D ) Heatmap of LFQ intensity of proteins enriched at DNA baits . Telomere interactors are indicated on the right side of the heatmap . Color code indicates measured log2 LFQ intensities in the telomere pull - down . RNA binding proteins are marked with a black box on the left side of the figure . Abbreviations : LFQ : Label free quantification RBP : RNA binding

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

D) Heatmap of LFQ intensity of proteins enriched at DNA baits. Telomere interactors are indicated on the right side of the heatmap. Color code indicates measured log2 LFQ intensities in the telomere pull-down. RNA binding proteins are marked with a black box on the left side of the figure. Abbreviations: LFQ: Label free quantification RBP: RNA binding protein


E ) Critically short telomeres arise after 90 population doublings in the absence of telomerase ( tlc1 ) . Genomic DNA was extracted at indicated population doublings and digested with XhoI . A radioactive probe was used to recognize telomeric DNA in a southern blot . Abbreviations : PD : population doubling .

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E) Critically short telomeres arise after 90 population doublings in the absence of telomerase (tlc1). Genomic DNA was extracted at indicated population doublings and digested with XhoI. A radioactive probe was used to recognize telomeric DNA in a southern blot. Abbreviations: PD: population doubling.


F ) tlc1 cells reach senescence at PD 80 - 100 . % viability of indicated strains after propagation in YPD is indicated at different population doublings n = 3 . Asterisk indicates population doubling used in G ) to determine binding of Npl3 to short telomeres by TAP - ChIP . 3 independent spores from the indicated genotypes were used . Population doublings are counted from germination of meiotic products and hence begin at approx . PD40 . Abbreviations : TAP : tandem affinity purification

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F) tlc1 cells reach senescence at PD 80-100. %viability of indicated strains after propagation in YPD is indicated at different population doublings n=3. Asterisk indicates population doubling used in G) to determine binding of Npl3 to short telomeres by TAP-ChIP. 3 independent spores from the indicated genotypes were used. Population doublings are counted from germination of meiotic products and hence begin at approx. PD40. Abbreviations: TAP: tandem affinity purification tag


G ) Npl3 - TAP associates strongly to short telomeres . Cross - linked samples from indicated strains were used in a TAP - ChIP . Enrichment at telomeres was determined by quantitative PCR on indicated telomeres . Data represents mean % input + / - SEM n = 3 ( paired t - test one tailed * p < 0 . 05 , * * p < 0 . 01 ) . PD90 refers to 90 population doublings in the absence of TLC1 ( telomerase RNA subunit ) . The no tag control serves as an indicator of non - specific background signal . Abbreviations : TAP : tandem affinity purification tag , PD : population doubling .

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

G) Npl3-TAP associates strongly to short telomeres. Cross-linked samples from indicated strains were used in a TAP-ChIP. Enrichment at telomeres was determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM n=3 (paired t-test one tailed *p<0.05, **p<0.01). PD90 refers to 90 population doublings in the absence of TLC1 (telomerase RNA subunit). The no tag control serves as an indicator of non-specific background signal. Abbreviations: TAP: tandem affinity purification tag, PD: population doubling.


A ) Telomere interactors were identified and plotted as a volcano plot as in Figure 1 A , B and using tlc1 protein extracts . Interactors were identified using untreated protein extracts from tlc1 cells ( left panel ) or digested with RNase A and RNase H ( right panel ) .

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A) Telomere interactors were identified and plotted as a volcano plot as in Figure 1 A, B and using tlc1 protein extracts. Interactors were identified using untreated protein extracts from tlc1 cells (left panel) or digested with RNase A and RNase H (right panel).


B ) Npl3 associates to TERRA . Cross - linked samples from the indicated strains were subjected to Npl3 - TAP RIP . Enrichment of the indicated RNAs was determined by quantitative PCR on pulled - down reverse - transcribed RNA . Data represents mean % input + / - SEM n = 3 ( unpaired t - test two tailed * p < 0 . 05 , * * p < 0 . 01 ) .

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B) Npl3 associates to TERRA. Cross-linked samples from the indicated strains were subjected to Npl3-TAP RIP. Enrichment of the indicated RNAs was determined by quantitative PCR on pulled-down reverse-transcribed RNA. Data represents mean % input +/- SEM n=3 (unpaired t-test two tailed *p<0.05, **p<0.01).


Npl3 associates strongly to telomeres in the absence of Rat1 . C ) The Rat1 - AID tagged protein is strongly degraded following 1h of 1mM IAA treatment . Protein levels are determined using anti - FLAG ( Rat1 - AID ) and anti - actin antibodies . Abbreviations : IAA : indole - 3 - acetic acid , AID : Auxin inducible degron .

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

Npl3 associates strongly to telomeres in the absence of Rat1. C) The Rat1-AID tagged protein is strongly degraded following 1h of 1mM IAA treatment. Protein levels are determined using anti-FLAG (Rat1-AID) and anti-actin antibodies. Abbreviations: IAA: indole-3-acetic acid, AID: Auxin inducible degron.


Npl3 associates strongly to telomeres in the absence of Rat1 . D ) TERRA levels increase in cells expressing the Rat1 - AID variant after 1h treatment with 1mM IAA . TERRA levels were determined by quantitative PCR on reverse - transcribed RNAs after phenol - chloroform extraction . RNA levels are normalized to 7S ncRNA levels . Data represents fold increase to corresponding - IAA samples n = 2 . Abbreviations : IAA : indole - 3 - acetic acid , AID : Auxin inducible degron .

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

Npl3 associates strongly to telomeres in the absence of Rat1. D) TERRA levels increase in cells expressing the Rat1-AID variant after 1h treatment with 1mM IAA. TERRA levels were determined by quantitative PCR on reverse-transcribed RNAs after phenol-chloroform extraction. RNA levels are normalized to 7S ncRNA levels. Data represents fold increase to corresponding -IAA samples n=2. Abbreviations: IAA: indole-3-acetic acid, AID: Auxin inducible degron.


Npl3 associates strongly to telomeres in the absence of Rat1 . E ) Npl3 - TAP associates strongly to telomeres after 1mM IAA treatment in cells with Rat1 - AID . Cross - linked samples from indicated strains were used in a TAP - ChIP . Enrichment at telomeres was determined by quantitative PCR on indicated telomeres . Data represents mean % input + / - SEM n = 3 ( paired t - test one tailed * p < 0 . 05 , * * p < 0 . 01 ) . Abbreviations : IAA : indole - 3 - acetic acid , AID : Auxin inducible degron .

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

Npl3 associates strongly to telomeres in the absence of Rat1. E) Npl3-TAP associates strongly to telomeres after 1mM IAA treatment in cells with Rat1-AID. Cross-linked samples from indicated strains were used in a TAP-ChIP. Enrichment at telomeres was determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM n=3 (paired t-test one tailed *p<0.05, **p<0.01). Abbreviations: IAA: indole-3-acetic acid, AID: Auxin inducible degron.


F - G ) Npl3 associates to telomeres in an R - loop dependent manner . E ) RNH1 was overexpressed from a galactose - inducible promoter . Protein levels are determined using anti - HA and anti - actin antibodies . F ) Npl3 - TAP associates to telomeres in an R - loop - dependent manner . Cross - linked samples from indicated strains were used in a TAP - ChIP . Enrichment at telomeres was determined by quantitative PCR on indicated telomeres . Data represents mean % input + / - SEM n = 3 ( paired t - test one tailed * p < 0 . 05 , * * p < 0 . 01 ) . Abbreviations : eV : empty vector control , oE :

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

F-G) Npl3 associates to telomeres in an R-loop dependent manner. E) RNH1 was overexpressed from a galactose-inducible promoter. Protein levels are determined using anti-HA and anti-actin antibodies. F) Npl3-TAP associates to telomeres in an R-loop-dependent manner. Cross-linked samples from indicated strains were used in a TAP-ChIP. Enrichment at telomeres was determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM n=3 (paired t-test one tailed *p<0.05, **p<0.01). Abbreviations: eV: empty vector control, oE: overexpression


A - B ) Npl3 associates to telomeres in early S . A ) Cell cycle profiles of indicated strains . Cells were synchronized in G1 with alpha - factor for 2 . 5h and released into the cell cycle at 30 ° C in pre - warmed medium supplemented with the indicated HU concentrations . B ) Npl3 - TAP associates strongly to telomeres in early S ( 250 mM HU ) . Cross - linked samples from indicated strains were used in a TAP - ChIP . Enrichment at telomeres was determined by quantitative PCR on indicated telomeres . Data represents mean % input + / - SEM relative to cells arrested in alpha factor n = 3 ( paired t - test two tailed * p < 0 . 05 , * * p < 0 . 01 Abbreviations : ns : not significant HU : hydroxyurea . * p < 0 . 05 , * * p < 0 .

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

A-B) Npl3 associates to telomeres in early S. A) Cell cycle profiles of indicated strains. Cells were synchronized in G1 with alpha-factor for 2.5h and released into the cell cycle at 30°C in pre-warmed medium supplemented with the indicated HU concentrations. B) Npl3-TAP associates strongly to telomeres in early S (250 mM HU). Cross-linked samples from indicated strains were used in a TAP-ChIP. Enrichment at telomeres was determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM relative to cells arrested in alpha factor n=3 (paired t-test two tailed *p<0.05, **p<0.01 Abbreviations: ns: not significant HU: hydroxyurea. *p<0.05, **p<0.01


C ) NPL3 over expression stabilizes telomeric R - loops . The indicated strains were grown on 1 % raffinose 2 % galactose to induce NPL3 over expression . Cross - linked samples were subjected to DRIP . DNA - RNA hybrids were immunoprecipitated using the S9 . 6 antibody . R - loop levels were determined by quantitative PCR on indicated telomeres . Data represents mean % input + / - SEM relative to 250 mM HU eV n = 3 ( paired t - test two tailed * p < 0 . 05 , * * p < 0 . 01 ) . Abbreviations eV : empty vector control , oE : over expression HU : hydroxyurea . * p < 0 . 05 , * * p < 0 .

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

C) NPL3 over expression stabilizes telomeric R-loops. The indicated strains were grown on 1% raffinose 2% galactose to induce NPL3 over expression. Cross-linked samples were subjected to DRIP. DNA-RNA hybrids were immunoprecipitated using the S9.6 antibody. R-loop levels were determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM relative to 250 mM HU eV n=3 (paired t-test two tailed *p<0.05, **p<0.01). Abbreviations eV: empty vector control, oE: over expression HU: hydroxyurea. *p<0.05, **p<0.01


D ) Deletion of NPL3 decreases telomeric R - loops . The indicated strains were crosslinked and subjected to DRIP . DNA - RNA hybrids were immunoprecipitated using the S9 . 6 antibody . R - loop levels were determined by quantitative PCR on indicated telomeres . Data represents mean % input + / - SEM n = 3 . Specificity of S9 . 6 antibody was determined treating a fraction of the samples with RNase H .

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

D) Deletion of NPL3 decreases telomeric R-loops. The indicated strains were crosslinked and subjected to DRIP. DNA-RNA hybrids were immunoprecipitated using the S9.6 antibody. R-loop levels were determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM n=3. Specificity of S9.6 antibody was determined treating a fraction of the samples with RNase H.


E - G ) NPL3 stabilizes R - loops . E ) R - loop dot blot . The indicated strains were grown on 1 % raffinose 2 % galactose for 72h and subsequently released into 1 % raffinose 2 % galactose or 2 % glucose medium for 6 PDs . R - loop levels and dsDNA levels were determined using the S9 . 6 antibody and anti - dsDNA antibodies . Specificity of the S9 . 6 antibody was confirmed by treatment with RNase H . Loaded genomic DNA content is indicated at the bottom of the figure . F ) R - loop dot blot quantification . Data represents R - loop signal as the S9 . 6 antibody signal relative to corresponding dsDNA signal n = 3 ( unpaired t - test two tailed * p < 0 . 05 ) . G ) Serial dilutions of indicated strains were assayed on 1 % raffinose 2 % galactose media . Plates were imaged after 72h incubation at indicated temperatures . Abbreviations : V : empty vector control , oE : over expression , ds : double - strand ,

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

E-G) NPL3 stabilizes R-loops. E) R-loop dot blot. The indicated strains were grown on 1% raffinose 2% galactose for 72h and subsequently released into 1% raffinose 2% galactose or 2% glucose medium for 6 PDs. R-loop levels and dsDNA levels were determined using the S9.6 antibody and anti-dsDNA antibodies. Specificity of the S9.6 antibody was confirmed by treatment with RNase H. Loaded genomic DNA content is indicated at the bottom of the figure. F) R-loop dot blot quantification. Data represents R-loop signal as the S9.6 antibody signal relative to corresponding dsDNA signal n=3 (unpaired t-test two tailed *p<0.05). G) Serial dilutions of indicated strains were assayed on 1% raffinose 2% galactose media. Plates were imaged after 72h incubation at indicated temperatures. Abbreviations: V: empty vector control, oE: over expression, ds: double-strand,


A ) Serial dilutions of indicated strains were assayed on YPD media . Cells were plated after 30 - 50 generations propagated on YPD agar media . Two consecutive passages from one representative biological replicate are shown . Plates were imaged after 72h growth at 30 ° C .

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A) Serial dilutions of indicated strains were assayed on YPD media. Cells were plated after 30-50 generations propagated on YPD agar media. Two consecutive passages from one representative biological replicate are shown. Plates were imaged after 72h growth at 30°C.


B ) Serial dilutions of indicated strains were assayed on YPD media . Cells were plated after 30 - 50 generations propagated on YPD agar media . Two consecutive passages from one representative biological replicate are shown . Plates were imaged after 48h growth at 30 ° C .

B) Serial dilutions of indicated strains were assayed on YPD media. Cells were plated after 30-50 generations propagated on YPD agar media. Two consecutive passages from one representative biological replicate are shown. Plates were imaged after 48h growth at 30°C.


C ) Serial dilutions of indicated strains were assayed on SC - His media . Cells were plated after 30 - 50 generations propagated on SC - His agar media . Two consecutive passages from one representative biological replicate are shown . Plates were imaged after 72h growth at 30 ° C .

C) Serial dilutions of indicated strains were assayed on SC-His media. Cells were plated after 30-50 generations propagated on SC-His agar media. Two consecutive passages from one representative biological replicate are shown. Plates were imaged after 72h growth at 30°C.


( B ) Western blot analysis of Atxn1 levels in cerebellar granule neurons ( CGNs ) after knockdown of Zbtb7a and / or Zbtb7b ( n = 3 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , one - way ANOVA .

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(B) Western blot analysis of Atxn1 levels in cerebellar granule neurons (CGNs) after knockdown of Zbtb7a and/or Zbtb7b (n = 3). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA.


( C ) Western blot analysis of Atxn1 levels in the cerebella of wild - type ( n = 3 ) , Zbtb7a + / - ( n = 4 ) , Zbtb7b + / - ( n = 3 ) , Zbtb7a + / - ; Zbtb7b + / - ( n = 4 ) mice . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , one - way ANOVA .

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(C) Western blot analysis of Atxn1 levels in the cerebella of wild-type (n = 3), Zbtb7a+/- (n = 4), Zbtb7b+/- (n = 3), Zbtb7a+/-; Zbtb7b+/- (n = 4) mice. Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA.


( D ) Western blot analysis of Atxn1 levels in CGNs after overexpressing Zbtb7a and / or Zbtb7b ( n = 3 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , one - way ANOVA .

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(D) Western blot analysis of Atxn1 levels in CGNs after overexpressing Zbtb7a and/or Zbtb7b (n = 3). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, one-way ANOVA.


( E ) Genetic interaction of wild - type human ATXN1 ( 30Q ) with either ZBTB7A or ZBTB7B expressed in the Drosophila eyes . Co - expression of ATXN1 ( 30Q ) with ZBTB7A or ZBTB7B severely disrupted the external Drosophila eye structure and increased ATXN1 levels . Scale bar : 100

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(E) Genetic interaction of wild-type human ATXN1(30Q) with either ZBTB7A or ZBTB7B expressed in the Drosophila eyes. Co-expression of ATXN1(30Q) with ZBTB7A or ZBTB7B severely disrupted the external Drosophila eye structure and increased ATXN1 levels. Scale bar: 100 μm


( C ) Western blot analysis of ATXN1 levels after overexpression of ZBTB7B wild - type ( WT ) , zinc - finger domain mutant ( ZFmut ) or BTB domain mutants ( BTBmut1 and BTBmut2 ) ( n = 3 ) . Data information : Mean ± SD , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(C) Western blot analysis of ATXN1 levels after overexpression of ZBTB7B wild-type (WT), zinc-finger domain mutant (ZFmut) or BTB domain mutants (BTBmut1 and BTBmut2) (n = 3). Data information: Mean ± SD, ***P<0.001 ****P<0.0001, one-way ANOVA.


( D ) qRT - PCR results of Atxn1 mRNA levels in the cerebella of wild - type ( n = 3 ) , Zbtb7b + / - ( n = 4 ) , and Zbtb7b - / - mice ( n = 4 ) . Data information : Mean ± SD , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

(D) qRT-PCR results of Atxn1 mRNA levels in the cerebella of wild-type (n = 3), Zbtb7b+/- (n = 4), and Zbtb7b-/- mice (n = 4). Data information: Mean ± SD, ***P<0.001 ****P<0.0001, one-way ANOVA.


( E ) MA plot ( M : log ratio ; A : mean average ) of the differentially expressed genes ( DEG ) analysis from RNA - seq samples of the cells overexpressing either WT or ZFmut ZBTB7B . Four filters and the number of genes kept after applying each filter are described . Two genes that passed the 4th filter are labeled .

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(E) MA plot (M: log ratio; A: mean average) of the differentially expressed genes (DEG) analysis from RNA-seq samples of the cells overexpressing either WT or ZFmut ZBTB7B. Four filters and the number of genes kept after applying each filter are described. Two genes that passed the 4th filter are labeled.


( F ) Expression heatmap of the 16 genes that passed the 3rd filter . Color scale : row z - score .

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(F) Expression heatmap of the 16 genes that passed the 3rd filter. Color scale: row z-score.


( G ) qRT - PCR results of RSK3 mRNA levels across different CRISPR knockout cell lines ( n = 2 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(G) qRT-PCR results of RSK3 mRNA levels across different CRISPR knockout cell lines (n = 2). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.


( H ) Epistatic relationship between ZBTB7B and RSK3 in terms of ATXN1 regulation . ATXN1 levels were measured after overexpression of ZBTB7B in the RSK3 knockout cell line ( sgRSK3 ) and control cell lines ( WT and sgControl ) ( n = 3 ) . ZBTB7B + RSK3 : co - overexpression of both ZBTB7B and RSK3 . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(H) Epistatic relationship between ZBTB7B and RSK3 in terms of ATXN1 regulation. ATXN1 levels were measured after overexpression of ZBTB7B in the RSK3 knockout cell line (sgRSK3) and control cell lines (WT and sgControl) (n = 3). ZBTB7B+RSK3: co-overexpression of both ZBTB7B and RSK3. Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.


( I ) qRT - PCR results of RSK3 mRNA levels after knockdown of ZBTB7B in Daoy cells ( n = 2 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(I) qRT-PCR results of RSK3 mRNA levels after knockdown of ZBTB7B in Daoy cells (n = 2). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.


( J ) qRT - PCR results of Rsk3 mRNA levels in the cerebella of Zbtb7b conditional knockout mice ( n = 6 ) . Data information : Mean ± SD , * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed t - test .

(J) qRT-PCR results of Rsk3 mRNA levels in the cerebella of Zbtb7b conditional knockout mice (n = 6). Data information: Mean ± SD, *P<0.05, ***P<0.001, ****P<0.0001, two-tailed t-test.


( K ) Position frequency matrix of ZBTB7B binding DNA sequence and a table of the predicted ZBTB7B binding sites on human RSK3 promoter . Start and End position numbers are described based on TSS . The seventy base pairs upstream RSK3 promotor includes top two potential binding sites and was used for luciferase assays . Data information : Mean ± SD , * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed t - test .

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(K) Position frequency matrix of ZBTB7B binding DNA sequence and a table of the predicted ZBTB7B binding sites on human RSK3 promoter. Start and End position numbers are described based on TSS. The seventy base pairs upstream RSK3 promotor includes top two potential binding sites and was used for luciferase assays. Data information: Mean ± SD, *P<0.05, ***P<0.001, ****P<0.0001, two-tailed t-test.


( L ) Luciferase assay result of RSK3 promoter in HEK 293T and HeLa cells transfected with either an empty or ZBTB7B expressing vector ( n = 3 ) . Data information : Mean ± SD , * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed t - test .

(L) Luciferase assay result of RSK3 promoter in HEK 293T and HeLa cells transfected with either an empty or ZBTB7B expressing vector (n = 3). Data information: Mean ± SD, *P<0.05, ***P<0.001, ****P<0.0001, two-tailed t-test.


( A ) Western blot analysis of ATXN1 after knockdown of RSK3 in Daoy cells . Bottom left panel : qRT - PCR results of RSK3 mRNA . Bottom right panel : densitometry of the western blot image in the top panel ( n = 3 except for shATXN1 where n = 1 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(A) Western blot analysis of ATXN1 after knockdown of RSK3 in Daoy cells. Bottom left panel: qRT-PCR results of RSK3 mRNA. Bottom right panel: densitometry of the western blot image in the top panel (n = 3 except for shATXN1 where n = 1). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.


( B ) Western blot analysis of ATXN1 after overexpression of RSK3 , ZBTB7B , or eGFP in Daoy cells ( n = 3 ) . Data information : RSK3 antibody used in is only suitable for detecting overexpressed RSK3 but not for endogenous RSK3 . Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(B) Western blot analysis of ATXN1 after overexpression of RSK3, ZBTB7B, or eGFP in Daoy cells (n = 3). Data information: RSK3 antibody used in is only suitable for detecting overexpressed RSK3 but not for endogenous RSK3. Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.


( C ) Western blot analysis of ATXN1 ( 82Q ) after overexpression of either wild - type or inactive RSK3 in Daoy cells expressing ATXN1 ( 82Q ) ( n = 3 ) . Data information : RSK3 antibody used in is only suitable for detecting overexpressed RSK3 but not for endogenous RSK3 . Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(C) Western blot analysis of ATXN1(82Q) after overexpression of either wild-type or inactive RSK3 in Daoy cells expressing ATXN1(82Q) (n = 3). Data information: RSK3 antibody used in is only suitable for detecting overexpressed RSK3 but not for endogenous RSK3. Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.


( D ) In vitro kinase assay of RSK3 with ATXN1 . MSK1 was used as a positive control . Asterisk : auto - phosphorylated RSK3 .

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(D) In vitro kinase assay of RSK3 with ATXN1. MSK1 was used as a positive control. Asterisk: auto-phosphorylated RSK3.


( E ) In vitro kinase assay of four different RSK members with ATXN1 .

(E) In vitro kinase assay of four different RSK members with ATXN1.


( F ) In vitro kinase assay of RSK3 with N - terminal ( F1 ) , middle ( F2 ) , and C - terminal fragment ( F3 ) of ATXN1 .

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(F) In vitro kinase assay of RSK3 with N-terminal (F1), middle (F2), and C-terminal fragment (F3) of ATXN1.


( G ) Mass spectrometry analysis of phosphorylated residues in ATXN1 F3 after in vitro kinase assay with RSK3 .

(G) Mass spectrometry analysis of phosphorylated residues in ATXN1 F3 after in vitro kinase assay with RSK3.


( H ) Western blot analysis of ATXN1 after a treatment with RSK3 inhibitor ( BI - D1870 ) on SCA1 patient iPSC - derived neurons ( n = 3 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(H) Western blot analysis of ATXN1 after a treatment with RSK3 inhibitor (BI-D1870) on SCA1 patient iPSC-derived neurons (n = 3). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.


( I ) Western blot analysis of total and phospho - S776 ATXN1 after a co - treatment with MSK1 ( SB - 747651A ) and RSK3 inhibitor ( BI - D1870 ) on SCA1 patient iPSC - derived neurons ( n = 3 ) . Data information : Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(I) Western blot analysis of total and phospho-S776 ATXN1 after a co-treatment with MSK1 (SB-747651A) and RSK3 inhibitor (BI-D1870) on SCA1 patient iPSC-derived neurons (n = 3). Data information: Mean ± SD, *P<0.05, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA.


( A ) Images of Drosophila eyes expressing human ATXN1 ( 82Q ) with either knockdown or overexpression of the Drosophila RSK3 homolog Sk6II . Scale bar : 100 μm ( top ) and 20 μm ( bottom ) .

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(A) Images of Drosophila eyes expressing human ATXN1(82Q) with either knockdown or overexpression of the Drosophila RSK3 homolog Sk6II. Scale bar: 100 μm (top) and 20 μm (bottom).


( B ) Improvement of motor performance by the knockdown of Sk6II in Drosophila SCA1 model expressing ATXN1 ( 82Q ) in the central nervous system . Mean ± SEM , * P < 0 . 05 , Linear mixed - effect model ANOVA , n = 10 / genotype , four times of measurements .

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(B) Improvement of motor performance by the knockdown of Sk6II in Drosophila SCA1 model expressing ATXN1(82Q) in the central nervous system. Mean ± SEM, *P < 0.05, Linear mixed-effect model ANOVA, n = 10/genotype, four times of measurements.


( C ) A representative image of a mouse brain 4 weeks after AAV injection of shRsk3 into 12 weeks old SCA1 mice ( Atxn1154Q / 2Q ) cerebellum . Scale bar : 2 mm .

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(C) A representative image of a mouse brain 4 weeks after AAV injection of shRsk3 into 12 weeks old SCA1 mice (Atxn1154Q/2Q) cerebellum. Scale bar: 2 mm.


( D ) Western blot analysis of wild - type ( 2Q ) and mutant Atxn1 ( 154Q ) in the cerebella of 16 weeks old SCA1 mice dissected 4 weeks after the stereotaxic injection of AAV9 harboring shControl ( n = 3 ) , shRsk3 ( n = 3 ) , or shAtxn1 ( n = 2 ) . Mean ± SD , * P < 0 . 05 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA .

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(D) Western blot analysis of wild-type (2Q) and mutant Atxn1 (154Q) in the cerebella of 16 weeks old SCA1 mice dissected 4 weeks after the stereotaxic injection of AAV9 harboring shControl (n = 3), shRsk3 (n = 3), or shAtxn1 (n = 2). Mean ± SD, *P<0.05, ***P<0.001 ****P<0.0001, one-way ANOVA.


( E ) Western blot analysis of wild - type ( 2Q ) and mutant Atxn1 ( 154Q ) in the brainstem ( BS ) and cerebella ( CBM ) of 8 weeks old Atxn1154Q / 2Q ( n = 6 ) and Atxn1154Q / 2Q ; Rsk3 + / - mice ( n = 7 ) . Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , two - tailed t - test .

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(E) Western blot analysis of wild-type (2Q) and mutant Atxn1 (154Q) in the brainstem (BS) and cerebella (CBM) of 8 weeks old Atxn1154Q/2Q (n = 6) and Atxn1154Q/2Q; Rsk3+/- mice (n = 7). Mean ± SD, *P<0.05, **P<0.01, two-tailed t-test.


( F ) Rotarod motor performance test result of Atxn1154Q / 2Q ; Rsk3 + / - mice at 11 - 12 weeks old . Mean ± SEM , * P < 0 . 05 , two - way ANOVA , n = 35 , 31 , 34 , 38 for WT , Rsk3 + / - , Atxn1154Q / 2Q , Atxn1154Q / 2Q ; Rsk3 + / - genotype , respectively .

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(F) Rotarod motor performance test result of Atxn1154Q/2Q; Rsk3+/- mice at 11-12 weeks old. Mean ± SEM, *P<0.05, two-way ANOVA, n = 35, 31, 34, 38 for WT, Rsk3+/-, Atxn1154Q/2Q, Atxn1154Q/2Q; Rsk3+/- genotype, respectively.


( A ) Fluorescent western blot analysis of Atxn1 , Msk1 , and Rsk3 in seven different brain regions of wild - type mice at 9 weeks old ( n = 3 ) . Each protein levels were represented relative to the protein levels in brainstem . Mean ± SD , * * P < 0 . 01 , * * * P < 0 . 001 * * * * P < 0 . 0001 , one - way ANOVA . Data information : BS : brainstem , CBM : cerebellum , MB : midbrain , ST : striatum , HC : hippocampus , CTX : cortex , OB : olfactory bulbs .

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(A) Fluorescent western blot analysis of Atxn1, Msk1, and Rsk3 in seven different brain regions of wild-type mice at 9 weeks old (n = 3). Each protein levels were represented relative to the protein levels in brainstem. Mean ± SD, **P<0.01, ***P<0.001 ****P<0.0001, one-way ANOVA. Data information: BS: brainstem, CBM: cerebellum, MB: midbrain, ST: striatum, HC: hippocampus, CTX: cortex, OB: olfactory bulbs.


( B ) Representative BaseScope assay images of Rsk3 in the whole brain sagittal sections of wild - type ( top ) and Rsk3 - / - mice ( bottom ) at 5 - 6 weeks old . The areas within the black squares are magnified in the right panels in each genotype . Scale bar = 2 mm in whole brain images ; 50 μm in the magnified images . Data information : BS : brainstem , CBM :

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(B) Representative BaseScope assay images of Rsk3 in the whole brain sagittal sections of wild-type (top) and Rsk3-/- mice (bottom) at 5-6 weeks old. The areas within the black squares are magnified in the right panels in each genotype. Scale bar = 2 mm in whole brain images; 50 μm in the magnified images. Data information: BS: brainstem, CBM: cerebellum


( C ) Representative IF images of Msk1 in the whole brain sagittal sections of wild - type ( top ) and Msk1 - / - mice ( bottom ) at 6 months old . The areas within the white squares are magnified in the right panels in each genotype . Scale bar = 2 mm in whole brain images ; 50 μm in the magnified images . Data information : BS : brainstem , CBM :

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(C) Representative IF images of Msk1 in the whole brain sagittal sections of wild-type (top) and Msk1-/- mice (bottom) at 6 months old. The areas within the white squares are magnified in the right panels in each genotype. Scale bar = 2 mm in whole brain images; 50 μm in the magnified images. Data information: BS: brainstem, CBM: cerebellum


( D ) Western blot analysis of total and active form of Rsk3 and Msk1 in the cerebella and brainstem of 12 weeks old WT and SCA1 mice ( n = 4 ) . Relative Rsk3 and Msk1 levels were represented into log2 scale . The protein levels of the two kinases are significantly different between the two brain regions . Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , two - way ANOVA . Data information : BS : brainstem , CBM :

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(D) Western blot analysis of total and active form of Rsk3 and Msk1 in the cerebella and brainstem of 12 weeks old WT and SCA1 mice (n = 4). Relative Rsk3 and Msk1 levels were represented into log2 scale. The protein levels of the two kinases are significantly different between the two brain regions. Mean ± SD, *P<0.05, **P<0.01, ****P <0.0001, two-way ANOVA. Data information: BS: brainstem, CBM: cerebellum


( E ) Western blot analysis of ATXN1 , RSK3 , pRSK3 , MSK1 , and pMSK1 in the postmortem cerebella , pons , and medulla oblongata of human controls and SCA1 patients ( n = 3 per genotype ) . Total protein was used for normalization , and protein levels are represented relative to cerebellar protein levels in control group . C : Cerebellum , P : Pons , M : medulla oblongata . Values from P and M are combined and presented in BS . Mean ± SEM , * P < 0 . 05 , * * P < 0 . 01 , two - way ANOVA , n = 3 ( CBM ) and 6 ( BS ) . Data information : BS : brainstem , CBM :

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(E) Western blot analysis of ATXN1, RSK3, pRSK3, MSK1, and pMSK1 in the postmortem cerebella, pons, and medulla oblongata of human controls and SCA1 patients (n = 3 per genotype). Total protein was used for normalization, and protein levels are represented relative to cerebellar protein levels in control group. C: Cerebellum, P: Pons, M: medulla oblongata. Values from P and M are combined and presented in BS. Mean ± SEM, *P<0.05, **P<0.01, two-way ANOVA, n = 3 (CBM) and 6 (BS). Data information: BS: brainstem, CBM: cerebellum


( A ) Western blot analysis of total and pS776 wild - type and mutant Atxn1 in the brainstem of 8 weeks old SCA1 mice with heterozygous knockout of either one or both kinases . Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , one - way ANOVA , n = 7 , 6 , 4 , 7 following the order of genotypes in the western blot , respectively .

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(A) Western blot analysis of total and pS776 wild-type and mutant Atxn1 in the brainstem of 8 weeks old SCA1 mice with heterozygous knockout of either one or both kinases. Mean ± SD, *P<0.05, **P<0.01, ****P<0.0001, one-way ANOVA, n = 7, 6, 4, 7 following the order of genotypes in the western blot, respectively.


( B ) Plethysmography data measuring breathing ability ( total ventilated gas volume per minute ) of 34 weeks old mice with different genotypes . Mean ± SD , * P < 0 . 05 , two - tailed t - test , n = 12 , 13 , 15 , 13 , 15 , 9 , 15 , 13 following the order of genotypes in the graph , respectively .

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(B) Plethysmography data measuring breathing ability (total ventilated gas volume per minute) of 34 weeks old mice with different genotypes. Mean ± SD, *P<0.05, two-tailed t-test, n = 12, 13, 15, 13, 15, 9, 15, 13 following the order of genotypes in the graph, respectively.


( C ) Representative immunohistochemistry images of Iba1 + reactive microglia in the coronally sectioned hypoglossal nucleus of 36 weeks old mice with different genotypes . Yellow arrows indicates Iba1 + microglia , and larger pyramidal shaped cells are motoneurons . scale bar = 50 μm .

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(C) Representative immunohistochemistry images of Iba1+ reactive microglia in the coronally sectioned hypoglossal nucleus of 36 weeks old mice with different genotypes. Yellow arrows indicates Iba1+ microglia, and larger pyramidal shaped cells are motoneurons. scale bar = 50 μm.


( D ) Quantification of the number of Iba1 + cells shown in ( C ) . Mean ± SD , * P < 0 . 05 , * * * P < 0 . 001 , one - way ANOVA , n = 3 mice per genotype , each dot is an averaged measurement from six sections per mouse .

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(D) Quantification of the number of Iba1+ cells shown in (C). Mean ± SD, *P<0.05, ***P<0.001, one-way ANOVA, n = 3 mice per genotype, each dot is an averaged measurement from six sections per mouse.


( E ) Representative Hematoxylin and Eosin stained motoneurons in the coronally sectioned hypoglossal nucleus ( white dashed line ) of 36 weeks old mice with different genotypes . The motoneurons are dark purple with pyramidal shape within the nucleus . C : central canal . Scale bar = 100 μm .

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(E) Representative Hematoxylin and Eosin stained motoneurons in the coronally sectioned hypoglossal nucleus (white dashed line) of 36 weeks old mice with different genotypes. The motoneurons are dark purple with pyramidal shape within the nucleus. C: central canal. Scale bar = 100 μm.


( F ) Quantification of the number of motoneurons shown in ( E ) . Mean ± SD , * P < 0 . 05 , one - way ANOVA , n = 3 mice per genotype , each dot is an averaged measurement from six sections per mouse .

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(F) Quantification of the number of motoneurons shown in (E). Mean ± SD, *P<0.05, one-way ANOVA, n = 3 mice per genotype, each dot is an averaged measurement from six sections per mouse.


( G ) Kaplan - Meier survival graphs of SCA1 mice with heterozygous knockout of either one or both kinases . The data of Msk1 + / - , Rsk3 + / - , and Msk1 + / - ; Rsk3 + / - mice overlap those of wild - type mice . * P < 0 . 05 , Gehan - Breslow - Wilcoxon test , n = 15 , 15 , 12 , 13 , 22 , 11 , 20 , 19 following the order of genotypes in the legend on top of the figure , respectively .

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(G) Kaplan-Meier survival graphs of SCA1 mice with heterozygous knockout of either one or both kinases. The data of Msk1+/-, Rsk3+/-, and Msk1+/-; Rsk3+/- mice overlap those of wild-type mice. *P<0.05, Gehan-Breslow-Wilcoxon test, n = 15, 15, 12, 13, 22, 11, 20, 19 following the order of genotypes in the legend on top of the figure, respectively.


( A ) Western blot analysis of total and pS776 wild - type and mutant Atxn1 in the cerebella of 8 weeks old SCA1 mice with heterozygous knockout of either one or both kinases . Mean ± SD , * * P < 0 . 01 , * * * P < 0 . 001 , one - way ANOVA , n = 7 , 6 , 4 , 7 following the order of genotypes in the western blot , respectively .

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(A) Western blot analysis of total and pS776 wild-type and mutant Atxn1 in the cerebella of 8 weeks old SCA1 mice with heterozygous knockout of either one or both kinases. Mean ± SD, **P<0.01, ***P<0.001, one-way ANOVA, n = 7, 6, 4, 7 following the order of genotypes in the western blot, respectively.


( B ) Rotarod motor performance test result of 11 - 12 weeks old SCA1 mice with heterozygous knockout of either one or both kinases . Mean ± SEM , * P < 0 . 05 , two - way ANOVA , n = 18 , 12 , 13 , 11 , 19 , 13 , 15 , 13 following the order of genotypes in the legend on top of the figure , respectively .

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(B) Rotarod motor performance test result of 11-12 weeks old SCA1 mice with heterozygous knockout of either one or both kinases. Mean ± SEM, *P<0.05, two-way ANOVA, n = 18, 12, 13, 11, 19, 13, 15, 13 following the order of genotypes in the legend on top of the figure, respectively.


( C ) Representative immunofluorescent ( IF ) images of cerebellar molecular layer between the cerebellum lobule V and VI across different genotypes at 36 weeks old , stained with anti - Calbindin 1 antibody . Scale bar = 100 μm .

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(C) Representative immunofluorescent (IF) images of cerebellar molecular layer between the cerebellum lobule V and VI across different genotypes at 36 weeks old, stained with anti-Calbindin 1 antibody. Scale bar = 100 μm.


( D ) Quantification of the thickness of top and bottom molecular layers shown in ( C ) . Mean ± SD , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , one - way ANOVA , n = 3 mice per genotype , each dot is an averaged measurement from three sections per mouse .

(D) Quantification of the thickness of top and bottom molecular layers shown in (C). Mean ± SD, *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA, n = 3 mice per genotype, each dot is an averaged measurement from three sections per mouse.


Exponential growth of E . coli K - 12 , measured using optical density ( OD600 ) , in lysogenic broth ( LB , white circles ) or minimal medium supplemented with different carbon sources , listed in the legend on the bottom right in a greyscale . All the cultures are grown in batch mode , see Methods .

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Exponential growth of E. coli K-12, measured using optical density (OD600), in lysogenic broth (LB, white circles) or minimal medium supplemented with different carbon sources, listed in the legend on the bottom right in a greyscale. All the cultures are grown in batch mode, see Methods.


Bacterial viability in colony forming units ( CFU ) per ml of cultures from panel A , grown until OD600 0 . 5 , washed and re - suspended in carbon - free minimal medium . Death rates ( slopes of the exponential fits ) are listed in Table EV1 . Initial density for fast growth ( lighter shades ) is lower than for slow growth ( darker shades ) , due larger cell sizes that lead to lower cell densities per OD600 .

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Bacterial viability in colony forming units (CFU) per ml of cultures from panel A, grown until OD600 0.5, washed and re-suspended in carbon-free minimal medium. Death rates (slopes of the exponential fits) are listed in Table EV1. Initial density for fast growth (lighter shades) is lower than for slow growth (darker shades), due larger cell sizes that lead to lower cell densities per OD600.


Death rates of panel B plotted against growth rates of panel A shown as circles in a grey - scale . Squares show data obtained using eight other carbon substrates , also listed in Table EV1 . Color circles show data obtained by varying growth rate in a chemostat . The black line shows a linear weighted least square fit to log - transformed data , that takes uncertainty in data points into account ( see Methods ) . Data shown as mean ± ( standard deviation ) SD . Two or three replicates per condition .

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

Death rates of panel B plotted against growth rates of panel A shown as circles in a grey-scale. Squares show data obtained using eight other carbon substrates, also listed in Table EV1. Color circles show data obtained by varying growth rate in a chemostat. The black line shows a linear weighted least square fit to log-transformed data, that takes uncertainty in data points into account (see Methods). Data shown as mean ± (standard deviation) SD. Two or three replicates per condition.


Death rates of wild - type and GlpK22 mutants plotted versus growth rates . Wild type is grown in glycerol minimal medium in batch cultures ( black circle ) and in ' chemostat ' continuous cultures with growth rates coded in colors , see legend on the center right . GlpK22 mutants ( white circle ) are grown in glycerol minimal medium in batch culture . Data shown as mean ± ( standard deviation ) SD . Two replicates per condition .

Death rates of wild-type and GlpK22 mutants plotted versus growth rates. Wild type is grown in glycerol minimal medium in batch cultures (black circle) and in 'chemostat' continuous cultures with growth rates coded in colors, see legend on the center right. GlpK22 mutants (white circle) are grown in glycerol minimal medium in batch culture. Data shown as mean ± (standard deviation) SD. Two replicates per condition.


Example experiment of the assay sketched in panel C . Cells previously grown at 0 . 3 h − 1 in continuous culture are starved ( green circles ) . After 2 days of starvation , a sample is ( 1 ) extracted and ( 2 ) UV - sterilized , see dashed line , followed by ( 3 ) inoculation of 1 % of starved and ( 4 ) growth of the culture . The difference between the maximum viability after growth , and the initial viability at point ( 3 ) is the absolute yield .

Example experiment of the assay sketched in panel C. Cells previously grown at 0.3 h − 1 in continuous culture are starved (green circles). After 2 days of starvation, a sample is (1) extracted and (2) UV-sterilized, see dashed line, followed by (3) inoculation of 1% of starved and (4) growth of the culture. The difference between the maximum viability after growth, and the initial viability at point (3) is the absolute yield.


The absolute yield measured at three different time points for each of the growth conditions shown in panel A is plotted against the viability of the starvation culture at the extraction time . The recycling yield is extracted as the slope of the linear fits . Recycling yield from panel E plotted versus growth rate . Data shown as mean ± SD . Two replicates per condition .

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The absolute yield measured at three different time points for each of the growth conditions shown in panel A is plotted against the viability of the starvation culture at the extraction time. The recycling yield is extracted as the slope of the linear fits. Recycling yield from panel E plotted versus growth rate. Data shown as mean ± SD. Two replicates per condition.


Example experiment from a starved culture previously grown at a rate of 0 . 3 h − 1 with a viability after 5 days of starvation . After ( 1 ) extraction and ( 2 ) addition of 40 µM of glycerol the decay of viability is delayed ( black circles ) compared to a control without glycerol ( green symbols ) . Per viable cell , the glycerol addition in this experiment is 0 . 33 fmol CFU − 1 . After an initial period of survival , the culture with added glycerol ( black ) dies at the same rate as the control ( green ) . The ' lag time ' fitted to this data is 1 day .

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

Example experiment from a starved culture previously grown at a rate of 0.3 h − 1 with a viability after 5 days of starvation. After (1) extraction and (2) addition of 40 µM of glycerol the decay of viability is delayed (black circles) compared to a control without glycerol (green symbols). Per viable cell, the glycerol addition in this experiment is 0.33 fmol CFU − 1. After an initial period of survival, the culture with added glycerol (black) dies at the same rate as the control (green). The 'lag time' fitted to this data is 1 day.


Lag time of the experiments of panel B for different glycerol concentrations . Lag increases linearly with glycerol concentration and maintenance rate is extracted as the inverse of the slope of the linear fits .

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

Lag time of the experiments of panel B for different glycerol concentrations. Lag increases linearly with glycerol concentration and maintenance rate is extracted as the inverse of the slope of the linear fits.


Maintenance rate values extracted from panel I plotted versus growth rate . Data shown as mean ± SD . Two replicates per condition .

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Maintenance rate values extracted from panel I plotted versus growth rate. Data shown as mean ± SD. Two replicates per condition.


( K ) Consistency check of the equation in panel A . Fold changes of maintenance rate , FC ( β ) , divided by the fold changes of the recycling yield , FC ( α ) , plotted versus the fold changes of death rate , FC ( γ ) . All fold changes are relative to wild - type in batch , growing at 0 . 7 h − 1 . The dashed line shows the unity line FC ( γ ) = FC ( β ) / FC ( α ) .

(K) Consistency check of the equation in panel A. Fold changes of maintenance rate, FC(β), divided by the fold changes of the recycling yield, FC(α), plotted versus the fold changes of death rate, FC(γ). All fold changes are relative to wild-type in batch, growing at 0.7 h − 1. The dashed line shows the unity line FC(γ)= FC(β)/FC(α).


Cell size during starvation . Length and width of the cells are measured with phase - contrast microscopy and the volume is computed considering cell shape as a cylinder with two semi - spheres , as described in the graphical synopsis at the top ( see also Table EV3 and Methods ) . Each measurement is an average of 200 cells . ( A ) Measured length and width of wild type cells starved in batch cultures and previously grown in the chemostat at different steady - state growth rates and of GlpK22 cells starved and previously grown in batch culture at μ = 0 . 9 h − 1 ( see upper color legend ) . Note that , as shown in Schink et al . ( 2019 ) , cell widths do not change from steady state growth to starvation , while lengths decrease . Both length and width increase exponentially with growth rate . ( B ) Starvation volume of the cells described in panel A , computed as explained in the graphical synopsis ( see also Methods ) . In agreement with literature ( Schaechter et al . , 1958 ) , it increases exponentially with growth rate ( black line ) .

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

Cell size during starvation. Length and width of the cells are measured with phase-contrast microscopy and the volume is computed considering cell shape as a cylinder with two semi-spheres, as described in the graphical synopsis at the top (see also Table EV3 and Methods). Each measurement is an average of 200 cells. (A) Measured length and width of wild type cells starved in batch cultures and previously grown in the chemostat at different steady-state growth rates and of GlpK22 cells starved and previously grown in batch culture at μ = 0.9 h − 1 (see upper color legend). Note that, as shown in Schink et al. (2019), cell widths do not change from steady state growth to starvation, while lengths decrease. Both length and width increase exponentially with growth rate. (B) Starvation volume of the cells described in panel A, computed as explained in the graphical synopsis (see also Methods). In agreement with literature (Schaechter et al., 1958), it increases exponentially with growth rate (black line).


Recycling yield and maintenance rate per cell volume . ( C ) Recycling yields and ( D ) maintenance rate measured as described in Fig . 2 are normalized per cell volume and plotted versus the previous growth rate of the cultures they refer to ( see color legend at the top and Table EV3 ) . The normalized yield is constant within the uncertainty . Maintenance rate increases significantly with a slope of ( 0 . 99 ± 0 . 55 ) h , matching the increase of the death rate , ( 1 . 1 ± 0 . 4 ) h , shown in Fig 2B . Solid black lines show linear fits on log transformed data , using weighted least square fits .

Recycling yield and maintenance rate per cell volume. (C) Recycling yields and (D) maintenance rate measured as described in Fig. 2 are normalized per cell volume and plotted versus the previous growth rate of the cultures they refer to (see color legend at the top and Table EV3). The normalized yield is constant within the uncertainty. Maintenance rate increases significantly with a slope of (0.99±0.55) h, matching the increase of the death rate, (1.1±0.4) h, shown in Fig 2B. Solid black lines show linear fits on log transformed data, using weighted least square fits.


Death rates of ∆ rpoS mutants versus the growth rate ( grey symbols ) , compared to wild - type E . coli K - 12 ( white symbols , data from Fig . 1 ) . Death rate increases with growth rate with a smaller slope ( ∆ rpoS : 0 . 57 ± 0 . 13 h ) compared to wild - type ( WT : 0 . 88 ± 0 . 13 h ) . Black and grey lines show linear weighted least square fits to log - transformed data , which take uncertainty in data points into account ( see Methods ) . Data shown as mean ± SD . Two or three replicates per condition .

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Death rates of ∆rpoS mutants versus the growth rate (grey symbols), compared to wild-type E. coli K-12 (white symbols, data from Fig. 1). Death rate increases with growth rate with a smaller slope (∆rpoS: 0.57±0.13 h) compared to wild-type (WT: 0.88±0.13 h). Black and grey lines show linear weighted least square fits to log-transformed data, which take uncertainty in data points into account (see Methods). Data shown as mean ± SD. Two or three replicates per condition.


The optimal growth rate , i . e . the one that maximizes fitness over a cycle of famine and feast , depends on the ratio of feast and famine periods . For long periods of feast interrupted by short periods of famine , E . coli the optimal strategy is to grow fast . For long periods of starvation intermitted by short periods of growth , the optimal strategy is long survival and slow growth . For the scenario of panel A and B , $ \ frac { T _ { - } } { T _ { + } } = 48 $ , the optimum is μ = 0 . 86 h − 1 and γ = 0 . 5 d − 1 , see dashed line .

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The optimal growth rate, i.e. the one that maximizes fitness over a cycle of famine and feast, depends on the ratio of feast and famine periods. For long periods of feast interrupted by short periods of famine, E. coli the optimal strategy is to grow fast. For long periods of starvation intermitted by short periods of growth, the optimal strategy is long survival and slow growth. For the scenario of panel A and B, $\frac{T_{-}}{T_{+}} = 48$, the optimum is μ = 0.86 h − 1 and γ = 0.5 d − 1, see dashed line.


Adaptation at the end of growth . When the nutrient concentration decreases , the nutrient uptake rate slows down with a Michaelis - Menten type kinetics ( top ) . Depending on the uptake affinity , bacteria will experience either a sharp or smooth decrease of growth rate ( bottom left , bright orange : 10 µM , dark orange : 1 mM ) . During this slow down , the proteome adapts ( bottom center ) , with lower affinities ( dark orange ) showing a slight improvement of proteome adaptation , which results in a slightly lower death rate ( bottom right ) .

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

Adaptation at the end of growth. When the nutrient concentration decreases, the nutrient uptake rate slows down with a Michaelis-Menten type kinetics (top). Depending on the uptake affinity, bacteria will experience either a sharp or smooth decrease of growth rate (bottom left, bright orange: 10 µM, dark orange: 1 mM). During this slow down, the proteome adapts (bottom center), with lower affinities (dark orange) showing a slight improvement of proteome adaptation, which results in a slightly lower death rate (bottom right).


Adaptation in complex media . A medium supplemented with multiple nutrients ( one - red , two - green , ten - blue ) leads to a step - wise decrease of growth rate during nutrient depletion ( bottom left ) . Due to the extended periods of slow growth after exhaustion of primary nutrients , bacteria can adapt their proteome substantially ( bottom center ) , and decrease their death rate ( bottom right ) .

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

Adaptation in complex media. A medium supplemented with multiple nutrients (one - red, two - green, ten - blue) leads to a step-wise decrease of growth rate during nutrient depletion (bottom left). Due to the extended periods of slow growth after exhaustion of primary nutrients, bacteria can adapt their proteome substantially (bottom center), and decrease their death rate (bottom right).


Live cell microscopy of U - 2 OS - TetO cells expressing rTetR - GFP ( ctrl ) , or rTetR - GFP - Kin14VIb ( Kin14VIb ) ( B ) Representative stills showing the most frequently observed metaphase localization and daughter nuclei distribution of the duplicated TetO locus ( white arrowheads ) . Chromatin was visualized through expression of H2B - mCherry , GFP depicts the TetO locus . Due to the presence of a predicted NES sequence in Kin14VIb , the TetO locus only becomes visible at NEB in Kin14VIb - expressing cells . Time = h : min , scale bar = 5 µm . Schemes illustrating the metaphase localization of the TetO locus and its subsequent distribution over daughter nuclei are shown on the right .

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

Live cell microscopy of U-2 OS-TetO cells expressing rTetR-GFP (ctrl), or rTetR-GFP-Kin14VIb (Kin14VIb) (B) Representative stills showing the most frequently observed metaphase localization and daughter nuclei distribution of the duplicated TetO locus (white arrowheads). Chromatin was visualized through expression of H2B-mCherry, GFP depicts the TetO locus. Due to the presence of a predicted NES sequence in Kin14VIb, the TetO locus only becomes visible at NEB in Kin14VIb-expressing cells. Time = h:min, scale bar = 5 µm. Schemes illustrating the metaphase localization of the TetO locus and its subsequent distribution over daughter nuclei are shown on the right.


( C ) Quantification of the fraction of ctrl or Kin14VIb - expressing cells with the TetO locus observed outside the metaphase plate ( TetO out ) . ( D ) Deconstruction of ( C ) . Fraction of cells with the indicated orientation of the duplicated TetO locus and TetO chromosome . Data information : Bars represent the mean of 2 biological replicates , while dark - and light - colored dots represent the values of each experiment . ( C , D ) : N ≥ 29 cells per condition , per experiment , * * * * P < 0 . 0001 ; Fisher ' s exact test .

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(C) Quantification of the fraction of ctrl or Kin14VIb-expressing cells with the TetO locus observed outside the metaphase plate (TetO out). (D) Deconstruction of (C). Fraction of cells with the indicated orientation of the duplicated TetO locus and TetO chromosome. Data information: Bars represent the mean of 2 biological replicates, while dark- and light-colored dots represent the values of each experiment. (C, D): N ≥ 29 cells per condition, per experiment, ****P<0.0001; Fisher's exact test.


E . Quantification of the number of cells with Mad1 - positive KTs nearby the TetO locus . In case the KTs of the TetO chromosome were aligned , we scored whether KTs in the vicinity of the TetO locus were Mad1 + . In case the KTs of the TetO chromosome could be distinguished in rTetR - GFP - Kin14VIb - expressing cells ( with either orthogonal or parallel oriented KTs ) , we scored if at least one of the KTs was Mad1 + . Data information : Bars represent the mean of 2 biological replicates , while dark - and light - colored dots represent the values of each experiment . ( E ) : N ≥ 27 cells per condition , per experiment , ( ns ) not significant ; Fisher ' s exact test .

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E. Quantification of the number of cells with Mad1-positive KTs nearby the TetO locus. In case the KTs of the TetO chromosome were aligned, we scored whether KTs in the vicinity of the TetO locus were Mad1+. In case the KTs of the TetO chromosome could be distinguished in rTetR-GFP-Kin14VIb-expressing cells (with either orthogonal or parallel oriented KTs), we scored if at least one of the KTs was Mad1+. Data information: Bars represent the mean of 2 biological replicates, while dark- and light-colored dots represent the values of each experiment. (E): N ≥27 cells per condition, per experiment, (ns) not significant; Fisher's exact test.


A - D ( A , C ) Frequencies of the different types of segregation errors observed during anaphase ( A ) and late anaphase / telophase ( C ) . Schemes illustrating the observed types of anaphase errors are shown on the right of ( A ) . Cells within the main TetO locus distribution categories in control ( 1 - 1 ) , and Kin14VIb ( 2 - 0 ) conditions were scored ( see also Fig EV1A ) . The majority of Kin14VIb - expressing anaphases display at least one stretched arm ( CENP - C in line with TetO locus at the pole ) or a bridge in line with the TetO locus at the pole ( " stretched arm or bridge in line " ) , some of them with additional bridges ( + ) . ( B , D ) IF images for CENP - C , GFP , H3S10ph of U - 2 OS TetO cells in anaphase ( B ) and late anaphase or telophase ( D ) , expressing either rTetR - GFP or rTetR - GFP - Kin14VIb . Images ( maximum intensity projections ) represent the most frequently observed anaphase and telophase errors in cells with either a 1 - 1 ( majority of events in ctrl ) , or 2 - 0 ( majority of events in Kin14VIb ) TetO locus distribution . Scale bar = 5 µm . Yellow arrowheads indicate TetO locus , red arrowheads CENP - C . Data information : ( A , C ) : Means ± S . E . M . of 3 biological replicates are shown . Dots represent the values of each experiment . N = 41 - 89 cells per condition , per experiment * * * * P < 0 . 0001 , ( ns ) not significant ; Fisher ' s exact test . ( B , D ) : for optimal visibility of the TetO foci , the brightness and contrast of all channels were linearly adjusted for individual cells .

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

A-D (A, C) Frequencies of the different types of segregation errors observed during anaphase (A) and late anaphase/telophase (C). Schemes illustrating the observed types of anaphase errors are shown on the right of (A). Cells within the main TetO locus distribution categories in control (1-1), and Kin14VIb (2-0) conditions were scored (see also Fig EV1A). The majority of Kin14VIb-expressing anaphases display at least one stretched arm (CENP-C in line with TetO locus at the pole) or a bridge in line with the TetO locus at the pole ("stretched arm or bridge in line"), some of them with additional bridges (+). (B, D) IF images for CENP-C, GFP, H3S10ph of U-2 OS TetO cells in anaphase (B) and late anaphase or telophase (D), expressing either rTetR-GFP or rTetR-GFP-Kin14VIb. Images (maximum intensity projections) represent the most frequently observed anaphase and telophase errors in cells with either a 1-1 (majority of events in ctrl), or 2-0 (majority of events in Kin14VIb) TetO locus distribution. Scale bar = 5 µm. Yellow arrowheads indicate TetO locus, red arrowheads CENP-C. Data information: (A, C): Means ± S.E.M. of 3 biological replicates are shown. Dots represent the values of each experiment. N= 41-89 cells per condition, per experiment ****P<0.0001, (ns) not significant; Fisher's exact test. (B, D): for optimal visibility of the TetO foci, the brightness and contrast of all channels were linearly adjusted for individual cells.


A . Representative images of U - 2 OS TetO anaphases / telophases expressing rTetR - GFP or rTetR - GFP - Kin14VIb with FISH - marked centromeres of chromosome 1 ( chr1 - CEN ) . White and yellow arrowheads : chr1 - CEN segregated into the main masses of segregating chromosomes ; red arrowheads : chr1 - CEN FISH signal fragmented or splitted in a bridge ( bridging ) ; chr1 - CEN lagging between the two main masses of segregating chromosomes ( lagging ) ; chr1 - CEN late arrival ( late - equal : one CEN focus in but near the edge of a separated chromosome mass and connected to a bridge ) . For clarity , the maximum intensity projection of a subset of z stacks ( 6 - 12 / 64 ) is shown . Scale bar = 5 µm . B . Frequency of the different types of errors observed in A . The mean ( bars ) and individual values ( dots ) of 2 independent experiments are shown . Data information : In ( A ) , for optimal visibility of the chr1 - CEN FISH foci , the brightness and contrast were linearly adjusted for individual cells . ( B ) : n ≥ 45 cells per condition , per experiment . Bars represent the mean of 2 biological replicates , while dark - and light - colored dots represent the values of each experiment . * P < 0 . 05 , ( ns ) not significant . Fisher ' s exact test .

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A. Representative images of U-2 OS TetO anaphases/telophases expressing rTetR-GFP or rTetR-GFP-Kin14VIb with FISH-marked centromeres of chromosome 1 (chr1-CEN). White and yellow arrowheads: chr1-CEN segregated into the main masses of segregating chromosomes; red arrowheads: chr1-CEN FISH signal fragmented or splitted in a bridge (bridging); chr1-CEN lagging between the two main masses of segregating chromosomes (lagging); chr1-CEN late arrival (late-equal: one CEN focus in but near the edge of a separated chromosome mass and connected to a bridge). For clarity, the maximum intensity projection of a subset of z stacks (6-12/64) is shown. Scale bar = 5 µm. B. Frequency of the different types of errors observed in A. The mean (bars) and individual values (dots) of 2 independent experiments are shown. Data information: In (A), for optimal visibility of the chr1-CEN FISH foci, the brightness and contrast were linearly adjusted for individual cells. (B): n ≥ 45 cells per condition, per experiment. Bars represent the mean of 2 biological replicates, while dark- and light-colored dots represent the values of each experiment. * P<0.05, (ns) not significant. Fisher's exact test.


C . IF for Cas9 and CENP - C of metaphases of RPE1 - dCas9 - Kin14VIb cells transduced with Chr1 - telo sgRNA in the presence or absence of 500 nM rapalog . The Chr1 - telo loci ( Cas9 foci , white arrowheads ) are aligned on the metaphase plate in control condition ( - rapalog ) , while the loci are facing either a single or opposite poles after rapalog addition to induce Kin14VIb binding to dCas9 . Scale bar = 5 µm . For clarity , the maximum intensity projection of a subset of z stacks ( 18 - 50 / 100 ) is shown . Data information : ( C for optimal visibility of the Chr1 - telo foci , the brightness and contrast of all channels were linearly adjusted for individual cells .

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

C. IF for Cas9 and CENP-C of metaphases of RPE1-dCas9-Kin14VIb cells transduced with Chr1-telo sgRNA in the presence or absence of 500 nM rapalog. The Chr1-telo loci (Cas9 foci, white arrowheads) are aligned on the metaphase plate in control condition (- rapalog), while the loci are facing either a single or opposite poles after rapalog addition to induce Kin14VIb binding to dCas9. Scale bar = 5 µm. For clarity, the maximum intensity projection of a subset of z stacks (18-50/100) is shown. Data information: (C for optimal visibility of the Chr1-telo foci, the brightness and contrast of all channels were linearly adjusted for individual cells.


D , Live cell microscopy of asynchronously growing RPE1 - dCas9 - Kin14VIb cells transduced with Chr1 - telo sgRNA in the presence or absence of 500 nM rapalog to induce kinesin binding to the subtelomeric locus . ( D ) Stills showing the most frequently observed metaphase localization and daughter cell distribution ( white arrowheads ) of the duplicated Chr1 - telo loci . SiR - DNA was used to visualize the DNA , GFP depicts the Chr1 - telo loci . Time ( h : min ) . Scale bar = 5 µm . Note that in the upper panel , the cell was already in mitosis when imaging started ( t = 0 : 00 ) . Data information : ( D , Cells were derived from three independent imaging experiments . N = 23 cells ( - rapalog ) , n = 41 cells ( + rapalog ) . Sp = same pole , op = opposite pole , ss = same sisters . D ) : for optimal visibility of the Chr1 - telo foci , the brightness and contrast of all channels were linearly adjusted for individual cells .

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

D, Live cell microscopy of asynchronously growing RPE1-dCas9-Kin14VIb cells transduced with Chr1-telo sgRNA in the presence or absence of 500 nM rapalog to induce kinesin binding to the subtelomeric locus. (D) Stills showing the most frequently observed metaphase localization and daughter cell distribution (white arrowheads) of the duplicated Chr1-telo loci. SiR-DNA was used to visualize the DNA, GFP depicts the Chr1-telo loci. Time (h:min). Scale bar = 5 µm. Note that in the upper panel, the cell was already in mitosis when imaging started (t =0:00). Data information: (D, Cells were derived from three independent imaging experiments. N = 23 cells (- rapalog), n = 41 cells (+ rapalog). Sp = same pole, op = opposite pole, ss = same sisters. D): for optimal visibility of the Chr1-telo foci, the brightness and contrast of all channels were linearly adjusted for individual cells.


B , Live cell microscopy of asynchronously growing RPE1 - dCas9 - Kin14VIb cells transduced with Chr9 - cen sgRNA and plus / minus rapalog to induce kinesin binding to the pericentromeric locus ( Movies S4 - S8 ) . ( B ) Stills of the most frequently observed metaphase orientations and daughter cell distributions of the duplicated Chr9 - cen loci ( white arrowheads ) are shown . SiR - DNA was used to visualize the DNA , GFP depicts the Chr9 - cen loci . Time ( h : min ) . Scale bar = 5 µm . Data information : Cells were derived from two independent imaging experiments . . In ( B ) , for optimal visibility of the Chr9 - cen foci , the brightness and contrast of all channels were linearly adjusted for individual cells . See Movies EV4 - S8 for equally adjusted corresponding examples .

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

B, Live cell microscopy of asynchronously growing RPE1-dCas9-Kin14VIb cells transduced with Chr9-cen sgRNA and plus/minus rapalog to induce kinesin binding to the pericentromeric locus (Movies S4-S8). (B) Stills of the most frequently observed metaphase orientations and daughter cell distributions of the duplicated Chr9-cen loci (white arrowheads) are shown. SiR-DNA was used to visualize the DNA, GFP depicts the Chr9-cen loci. Time (h:min). Scale bar = 5 µm. Data information: Cells were derived from two independent imaging experiments.. In (B), for optimal visibility of the Chr9-cen foci, the brightness and contrast of all channels were linearly adjusted for individual cells. See Movies EV4- S8 for equally adjusted corresponding examples.


D . Time between nuclear envelop break down ( NEB ) and anaphase onset in the presence ( Kin14VIb bound to dCas9 ) or absence ( Kin14VIb expressed , but not bound to dCas9 ) of rapalog . Data information : Cells were derived from two independent imaging experiments . . ( D ) : Mean and S . D . are shown .

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

D. Time between nuclear envelop break down (NEB) and anaphase onset in the presence (Kin14VIb bound to dCas9) or absence (Kin14VIb expressed, but not bound to dCas9) of rapalog. Data information: Cells were derived from two independent imaging experiments.. (D): Mean and S.D. are shown.


A . Representative IF images of RPE1 - dCas9 - Kin14VIb cells , showing the localization of the Chr9 - cen loci in metaphase in the absence or presence or rapalog . Magnifications of the white boxed regions ( each region showing one Chr9 - cen locus ) are shown in the corners ( scale bars = 2 µm ) . The fraction of cells with Chr9 mis - aligned and CENP - C near the locus ( Cas9 ) is indicated below the image . For clarity , the maximum intensity projection of a subset of z stacks in which the Chr9 - cen loci are in focus ( 2 - 10 / 100 ) is shown . B . Frequency of the observed metaphase localizations of the Chr9 - cen loci as shown in ( A ) . Data information : In all cases , bars represent the mean of 2 independent experiments , while dark - and light - colored dots represent the values of each experiment . ( B ) : * * * * P < 0 . 0001 , Fisher ' s exact test . N ≥ 31 cells per condition , per experiment . ( A , For optimal visibility of the Chr9 - cen foci the brightness and contrast of all channels were linearly adjusted for individual cells .

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

A. Representative IF images of RPE1-dCas9-Kin14VIb cells, showing the localization of the Chr9-cen loci in metaphase in the absence or presence or rapalog. Magnifications of the white boxed regions (each region showing one Chr9-cen locus) are shown in the corners (scale bars = 2 µm). The fraction of cells with Chr9 mis-aligned and CENP-C near the locus (Cas9) is indicated below the image. For clarity, the maximum intensity projection of a subset of z stacks in which the Chr9-cen loci are in focus (2-10/100) is shown. B. Frequency of the observed metaphase localizations of the Chr9-cen loci as shown in (A). Data information: In all cases, bars represent the mean of 2 independent experiments, while dark- and light-colored dots represent the values of each experiment. (B): **** P<0.0001, Fisher's exact test. N ≥ 31 cells per condition, per experiment. (A, For optimal visibility of the Chr9-cen foci the brightness and contrast of all channels were linearly adjusted for individual cells.


C . Representative IF images of RPE1 - dCas9 - Kin14VIb cells , showing the segregation and distribution of Chr9 - cen loci in anaphase ( white arrowheads ) . Red arrowheads indicate H3S10ph - positive chromatin bridges . For clarity , the maximum intensity projection of a subset of z stacks in which the chr9 - cen loci are in focus ( 30 - 62 / 100 ) is shown . Scale bar = 2 µm . Data information : C ) : For optimal visibility of the Chr9 - cen foci H3S10ph - positive chromatin bridges , the brightness and contrast of all channels were linearly adjusted for individual cells .

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

C. Representative IF images of RPE1-dCas9-Kin14VIb cells, showing the segregation and distribution of Chr9-cen loci in anaphase (white arrowheads). Red arrowheads indicate H3S10ph-positive chromatin bridges. For clarity, the maximum intensity projection of a subset of z stacks in which the chr9-cen loci are in focus (30-62/100) is shown. Scale bar = 2 µm. Data information: C): For optimal visibility of the Chr9-cen foci H3S10ph-positive chromatin bridges, the brightness and contrast of all channels were linearly adjusted for individual cells.


E . Frequency of cells with a 2 - 2 distribution of the Chr9 - telo loci . Data information : In all cases , bars represent the mean of 2 independent experiments , while dark - and light - colored dots represent the values of each experiment . : * * * * P < 0 . 0001 , Fisher ' s exact test . N ≥ 29 cells per condition , per experiment .

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

E. Frequency of cells with a 2-2 distribution of the Chr9-telo loci. Data information: In all cases, bars represent the mean of 2 independent experiments, while dark- and light-colored dots represent the values of each experiment. : **** P<0.0001, Fisher's exact test. N ≥ 29 cells per condition, per experiment.


F . Frequency of the observed segregation / distribution errors of the Chr9 - cen loci as shown in ( C ) . Data information : In all cases , bars represent the mean of 2 independent experiments , while dark - and light - colored dots represent the values of each experiment . F ) : * * * * P < 0 . 0001 , Fisher ' s exact test . N ≥ 29 cells per condition , per experiment .

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

F. Frequency of the observed segregation/distribution errors of the Chr9-cen loci as shown in (C). Data information: In all cases, bars represent the mean of 2 independent experiments, while dark- and light-colored dots represent the values of each experiment. F): **** P<0.0001, Fisher's exact test. N ≥ 29 cells per condition, per experiment.


C . Average percentage of the bases of the p or q arm that are ( classified as ) 0 , 1 , 3 or 4 - somy . Percentages are depicted as stacked bar plots and are determined for all copy numbers other than 2 - somy ( euploid ) .

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

C. Average percentage of the bases of the p or q arm that are (classified as) 0, 1, 3 or 4-somy. Percentages are depicted as stacked bar plots and are determined for all copy numbers other than 2-somy (euploid).


( a - d ) Degradation of long - lived proteins in MEFs from wild - type ( 18Q - htt ) and mutant huntingtin knock - in ( 111Q - htt ) mice . ( a , b ) Rates of protein degradation after serum removal ( a )

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

(a-d) Degradation of long-lived proteins in MEFs from wild-type (18Q-htt) and mutant huntingtin knock-in (111Q-htt) mice. (a,b) Rates of protein degradation after serum removal (a)


( a - d ) Degradation of long - lived proteins in MEFs from wild - type ( 18Q - htt ) and mutant huntingtin knock - in ( 111Q - htt ) mice . ( a , b ) Rates of protein degradation after treatment with rapamycin ( rapam ) or thapsigargin ( thapsig ) ( b ) .

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

(a-d) Degradation of long-lived proteins in MEFs from wild-type (18Q-htt) and mutant huntingtin knock-in (111Q-htt) mice. (a,b) Rates of protein degradation after treatment with rapamycin (rapam) or thapsigargin (thapsig) (b).


( c ) Lysosomal degradation calculated as percentage of protein degradation sensitive to NH4Cl .

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

(c) Lysosomal degradation calculated as percentage of protein degradation sensitive to NH4Cl.


( d ) Contribution of macroautophagy calculated as percentage of protein degradation sensitive to 3 - methyladenine ( 3 - MA ) .

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

(d) Contribution of macroautophagy calculated as percentage of protein degradation sensitive to 3-methyladenine (3-MA).


( e ) Degradation of long - lived proteins in striatal cells from wild - type ( 7Q - htt ) and mutant huntingtin knock - in mice ( 111Q - htt ) in response to different autophagic stimuli .

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

(e) Degradation of long-lived proteins in striatal cells from wild-type (7Q-htt) and mutant huntingtin knock-in mice (111Q-htt) in response to different autophagic stimuli.


( f , g ) Rates of protein degradation in wild - type and HD MEFs ( f ) and striatal cells ( g ) in the absence ( control ) or presence ( Atg7 − ) of Atg7 RNA interference .

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

(f,g) Rates of protein degradation in wild-type and HD MEFs (f) and striatal cells (g) in the absence (control) or presence (Atg7−) of Atg7 RNA interference.


( h ) Contribution of macroautophagy ( macroaut ) to the degradation of long - lived proteins in lymphoblasts from unaffected controls ( UC ) and people with HD . Mean + s . d . of values from two to four different individuals and three or four different experiments . * P 0 . 05 versus untreated , # P 0 . 05 versus control .

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

(h) Contribution of macroautophagy (macroaut) to the degradation of long-lived proteins in lymphoblasts from unaffected controls (UC) and people with HD. Mean + s.d. of values from two to four different individuals and three or four different experiments. *P 0.05 versus untreated, #P 0.05 versus control.


( a ) Top : LC3 immunostaining of 18Q - htt and 111Q - htt MEFs maintained in the presence ( + ) or absence ( - ) of serum and inhibitors of lysosomal proteolysis ( inhib + ) . Bottom : mean number of LC3 + vesicles per cell in cells maintained in the presence ( left ) or absence ( right ) of serum . n = 4 .

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

(a) Top: LC3 immunostaining of 18Q-htt and 111Q-htt MEFs maintained in the presence (+) or absence (-) of serum and inhibitors of lysosomal proteolysis (inhib+). Bottom: mean number of LC3+ vesicles per cell in cells maintained in the presence (left) or absence (right) of serum. n = 4.


( b ) Top : LC3 immunoblot of the same cells after serum ( S ) removal or thapsigargin ( TG ) treatment . PI , protease inhibitors . Bottom : LC3 - II levels and LC3 - II flux ( calculated as a multiple of LC3 - II value in absence of PI ; lateral numbers ) . n = 4 .

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

(b) Top: LC3 immunoblot of the same cells after serum (S) removal or thapsigargin (TG) treatment. PI, protease inhibitors. Bottom: LC3-II levels and LC3-II flux (calculated as a multiple of LC3-II value in absence of PI; lateral numbers). n = 4.


( c ) LC3 - II values and LC3 - II flux in neuronal cultures from wild - type ( WT ) or HD94 mice ( HD ) grown on wild - type rat astrocyte monolayers , analyzed as in b . n = 4 .

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

(c) LC3-II values and LC3-II flux in neuronal cultures from wild-type (WT) or HD94 mice (HD) grown on wild-type rat astrocyte monolayers, analyzed as in b. n = 4.


( d ) LC3 staining of lymphoblasts , from three unaffected controls ( UC ) and people with HD , maintained in the presence or absence of serum . Accession numbers are indicated above panels . Right : mean number of LC3 + vesicles . Extended study in Supplementary Figure 8b , c .

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

(d) LC3 staining of lymphoblasts, from three unaffected controls (UC) and people with HD, maintained in the presence or absence of serum. Accession numbers are indicated above panels. Right: mean number of LC3+ vesicles. Extended study in Supplementary Figure 8b,c.


( e ) Immunoblots for LC3 - II in UC and HD lymphoblasts treated or not treated with protease inhibitors . Right : LC3 - II values and LC3 - II flux . Extended study in Supplementary Figure 8a . Mean + s . d . ; # P 0 . 05 . Full - length blots in Supplementary Figure 20 .

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

(e) Immunoblots for LC3-II in UC and HD lymphoblasts treated or not treated with protease inhibitors. Right: LC3-II values and LC3-II flux. Extended study in Supplementary Figure 8a. Mean + s.d.; #P 0.05. Full-length blots in Supplementary Figure 20.


( a ) Electron microscopy of striatal neurons from control and HD94 mice grown over a wild - type ratastrocyte monolayer . Higher magnification fields show the double membrane and clear content of the cytosolic vesicles . Top right : number of vacuoles per cell profile ( 13 - 17 cell profiles in triplicate experiments ; * P 0 . 05 ) . Arrows , enlarged electron - clear vesicles .

(a) Electron microscopy of striatal neurons from control and HD94 mice grown over a wild-type ratastrocyte monolayer. Higher magnification fields show the double membrane and clear content of the cytosolic vesicles. Top right: number of vacuoles per cell profile (13-17 cell profiles in triplicate experiments; *P 0.05). Arrows, enlarged electron-clear vesicles.


( b ) Electron micrographs of striatal cells from mutant huntingtin knock - in ( 111Q - htt ) and wild - type ( 7Q - htt ) mice .

O O O O O O 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) Electron micrographs of striatal cells from mutant huntingtin knock-in (111Q-htt) and wild-type (7Q-htt) mice.


( c ) Electron micrographs of lymphoblasts from unaffected controls ( UC ) and people with HD , maintained in the presence or absence of serum . Arrows , enlarged electron - clear vesicles .

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

(c) Electron micrographs of lymphoblasts from unaffected controls (UC) and people with HD, maintained in the presence or absence of serum. Arrows, enlarged electron-clear vesicles.


( d ) Immunogold for LC3 in striatal cells from mutant huntingtin knock - in mice ( 111Q - htt ) , striatal neurons from HD94 mice and lymphoblasts ( lymph ) from people with HD . Full fields and more details of autophagic vacuoles in Supplementary Figures 10 and 11 .

(d) Immunogold for LC3 in striatal cells from mutant huntingtin knock-in mice (111Q-htt), striatal neurons from HD94 mice and lymphoblasts (lymph) from people with HD. Full fields and more details of autophagic vacuoles in Supplementary Figures 10 and 11.


( a ) Electron micrographs of fractions enriched in autophagosomes and autophagolysosomes isolated from liver of 18Q - htt and 111Q - htt mice . Insets : higher magnification of single vesicles . Right : percentage of vesicles with electron - clear ( light ) , vesiculated ( multivesicular ) or electron - dense ( dark ) contents . Mean + s . e . m . of three different isolations ( > ∼ 1 , 000 autophagic vacuoles ) .

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

(a) Electron micrographs of fractions enriched in autophagosomes and autophagolysosomes isolated from liver of 18Q-htt and 111Q-htt mice. Insets: higher magnification of single vesicles. Right: percentage of vesicles with electron-clear (light), vesiculated (multivesicular) or electron-dense (dark) contents. Mean + s.e.m. of three different isolations (> ∼1,000 autophagic vacuoles).


( b ) Bidimensional electrophoresis and Sypro Ruby staining of these fractions . Left , intact vacuoles ; right , vacuole contents . Horizontal dimension , isoelectric point ( pH ) ; vertical , molecular mass ( kDa ) . * P 0 . 05 .

(b) Bidimensional electrophoresis and Sypro Ruby staining of these fractions. Left, intact vacuoles; right, vacuole contents. Horizontal dimension, isoelectric point (pH); vertical, molecular mass (kDa). *P 0.05.