( C ) Correlation of poison isoform inclusion and GE levels for all RBPs with temperature - dependent AS - decay skipped exon events . Shown are the log2 fold change ( FC ) in GE versus the ∆ PSI of the poison isoform between the CHX samples . Top shows the GE change between the DMSO samples and bottom between the CHX samples . R² and P ( deviation from zero slope ) are indicated . N = 24 .

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(C) Correlation of poison isoform inclusion and GE levels for all RBPs with temperature-dependent AS-decay skipped exon events. Shown are the log2 fold change (FC) in GE versus the ∆PSI of the poison isoform between the CHX samples. Top shows the GE change between the DMSO samples and bottom between the CHX samples. R² and P (deviation from zero slope) are indicated. N=24.


( E ) Splicing reporters , containing only an intron ( GFP ctrl , # 1 ) or additionally temperature - dependent exons with ~ 1kB surrounding introns . Representative radioactive RT - PCRs ( bottom ) confirm cold - induced inclusion of Ythdf3 exon 7 ( # 2 ) and human Synerg exon 21 ( # 3 ) after 24 hours at the indicated temperatures . Representative images are derived from three independent gels . See SourceDataForFigure2E for uncropped versions of these gels .

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(E) Splicing reporters, containing only an intron (GFP ctrl, #1) or additionally temperature-dependent exons with ~1kB surrounding introns. Representative radioactive RT-PCRs (bottom) confirm cold-induced inclusion of Ythdf3 exon 7 (#2) and human Synerg exon 21 (#3) after 24 hours at the indicated temperatures. Representative images are derived from three independent gels. See SourceDataForFigure2E for uncropped versions of these gels.


( F - G ) GFP expression was quantified using FACS and is shown relative to GFP ctrl . In ( F ) transfected cells were incubated at the indicated temperatures for 24 hours ( n = 3 , mean + / - SD ) . In ( G ) cells were incubated directly after transfection for 12 hours at 39 ° C ( blue ) or 33 ° C ( red ) and then shifted for 2 , 4 , 8 and 12 hours ( n = 3 , mean + / - SD ) .

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(F-G) GFP expression was quantified using FACS and is shown relative to GFP ctrl. In (F) transfected cells were incubated at the indicated temperatures for 24 hours (n=3, mean +/- SD). In (G) cells were incubated directly after transfection for 12 hours at 39°C (blue) or 33°C (red) and then shifted for 2, 4, 8 and 12 hours (n=3, mean +/- SD).


( A ) SR protein specific temperature - dependent AS - decay . Sashimi plots are shown for heat - induced poison exon inclusion for Srsf2 ( left ) and cold - induced poison isoform formation for Srsf10 ( right ) . Quantification of GE and AS are presented in the center ( as in Figure 2B ) , lines represent mean .

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(A) SR protein specific temperature-dependent AS-decay. Sashimi plots are shown for heat-induced poison exon inclusion for Srsf2 (left) and cold-induced poison isoform formation for Srsf10 (right). Quantification of GE and AS are presented in the center (as in Figure 2B), lines represent mean.


( B ) Poison exon inclusion for Srsf2 ( left ) and Srsf10 ( right ) in a 24h temperature - rhythm in human cells . Hek293 cells were pre - entrained with square - wave temperature cycles ( 12h 34 ° C / 12h 38 ° C ) for 48h . For the last 24h , cells were treated with DMSO or CHX every 4h and harvested after 4h and analyzed by splicing sensitive RT - PCR ( n = 3 , mean ± SD ) . White area : 34 ° C ; Red area : 38 ° C . In Hek293 cells , inclusion of Srsf10 exon 3 is coupled to polyadenylation making it a weak decay target .

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(B) Poison exon inclusion for Srsf2 (left) and Srsf10 (right) in a 24h temperature-rhythm in human cells. Hek293 cells were pre-entrained with square-wave temperature cycles (12h 34°C/ 12h 38°C) for 48h. For the last 24h, cells were treated with DMSO or CHX every 4h and harvested after 4h and analyzed by splicing sensitive RT-PCR (n=3, mean ± SD). White area: 34°C; Red area: 38°C. In Hek293 cells, inclusion of Srsf10 exon 3 is coupled to polyadenylation making it a weak decay target.


( C ) AS - decay of human targets at 34 ° C ( blue ) and 38 ° C ( red ) was investigated after a square - wave temperature regime ( see B ) . RNAs were harvested after 56 and 68 hours , respectively ( n = 3 , mean ± SD ) . Statistical significance was determined by 2 - way ANOVA and is indicated by asterisks : adjusted p values : not significant ( ns ) , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Box plots show median PSI of validation PCRs , whiskers show min to max . Individual data points are shown as circles .

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(C) AS-decay of human targets at 34°C (blue) and 38°C (red) was investigated after a square-wave temperature regime (see B). RNAs were harvested after 56 and 68 hours, respectively (n=3, mean ± SD). Statistical significance was determined by 2-way ANOVA and is indicated by asterisks: adjusted p values: not significant (ns), **p<0.01, ***p<0.001, ****p<0.0001. Box plots show median PSI of validation PCRs, whiskers show min to max. Individual data points are shown as circles.


( E ) Quantification of Srsf2 AS - decay in hamster , rabbit and chicken cells ( n = 2 , mean ± SD ) . Statistical significance was determined by 2 - way ANOVA , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . Box plots show median PSI of validation PCRs , whiskers show min to max . Individual data points are shown as circles .

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(E) Quantification of Srsf2 AS-decay in hamster, rabbit and chicken cells (n=2, mean ± SD). Statistical significance was determined by 2-way ANOVA, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Box plots show median PSI of validation PCRs, whiskers show min to max. Individual data points are shown as circles.


Rhythmic Srsf2 productive mRNA levels ( relative to Gapdh ) were analyzed by RT - qPCR . For each clone , expression is normalized to time point 52h ,

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Rhythmic Srsf2 productive mRNA levels (relative to Gapdh) were analyzed by RT-qPCR. For each clone, expression is normalized to time point 52h,


Rhythmic Srsf10 productive mRNA levels ( relative to Gapdh ) were analyzed by RT - qPCR . For each clone , expression is normalized to time point 52h For Srsf10 two independently generated clones were investigated ( ∆ E3 A and B ) . Statistical significance was determined by unpaired t - test and is indicated by asterisks : p values : * * p < 0 . 01 , * * * * p < 0 . 0001 .

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Rhythmic Srsf10 productive mRNA levels (relative to Gapdh) were analyzed by RT-qPCR. For each clone, expression is normalized to time point 52h For Srsf10 two independently generated clones were investigated (∆E3 A and B). Statistical significance was determined by unpaired t-test and is indicated by asterisks: p values: **p<0.01, ****p<0.0001.


Comparing temperature - induced expression of the core clock components Bmal1 in WT and Srsf10 ∆ E3 cells ( relative to Gapdh , normalized to time point 52h ) . Statistical significance was determined by unpaired t - test and is indicated by asterisks : p values : * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . n = 3 , mean ± SEM .

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Comparing temperature-induced expression of the core clock components Bmal1 in WT and Srsf10 ∆E3 cells (relative to Gapdh, normalized to time point 52h). Statistical significance was determined by unpaired t-test and is indicated by asterisks: p values: *p<0.05, ***p<0.001, ****p<0.0001. n=3, mean ± SEM.


Comparing temperature - induced expression of the core clock components Per1 in WT and Srsf10 ∆ E3 cells ( relative to Gapdh , normalized to time point 52h ) . Statistical significance was determined by unpaired t - test and is indicated by asterisks : p values : * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . n = 3 , mean ± SEM .

Comparing temperature-induced expression of the core clock components Per1 in WT and Srsf10 ∆E3 cells (relative to Gapdh, normalized to time point 52h). Statistical significance was determined by unpaired t-test and is indicated by asterisks: p values: *p<0.05, ***p<0.001, ****p<0.0001. n=3, mean ± SEM.


( A ) Normalized GE values of plant SR proteins in a 48 - hour time course . In the first day , plants were kept at 20 ° C ( red ) and shifted to 4 ° C ( blue ) for the second day . Plants were under a 12 - hour dark / light regimen ( gray / yellow bars ) . Warm - and cold - expressed genes are indicated .

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(A) Normalized GE values of plant SR proteins in a 48-hour time course. In the first day, plants were kept at 20°C (red) and shifted to 4°C (blue) for the second day. Plants were under a 12-hour dark/light regimen (gray/yellow bars). Warm- and cold-expressed genes are indicated.


( C ) Sashimi plots as shown in Figure 2A ( without conservation score ) for decay - inducing AS events upon cold ( At - SCL33 , left ) or warm temperature ( At - SR34 , right ) . Data shown represent , from top to bottom , warm - dark , warm - light , cold - dark and cold - light conditions as indicated on the right of the plots . ( D ) For validation , A . thaliana were incubated for 3 days either at 20 ° C or 4 ° C and RNA was investigated for gene expression ( green , left y - axis ) and poison isoform formation ( orange , right y - axis ) . Gene expression of At - SCL33 ( left ) or At - SR34 ( right ) is shown relative to Ipp2 . Lines represent mean , individual data points are shown .

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(C) Sashimi plots as shown in Figure 2A (without conservation score) for decay-inducing AS events upon cold (At-SCL33, left) or warm temperature (At-SR34, right). Data shown represent, from top to bottom, warm-dark, warm-light, cold-dark and cold-light conditions as indicated on the right of the plots. (D) For validation, A. thaliana were incubated for 3 days either at 20°C or 4°C and RNA was investigated for gene expression (green, left y-axis) and poison isoform formation (orange, right y-axis). Gene expression of At-SCL33 (left) or At-SR34 (right) is shown relative to Ipp2. Lines represent mean, individual data points are shown.


( C ) Sashimi plots as shown in Figure 2A ( without conservation score ) for decay - inducing AS events upon cold ( At - SCL33 , left ) or warm temperature ( At - SR34 , right ) . Data shown represent , from top to bottom , warm - dark , warm - light , cold - dark and cold - light conditions as indicated on the right of the plots .

(C) Sashimi plots as shown in Figure 2A (without conservation score) for decay-inducing AS events upon cold (At-SCL33, left) or warm temperature (At-SR34, right). Data shown represent, from top to bottom, warm-dark, warm-light, cold-dark and cold-light conditions as indicated on the right of the plots.


( E ) Correlation of poison isoform inclusion and GE levels for temperature - dependent poison skipped exon events . Shown are the log2 fold change ( FC ) in GE versus the ∆ PSI of the poison isoform . Decay targets were identified by using data from CHX - treated plants . R² and P ( deviation from zero slope ) are indicated . N = 80

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(E) Correlation of poison isoform inclusion and GE levels for temperature-dependent poison skipped exon events. Shown are the log2 fold change (FC) in GE versus the ∆PSI of the poison isoform. Decay targets were identified by using data from CHX-treated plants. R² and P(deviation from zero slope) are indicated. N=80


( E ) Correlation of poison isoform inclusion and GE levels for temperature - dependent poison skipped exon events . Shown are the log2 fold change ( FC ) in GE versus the ∆ PSI of the poison isoform . Decay targets were identified by using data from CHX - treated plants . R² and P ( deviation from zero slope ) are indicated . N =

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( B ) Correlation of rhythmic Srsf10 expression ( orange , left y - axis ) and exon 3 inclusion ( black , right y - axis ) in mouse liver samples from the indicated ZTs ( n = 4 , mean + / - SEM ) . Quantifications were obtained by RNA - seq analysis of datasets from ( Atger et al . , 2015 ) .

(B) Correlation of rhythmic Srsf10 expression (orange, left y-axis) and exon 3 inclusion (black, right y-axis) in mouse liver samples from the indicated ZTs (n=4, mean +/- SEM). Quantifications were obtained by RNA-seq analysis of datasets from (Atger et al., 2015).


( C ) Representative Western blot of SRSF10 protein levels from mouse liver nuclear extracts ( NX ) from different ZTs . The different SRSF10 variants are highlighted on the right . SRSF10 - fl and SRSF10 - 2 are the result of differential last exon usage . hnRNP L was used as a loading control . The asterisks could represent hyperphosphorylated SRSF10 - fl through higher CLK activity during the day . ( D ) Quantification of SRSF10 - fl + SRSF10 - 2 relative to ZT0 and hnRNP L ( mean of at least 3 mice ± SEM ) . Student ' s unpaired t test - derived p values * * * p < 0 . 001 .

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(C) Representative Western blot of SRSF10 protein levels from mouse liver nuclear extracts (NX) from different ZTs. The different SRSF10 variants are highlighted on the right. SRSF10-fl and SRSF10-2 are the result of differential last exon usage. hnRNP L was used as a loading control. The asterisks could represent hyperphosphorylated SRSF10-fl through higher CLK activity during the day. (D) Quantification of SRSF10-fl + SRSF10-2 relative to ZT0 and hnRNP L (mean of at least 3 mice ± SEM). Student's unpaired t test-derived p values ***p<0.001.


( C ) Representative Western blot of SRSF10 protein levels from mouse liver nuclear extracts ( NX ) from different ZTs . The different SRSF10 variants are highlighted on the right . SRSF10 - fl and SRSF10 - 2 are the result of differential last exon usage . hnRNP L was used as a loading control . The asterisks could represent hyperphosphorylated SRSF10 - fl through higher CLK activity during the day .

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(C) Representative Western blot of SRSF10 protein levels from mouse liver nuclear extracts (NX) from different ZTs. The different SRSF10 variants are highlighted on the right. SRSF10-fl and SRSF10-2 are the result of differential last exon usage. hnRNP L was used as a loading control. The asterisks could represent hyperphosphorylated SRSF10-fl through higher CLK activity during the day.


( A ) Dose - response curves ( normalized growth rate as a function of drug concentration ) for different antibiotics . Growth rate was measured via optical density measurements over time Antibiotics used : Trimethoprim ( TMP ) , tetracycline ( TET ) , chloramphenicol ( CHL ) , ciprofloxacin ( CPR ) , lincomycin ( LIN ) , nitrofurantoin ( NIT ) , and mecillinam ( MEC ) . The TMP dose - response curve ( dark blue ) is by far the shallowest . Lines are fits of the Hill function $ \ frac { g \ left ( c \ right ) } { g ( 0 ) } = \ frac { 1 } { 1 + \ left ( \ frac { c } { IC _ { 50 } } \ right ) ^ { n } } \ $ to the data . Drug concentrations were arbitrarily rescaled to better visualize dose - response curve steepness ; Error bars show standard deviation of 12 biological replicates .

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(A) Dose-response curves (normalized growth rate as a function of drug concentration) for different antibiotics. Growth rate was measured via optical density measurements over time Antibiotics used: Trimethoprim (TMP), tetracycline (TET), chloramphenicol (CHL), ciprofloxacin (CPR), lincomycin (LIN), nitrofurantoin (NIT), and mecillinam (MEC). The TMP dose-response curve (dark blue) is by far the shallowest. Lines are fits of the Hill function $\frac{g\left( c \right)}{g(0)} = \frac{1}{1 + \left( \frac{c}{IC_{50}} \right)^{n}}\ $to the data. Drug concentrations were arbitrarily rescaled to better visualize dose-response curve steepness; Error bars show standard deviation of 12 biological replicates.


( C ) Density scatterplot showing growth response to TMP versus normalized drug - free growth rate for 3 , 913 gene deletion strains these are essentially all viable gene deletion strains in E . coli , no selection of strains was made . These gene deletion strains exhibit diverse growth rates , offering an unbiased way to test the relation between the drug - free growth rate and the response to antibiotics . Response is defined as growth rate in the presence of TMP normalized to the drug - free growth rate of the respective deletion strain . TMP was used at a fixed concentration that inhibits wild type growth by about 30 % Spearman correlation coefficient ρs is shown .

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(C) Density scatterplot showing growth response to TMP versus normalized drug-free growth rate for 3,913 gene deletion strains these are essentially all viable gene deletion strains in E. coli, no selection of strains was made. These gene deletion strains exhibit diverse growth rates, offering an unbiased way to test the relation between the drug-free growth rate and the response to antibiotics. Response is defined as growth rate in the presence of TMP normalized to the drug-free growth rate of the respective deletion strain. TMP was used at a fixed concentration that inhibits wild type growth by about 30% Spearman correlation coefficient ρs is shown.


( D ) Bar chart showing negative Spearman correlation coefficients − ρs compared across antibiotics Error bars show bootstrap standard error of ρs . TMP ( blue ) exhibits by far the strongest negative correlation , indicating the existence of a particularly strong growth - mediated negative feedback loop for this antibiotic .

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(D) Bar chart showing negative Spearman correlation coefficients  − ρs compared across antibiotics Error bars show bootstrap standard error of ρs. TMP (blue) exhibits by far the strongest negative correlation, indicating the existence of a particularly strong growth-mediated negative feedback loop for this antibiotic.


( A ) Growth rate under glucose limitation achieved by adding the non - metabolizable structural glucose analog α - methyl glucoside ( αMG ) at different ratios to glucose in a minimal medium Data information : Error bars in A , show standard deviation from 6 , 12 , and 18 biological replicates , respectively ; day - to - day reproducibility of growth rate measurements is

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(A) Growth rate under glucose limitation achieved by adding the non-metabolizable structural glucose analog α-methyl glucoside (αMG) at different ratios to glucose in a minimal medium Data information: Error bars in A, show standard deviation from 6, 12, and 18 biological replicates, respectively; day-to-day reproducibility of growth rate measurements is high


( B ) Normalized growth rate ( gray scale ) from a checkerboard assay in a two - dimensional concentration gradient of TMP and αMG . Dashed black line shows contour line of 90 % growth inhibition ( IC90 line ) . Red arrow shows increase in IC90 as growth is lowered . Inset : Normalized growth rate as a function of TMP concentration along the column marked in blue .

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(B) Normalized growth rate (gray scale) from a checkerboard assay in a two-dimensional concentration gradient of TMP and αMG. Dashed black line shows contour line of 90% growth inhibition (IC90 line). Red arrow shows increase in IC90 as growth is lowered. Inset: Normalized growth rate as a function of TMP concentration along the column marked in blue.


( C ) Fold - change in IC90 at αMG / glucose ratio 2 . 5 in assays as in B for different antibiotics Lowering growth rate increases IC90 for TMP but not for other antibiotics . Data information : Error bars in C show standard deviation from three neighboring αMG / glucose

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(C) Fold-change in IC90 at αMG/glucose ratio 2.5 in assays as in B for different antibiotics Lowering growth rate increases IC90 for TMP but not for other antibiotics. Data information: Error bars in C show standard deviation from three neighboring αMG/glucose ratios


( D ) Growth rate in rich medium ( LB ) under different levels of overexpression of a gratuitous protein from a T5 - lac promoter ; overexpression burden is controlled by IPTG concentration Data information : Error bars in D , show standard deviation from 6 , 12 , and 18 biological replicates , respectively ; day - to - day reproducibility of growth rate measurements is

(D) Growth rate in rich medium (LB) under different levels of overexpression of a gratuitous protein from a T5-lac promoter; overexpression burden is controlled by IPTG concentration Data information: Error bars in D, show standard deviation from 6, 12, and 18 biological replicates, respectively; day-to-day reproducibility of growth rate measurements is high


( E ) As B but for growth rate reduction by protein overexpression in a two - dimensional concentration gradient of TMP and IPTG .

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(E) As B but for growth rate reduction by protein overexpression in a two-dimensional concentration gradient of TMP and IPTG.


( F ) Fold - change in IC90 at 1 . 25 mM IPTG in assays as in E for different antibiotics Overexpression of unnecessary protein increases IC90 for TMP by almost five - fold ; no comparable increase occurs for other antibiotics . Data information : Error bars in F show standard deviation from IPTG concentrations centered at 2 . 5 and 1 . 25 mM , respectively . CHL was not used in the protein overexpression assay in F since the plasmid used for overexpression has a CHL - resistance

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(F) Fold-change in IC90 at 1.25 mM IPTG in assays as in E for different antibiotics Overexpression of unnecessary protein increases IC90 for TMP by almost five-fold; no comparable increase occurs for other antibiotics. Data information: Error bars in F show standard deviation from IPTG concentrations centered at 2.5 and 1.25 mM, respectively. CHL was not used in the protein overexpression assay in F since the plasmid used for overexpression has a CHL-resistance marker


( G ) Growth rate in minimal medium containing different carbon sources Glucose ( GLU ) , fructose ( FRU ) , mannose ( MAN ) , galactose ( GAL ) , and glycerol ( GLY ) . Data information : Error bars in show standard deviation from 6 , 12 , and 18 biological replicates , respectively ; day - to - day reproducibility of growth rate measurements is

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(G) Growth rate in minimal medium containing different carbon sources Glucose (GLU), fructose (FRU), mannose (MAN), galactose (GAL), and glycerol (GLY). Data information: Error bars in show standard deviation from 6, 12, and 18 biological replicates, respectively; day-to-day reproducibility of growth rate measurements is high


( H ) Normalized growth rates ( gray scale ) on different carbon sources ( x - axis ) at different TMP concentrations ( y - axis ) .

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(H) Normalized growth rates (gray scale) on different carbon sources (x-axis) at different TMP concentrations (y-axis).


( I ) Fold - change in IC90 in assays as in H for different antibiotics Data information : Error bars in I show standard deviation from three biological replicates .

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(I) Fold-change in IC90 in assays as in H for different antibiotics Data information: Error bars in I show standard deviation from three biological replicates.


( A ) TMP dose - response curve in minimal medium with glucose as carbon source and at lower drug - free growth rate due to glucose limitation , achieved by increasing the αMG / glucose ratio from 0 ( black ) to 5 ( gray ) . Glucose limitation results in a steeper dose - response curve . Lines show Hill function fits ( B ) Steepness of TMP dose - response curves ( dose - sensitivity n ) versus drug - free growth rate at different αMG concentrations Numbers next to data points show αMG / glucose ratio . Data information : Growth rate error bars show standard deviation of three replicates ; vertical error bars in B show standard deviation of parameter estimates from Hill function fit .

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

(A) TMP dose-response curve in minimal medium with glucose as carbon source and at lower drug-free growth rate due to glucose limitation, achieved by increasing the αMG /glucose ratio from 0 (black) to 5 (gray). Glucose limitation results in a steeper dose-response curve. Lines show Hill function fits (B) Steepness of TMP dose-response curves (dose-sensitivity n) versus drug-free growth rate at different αMG concentrations Numbers next to data points show αMG/glucose ratio. Data information: Growth rate error bars show standard deviation of three replicates; vertical error bars in B show standard deviation of parameter estimates from Hill function fit.


( C , D ) As A , B but for growth limitation by gratuitous protein overexpression in rich growth medium Inducing overexpression with IPTG at 10 mM ( light gray ) steepens the dose - response curve compared to no induction ( black ) . Numbers next to data points in D show IPTG concentration in mM . Data information : Growth rate error bars show standard deviation of three replicates ; vertical error bars in D , show standard deviation of parameter estimates from Hill function fit .

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(C,D) As A,B but for growth limitation by gratuitous protein overexpression in rich growth medium Inducing overexpression with IPTG at 10 mM (light gray) steepens the dose-response curve compared to no induction (black). Numbers next to data points in D show IPTG concentration in mM. Data information: Growth rate error bars show standard deviation of three replicates; vertical error bars in D, show standard deviation of parameter estimates from Hill function fit.


( E , F ) As A , B but for growth limitation by varying the carbon source in a minimal medium Data information : Growth rate error bars show standard deviation of three replicates ; vertical error bars in F show standard deviation of parameter estimates from Hill function fit .

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(E,F) As A,B but for growth limitation by varying the carbon source in a minimal medium Data information: Growth rate error bars show standard deviation of three replicates; vertical error bars in F show standard deviation of parameter estimates from Hill function fit.


( A ) Dependence of growth rate ( black ) and folA expression ( green ) on TMP concentration . Schematic : FolA expression was measured using a promoter - GFP reporter inserted at a neutral site in the genome Data information : Black line in A shows Hill function fit other lines show polynomial fits of second order ( A to guide the eye . Horizontal dotted green line shows folA expression level in the absence of TMP . Error bars show standard deviation of three biological replicates .

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(A) Dependence of growth rate (black) and folA expression (green) on TMP concentration. Schematic: FolA expression was measured using a promoter-GFP reporter inserted at a neutral site in the genome Data information: Black line in A shows Hill function fit other lines show polynomial fits of second order (A to guide the eye. Horizontal dotted green line shows folA expression level in the absence of TMP. Error bars show standard deviation of three biological replicates.


( B ) Growth rate ( black ) and folA expression ( green ) in the absence of TMP at different growth rates achieved by different ratios of αMG / glucose . Data information : other lines show polynomial fits of second order B ) to guide the eye . Horizontal dotted green line shows folA expression level in the absence of TMP . Error bars show standard deviation of three biological replicates .

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(B) Growth rate (black) and folA expression (green) in the absence of TMP at different growth rates achieved by different ratios of αMG/glucose. Data information: other lines show polynomial fits of second order B) to guide the eye. Horizontal dotted green line shows folA expression level in the absence of TMP. Error bars show standard deviation of three biological replicates.


( C ) Scatterplot of folA expression level with growth rate across all combinations of TMP concentrations and αMG / glucose ratios Pearson ' s correlation coefficient ρ and p - value from two - sided t - test are shown . FolA expression is largely determined by growth rate , suggesting that this regulation is growth rate - dependent and not mediated by a specific molecular mechanism . Data information : ; other lines show polynomial fits of first ( C ) order to guide the eye . Horizontal dotted green line shows folA expression level in the absence of TMP . Error bars show standard deviation of three biological replicates .

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

(C) Scatterplot of folA expression level with growth rate across all combinations of TMP concentrations and αMG/glucose ratios Pearson's correlation coefficient ρ and p-value from two-sided t-test are shown. FolA expression is largely determined by growth rate, suggesting that this regulation is growth rate-dependent and not mediated by a specific molecular mechanism. Data information: ; other lines show polynomial fits of first (C) order to guide the eye. Horizontal dotted green line shows folA expression level in the absence of TMP. Error bars show standard deviation of three biological replicates.


( D ) Dependence of folA expression on TMP concentration at four different αMG / glucose ratios ( 0 , 2 . 5 , 5 , and 10 as shown ) . Darker green indicates greater αMG / glucose ratio . FolA expression converges approximately to the same level at high TMP concentrations . Data information : Horizontal dotted green line shows folA expression level in the absence of TMP . Error bars show standard deviation of three biological replicates .

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

(D) Dependence of folA expression on TMP concentration at four different αMG/glucose ratios (0, 2.5, 5, and 10 as shown). Darker green indicates greater αMG/glucose ratio. FolA expression converges approximately to the same level at high TMP concentrations. Data information: Horizontal dotted green line shows folA expression level in the absence of TMP. Error bars show standard deviation of three biological replicates.


( B ) Growth rate as a function of TMP concentration for different paths through IPTG - TMP concentration space Constant FolA is shown in black and inverted FolA regulation in red . Wild - type dose - response curve ( blue line ; is shown for comparison . ( C ) Steepness of the dose response curve ( quantified as dose - sensitivity n ) for the three cases in B . Inset : Normalized FolA expression level as a function of TMP concentration for the three cases in B ; WT ( blue ) colors as in the bar chart and in B . Data information : Error bars in B show standard deviation of the measured growth rates used for interpolating the values shown ; the entire experiment was replicated once Error bars in C show standard deviation of parameter estimates from Hill function fit .

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

(B) Growth rate as a function of TMP concentration for different paths through IPTG-TMP concentration space Constant FolA is shown in black and inverted FolA regulation in red. Wild-type dose-response curve (blue line; is shown for comparison. (C) Steepness of the dose response curve (quantified as dose-sensitivity n) for the three cases in B. Inset: Normalized FolA expression level as a function of TMP concentration for the three cases in B; WT (blue) colors as in the bar chart and in B. Data information: Error bars in B show standard deviation of the measured growth rates used for interpolating the values shown; the entire experiment was replicated once Error bars in C show standard deviation of parameter estimates from Hill function fit.


( E , F ) Kymograph and individual images of time - lapse observation of Mal3 - GFP ( green ) and Dli1 - 3mCherry ( magenta ) along the longitudinal axis . Yellow arrowheads indicate temporary accumulation of growing plus tips during the SPB is turning . Blue asterisks indicate dynein accumulating at the distal cell end cortex in the direction of the SPB movement . Individual images indicated by ( a - d ) are shown in ( F ) . Data information : Scale bars , 5 µm .

(E, F) Kymograph and individual images of time-lapse observation of Mal3-GFP (green) and Dli1-3mCherry (magenta) along the longitudinal axis. Yellow arrowheads indicate temporary accumulation of growing plus tips during the SPB is turning. Blue asterisks indicate dynein accumulating at the distal cell end cortex in the direction of the SPB movement. Individual images indicated by (a-d) are shown in (F). Data information: Scale bars, 5 µm.


( I ) Kymographs of the SPB ( Sfi1 - mCherry , yellow ) and histone H2B ( Htb1 - CFP , blue ) in dhc1 ∆ mutant . At 20 min ( white solid lines ) , dimethyl sulfoxide ( DMSO , as a negative control ) or MBC ( final concentration of 20 µg / mL ) were added . Dashed lines indicate cell shape . Data information : Scale bars , 5 µm .

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(I) Kymographs of the SPB (Sfi1-mCherry, yellow) and histone H2B (Htb1-CFP, blue) in dhc1∆ mutant. At 20 min (white solid lines), dimethyl sulfoxide (DMSO, as a negative control) or MBC (final concentration of 20 µg/mL) were added. Dashed lines indicate cell shape. Data information: Scale bars, 5 µm.


( E - H ) Spectrograms indicating changes in the power spectral density for variable parameters in the pulling - only model . The gray scale ( black to white ) corresponds to the values of the power spectral density ( low to high ) . The default parameters are indicated by the arrowheads on the horizontal axes .

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(E-H) Spectrograms indicating changes in the power spectral density for variable parameters in the pulling-only model. The gray scale (black to white) corresponds to the values of the power spectral density (low to high). The default parameters are indicated by the arrowheads on the horizontal axes.


( D ) Scatter plot of cell length versus peak frequency during the azygotic oscillation . n = 45 cells .

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(D) Scatter plot of cell length versus peak frequency during the azygotic oscillation. n = 45 cells.


( C ) Tracks of the SPB along the longitudinal ( x , blue line ) and lateral ( y , red line ) axes . The 64 - minute period that gives the fastest movement of the SPB is indicated by the green bar and analyzed in ( D ) .

(C) Tracks of the SPB along the longitudinal (x, blue line) and lateral (y, red line) axes. The 64-minute period that gives the fastest movement of the SPB is indicated by the green bar and analyzed in (D).


( A ) Time - lapse observation of microtubules ( GFP - Atb2 , green ) , dynein ( Dli1 - 3mCherry , red ) and the SPB ( Sad1 - CFP , blue ) in wild - type and klp6 ∆ cells . Arrowheads indicate the SPB . The arrow shows an extensively elongated microtubule along the cell cortex in a klp6 ∆ cell . Data information : Scale bars , 5 µm .

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(A) Time-lapse observation of microtubules (GFP-Atb2, green), dynein (Dli1-3mCherry, red) and the SPB (Sad1-CFP, blue) in wild-type and klp6∆ cells. Arrowheads indicate the SPB. The arrow shows an extensively elongated microtubule along the cell cortex in a klp6∆ cell. Data information: Scale bars, 5 µm.


( B and C ) Time - lapse observation of Klp6 ( Klp6 - GFP , green ) , dynein ( Dli1 - 3mCherry , red ) and microtubules ( CFP - Atb2 , blue ) . A magnified view of the square area is shown in ( C ) . Arrowheads indicate accumulation of Klp6 on microtubules extending along the cell cortex . Data information : Scale bars , 5 µm .

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(B and C) Time-lapse observation of Klp6 (Klp6-GFP, green), dynein (Dli1-3mCherry, red) and microtubules (CFP-Atb2, blue). A magnified view of the square area is shown in (C). Arrowheads indicate accumulation of Klp6 on microtubules extending along the cell cortex. Data information: Scale bars, 5 µm.


F . Dopaminergic neuronal cultures from control lines and glucocerebrosidase ( GBA ) N370S PD patients were immunoblotted for the presence of LAG3 . No band for LAG3 could be observed in neurons .

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F. Dopaminergic neuronal cultures from control lines and glucocerebrosidase (GBA) N370S PD patients were immunoblotted for the presence of LAG3. No band for LAG3 could be observed in neurons.


G . Using high power , high resolution laser scanning confocal microscopy , no human LAG3 signal could be detected in human neurons ( Auto - hLAG3 transduced , DOX OFF ) by two different anti - human LAG3 antibodies ( 17B4 and D2G40 ; left panel and zoomed - in insets ) whereas LAG3 was clearly detected in human neurons induced to express hLAG3 ( DOX ON ; right panel and zoomed - in insets ) . Scale bars 25 µm .

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G. Using high power, high resolution laser scanning confocal microscopy, no human LAG3 signal could be detected in human neurons (Auto-hLAG3 transduced, DOX OFF) by two different anti-human LAG3 antibodies (17B4 and D2G40; left panel and zoomed-in insets) whereas LAG3 was clearly detected in human neurons induced to express hLAG3 (DOX ON; right panel and zoomed-in insets). Scale bars 25 µm.


H . Human brain homogenates from autopsy material were immunoblotted for the presence of LAG3 . No band for LAG3 could be evidenced in any of the brain homogenates . Control samples ( tonsils and activated human T cells ) show expected bands .

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H. Human brain homogenates from autopsy material were immunoblotted for the presence of LAG3. No band for LAG3 could be evidenced in any of the brain homogenates. Control samples (tonsils and activated human T cells) show expected bands.


B . Mixed neuroglial , neuron - enriched , and glial cortical cultures were assayed for the presence of LAG3 where no band could be found . Activated T cells display band at expected size .

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B. Mixed neuroglial, neuron-enriched, and glial cortical cultures were assayed for the presence of LAG3 where no band could be found. Activated T cells display band at expected size.


D . Using high power , high resolution laser scanning confocal microscopy , no mouse LAG3 signal could be detected in murine primary neuronal cultures ( Auto - mLAG3 transduced , DOX OFF and non - transduced neurons ) using anti - mouse LAG3 antibody ( MABF954 ) whereas LAG3 was clearly detected in primary neurons induced to express mLAG3 ( Auto - mLAG3 transduced , DOX ON ) . Scale bars 20 µm .

D. Using high power, high resolution laser scanning confocal microscopy, no mouse LAG3 signal could be detected in murine primary neuronal cultures (Auto-mLAG3 transduced, DOX OFF and non-transduced neurons) using anti-mouse LAG3 antibody (MABF954) whereas LAG3 was clearly detected in primary neurons induced to express mLAG3 (Auto-mLAG3 transduced, DOX ON). Scale bars 20 µm.


E . RT - qPCR - derived CT values to measure the cDNA copy number . Mixed neuroglial cultures , glial cultures , and neuron - enriched cultures show transcript levels that are close to indistinguishable to those from LAG3 ‑ / ‑ BH . Conversely , around 1600 transcripts / ng of RNA were counted in activated T cells .

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E. RT-qPCR-derived CT values to measure the cDNA copy number. Mixed neuroglial cultures, glial cultures, and neuron-enriched cultures show transcript levels that are close to indistinguishable to those from LAG3‑/‑ BH. Conversely, around 1600 transcripts/ng of RNA were counted in activated T cells.


A . and B . Binding of ASC filaments ( A ) or α - synuclein PFFs ( B ) to multiple proteins . Serial dilutions of ASC filaments and α - synuclein PFFs were performed and bound fractions were detected using anti - ASC or anti - α - synuclein ( MJFR1 ) antibodies . ASC filaments did not interact with any of the proteins ( A ) . Conversely , α - synuclein PFFs strongly bound LAG3 , CD4 , and MTBD tau and showed moderate binding properties to PrPC , APOE3 , and TDP - 43 . No binding between α - synuclein PFFs and nAra h 2 or BSA could be detected . Values represent means ± SD of technical duplicates .

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A. and B. Binding of ASC filaments (A) or α-synuclein PFFs (B) to multiple proteins. Serial dilutions of ASC filaments and α-synuclein PFFs were performed and bound fractions were detected using anti-ASC or anti-α-synuclein (MJFR1) antibodies. ASC filaments did not interact with any of the proteins (A). Conversely, α-synuclein PFFs strongly bound LAG3, CD4, and MTBD tau and showed moderate binding properties to PrPC, APOE3, and TDP-43. No binding between α-synuclein PFFs and nAra h 2 or BSA could be detected. Values represent means ± SD of technical duplicates.


C . and D . Diffusional sizing of α - synuclein PFFs with fluorescently conjugated LAG3 , CD4 , BSA , and MJFR1 α - synuclein antibody ( C ) and of fluorescently labelled LAG3 with monomeric α - synuclein , α - synuclein PFFs , FGL1 , and Relatlimab anti - LAG3 antibody ( D ) . An increase in the hydrodynamic radius ( Rh ) of α - synuclein PFFs is seen with increasing concentrations for MJFR1 but not for LAG3 or any of the control proteins , also not at a lower scale ( see insert ) ( C ) . Similarly , the hydrodynamic radius of labelled LAG3 increases with higher concentrations of Relatlimab and FGL1 but does not change with α - synuclein PFFs or monomeric α - synuclein ( D ) . Values are given as means ± SD of technical triplicates .

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C. and D. Diffusional sizing of α-synuclein PFFs with fluorescently conjugated LAG3, CD4, BSA, and MJFR1 α-synuclein antibody (C) and of fluorescently labelled LAG3 with monomeric α-synuclein, α-synuclein PFFs, FGL1, and Relatlimab anti-LAG3 antibody (D). An increase in the hydrodynamic radius (Rh) of α-synuclein PFFs is seen with increasing concentrations for MJFR1 but not for LAG3 or any of the control proteins, also not at a lower scale (see insert) (C). Similarly, the hydrodynamic radius of labelled LAG3 increases with higher concentrations of Relatlimab and FGL1 but does not change with α-synuclein PFFs or monomeric α-synuclein (D). Values are given as means ± SD of technical triplicates.


E . and F . Co - immunoprecipitation of LAG3 and subsequent immunoblotting with anti α - synuclein antibody ( MJFR1 ) ( E ) and co - immunoprecipitation of α - synuclein ( monomeric or PFFs ) and subsequent immunoblotting with anti - LAG3 antibody ( D2G40 ) ( F ) . Using the anti - α - synuclein antibody for pulldown resulted in LAG3 bands with both monomeric as well as fibrillar α - synuclein . The usage of anti - LAG3 for pulldown led to the specific detection of α - synuclein PFFs , although the PFF fraction predominates also in the input and supernatant . Bands for α - synuclein ( E ) and LAG3 ( F ) are indicated by arrows .

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E. and F. Co-immunoprecipitation of LAG3 and subsequent immunoblotting with anti α-synuclein antibody (MJFR1) (E) and co-immunoprecipitation of α-synuclein (monomeric or PFFs) and subsequent immunoblotting with anti-LAG3 antibody (D2G40) (F). Using the anti-α-synuclein antibody for pulldown resulted in LAG3 bands with both monomeric as well as fibrillar α-synuclein. The usage of anti-LAG3 for pulldown led to the specific detection of α-synuclein PFFs, although the PFF fraction predominates also in the input and supernatant. Bands for α-synuclein (E) and LAG3 (F) are indicated by arrows.


Human neural cultures transduced by Auto - hLAG3 were treated by α - synuclein PFFs 4 days post LAG3 expression induction by DOX and kept in culture for 2 or 4 weeks . Both transgenic ( hLAG3 D2G40 - positive in ( A ) and 17B4 - positive in ( B ) ) and non - transduced , wild - type neurons ( selected neurons in zoomed - in insets ) propagated α - synuclein and developed characteristic pS129 - positive ( 81A - positive in ( A ) and EP1536Y - positive in ( B ) ) α - synuclein aggregates . Scale bars 25 µm . Trained ilastik algorithms were used to segment pixels of 81A , EP1536Y and MAP2 stainings imaged by high - content wide - field microscope , which were used to quantify the signal of 81A - positive ( C ) and EP1536Y - positive ( D ) α - synuclein aggregates expressed as % of MAP2 - positive area . Almost the entire wells ( 182 fields per well ) were imaged for every condition and replicate and each datapoint in the plot represents the entire well . Error bars indicate mean ± SD of biological replicates ( duplicates ) . For few conditions , unicates were used , shown as one dot . One - way ANOVA followed by Tukey ' s multiple comparison test demonstrated that neurons that did not express LAG3 ( DOX OFF ) showed no difference in α - synuclein propagation when compared to LAG3 - expressing neurons ( DOX ON ) as demonstrated by two different pS129 α - synuclein antibodies ( p = 0 . 9998 for 81A at 2 weeks and p = 0 . 2522 at 4 weeks ; p = 0 . 9986 for EP1536Y at 2 weeks and p = 0 . 3042 at 4 weeks ) .

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Human neural cultures transduced by Auto-hLAG3 were treated by α-synuclein PFFs 4 days post LAG3 expression induction by DOX and kept in culture for 2 or 4 weeks. Both transgenic (hLAG3 D2G40-positive in (A) and 17B4-positive in (B)) and non-transduced, wild-type neurons (selected neurons in zoomed-in insets) propagated α-synuclein and developed characteristic pS129-positive (81A-positive in (A) and EP1536Y-positive in (B)) α-synuclein aggregates. Scale bars 25 µm. Trained ilastik algorithms were used to segment pixels of 81A, EP1536Y and MAP2 stainings imaged by high-content wide-field microscope, which were used to quantify the signal of 81A-positive (C) and EP1536Y-positive (D) α-synuclein aggregates expressed as % of MAP2-positive area. Almost the entire wells (182 fields per well) were imaged for every condition and replicate and each datapoint in the plot represents the entire well. Error bars indicate mean ± SD of biological replicates (duplicates). For few conditions, unicates were used, shown as one dot. One-way ANOVA followed by Tukey's multiple comparison test demonstrated that neurons that did not express LAG3 (DOX OFF) showed no difference in α-synuclein propagation when compared to LAG3-expressing neurons (DOX ON) as demonstrated by two different pS129 α-synuclein antibodies (p=0.9998 for 81A at 2 weeks and p=0.2522 at 4 weeks; p=0.9986 for EP1536Y at 2 weeks and p=0.3042 at 4 weeks).


A . Survival curves of A53T α - synuclein TG mice , with LAG3 knockout , heterozygous LAG3 expression , or LAG3 WT . Median survival was shortest for α - synuclein TG LAG3 ‑ / ‑ at 239 d , followed by α - synuclein TG LAG3 + / - at 260 d and α - synuclein TG LAG3 + / + at 268 d . The survivals of ASYNA53T LAG3 ‑ / ‑ , LAG3 + / - and LAG3 + / + mice were similar ( Mantel - Cox log - rank test , p - value = 0 . 165 ) . Mice were euthanized and dissected in the late - stage symptomatic phase , which would inevitably result in death within a week . None of the control LAG3 ‑ / ‑ α - synuclein WT mice died over the course of the experiment .

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A. Survival curves of A53T α-synuclein TG mice, with LAG3 knockout, heterozygous LAG3 expression, or LAG3 WT. Median survival was shortest for α-synuclein TG LAG3‑/‑ at 239 d, followed by α-synuclein TG LAG3+/- at 260 d and α-synuclein TG LAG3+/+ at 268 d. The survivals of ASYNA53T LAG3‑/‑, LAG3+/- and LAG3+/+ mice were similar (Mantel-Cox log-rank test, p-value = 0.165). Mice were euthanized and dissected in the late-stage symptomatic phase, which would inevitably result in death within a week. None of the control LAG3‑/‑ α-synuclein WT mice died over the course of the experiment.


B . Immunostaining for hyperphosphorylated α - synuclein aggregates ( pS129 ) and aggregated protein ( Thioflavin S ) in sections of end - stage symptomatic mice ; no obvious difference in terms of staining pattern or number of stained cells were apparent in midbrain or brain stem . Scale bars represent 1 mm ( sagittal section ) and 100 µm ( panel ) .

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B. Immunostaining for hyperphosphorylated α-synuclein aggregates (pS129) and aggregated protein (Thioflavin S) in sections of end-stage symptomatic mice; no obvious difference in terms of staining pattern or number of stained cells were apparent in midbrain or brain stem. Scale bars represent 1 mm (sagittal section) and 100 µm (panel).


C . Immunofluorescence staining and quantification of pS129 - positive α - synuclein inclusions in hippocampal slice cultures ( HSCs ) at 5 weeks post - seeding with 350 µM PFF . Scale bars represent 500 µm ( overview ) , and 100 µm ( insets ) . Shown is mean ± SEM and individual values are shown ; n = 6 cultures per group ; two - way - ANOVA revealed for LAG3 F ( 1 , 20 ) = 0 . 0294 ; p = 0 . 8656 ; A53T F ( 1 , 20 ) = 36 . 42 , p < 0 . 0001 ; interaction F ( 1 , 20 ) = 0 . 0062 , p = 0 . 9382 . Bonferroni ' s correction for multiple comparisons revealed p = 0 . 9973 for LAG3 WT vs . KO in A53T TG and p > 0 . 9808 for LAG3 WT vs . KO in A53T WT .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunofluorescence staining and quantification of pS129-positive α-synuclein inclusions in hippocampal slice cultures (HSCs) at 5 weeks post-seeding with 350 µM PFF. Scale bars represent 500 µm (overview), and 100 µm (insets). Shown is mean ± SEM and individual values are shown; n = 6 cultures per group; two-way-ANOVA revealed for LAG3 F(1, 20) = 0.0294; p = 0.8656; A53T F(1, 20) = 36.42, p < 0.0001; interaction F(1, 20) = 0.0062, p = 0.9382. Bonferroni's correction for multiple comparisons revealed p = 0.9973 for LAG3 WT vs. KO in A53T TG and p > 0.9808 for LAG3 WT vs. KO in A53T WT.


Solubilized mitochondria from wild type HEK293T cells were subjected to immunoisolation with MITRAC12 antiserum . Eluates were analysed by SDS - PAGE and western botting . Total 1 . 5 % , Eluate , 100 % .

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Solubilized mitochondria from wild type HEK293T cells were subjected to immunoisolation with MITRAC12 antiserum. Eluates were analysed by SDS-PAGE and western botting. Total 1.5 %, Eluate, 100 %.


Wild type mitochondria were solubilized and subjected to 2D - BN / SDS - PAGE and western blotting with indicated antibodies .

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

Wild type mitochondria were solubilized and subjected to 2D-BN/SDS-PAGE and western blotting with indicated antibodies.


Solubilized mitochondria from MITRAC12FLAG - or ACAD9FLAG - expressing cells were subjected to anti - FLAG immunoisolation and native elution . Eluates were analysed by 2D - BN / SDS - PAGE and western blot analysis . Total 0 . 8 % ( MITRAC12FLAG isolation ) , 0 . 35 % ( ACAD9FLAG isolation ) , Eluate 100 % .

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

Solubilized mitochondria from MITRAC12FLAG- or ACAD9FLAG-expressing cells were subjected to anti-FLAG immunoisolation and native elution. Eluates were analysed by 2D-BN/SDS-PAGE and western blot analysis. Total 0.8 % (MITRAC12FLAG isolation), 0.35 % (ACAD9FLAG isolation), Eluate 100 %.


Solubilized mitochondria from MITRAC15FLAG - expressing cells were first subjected to native anti - FLAG immunoisolation ( Total 0 . 1 % , Eluate 10 % , lane 4 ) . Purified complexes were applied to anti - MITRAC12 immunoisolation ( Eluates 100 % , lane 5 ) . All samples were analyzed by SDS - PAGE and immunoblotting .

Solubilized mitochondria from MITRAC15FLAG-expressing cells were first subjected to native anti-FLAG immunoisolation (Total 0.1 %, Eluate 10 %, lane 4). Purified complexes were applied to anti-MITRAC12 immunoisolation (Eluates 100 %, lane 5). All samples were analyzed by SDS-PAGE and immunoblotting.


Mitochondrial translational products were labelled with [ 35S ] methionine and anti - FLAG immunoisolation was performed as described in Fig . 1D . Eluates were analyzed by SDS - PAGE and digital autoradiography . Total 1 . 5 % , Eluate 100 % .

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Mitochondrial translational products were labelled with [35S]methionine and anti-FLAG immunoisolation was performed as described in Fig. 1D. Eluates were analyzed by SDS-PAGE and digital autoradiography. Total 1.5 %, Eluate 100 %.


Solubilized mitochondria from MITRAC15FLAG - or TIM21FLAG - expressing cells were subjected to anti - FLAG immunoisolation and analyzed by 2D - BN / SDS - PAGE and western blotting . MITRAC15FLAG isolation : Total 0 . 1 % , Eluate 100 % . TIM21FLAG isolation : Total 0 . 2 % , Eluate 66 % ( TIM21FLAG decoration ) , 33 % ( MITRAC15 , ACAD9 , MITRAC12 , COX1 decoration ) .

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Solubilized mitochondria from MITRAC15FLAG- or TIM21FLAG-expressing cells were subjected to anti-FLAG immunoisolation and analyzed by 2D-BN/SDS-PAGE and western blotting. MITRAC15FLAG isolation: Total 0.1 %, Eluate 100 %. TIM21FLAG isolation: Total 0.2 %, Eluate 66 % (TIM21FLAG decoration), 33 % (MITRAC15, ACAD9, MITRAC12, COX1 decoration).


Solubilized mitochondria from MITRAC15FLAG - or C12ORF62FLAG - expressing cells were subjected to anti - FLAG immunoisolation and analyzed by SDS - PAGE and immunoblotting . Total 0 . 4 % , Eluate 100 % ( MITRAC15FLAG isolation ) , 70 % ( C12ORF62FLAG isolation ) .

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Solubilized mitochondria from MITRAC15FLAG- or C12ORF62FLAG- expressing cells were subjected to anti-FLAG immunoisolation and analyzed by SDS-PAGE and immunoblotting. Total 0.4 %, Eluate 100 % (MITRAC15FLAG isolation), 70 % (C12ORF62FLAG isolation).


Solubilized mitochondria from ACAD9FLAG - or MITRAC15FLAG - expressing cells were subjected to anti - FLAG immunoisolation and analyzed by SDS - PAGE and immunoblotting . Total 0 . 5 % ( ACAD9FLAG isolation ) , 0 . 4 % ( MITRAC15FLAG isolation ) Eluate 100 % . Asterisk ( * ) indicates TIM21 signal . ACAD9 / ACAD9FLAG could not be separated with the used gel system , both proteins are detected with ACAD9 antiserum .

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

Solubilized mitochondria from ACAD9FLAG-or MITRAC15FLAG- expressing cells were subjected to anti-FLAG immunoisolation and analyzed by SDS-PAGE and immunoblotting. Total 0.5 % (ACAD9FLAG isolation), 0.4 % (MITRAC15FLAG isolation) Eluate 100 %. Asterisk (*) indicates TIM21 signal. ACAD9/ACAD9FLAG could not be separated with the used gel system, both proteins are detected with ACAD9 antiserum.


Mitochondria from wild type and MITRAC15 - / - cells were isolated and subjected to steady - state protein analyses by western blotting .

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

Mitochondria from wild type and MITRAC15-/- cells were isolated and subjected to steady-state protein analyses by western blotting.


Mitochondria from wild type and MITRAC15 - / - cells were isolated and analyzed by BN - PAGE . Triton X - 100 ( left site ) or digitonin ( right site ) were used for solubilization . The amount of complex I was quantified utilizing the NDUFS1 signal and normalized against SDHA levels ( error bars indicate SEM from three biological replicates ) .

Mitochondria from wild type and MITRAC15-/- cells were isolated and analyzed by BN-PAGE. Triton X-100 (left site) or digitonin (right site) were used for solubilization. The amount of complex I was quantified utilizing the NDUFS1 signal and normalized against SDHA levels (error bars indicate SEM from three biological replicates).


Complex I and citrate synthase activity were measured in WT and MITRAC15 - / - cells ( error bars indicate SEM from three biological replicates ; * p < 0 , 05 ; unpaired t - test ) .

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Complex I and citrate synthase activity were measured in WT and MITRAC15-/- cells (error bars indicate SEM from three biological replicates; *p < 0,05; unpaired t-test).


Pulse - chase [ 35S ] methionine radiolabeling of mitochondrial translation products in wild type and MITRAC15 - / - cells . Samples were analyzed by SDS - PAGE and digital autoradiography . Signal intensity of ND2 was normalized to the ATP6 signal and the relative ND2 stability was calculated by relative chase ND2 signal / relative pulse ND2 signal ( error bars indicate SEM from three biological replicates ; * p < 0 , 05 ; unpaired t - test ) .

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

Pulse-chase [35S]methionine radiolabeling of mitochondrial translation products in wild type and MITRAC15-/- cells. Samples were analyzed by SDS-PAGE and digital autoradiography. Signal intensity of ND2 was normalized to the ATP6 signal and the relative ND2 stability was calculated by relative chase ND2 signal/relative pulse ND2 signal (error bars indicate SEM from three biological replicates; *p < 0,05; unpaired t-test).


Pulse radiolabeling after re - expressing MITRAC15FLAG in MITRAC15 - / - cells . Samples were analyzed by SDS - PAGE and digital autoradiography . Relative pulse signal intensity of ND2 was normalized to ATP6 ( error bars indicate SEM from three biological replicates ; * p < 0 , 05 ; * * p < 0 . 01 ; unpaired t - test ) .

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

Pulse radiolabeling after re-expressing MITRAC15FLAG in MITRAC15-/- cells. Samples were analyzed by SDS-PAGE and digital autoradiography. Relative pulse signal intensity of ND2 was normalized to ATP6 (error bars indicate SEM from three biological replicates; *p < 0,05; **p < 0.01; unpaired t-test).


Mitochondrial translation products were radiolabelled with [ 35S ] methionine after siRNA - mediated depletion of ACAD9 in wild type and MITRAC15 - / - cells . Cell lysates were analyzed by SDS - PAGE and digital autoradiography . For quantifications the signal intensity of ND2 was normalized to ATP6 and the relative ND2 stability was calculated by relative chase ND2 signal / relative pulse ND2 signal . ( error bars indicate SEM from three biological replicates ; * p < 0 , 05 ; * * p < 0 . 01 * * * p < 0 . 001 ; unpaired t - test ) .

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

Mitochondrial translation products were radiolabelled with [35S]methionine after siRNA-mediated depletion of ACAD9 in wild type and MITRAC15-/- cells. Cell lysates were analyzed by SDS-PAGE and digital autoradiography. For quantifications the signal intensity of ND2 was normalized to ATP6 and the relative ND2 stability was calculated by relative chase ND2 signal/relative pulse ND2 signal. (error bars indicate SEM from three biological replicates; *p < 0,05; **p < 0.01 ***p < 0.001; unpaired t-test).


FLAG - immunoprecipitation was performed in mitochondrial extracts from MITRAC15FLAG - expressing cells after siRNA - mediated down regulation of ACAD9 and pulse radiolabeling . SDS - PAGE followed by autoradiography was used to analyze eluates . Relative signal intensity of ND2 , ND3 and ND4L were normalized to ATP6 ( total ) or isolation efficiency ( eluate ) . Total 2 % , Eluate 100 % . Error bars indicate SEM from three biological replicates ; * * p < 0 . 01 ; unpaired t - test .

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

FLAG-immunoprecipitation was performed in mitochondrial extracts from MITRAC15FLAG-expressing cells after siRNA-mediated down regulation of ACAD9 and pulse radiolabeling. SDS-PAGE followed by autoradiography was used to analyze eluates. Relative signal intensity of ND2, ND3 and ND4L were normalized to ATP6 (total) or isolation efficiency (eluate). Total 2 %, Eluate 100 %. Error bars indicate SEM from three biological replicates; **p < 0.01; unpaired t-test.


After C12orf62 down regulation , [ 35S ] methionine labelling was performed . During labeling mitochondrial translation was inhibited with puromycin ( pur ) ( 2 µg / ml ) . MITRAC15FLAG interacting nascent chains were isolated by FLAG - immunoprecipitation from cell lysates . Eluates were subjected to SDS - PAGE , analyzed by digital autoradiography . Total 2 % , MITRAC15FLAG eluates 100 % ( in case of C12ORF62FLAG , 25 % of eluate was loaded as a standard ) . Red asterisks , accumulating nascent chains of ND2 . The accumulating nascent chains of ND2 of lane 12 is presented in a magnification . F1 - F3 mark nascent chains of COX1 [ 18 ] .

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

After C12orf62 down regulation, [35S]methionine labelling was performed. During labeling mitochondrial translation was inhibited with puromycin (pur) (2 µg/ml). MITRAC15FLAG interacting nascent chains were isolated by FLAG-immunoprecipitation from cell lysates. Eluates were subjected to SDS-PAGE, analyzed by digital autoradiography. Total 2 %, MITRAC15FLAG eluates 100 % (in case of C12ORF62FLAG, 25% of eluate was loaded as a standard). Red asterisks, accumulating nascent chains of ND2. The accumulating nascent chains of ND2 of lane 12 is presented in a magnification. F1-F3 mark nascent chains of COX1 [18].


HEK293T cells were pulse labelled with [ 35S ] methionine in the presence of puromycin ( 2 µg / ml ) . During puromycin treatment , cells were incubated for 5 min and 10 min in medium lacking [ 35S ] methionine . Anti - FLAG immunoisolations were performed as described and eluates analyzed as described in Fig . 4C . Total 1 % , Eluates 100 % . The signal of ND2 was graphed after normalization against the COX2 signal and the signal of mature ND2 in the 5 min chase sample was set as 100 % ( error bars indicate SEM from three biological replicates ; * p < 0 , 05 ; paired t - test ) . Red asterisks , accumulating nascent chains of ND2 .

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

HEK293T cells were pulse labelled with [35S]methionine in the presence of puromycin (2 µg/ml). During puromycin treatment, cells were incubated for 5 min and 10 min in medium lacking [35S]methionine. Anti-FLAG immunoisolations were performed as described and eluates analyzed as described in Fig. 4C. Total 1 %, Eluates 100 %. The signal of ND2 was graphed after normalization against the COX2 signal and the signal of mature ND2 in the 5 min chase sample was set as 100 % (error bars indicate SEM from three biological replicates; *p < 0,05; paired t-test). Red asterisks, accumulating nascent chains of ND2.


Radiolabeling with [ 35S ] methionine , anti - FLAG immunoisolation and sample analyses were performed Total 1 % , Eluates 100 % . Red asterisks , accumulating nascent chains of ND2 .

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Radiolabeling with [35S]methionine, anti-FLAG immunoisolation and sample analyses were performed Total 1 %, Eluates 100 %. Red asterisks, accumulating nascent chains of ND2.


Depletion of ACAD9 by siRNA was performed Total 1 % , Eluates 100 % . Red asterisks , accumulating nascent chains of ND2 .

Depletion of ACAD9 by siRNA was performed Total 1 %, Eluates 100 %. Red asterisks, accumulating nascent chains of ND2.


( A - F ) Immunocytochemistry of DRG neurons and the corresponding quantifications of the indicated genotypes for surface TrkA ( A ) , surface TRPV1 ( B ) , Rab3A ( C ) , synaptotagmin ( D ) , DENN / MADD ( E ) and α - tubulin ( F ) . TrkA and TRPV1 were visualized without permeabilization . ns , p > 0 . 05 ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; Welch ' s t test from at least three independent cultures . Error bars show SEM . Scale bars , 2 µm .

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(A-F) Immunocytochemistry of DRG neurons and the corresponding quantifications of the indicated genotypes for surface TrkA (A), surface TRPV1 (B), Rab3A (C), synaptotagmin (D), DENN/MADD (E) and α-tubulin (F). TrkA and TRPV1 were visualized without permeabilization. ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; Welch's t test from at least three independent cultures. Error bars show SEM. Scale bars, 2 µm.


Time - lapse fluorescence images of DRG neurons expressing KIF1A - EGFP and KIF1A ( E239K ) - EGFP ( B ) Arrowheads in ( B ) show the anterogradely moving particles . Scale bar in ( B ) , 2 μm . See Movies EV1 and 2 .

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Time-lapse fluorescence images of DRG neurons expressing KIF1A-EGFP and KIF1A(E239K)-EGFP (B) Arrowheads in (B) show the anterogradely moving particles. Scale bar in (B), 2 μm. See Movies EV1 and 2.


( F ) Movements of KIF1A - EGFP and KIF1A ( E239K ) - EGFP expressed in KIF1A knockdown DRG and the scrambled control . White scale bar , 1 μm . ( G ) Anterograde velocities of the indicated genotypes calculated from the kymograph slopes . Data are shown as mean and SD . KD + KIF1A - EGFP , n = 29 ; KD + KIF1A ( E239K ) - EGFP , n = 32 ; scrambled + KIF1A - EGFP , n = 44 ; scrambled + KIF1A ( E239K ) - EGFP , n = 42 from three independent cultures . ns , p = 0 . 3329 ; * * , p < 0 . 01 ; * * * * , p < 0 . 0001 ; Unpaired t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Movements of KIF1A-EGFP and KIF1A(E239K)-EGFP expressed in KIF1A knockdown DRG and the scrambled control. White scale bar, 1 μm. (G) Anterograde velocities of the indicated genotypes calculated from the kymograph slopes. Data are shown as mean and SD. KD+KIF1A-EGFP, n = 29; KD+KIF1A(E239K)-EGFP, n = 32; scrambled+KIF1A-EGFP, n = 44; scrambled+KIF1A(E239K)-EGFP, n = 42 from three independent cultures. ns, p = 0.3329; **, p < 0.01; ****, p < 0.0001; Unpaired t test.


( A ) Representative kymographs of tetramethylrhodamine - labelled microtubule movement driven by the WT and the E239K mutant . The arrowheads represent the end positions of the WT and E239K .

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(A) Representative kymographs of tetramethylrhodamine-labelled microtubule movement driven by the WT and the E239K mutant. The arrowheads represent the end positions of the WT and E239K.


( D ) ATPase activity of WT ( green ) and E239K mutant ( magenta ) activated by microtubules . Data are represented as the mean ± SD . The data from at least three independent experiments were analysed . * * * * , p < 0 . 0001 , two - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ATPase activity of WT (green) and E239K mutant (magenta) activated by microtubules. Data are represented as the mean ± SD. The data from at least three independent experiments were analysed. ****, p < 0.0001, two-way ANOVA.


( F ) Rate constants ( kobs ) determined for the reaction of mant - ATP with nucleotide - free KIF1A ( WT ) and KIF1A ( E239K ) were plotted against concentration of mant - ATP . Data are represented as the mean ± SD . The data from three independent measurements were analysed .

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 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

(F) Rate constants (kobs) determined for the reaction of mant-ATP with nucleotide-free KIF1A(WT) and KIF1A(E239K) were plotted against concentration of mant-ATP. Data are represented as the mean ± SD. The data from three independent measurements were analysed.


( A ) The positions of the β6 - L10 - β7 and neck - coil peptides are shown in brown and green . The positions of the neck - linker peripheral peptides ( α1 - β3 - P loop , α4 - L12 and L12 - α5 ) are shown in light blue . ( B - D ) Quantitation of site - specific intramolecular interactions . Representative MS / MS spectra of the indicated peptides , including the mutated amino acid ( β6 - L10 - β7 ) ( B and C ) and neck - coil ( D ) . In the inserted graphs , the intensities in the ADP - BeFx and ADP - AlFx states were measured with respect to the nucleotide - free state as 1 . 0 . Grey bar graphs , WT ; black bar graphs , E239K ; ( - ) , nucleotide - free .

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

(A) The positions of the β6-L10-β7 and neck-coil peptides are shown in brown and green. The positions of the neck-linker peripheral peptides (α1-β3-P loop, α4-L12 and L12-α5) are shown in light blue. (B-D) Quantitation of site-specific intramolecular interactions. Representative MS/MS spectra of the indicated peptides, including the mutated amino acid (β6-L10-β7) (B and C) and neck-coil (D). In the inserted graphs, the intensities in the ADP-BeFx and ADP-AlFx states were measured with respect to the nucleotide-free state as 1.0. Grey bar graphs, WT; black bar graphs, E239K; (-), nucleotide-free.


B - G . ( B , D , and F ) E histograms of the RDH substrate in the absence of EXD2 ( gray ) and after 0 - ( black ) , 100 - ( purple ) , and 200 - s ( blue ) incubation with 200 ( B ) , 100 ( D ) , and 5 ( F ) nM EXD2 . ( C , E , and G ) Representative smFRET trajectories showing EXD2 at 200 ( C ) , 100 ( E ) , and 5 ( G ) nM cleaving RDH in real time . The fluorescence intensities of the donor and acceptor are colored green and red , respectively . The corresponding FRET efficiencies are shown below in black . The duration between two Cambridge blue lines represents the time for the FRET efficiency to increase from ~ 0 . 2 to ~ 0 . 9 .

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B-G. (B, D, and F) E histograms of the RDH substrate in the absence of EXD2 (gray) and after 0- (black), 100- (purple), and 200-s (blue) incubation with 200 (B), 100 (D), and 5 (F) nM EXD2. (C, E, and G) Representative smFRET trajectories showing EXD2 at 200 (C), 100 (E), and 5 (G) nM cleaving RDH in real time. The fluorescence intensities of the donor and acceptor are colored green and red, respectively. The corresponding FRET efficiencies are shown below in black. The duration between two Cambridge blue lines represents the time for the FRET efficiency to increase from ~ 0.2 to ~ 0.9.


H . The FRET trajectories ' percentages show the FRET values ending above 0 . 9 and between 0 . 2 and 0 . 9 . The word " Surface " represents the experiment with EXD2 anchored on the glass surface . I . Statistics of the time for the FRET efficiency to increase from ~ 0 . 2 to ~ 0 . 9 under indicated experimental conditions . In violin plots , light - blue dots represent mean , and black bars show the interquartile range ( IQR ) ( thick bars ) and 1 . 5 times IQR ( thin bars ) ( n = 122 , 91 , 78 , and 101 from left to right ) .

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H. The FRET trajectories' percentages show the FRET values ending above 0.9 and between 0.2 and 0.9. The word "Surface" represents the experiment with EXD2 anchored on the glass surface. I. Statistics of the time for the FRET efficiency to increase from ~ 0.2 to ~ 0.9 under indicated experimental conditions. In violin plots, light-blue dots represent mean, and black bars show the interquartile range (IQR) (thick bars) and 1.5 times IQR (thin bars) (n = 122, 91, 78, and 101 from left to right).


D . A representative gel showing that EXD2 ( 500 nM ) cleaves the Cy5 - RNA and Cy5 - RDH substrates .

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D. A representative gel showing that EXD2 (500 nM) cleaves the Cy5-RNA and Cy5-RDH substrates.


F . The kinetic rates k1 and k2 obtained from the double exponential fitting to the dwell - time histograms of Pauses 1 - 3 and their combination ( Total ) . Fit parameters and 95 % confidence intervals are shown .

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F. The kinetic rates k1 and k2 obtained from the double exponential fitting to the dwell-time histograms of Pauses 1-3 and their combination (Total). Fit parameters and 95% confidence intervals are shown.


A - F . ( A and D ) Representative smFRET trajectories showing a stepwise FRET increase during EXD2 cleavage of the GC - RDH substrate under 1 mM MnCl2 ( A ) or the RDH substrate under 0 . 2 mM MnCl2 ( D ) . The solid red lines are the step - fitting results from an automated step - finding algorithm . ( B and E ) The corresponding TDP of EXD2 cleavage of GC - RDH under 1 mM MnCl2 ( B ) or RDH under 0 . 2 mM MnCl2 ( E ) . ( C and F ) The dwell - time histograms of combined pauses ( Total ) in the process of EXD2 cleavage of GC - RDH under 1 mM MnCl2 ( C ) or RDH under 0 . 2 mM MnCl2 ( F ) . The histogram fittings to a double exponential function are shown in red . The R2 of the double exponential function are 0 . 94 and 0 . 95 , respectively .

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A-F. (A and D) Representative smFRET trajectories showing a stepwise FRET increase during EXD2 cleavage of the GC-RDH substrate under 1 mM MnCl2 (A) or the RDH substrate under 0.2 mM MnCl2 (D). The solid red lines are the step-fitting results from an automated step-finding algorithm. (B and E) The corresponding TDP of EXD2 cleavage of GC-RDH under 1 mM MnCl2 (B) or RDH under 0.2 mM MnCl2 (E). (C and F) The dwell-time histograms of combined pauses (Total) in the process of EXD2 cleavage of GC-RDH under 1 mM MnCl2 (C) or RDH under 0.2 mM MnCl2 (F). The histogram fittings to a double exponential function are shown in red. The R2 of the double exponential function are 0.94 and 0.95, respectively.


Immunoblot comparing UFL1 autoUFMylation ( Top ) and UFM1 chain synthesis ( Middle ) in the presence of UFL1 expressed alone and in complex with UFBP1 . ( Bottom ) Assays to monitor UFMylation of substrates using purified TRIP4 . Reaction products were separated on a 4 - 12 % SDS - PAGE gel under reducing conditions and analysed by immunoblotting using indicated antibodies .

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Immunoblot comparing UFL1 autoUFMylation (Top) and UFM1 chain synthesis (Middle) in the presence of UFL1 expressed alone and in complex with UFBP1. (Bottom) Assays to monitor UFMylation of substrates using purified TRIP4. Reaction products were separated on a 4-12% SDS-PAGE gel under reducing conditions and analysed by immunoblotting using indicated antibodies.


In vitro reconstitution of 60S ribosome UFMylation . Purified 60S ribosome was incubated with either UFL1 on its own or UFL1 / UFBP1 complex together with UBA5 , UFC1 and UFM1 for indicated time points at 30 ° C .

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In vitro reconstitution of 60S ribosome UFMylation. Purified 60S ribosome was incubated with either UFL1 on its own or UFL1/UFBP1 complex together with UBA5, UFC1 and UFM1 for indicated time points at 30°C.


Immunoblot showing formation of free UFM1 chains in the presence ( Lane 3 , 4 ) and absence of UFL1 / UFBP1 ( Lane 1 , 2 ) . ( Bottom ) Graphical representation of the linkage composition of di - UFM1 chains obtained from LC - MS / MS analysis .

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Immunoblot showing formation of free UFM1 chains in the presence (Lane 3,4) and absence of UFL1/UFBP1 (Lane 1,2). (Bottom) Graphical representation of the linkage composition of di-UFM1 chains obtained from LC-MS/MS analysis.


Immunoblot analysing polyUFMylation ( top ) and UFL1 autoUFMylation ( bottom ) in the presence of indicated Lys to Arg ( K - R ) and Lys - less ( K0 ) mutants of UFM1 .

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Immunoblot analysing polyUFMylation (top) and UFL1 autoUFMylation (bottom) in the presence of indicated Lys to Arg (K-R) and Lys-less(K0) mutants of UFM1.


Immunoblot showing in vitro UFMylation assay to check for formation of di - UFM1 chains in the presence of WT , Lys - less ( K0 ) and single lys mutants ( K only ) of UFM1 .

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Immunoblot showing in vitro UFMylation assay to check for formation of di-UFM1 chains in the presence of WT, Lys-less (K0) and single lys mutants (K only) of UFM1.


Coomassie stained gels monitoring discharge of UFM1 from UFC1 in the presence of indicated free amino acids . UBE2D3 is used as a positive control for lysine and cysteine discharge .

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Coomassie stained gels monitoring discharge of UFM1 from UFC1 in the presence of indicated free amino acids. UBE2D3 is used as a positive control for lysine and cysteine discharge.


Immunoblot showing in vitro UFMylation assays in the presence of Cys to Ala ( indicated as C - A ) mutants of UFL1 to check for UFBP1 UFMylation ( top ) , UFL1 autoUFMylation ( middle ) and substrate UFMylation ( bottom ) .

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Immunoblot showing in vitro UFMylation assays in the presence of Cys to Ala (indicated as C-A) mutants of UFL1 to check for UFBP1 UFMylation (top), UFL1 autoUFMylation (middle) and substrate UFMylation (bottom).


Single - turnover lysine discharge assays in the presence and absence of full length UFL1 / UFBP1 complex . The reaction was stopped by addition of non - reducing SDS loading buffer ( 1x final ) and analysed for discharge on a 4 - 12 % SDS PAGE gel under non - reducing conditions . Top gel - LI - COR scan of fluorescently labelled UFM1 ( UFM1 * ) ; bottom gel - Coomassie

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Single-turnover lysine discharge assays in the presence and absence of full length UFL1/UFBP1 complex. The reaction was stopped by addition of non-reducing SDS loading buffer (1x final) and analysed for discharge on a 4-12% SDS PAGE gel under non-reducing conditions. Top gel - LI-COR scan of fluorescently labelled UFM1(UFM1*); bottom gel - Coomassie stained


Immunoblot from immunoprecipitation ( IP ) assays to validate the predicted model of UFL1 / UFBP1 interaction in cells . UFL1WT - 3xFLAG and UFL1L45R - 3xFLAG were transiently overexpressed along with UFBP1 - SBP in HEK293T - UFL1 KO cells and subjected to separate pulldowns using anti - FLAG antibody or streptavidin . Immunoprecipitated material was run on a 4 - 12 % SDS PAGE gel and analysed by immunoblotting using indicated antibodies .

Immunoblot from immunoprecipitation (IP) assays to validate the predicted model of UFL1/UFBP1 interaction in cells. UFL1WT-3xFLAG and UFL1L45R-3xFLAG were transiently overexpressed along with UFBP1-SBP in HEK293T-UFL1 KO cells and subjected to separate pulldowns using anti-FLAG antibody or streptavidin. Immunoprecipitated material was run on a 4-12% SDS PAGE gel and analysed by immunoblotting using indicated antibodies.


Immunoblot showing in vitro UFMylation assay to monitor formation of free di - UFM1 chains in the presence of UFL1 / UFBP1 complexes bearing different domain boundaries .

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Immunoblot showing in vitro UFMylation assay to monitor formation of free di-UFM1 chains in the presence of UFL1/UFBP1 complexes bearing different domain boundaries.


Immunoblot of in vitro assay monitoring UFMylation of 60S Ribosome in the presence of different UFL1 / UFBP1 truncations as depicted in ( E ) .

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Immunoblot of in vitro assay monitoring UFMylation of 60S Ribosome in the presence of different UFL1/UFBP1 truncations as depicted in (E).


In vitro UFMylation assay in the presence of increasing concentrations of CDK5RAP3 to monitor the E3 ligase activity of UFL1 / UFBP1 complex . The reaction products were run on a 4 - 12 % SDS - PAGE gel under reducing conditions and immunoblotting was performed using indicated antibodies .

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In vitro UFMylation assay in the presence of increasing concentrations of CDK5RAP3 to monitor the E3 ligase activity of UFL1/UFBP1 complex. The reaction products were run on a 4-12% SDS-PAGE gel under reducing conditions and immunoblotting was performed using indicated antibodies.


Single turnover lysine discharge assays to check for activation of UFC1 by UFL1 / UFBP1 in the presence and absence of CDK5RAP3 . The reaction products were run on a 4 - 12 % SDS PAGE and visualized by Oriole staining . Quantification of discharge of UFM1 from UFC1 in the presence and absence of CDK5RAP3 as seen in Fig 4D , n = 3 biological replicates ,

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Single turnover lysine discharge assays to check for activation of UFC1 by UFL1/UFBP1 in the presence and absence of CDK5RAP3. The reaction products were run on a 4-12% SDS PAGE and visualized by Oriole staining.   Quantification of discharge of UFM1 from UFC1 in the presence and absence of CDK5RAP3 as seen in Fig 4D, n=3 biological replicates,


Substrate UFMylation assays using purified 60S Ribosomes in the presence of increasing concentration of CDK5RAP3 . Reaction was performed for 10 min , stopped by addition of SDS Loading dye and analysed on an SDS - PAGE gel under reducing conditions followed by immunoblotting with indicated antibodies .

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Substrate UFMylation assays using purified 60S Ribosomes in the presence of increasing concentration of CDK5RAP3. Reaction was performed for 10 min, stopped by addition of SDS Loading dye and analysed on an SDS-PAGE gel under reducing conditions followed by immunoblotting with indicated antibodies.


Immunoblot analysis monitoring UFMylation of Histone H4 in the presence of increasing concentrations of CDK5RAP3 ( 1 µM , 2 µM , 3 µM ) . UFL1 / UFBP1 concentrations - 1 µM .

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Immunoblot analysis monitoring UFMylation of Histone H4 in the presence of increasing concentrations of CDK5RAP3 (1 µM, 2 µM, 3 µM). UFL1/UFBP1 concentrations - 1 µM.


Immunoblot showing the in vitro UFMylation assay to compare the activity of UFC1 Wild type and mutants in the presence of UFL1 / UFBP1 complex . In vitro UFMylation assay to compare the activity of UFC1 Wild type and UFC1 T106I in the presence of UFL1 / UFBP1 complex .

Immunoblot showing the in vitro UFMylation assay to compare the activity of UFC1 Wild type and mutants in the presence of UFL1/UFBP1 complex.   In vitro UFMylation assay to compare the activity of UFC1 Wild type and UFC1 T106I in the presence of UFL1/UFBP1 complex.


Substrate UFMylation assays using purified 60S Ribosomes in the presence of UFC1 Wild type and different mutants of UFC1 .

Substrate UFMylation assays using purified 60S Ribosomes in the presence of UFC1 Wild type and different mutants of UFC1.


In vitro UFMylation assays in the presence of Wild type UFC1 ( UFC1WT ) and UFC1 lacking the N - terminal helix ( UFC1ΔN ) .

In vitro UFMylation assays in the presence of Wild type UFC1 (UFC1WT) and UFC1 lacking the N-terminal helix (UFC1ΔN).


Assay to compare Lys discharge activities of UFC1WT and UFC1ΔN on its own , in the presence of UFL1 / UFBP1 or in the presence of preassembled UFL1 / UFBP1 / CDK5RAP3 complex . Top gel : LI - COR scan of fluorescently labelled UFM1 ( UFM1 * ) ; bottom gel - Coomassie stained ( Representative of three independent experiments ) .

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Assay to compare Lys discharge activities of UFC1WT and UFC1ΔN on its own, in the presence of UFL1/UFBP1 or in the presence of preassembled UFL1/UFBP1/CDK5RAP3 complex. Top gel: LI-COR scan of fluorescently labelled UFM1(UFM1*); bottom gel - Coomassie stained (Representative of three independent experiments).


H . Effect of deletion of N - term helix of UFC1 in 60S ribosome UFMylation . Purified 60S ribosomes ( 50 nM ) were incubated with 0 . 5 µM UBA5 , 1 µM UFM1 , 100 nM UFL1 / UFBP1 complex in the presence of either 1 µM UFC1WT or 1 µM UFC1ΔN at 30 ° C . The reaction was stopped at indicated time points and run on a 4 - 12 % SDS PAGE gel under reducing conditions followed by immunoblotting using indicated antibodies . ( Right - shorter time duration . Left - Longer time duration ) .

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H. Effect of deletion of N -term helix of UFC1 in 60S ribosome UFMylation. Purified 60S ribosomes (50 nM) were incubated with 0.5 µM UBA5, 1 µM UFM1, 100 nM UFL1/UFBP1 complex in the presence of either 1 µM UFC1WT or 1 µM UFC1ΔN at 30°C. The reaction was stopped at indicated time points and run on a 4-12% SDS PAGE gel under reducing conditions followed by immunoblotting using indicated antibodies. (Right - shorter time duration. Left - Longer time duration).


A Combined IF and DNA FISH analysis of RAD54 ( IF ) and telomeres ( FISH ) in ALT and non - ALT cell lines . White arrows indicate RAD54 foci that colocalize with telomeres . Scale bars = 10 µm B Quantification of data in A . A cell was counted positive if it contained 1 or more colocalization event between RAD54 and the telomere . At least 100 cells were counted per cell line per repeat . For SaOS2 , NOS , SJSA1 , HeLa LT n = 3 . For Cal72 , U2OS , HuO9 n = 4 . Values shown are mean ± sd

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A Combined IF and DNA FISH analysis of RAD54 (IF) and telomeres (FISH) in ALT and non-ALT cell lines. White arrows indicate RAD54 foci that colocalize with telomeres. Scale bars = 10 µm B Quantification of data in A. A cell was counted positive if it contained 1 or more colocalization event between RAD54 and the telomere. At least 100 cells were counted per cell line per repeat. For SaOS2, NOS, SJSA1, HeLa LT n = 3. For Cal72, U2OS, HuO9 n = 4. Values shown are mean±sd


Combined IF and DNA FISH analysis of RAD54 ( IF ) , PML ( IF ) and telomeres ( FISH ) in ALT cell lines SaOS2 and HuO9 . Scale bars = 10

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Combined IF and DNA FISH analysis of RAD54 (IF), PML (IF) and telomeres (FISH) in ALT cell lines SaOS2 and HuO9. Scale bars = 10 µm


Western blot showing expression of TRF1 - FOK1 - mCherry fusion protein following doxycycline induction , both WT and D450A ( nuclease dead ) . Cells were treated with 40 ng / mL doxycycline for 16 hrs and then 1µM Shield - 1 and 1µM 4 - OHT for 4

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Western blot showing expression of TRF1-FOK1-mCherry fusion protein following doxycycline induction, both WT and D450A (nuclease dead). Cells were treated with 40 ng/mL doxycycline for 16 hrs and then 1µM Shield-1 and 1µM 4-OHT for 4 hrs