numbers of parvalbumin (PV)-positive interneurons are similar between both genotypes. Anti-GluA1 immunosignal in all hippocampal layers is comparable between brain sections of Grin2aS/S mice and control littermates. CA1 cornu ammonis region 1, CA3 cornu ammonis region 3, DG dentate gyrus, mol stratum moleculare, ori stratum oriens, rad, stratum radiatum. Scale bars in d-g are in mm. The number of animals is given below the bars. For the Nissl stain, Tunnel test and Timm stain 3 mice were used per genotype. For the immunohistological analysis of glial fibrillary acidic protein (GFAP), neuronal nuclear antigen (NeuN), Calbindin (CB), and Parvalbumin (PV) five mice and for the GluA1 immunofluorescence stain, three mice were used (for statistics: Supplementary Statistics to Fig. 3).


Fig . 4 a Mortality curve showing that none of the Grin2a + / + controls were affected , while audiogenic seizures ( AGSs ) followed by respiratory arrest ( RA ) were induced in all Grin2aS / S mice and in a subset of heterozygous Grin2a + / S mutants during the 11 kHz tone exposure [ 4 repetitions × 20 s tone , 2 s brake ; pink squares ; p < 0 . 0001 by Log - rank ( Mantel - Cox ) test ] . b The c - Fos immunoreactivity was specifically increased in the hypothalamus ( VMH ) , the inferior colliculus ( IC ) , and periaqueductal gray ( PAG ) but not in the hippocampus ( HPC ) of a resuscitated Grin2aS / S mouse 90 min after AGS when compared to tone - exposed Grin2a + / + littermates . c Memantine i . p . injection in Grin2aS / S mice , 3 h before tone exposure , rescued AGS susceptibility in Grin2aS / S mice . The rescue effect could still be observed in four out of eight , and in two out of eight , mice 27 and 51 h after memantine treatment , respectively . d Increased c - Fos immunofluorescence ( in grayscale ) in the medial amygdala ( MeA ) and the VMH of a Grin2aS / S animal with AGS ( saline injection ) compared to a memantine rescued Grin2aS / S littermate . In the paraventricular nucleus of the thalamus ( PVT ) , the hippocampus ( HPC ) and cortex ( Cx ) there was no difference in c - Fos expression between memantine - injected and saline - injected animals ( for fluorescence images , see Supplementary Fig . 5 ) . e As in Fig . 4c but now MK - 801 and saline are used for pre - treatment . The MK - 801 effect was longer lasting compared to memantine and one out of four animals showed resistance to tone exposure even 4 days after MK - 801 injection . f Decreased c - Fos DAB immunosignal in an MK - 801 AGS - rescued Grin2aS / S animal ( bottom ) in the medial amygdala ( MeA ) , the VMH and the PVT when compared to saline - injected Grin2aS / S littermate ( top ) . Scale bars are in mm ( for statistics : Supplementary Statistics to Fig . 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fig. 4 a Mortality curve showing that none of the Grin2a+/+ controls were affected, while audiogenic seizures (AGSs) followed by respiratory arrest (RA) were induced in all Grin2aS/S mice and in a subset of heterozygous Grin2a+/S mutants during the 11 kHz tone exposure [4 repetitions × 20 s tone, 2 s brake; pink squares; p < 0.0001 by Log-rank (Mantel-Cox) test]. b The c-Fos immunoreactivity was specifically increased in the hypothalamus (VMH), the inferior colliculus (IC), and periaqueductal gray (PAG) but not in the hippocampus (HPC) of a resuscitated Grin2aS/S mouse 90 min after AGS when compared to tone-exposed Grin2a+/+ littermates. c Memantine i.p. injection in Grin2aS/S mice, 3 h before tone exposure, rescued AGS susceptibility in Grin2aS/S mice. The rescue effect could still be observed in four out of eight, and in two out of eight, mice 27 and 51 h after memantine treatment, respectively.  d Increased c-Fos immunofluorescence (in grayscale) in the medial amygdala (MeA) and the VMH of a Grin2aS/S animal with AGS (saline injection) compared to a memantine rescued Grin2aS/S littermate. In the paraventricular nucleus of the thalamus (PVT), the hippocampus (HPC) and cortex (Cx) there was no difference in c-Fos expression between memantine-injected and saline-injected animals (for fluorescence images, see Supplementary Fig. 5). e As in Fig. 4c but now MK-801 and saline are used for pre-treatment. The MK-801 effect was longer lasting compared to memantine and one out of four animals showed resistance to tone exposure even 4 days after MK-801 injection. f Decreased c-Fos DAB immunosignal in an MK-801 AGS-rescued Grin2aS/S animal (bottom) in the medial amygdala (MeA), the VMH and the PVT when compared to saline-injected Grin2aS/S littermate (top). Scale bars are in mm (for statistics: Supplementary Statistics to Fig. 4).


Fig . 5 C - Fos expression 120 min after i . p . injection of MK - 801 . a C - Fos DAB immunosignals are depicted for one representative brain section of the different genotypes as indicated . The original images , including the cortical regions and results of PBS injected animals , are presented in Supplementary Fig . 6 . The number of mice analyzed is given in brackets . b For quantitative comparisons the total numbers of c - Fos - positive cells in the cellular layers of the hippocampus were counted . C - Fos - positive cells in extra - hippocampal regions are given per mm2 . Slices from six mice per genotype were used for the statistical analyses ( two - tailed t - tests ) and error bars represent mean ± SEM . CA1 and CA3 cornu ammonis regions 1 and 3 , DG dentate gyrus , PC piriform cortex , RSC retrosplenial cortex , Neo neocortex , MTN midline thalamic nuclei , BLA basolateral amygdala . All scale bars : 0 . 2 mm .

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

Fig. 5 C-Fos expression 120 min after i.p. injection of MK-801. a C-Fos DAB immunosignals are depicted for one representative brain section of the different genotypes as indicated. The original images, including the cortical regions and results of PBS injected animals, are presented in Supplementary Fig. 6. The number of mice analyzed is given in brackets. b For quantitative comparisons the total numbers of c-Fos-positive cells in the cellular layers of the hippocampus were counted. C-Fos-positive cells in extra-hippocampal regions are given per mm2. Slices from six mice per genotype were used for the statistical analyses (two-tailed t-tests) and error bars represent mean ± SEM. CA1 and CA3 cornu ammonis regions 1 and 3, DG dentate gyrus, PC piriform cortex, RSC retrosplenial cortex, Neo neocortex, MTN midline thalamic nuclei, BLA basolateral amygdala. All scale bars: 0.2 mm.


Fig . 6 a ( left ) Peak frequencies during exploration and rapid eye movement ( REM ) sleep for theta ( 0 - 15 Hz , left diagrams ) and gamma oscillations ( 15 - 200 Hz , right diagrams ) in all genotypes . During exploration , the average theta range peak frequencies were at 8 . 61 ± 0 . 07 and 8 . 75 ± 0 . 28 Hz for Grin2a + / + mice and Grin2a + / S mice , respectively . In Grin2aS / S mice the theta range peak frequency was significantly slower , measuring 7 . 43 ± 0 . 24 Hz ( Kruskal Wallis test , p = 0 . 0159 ) . During REM sleep the average frequency ranges of Grin2a + / + and Grin2a + / S mice were around 7 . 00 ± 0 . 20 Hz whereas Grin2aS / S mice had a lower but not significantly different range 6 . 71 ± 0 . 22 Hz . a ( right ) No significant differences on the low and high gamma frequency bands - ranges between 20 - 100 Hz and 100 - 200 Hz , respectively111 - could be detected between genotypes , neither during exploration nor in REM sleep . The number of mice used in this experiment are given with the name of the genotypes . b ( left ) Grin2aS / S and Grin2a + / S mice show reduced theta - gamma phase - amplitude coupling as determined by the modulation index ( MI ) in the low gamma component in CA1 ( white arrowheads ) during awake and REM sleep states . b ( right ) For Grin2aS / S the MI value dropped significantly during exploration and REM sleep although the MI reduction reached significance only during exploration ( one - way ANOVA , Kruskal - Wallis test ) . Numbers below bars of bar graphs indicate the number of animals . Error bars represent mean ± SEM .

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

Fig. 6 a (left) Peak frequencies during exploration and rapid eye movement (REM) sleep for theta (0-15 Hz, left diagrams) and gamma oscillations (15-200 Hz, right diagrams) in all genotypes. During exploration, the average theta range peak frequencies were at 8.61 ± 0.07 and 8.75 ± 0.28 Hz for Grin2a+/+ mice and Grin2a+/S mice, respectively. In Grin2aS/S mice the theta range peak frequency was significantly slower, measuring 7.43 ± 0.24 Hz (Kruskal Wallis test, p = 0.0159). During REM sleep the average frequency ranges of Grin2a+/+ and Grin2a+/S mice were around 7.00 ± 0.20 Hz whereas Grin2aS/S mice had a lower but not significantly different range 6.71 ± 0.22 Hz. a (right) No significant differences on the low and high gamma frequency bands-ranges between 20-100 Hz and 100-200 Hz, respectively111-could be detected between genotypes, neither during exploration nor in REM sleep. The number of mice used in this experiment are given with the name of the genotypes.  b (left) Grin2aS/S and Grin2a+/S mice show reduced theta-gamma phase-amplitude coupling as determined by the modulation index (MI) in the low gamma component in CA1 (white arrowheads) during awake and REM sleep states. b (right) For Grin2aS/S the MI value dropped significantly during exploration and REM sleep although the MI reduction reached significance only during exploration (one-way ANOVA, Kruskal-Wallis test). Numbers below bars of bar graphs indicate the number of animals. Error bars represent mean ± SEM.


Fig . 7 a The total locomotor activity during the entire 2 h session in a novel environment was enhanced in Grin2aS / S mice . b Similarly , the exploratory behavior of Grin2aS / S mice remained high in all three successive 6 min exposure sessions to five novel objects ( ITI = 4 min ) , as exemplified by traces of the animal ' s movement on the right . c ( left ) The time of first fall ( Cliff Avoidance Reflex , CAR ) was significantly reduced for Grin2aS / S and Grin2a + / S mice . c ( right ) In a 60 - min session Grin2aS / S mice showed a significantly increased number of falls compared to wild - type mice . d ( left ) In the three - chamber sociability test Grin2aS / S mutants had a significantly reduced preference to explore the stimulus mouse over the object compared to Grin2a + / S and Grin2a + / + littermates . d ( right ) Occupancy heat maps in the three - chamber sociability test show a representative example of a Grin2aS / S mouse with a reduced preference for another mouse versus an inanimate object . The social preference is visible for Grin2a + / + and Grin2a + / S mice . The occupancy is color - coded separately for each group and translates to a % given in white numbers on the key for each genotype . f During the sample run in the Y - maze Grin2aS / S mice made significantly more total arm entries . In the test run , Grin2a + / + and Grins2a + / S showed a preference for the unexplored novel Y - maze arm , whereas Grin2aS / S mice showed a lack of novelty preference . The numbers of mice are shown in brackets or below the bars of bar graphs . Chance levels ( Ch . ) are indicated by dashed lines . Error bars represent mean ± SEM ( for statistics : Supplementary Statistics to Fig . 7 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fig. 7 a The total locomotor activity during the entire 2 h session in a novel environment was enhanced in Grin2aS/S mice.  b Similarly, the exploratory behavior of Grin2aS/S mice remained high in all three successive 6 min exposure sessions to five novel objects (ITI = 4 min), as exemplified by traces of the animal's movement on the right.  c (left) The time of first fall (Cliff Avoidance Reflex, CAR) was significantly reduced for Grin2aS/S and Grin2a+/S mice. c (right) In a 60-min session Grin2aS/S mice showed a significantly increased number of falls compared to wild-type mice. d (left) In the three-chamber sociability test Grin2aS/S mutants had a significantly reduced preference to explore the stimulus mouse over the object compared to Grin2a+/S and Grin2a+/+ littermates. d (right) Occupancy heat maps in the three-chamber sociability test show a representative example of a Grin2aS/S mouse with a reduced preference for another mouse versus an inanimate object. The social preference is visible for Grin2a+/+ and Grin2a+/S mice. The occupancy is color-coded separately for each group and translates to a % given in white numbers on the key for each genotype. f During the sample run in the Y-maze Grin2aS/S mice made significantly more total arm entries. In the test run, Grin2a+/+ and Grins2a+/S showed a preference for the unexplored novel Y-maze arm, whereas Grin2aS/S mice showed a lack of novelty preference. The numbers of mice are shown in brackets or below the bars of bar graphs. Chance levels (Ch.) are indicated by dashed lines. Error bars represent mean ± SEM (for statistics: Supplementary Statistics to Fig. 7).


Fig . 8 c Morris watermaze . In the standard Morris watermaze task ( left graph ) the path length to reach the hidden platform decreased across training blocks for Grin2a + / + and Grin2a + / S mice but not for Grin2aS / S mice . Two probe trials were conducted after 12 and 24 training trials ( P1 and P2 , respectively ) during which the platform was removed from the pool . At both P1 and P2 ( right bar graphs ) the Grin2aS / S mice failed to search for the platform in the target quadrant . AdjL adjacent left , Target fixed location of the hidden escape platform during acquisition , AdjR adjacent right , Opp opposite of target quadrant . Dashed lines indicate chance levels . d Rewarded alternation . ( left ) In the T - maze rewarded alternation task ( right ) spatial working memory performance was substantially impaired in Grin2aS / S mice . e Contiguous task . In the contiguous version of the conditional T - maze task ( with floor inserts covering the entire T - maze ; white Perspex versus gray wire mesh ) , Grin2a + / + and Grin2aS / S mice were able to associate a particular floor insert with the location of the reward in either the left or the right goal arm . f Discontiguous task . Separate groups of mice were trained in the discontiguous version of this conditional task , in which the floor insert cues were now limited to the start arm only . Grin2a + / + mice readily acquired the task , but Grin2aS / S mice failed to learn . The numbers of mice are shown in brackets or below the bars of bar graphs . Chance levels ( Ch . ) are indicated by dashed lines . Error bars represent mean ± SEM ( for statistics : Supplementary Statistics to Fig . 8 ) .

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

Fig. 8 c Morris watermaze. In the standard Morris watermaze task (left graph) the path length to reach the hidden platform decreased across training blocks for Grin2a+/+ and Grin2a+/S mice but not for Grin2aS/S mice. Two probe trials were conducted after 12 and 24 training trials (P1 and P2, respectively) during which the platform was removed from the pool. At both P1 and P2 (right bar graphs) the Grin2aS/S mice failed to search for the platform in the target quadrant. AdjL adjacent left, Target fixed location of the hidden escape platform during acquisition, AdjR adjacent right, Opp opposite of target quadrant. Dashed lines indicate chance levels.  d Rewarded alternation. (left) In the T-maze rewarded alternation task (right) spatial working memory performance was substantially impaired in Grin2aS/S mice.  e Contiguous task. In the contiguous version of the conditional T-maze task (with floor inserts covering the entire T-maze; white Perspex versus gray wire mesh), Grin2a+/+ and Grin2aS/S mice were able to associate a particular floor insert with the location of the reward in either the left or the right goal arm.  f Discontiguous task. Separate groups of mice were trained in the discontiguous version of this conditional task, in which the floor insert cues were now limited to the start arm only. Grin2a+/+ mice readily acquired the task, but Grin2aS/S mice failed to learn. The numbers of mice are shown in brackets or below the bars of bar graphs. Chance levels (Ch.) are indicated by dashed lines. Error bars represent mean ± SEM (for statistics: Supplementary Statistics to Fig. 8).


Figure 1A , HeLa cells were treated with H2O2 ( 5 mM , 1 min ) or PR - 619 ( 10 µM , 10 min ) and analysed by immunoblotting for endogenous p62 , NDP52 and OPTN in either reducing ( 2 . 5 % β - mE ) or non - reducing conditions . Data information : # , non - specific band or a post - translational modification . B , NDP52 and OPTN are reducible by the thioredoxin antioxidant system . HeLa cells were pre - treated with TrxR inhibitors , curcumin ( 50 μM ) or auranofin ( 5 μM ) for 30 min , then treated with H2O2 ( 500 μM ) at different time points as indicated . Immunoblotting was performed to detect NDP52 , OPTN and PRX - 3 ( dimer formation reflecting oxidation of the protein ) in non - reduced conditions and PRX - SO3 ( marker of oxidative stress and actin in reduced conditions . C , D , Immunoblotting for NDP52 in either reducing or non - reducing conditions of whole cell samples ( C ) and mitochondrial fractions ( D ) from HeLa cells treated with the combination of 4 µM antimycin A1 and 10 µM oligomycin ( AO ) in the presence or absence of 400 nM bafilomycin A1 ( Baf ) for 2 h . NDP52 DLC in mitochondrial fraction was quantified ( D ) . Data are mean ± s . e . m ( D ) . P values were calculated by one - way ANOVA followed by Sidak test on four independent experiments ( D ) . * * * , p < 0 . 001 ; ns ( non - significant ) . Data information : # , non - specific band or a post - translational modification .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A, HeLa cells were treated with H2O2 (5 mM, 1 min) or PR-619 (10 µM, 10 min) and analysed by immunoblotting for endogenous p62, NDP52 and OPTN in either reducing (2.5% β-mE) or non-reducing conditions. Data information: #, non-specific band or a post-translational modification.B, NDP52 and OPTN are reducible by the thioredoxin antioxidant system. HeLa cells were pre-treated with TrxR inhibitors, curcumin (50 μM) or auranofin (5 μM) for 30 min, then treated with H2O2 (500 μM) at different time points as indicated. Immunoblotting was performed to detect NDP52, OPTN and PRX-3 (dimer formation reflecting oxidation of the protein) in non-reduced conditions and PRX-SO3 (marker of oxidative stress and actin in reduced conditions.C, D, Immunoblotting for NDP52 in either reducing or non-reducing conditions of whole cell samples (C) and mitochondrial fractions (D) from HeLa cells treated with the combination of 4 µM antimycin A1 and 10 µM oligomycin (AO) in the presence or absence of 400 nM bafilomycin A1 (Baf) for 2 h. NDP52 DLC in mitochondrial fraction was quantified (D). Data are mean ± s.e.m (D). P values were calculated by one-way ANOVA followed by Sidak test on four independent experiments (D). ***, p<0.001; ns (non-significant). Data information: #, non-specific band or a post-translational modification.


Figure 2A - D , Single ( A ) , double ( B ) , triple ( C ) , and quadruple ( D ) cysteine mutant NDP52 plasmids were generated and transfected into HeLa PentaKO cells . Cells were treated with 20 µM PR - 619 for 10 min and analysed by immunoblotting for NDP52 in either reducing or non - reducing conditions . The constructs highlighted in red were subjected to the next round of Cys - Ala screen or further analyses .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A-D, Single (A), double (B), triple (C), and quadruple (D) cysteine mutant NDP52 plasmids were generated and transfected into HeLa PentaKO cells. Cells were treated with 20 µM PR-619 for 10 min and analysed by immunoblotting for NDP52 in either reducing or non-reducing conditions. The constructs highlighted in red were subjected to the next round of Cys-Ala screen or further analyses.


Figure 3B , Recombinant proteins of NDP52 WT and C18 , 153 , 163 , 321S mutant ( Mut ) were exposed to the indicated concentrations of H2O2 for 5 min and subjected to SDS - PAGE and CBB gel staining . C , Analytical size - exclusion chromatography ( SEC ) of the NDP52 WT and Mut proteins . D , Recombinant NDP52 WT and Mut proteins ( 5 ng ) were subjected to NATIVE - PAGE analysis . S . E , short exposure ; L . E . long exposure .

O B-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B, Recombinant proteins of NDP52 WT and C18,153,163,321S mutant (Mut) were exposed to the indicated concentrations of H2O2 for 5 min and subjected to SDS-PAGE and CBB gel staining.C, Analytical size-exclusion chromatography (SEC) of the NDP52 WT and Mut proteins.D, Recombinant NDP52 WT and Mut proteins (5 ng) were subjected to NATIVE-PAGE analysis. S.E, short exposure; L.E. long exposure.