Endogenous levels of free cholesterol revealed by filipin staining of ( B ) WT cells under the indicated treatments . Graphs show the filipin fluorescence intensity as a percentage over VEH .

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Endogenous levels of free cholesterol revealed by filipin staining of (B) WT cells under the indicated treatments. Graphs show the filipin fluorescence intensity as a percentage over VEH.


Endogenous levels of free cholesterol revealed by filipin staining of ( C ) PS - DKO cells under the indicated treatments . Graphs show the filipin fluorescence intensity as a percentage over VEH .

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Endogenous levels of free cholesterol revealed by filipin staining of (C) PS-DKO cells under the indicated treatments. Graphs show the filipin fluorescence intensity as a percentage over VEH.


( A ) Crude membrane fractions from APP - DKO cells expressing C99WT or C99MUT were treated with 0 . 2 % Triton - X - 100 , loaded onto continuous density sucrose gradients and centrifuged for 16h . Fractions from these gradients were analyzed by western blot to determine the migration of the indicated proteins [ two parallel gels ( bold vertical line ) ] .

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(A) Crude membrane fractions from APP-DKO cells expressing C99WT or C99MUT were treated with 0.2% Triton-X-100, loaded onto continuous density sucrose gradients and centrifuged for 16h. Fractions from these gradients were analyzed by western blot to determine the migration of the indicated proteins [two parallel gels (bold vertical line)].


( B - G ) Graphs represent the relative abundance of the indicated proteins in each fraction of the gradient , as measured by densitometry analysis of western blot signals ( ImageJ ) . Note the reduced degree of co - migration of C99MUT with MAM markers ( Acsl4 ) , compared to C99WT .

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(B-G) Graphs represent the relative abundance of the indicated proteins in each fraction of the gradient, as measured by densitometry analysis of western blot signals (ImageJ). Note the reduced degree of co-migration of C99MUT with MAM markers (Acsl4), compared to C99WT.


( A ) Representative confocal images of APP - DKO cells expressing GFP - tagged C99WT or C99MUT ( in green ) and fluorescent markers of mitochondria ( MitoDsRed , in red ) and ER ( Sec61β - BFP , in blue ) , and treated with DAPT to prevent C99 cleavage . Note the different distribution of C99WT or C99MUT forms . Scale bar = 10 µm . Insets show 5X amplifications of individual ( C99 in green , and mitochondria in red ) and merged images . Black and white bottom panels represent the areas where C99 and mitochondria signals colocalize . Upon thresholding each channel , a mask for mitochondria was generated and the C99 channel was superimposed on the mitochondria mask , so the positive pixels found in both channels are shown in black .

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(A) Representative confocal images of APP-DKO cells expressing GFP-tagged C99WT or C99MUT (in green) and fluorescent markers of mitochondria (MitoDsRed, in red) and ER (Sec61β-BFP, in blue), and treated with DAPT to prevent C99 cleavage. Note the different distribution of C99WT or C99MUT forms. Scale bar = 10 µm. Insets show 5X amplifications of individual (C99 in green, and mitochondria in red) and merged images. Black and white bottom panels represent the areas where C99 and mitochondria signals colocalize. Upon thresholding each channel, a mask for mitochondria was generated and the C99 channel was superimposed on the mitochondria mask, so the positive pixels found in both channels are shown in black.


( B ) Quantification of the degree of colocalization of C99WT or C99MUT forms with mitochondria or ER measured by ImageJ . Note the significantly decreased colocalization between C99MUT and mitochondria , when compared to that of C99WT . Unpaired t - test . ( 20 - 30 cells / condition from 3 independent experiments and 4 - 5 images / experiment ; * p < 0 . 05 ; n . s . , not significant ) .

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(B) Quantification of the degree of colocalization of C99WT or C99MUT forms with mitochondria or ER measured by ImageJ. Note the significantly decreased colocalization between C99MUT and mitochondria, when compared to that of C99WT. Unpaired t-test. (20-30 cells/condition from 3 independent experiments and 4-5 images/experiment; * p<0.05; n.s., not significant).


( C ) Representative confocal images of APP - DKO cells expressing GFP - tagged C99WT or C99MUT and ER and mitochondria markers as in ( A ) ( upper panel ) . Individual zoomed single channels ( middle panel ) as well as areas of ER - mitochondria colocalization ( bottom panel ) are shown for each condition . Scale bar = 10 µm .

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(C) Representative confocal images of APP-DKO cells expressing GFP-tagged C99WT or C99MUT and ER and mitochondria markers as in (A) (upper panel). Individual zoomed single channels (middle panel) as well as areas of ER-mitochondria colocalization (bottom panel) are shown for each condition. Scale bar = 10 µm.


( D ) Quantification of ER - mitochondria colocalization per cell analyzed . Upon thresholding each channel , a mask for ER was generated and the mitochondria channel was superimposed on the ER mask , so the positive pixels found in both channels were quantified and referenced over the total area of the ER mask . The number of cells analyzed is indicated in white text within each column . Levels of ER - mitochondria colocalization in APP - WT cells are shown for reference as a dashed line . EV , empty vector . One - way ANOVA ( n = 3 independent experiments and 5 - 8 images / experiment ; * p < 0 . 05 ) .

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(D) Quantification of ER-mitochondria colocalization per cell analyzed. Upon thresholding each channel, a mask for ER was generated and the mitochondria channel was superimposed on the ER mask, so the positive pixels found in both channels were quantified and referenced over the total area of the ER mask. The number of cells analyzed is indicated in white text within each column. Levels of ER-mitochondria colocalization in APP-WT cells are shown for reference as a dashed line. EV, empty vector. One-way ANOVA (n=3 independent experiments and 5-8 images/experiment; * p< 0.05).


( B ) Representative immunoblot to reveal C99 levels in total homogenate ( TH ) and MAM fractions of APP - DKO cells expressing C99WT or C99MUT before ( input ) and after cholesterol pull - down . Acsl4 was used as a MAM marker . Streptavidin - HRP was used to detect total biotinylation ( biotin conjugated to PhotoClick cholesterol ) . ( C ) Quantification of pulled - down C99 levels versus input from the experiment in B . Two - way repeated measures ANOVA ( Fraction , mutation ) ( n = 5 ; * p < 0 . 05 , * * p < 0 . 01 ) .

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(B) Representative immunoblot to reveal C99 levels in total homogenate (TH) and MAM fractions of APP-DKO cells expressing C99WT or C99MUT before (input) and after cholesterol pull-down. Acsl4 was used as a MAM marker. Streptavidin-HRP was used to detect total biotinylation (biotin conjugated to PhotoClick cholesterol). (C) Quantification of pulled-down C99 levels versus input from the experiment in B. Two-way repeated measures ANOVA (Fraction, mutation) (n=5; * p< 0.05, ** p< 0.01).


( D ) Immunoblot showing the levels of pulled - down PhotoClick Cholesterol ( streptavidin - HRP ) in ER and MAM fractions . Note how the levels of pulled - down C99 in the ER are negligible when compared to those from MAM . Isolated MAM and ER fractions were assessed by western blot ( shown in AppendixFig S1E ) .

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(D) Immunoblot showing the levels of pulled-down PhotoClick Cholesterol (streptavidin-HRP) in ER and MAM fractions. Note how the levels of pulled-down C99 in the ER are negligible when compared to those from MAM. Isolated MAM and ER fractions were assessed by western blot (shown in AppendixFig S1E).


( E ) Quantification of free cholesterol levels ( FC ) analyzed by lipidomics after subcellular fractionation to obtain MAM from the indicated cells . Lipid units are represented as molar mass over total moles of lipids analyzed ( mol % ) . Graphs represent percentage over controls . Dashed line represents control levels . One - way ANOVA . ( n = 4 ; * p < 0 . 05 )

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(E) Quantification of free cholesterol levels (FC) analyzed by lipidomics after subcellular fractionation to obtain MAM from the indicated cells. Lipid units are represented as molar mass over total moles of lipids analyzed (mol%). Graphs represent percentage over controls. Dashed line represents control levels. One-way ANOVA. (n=4; * p< 0.05)


( F - G ) Quantification of cholesterol uptake and esterification in the indicated cells was measured by 4h incubation with 3H - cholesterol and subsequent analysis of radiolabel incorporation . The dashed line indicates control levels . Graphs represent fold change over controls . Treatment with Sandoz 58 - 035 , a specific ACAT1 inhibitor , caused a ~ 95 % reduction in cholesterol esterification . EV , empty vector . One - sample t - test . ( n = 3 - 4 ; * p < 0 . 05 ) .

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(F-G) Quantification of cholesterol uptake and esterification in the indicated cells was measured by 4h incubation with 3H-cholesterol and subsequent analysis of radiolabel incorporation. The dashed line indicates control levels. Graphs represent fold change over controls. Treatment with Sandoz 58-035, a specific ACAT1 inhibitor, caused a ~95% reduction in cholesterol esterification. EV, empty vector. One-sample t-test. (n=3-4; * p< 0.05).


( H ) Ratio of cholesteryl esters : free cholesterol ( CE : FC ) in the indicated cells .

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(H) Ratio of cholesteryl esters:free cholesterol (CE:FC) in the indicated cells.


After 2 hour starvation , E . coli cultures were pulse fed 10 µM glucose at varying frequencies using the spin flask and plate reader systems , and optical density ( OD ) was measured over time ( inset example figures ) . Grey dots are OD measurements and the black lines are an empirical fit ( see Methods ) . For separate experiments ( n = 18 ) , the lag time is plotted against the frequency , represented as the time - integrated ( TI ) feedrate f ( mmol glucose / g dry cell weight / hour ) . An empirical fit ( grey solid line , see Methods ) was used to separate the lag ( non - dividing ) and dividing

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After 2 hour starvation, E. coli cultures were pulse fed 10 µM glucose at varying frequencies using the spin flask and plate reader systems, and optical density (OD) was measured over time (inset example figures). Grey dots are OD measurements and the black lines are an empirical fit (see Methods). For separate experiments (n = 18), the lag time is plotted against the frequency, represented as the time-integrated (TI) feedrate f (mmol glucose/g dry cell weight/hour). An empirical fit (grey solid line, see Methods) was used to separate the lag (non-dividing) and dividing phases


Normalized absolute cell counts versus time show linear increases after lag time for exemplary feedrates . Data are mean ± standard error of technical replicates ( n = 2 - 3 ) . Lag predicted from the empirical fit is indicated by vertical dotted

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Normalized absolute cell counts versus time show linear increases after lag time for exemplary feedrates. Data are mean ± standard error of technical replicates (n = 2-3). Lag predicted from the empirical fit is indicated by vertical dotted lines


The average size of cells decreased after lag . Micrographs and cell length distributions ( n > 400 per distribution ) are shown for specific time points , with f = 0 . 6 mmol / g /

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The average size of cells decreased after lag. Micrographs and cell length distributions (n > 400 per distribution) are shown for specific time points, with f = 0.6 mmol/g/h


Immobilized cells in the microfluidic experiment divided after a lag time that decreased with increasing glucose pulse frequency . The labeled times indicate the period , time between pulses , for a given

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Immobilized cells in the microfluidic experiment divided after a lag time that decreased with increasing glucose pulse frequency. The labeled times indicate the period, time between pulses, for a given experiment


Time course of the distribution of cellular DNA content . Sampled cells were stained with SYBR Green I and measured with flow cytometry over the course of a pulsing experiment ( f = 0 . 6 mmol / g / h ) The DNA content distribution over time is shown on the left side , and three specific time points are shown on the right . Within the first time point ( t = 0 ) , the highest distribution is taken to be high DNA content ( 2N ) , and the distribution at half of the 2N average was taken ( 1N ) as medium DNA . DNA distributions were separated into medium ( 1N ) and high DNA ( 2N ) . Distribution - specific estimated counts ( see Methods ) over time ( f = 0 . 6 mmol / g / h ) suggested that net division from high to medium DNA cells can explain the increase in cell counts and OD

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Time course of the distribution of cellular DNA content. Sampled cells were stained with SYBR Green I and measured with flow cytometry over the course of a pulsing experiment (f = 0.6 mmol/g/h) The DNA content distribution over time is shown on the left side, and three specific time points are shown on the right. Within the first time point (t = 0), the highest distribution is taken to be high DNA content (2N), and the distribution at half of the 2N average was taken (1N) as medium DNA. DNA distributions were separated into medium (1N) and high DNA (2N). Distribution-specific estimated counts (see Methods) over time (f = 0.6 mmol/g/h) suggested that net division from high to medium DNA cells can explain the increase in cell counts and OD increase


The spin flask system for glucose pulsing was connected to a real - time metabolomics platform . Traces of exemplary ions are shown that correspond to hexose phosphate , guanine , phenylalanine , and hypoxanthine for pulsing at non - division supporting frequencies of 0 . 06 , 0 . 12 , and 0 . 18 mmol / g / h . The TI feedrate is abbreviated as f ( units : mmol glucose / g dry cell weight / hour ) . Glucose pulses are indicated by the grey bars , and the pink region shows a no pulse control . Dots are ion intensity measurements . Solid lines are a moving average filter of the measured ion intensity . For clarity , dots are not shown for f = 0 mmol / g / h condition A metabolic scheme describing the propagation of fed glucose . Pulsed glucose is hypothesized to pass through central carbon metabolism and then be converted to downstream pathways including amino acid synthesis and nucleic acid synthesis . For nucleic acid synthesis , glucose is converted to the intermediate PRPP , which then can combine with nitrogen bases to form nucleotides for nucleic acid synthesis . Different pathways can be blocked with

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The spin flask system for glucose pulsing was connected to a real-time metabolomics platform. Traces of exemplary ions are shown that correspond to hexose phosphate, guanine, phenylalanine, and hypoxanthine for pulsing at non-division supporting frequencies of 0.06, 0.12, and 0.18 mmol/g/h. The TI feedrate is abbreviated as f (units: mmol glucose/g dry cell weight/hour). Glucose pulses are indicated by the grey bars, and the pink region shows a no pulse control. Dots are ion intensity measurements. Solid lines are a moving average filter of the measured ion intensity. For clarity, dots are not shown for f = 0 mmol/g/h condition A metabolic scheme describing the propagation of fed glucose. Pulsed glucose is hypothesized to pass through central carbon metabolism and then be converted to downstream pathways including amino acid synthesis and nucleic acid synthesis. For nucleic acid synthesis, glucose is converted to the intermediate PRPP, which then can combine with nitrogen bases to form nucleotides for nucleic acid synthesis. Different pathways can be blocked with antibiotics


Influence of antibiotics that inhibit macromolecular synthesis at the non - division TI feedrate of 0 . 18 mmol / g / h . Antibiotics were added one minute after the second pulse ( yellow region ) . Four different ions are shown corresponding to glutamate , phenylalanine , guanine , and thymine . Chloramphenicol ( blue ) inhibits protein biosynthesis , rifamycin ( orange ) inhibits RNA polymerase , and azidothymidine ( AZT ; red ) inhibits DNA

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Influence of antibiotics that inhibit macromolecular synthesis at the non-division TI feedrate of 0.18 mmol/g/h. Antibiotics were added one minute after the second pulse (yellow region). Four different ions are shown corresponding to glutamate, phenylalanine, guanine, and thymine. Chloramphenicol (blue) inhibits protein biosynthesis, rifamycin (orange) inhibits RNA polymerase, and azidothymidine (AZT; red) inhibits DNA synthesis


Percentage of labeled threonine and deoxyribose from protein and DNA hydrolysate shows de novo protein and DNA synthesis in non - dividing cells . After 6 hours of pulsing uniformly labeled 13C - glucose , cultures were lysed , and their macromolecules were washed free of latent metabolites and hydrolyzed to monomers . Labeling data are presented as the mean ± standard error of independent biological replicates ( n = 3 , all pairwise P < 0 . 02 as determined by one sided Student " s t -

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Percentage of labeled threonine and deoxyribose from protein and DNA hydrolysate shows de novo protein and DNA synthesis in non-dividing cells. After 6 hours of pulsing uniformly labeled 13C-glucose, cultures were lysed, and their macromolecules were washed free of latent metabolites and hydrolyzed to monomers. Labeling data are presented as the mean ± standard error of independent biological replicates (n = 3, all pairwise P < 0.02 as determined by one sided Student"s t-


Pulsing experiment was repeated in the presence of protease inhibitor ( PI ) that reduced the lag time for a given TI feedrate ( f = 0 . 28 mmol / g / h ) . The TI feedrate is abbreviated as f ( units : mmol glucose / g dry cell weight / hour ) . Wild - type lag ( from Figure 2A empirical fit ) is indicated by the dotted grey

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Pulsing experiment was repeated in the presence of protease inhibitor (PI) that reduced the lag time for a given TI feedrate (f = 0.28 mmol/g/h). The TI feedrate is abbreviated as f (units: mmol glucose/g dry cell weight/hour). Wild-type lag (from Figure 2A empirical fit) is indicated by the dotted grey line


Cells expressing a nearly functional FtsZ - mVenus fluorescent fusion as their sole copy of ftsZ were monitored after transition into carbon starvation . Two low motile MG1655 strains , one ( wild - type ) containing clpX and one without ( ΔclpX ) , were grown in M9 glucose to steady - state . At time zero , M9 media without glucose was flushed through device . Cells rapidly ceased elongation as measured by phase contrast imaging at 2 minute intervals . Fluorescent images were taken every hour throughout to measure both total Ftsz - mVenus per cell and localization patterns Thin individual lines show the total fluorescent per cell in a single lineage ( wild - type in solid blue , n = 154 . ΔclpX in dotted red , n = 144 ) . Thick solid lines are the time average across individual lineages for each subset ( wild - type in blue , ΔclpX in red ) . After starvation , cells containing clpX degrade FtsZ faster than cells which do not . Fluorescent signals are normalized to the average value for their respective subset at time 0 Distributions of cellular fluorescence for each subset at times corresponding to the top panel . Cellular fluorescence varies widely as total FtsZ per cell is a function of cell size ; a typical size distribution is shown at left . FtsZ concentration is roughly constant at steady - stat Student " s t - test P value shown for when distributions differ with significance level α = 0 . 01 . Representative images from cells in a single lineage with and without clpX . Wild - type strains may contain the characteristic FtsZ ring at midcell after shift down but it dissipates after several hours . ΔclpX strains display an abnormal FtsZ localization pattern even in steady - state . After shift down , the FtsZ may disassemble and reform along the cell body in distinct puncta . Image timing corresponds to the abscissa in the

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Cells expressing a nearly functional FtsZ-mVenus fluorescent fusion as their sole copy of ftsZ were monitored after transition into carbon starvation. Two low motile MG1655 strains, one (wild-type) containing clpX and one without (ΔclpX), were grown in M9 glucose to steady-state. At time zero, M9 media without glucose was flushed through device. Cells rapidly ceased elongation as measured by phase contrast imaging at 2 minute intervals. Fluorescent images were taken every hour throughout to measure both total Ftsz-mVenus per cell and localization patterns Thin individual lines show the total fluorescent per cell in a single lineage (wild-type in solid blue, n = 154. ΔclpX in dotted red, n = 144). Thick solid lines are the time average across individual lineages for each subset (wild-type in blue, ΔclpX in red). After starvation, cells containing clpX degrade FtsZ faster than cells which do not. Fluorescent signals are normalized to the average value for their respective subset at time 0 Distributions of cellular fluorescence for each subset at times corresponding to the top panel. Cellular fluorescence varies widely as total FtsZ per cell is a function of cell size; a typical size distribution is shown at left. FtsZ concentration is roughly constant at steady-stat Student"s t-test P value shown for when distributions differ with significance level α = 0.01. Representative images from cells in a single lineage with and without clpX. Wild-type strains may contain the characteristic FtsZ ring at midcell after shift down but it dissipates after several hours. ΔclpX strains display an abnormal FtsZ localization pattern even in steady-state. After shift down, the FtsZ may disassemble and reform along the cell body in distinct puncta. Image timing corresponds to the abscissa in the top


Genetically induced titration of PdhR in a pdhR mutant reintroduced lag commensurate with expression level for a given TI feedrate ( f = 0 . 38 mmol / g / h ) . Induction level corresponds to the amount of doxycycline ( max - 50 ng / uL , half - 10 ng / uL , and zero 0 ng / uL ) added at the onset of starvation . The TI feedrate is abbreviated as f ( units : mmol glucose / g dry cell weight / hour ) . Wild - type lag ( from Figure 2A empirical fit ) is indicated by the dotted grey line . Lag time reduced with synthesis levels of titrated FtsZ in the wild - type strain at f = 0 . 31 - 0 . 32 mmol / g / h . Lag time increased with titrated synthesis of ClpX in wild - type cells at f = 0 . 43 - 0 . 45 mmol / g /

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Genetically induced titration of PdhR in a pdhR mutant reintroduced lag commensurate with expression level for a given TI feedrate (f = 0.38 mmol/g/h). Induction level corresponds to the amount of doxycycline (max - 50 ng/uL, half - 10 ng/uL, and zero 0 ng/uL) added at the onset of starvation. The TI feedrate is abbreviated as f (units: mmol glucose/g dry cell weight/hour). Wild-type lag (from Figure 2A empirical fit) is indicated by the dotted grey line. Lag time reduced with synthesis levels of titrated FtsZ in the wild-type strain at f = 0.31-0.32 mmol/g/h. Lag time increased with titrated synthesis of ClpX in wild-type cells at f = 0.43-0.45 mmol/g/h


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


a , Cells ( wt , ∆ atg18 or ∆ atg18 / ∆ atg21 / ∆ hsv2 ( ) ) , with and without a plasmid expressing wt or a mutant form of Atg18 ) were grown in YPD , vacuoles were stained with FM4 - 64 and imaged by confocal microscopy . At least 10 confocal sections spaced at 300 nm were assembled into maximum projections . Images show cells before ( 0 min ) and 15 , 30 and 60 min after induction of vacuolar fragmentation by addition of 0 . 4 M NaCl . Red : FM4 - 64 . Gray : DIC . Scale bar 5 µm .

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a, Cells (wt, ∆atg18 or ∆atg18/∆atg21/∆hsv2 ()), with and without a plasmid expressing wt or a mutant form of Atg18) were grown in YPD, vacuoles were stained with FM4-64 and imaged by confocal microscopy. At least 10 confocal sections spaced at 300 nm were assembled into maximum projections. Images show cells before (0 min) and 15, 30 and 60 min after induction of vacuolar fragmentation by addition of 0.4 M NaCl. Red: FM4-64. Gray: DIC. Scale bar 5 µm.


b , The number of vacuoles per cell was quantified for the experiment in a . 150 cells from three independent experiments were counted for each condition . Values represent the mean , error bars the SEM .

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b, The number of vacuoles per cell was quantified for the experiment in a. 150 cells from three independent experiments were counted for each condition. Values represent the mean, error bars the SEM.


atg18 cells expressing plasmids carrying Atg18 - GFP or Atg18FGGG - GFP , a mutant in the lipid binding sites , were stained with FM4 - 64 . a , Confocal z - stacks were taken before and 2 , 6 and 10 min after addition of 0 . 4 M NaCl . Stacks were processed into maximum projections using ImageJ . Scale bar : 5 µm .

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atg18 cells expressing plasmids carrying Atg18-GFP or Atg18FGGG-GFP, a mutant in the lipid binding sites, were stained with FM4-64. a, Confocal z-stacks were taken before and 2, 6 and 10 min after addition of 0.4 M NaCl. Stacks were processed into maximum projections using ImageJ. Scale bar: 5 µm.


b , Same experiment as in a , but in a fab1 or vps34 background .

b, Same experiment as in a, but in a fab1 or vps34 background.


d , Localisation of Atg18 - GFP 5 min after salt shock . Experiment as in a . Scale bar : 1 µm .

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d, Localisation of Atg18-GFP 5 min after salt shock. Experiment as in a. Scale bar: 1 µm.


b , c Their secondary structure was analyzed by circular dichroism spectroscopy in the presence of increasing concentrations of hexafluoro - 2 - propanol ( HFIP ) , in the presence of 6 mM small unilamellar vesicles ( SUVs , 70 % egg PC and 30 % PS , total phospholipid concentration approx . 6 mM ) , or of or control buffer only ( blue ) . Spectra are shown for b , the wildtype

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b,c Their secondary structure was analyzed by circular dichroism spectroscopy in the presence of increasing concentrations of hexafluoro-2-propanol (HFIP), in the presence of 6 mM small unilamellar vesicles (SUVs, 70% egg PC and 30 % PS, total phospholipid concentration approx. 6 mM), or of or control buffer only (blue). Spectra are shown for b, the wildtype peptide


b , c Their secondary structure was analyzed by circular dichroism spectroscopy in the presence of increasing concentrations of hexafluoro - 2 - propanol ( HFIP ) , in the presence of 6 mM small unilamellar vesicles ( SUVs , 70 % egg PC and 30 % PS , total phospholipid concentration approx . 6 mM ) , or of or control buffer only ( blue ) . Spectra are shown for c , the SLoop peptide .

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b,c Their secondary structure was analyzed by circular dichroism spectroscopy in the presence of increasing concentrations of hexafluoro-2-propanol (HFIP), in the presence of 6 mM small unilamellar vesicles (SUVs, 70% egg PC and 30 % PS, total phospholipid concentration approx. 6 mM), or of or control buffer only (blue). Spectra are shown for c, the SLoop peptide.


d , Vacuole morphology of cells expressing Atg18Sloop - GFP , imaged before and after osmotic shock as in Fig 1a . Scale bar : 5 µm .

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d, Vacuole morphology of cells expressing Atg18Sloop-GFP, imaged before and after osmotic shock as in Fig 1a. Scale bar: 5 µm.


e , Number of vacuoles per cell in d , assayed as in Figure 1b . n = 3 .

e, Number of vacuoles per cell in d, assayed as in Figure 1b. n=3.


f , Vacuole morphology of cells expressing Atg18DLoop - GFP , imaged as in d .

f, Vacuole morphology of cells expressing Atg18DLoop-GFP, imaged as in d.


g , Number of vacuoles per cell in e , assayed as in Fig . 1b . n = 3 .

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g, Number of vacuoles per cell in e, assayed as in Fig. 1b. n=3.


Binding of recombinant Atg18 variants to liposomes . SUVs were made of EPC with 5 % of the indicated phosphoinositides , or of EPC and the indicated fraction of PS . Cholesterol had been added to 20 mol % of the total phospholipid amount . Liposomes were incubated with the purified proteins for 30 min at room temperature ( 25 ° C ) , centrifuged and the supernatants ( S ) and pellets ( P ) analyzed by SDS - PAGE and Coomassie staining . Binding is shown for a ,

Binding of recombinant Atg18 variants to liposomes. SUVs were made of EPC with 5% of the indicated phosphoinositides, or of EPC and the indicated fraction of PS. Cholesterol had been added to 20 mol% of the total phospholipid amount. Liposomes were incubated with the purified proteins for 30 min at room temperature (25°C), centrifuged and the supernatants (S) and pellets (P) analyzed by SDS-PAGE and Coomassie staining. Binding is shown for a, Atg18wt


Binding of recombinant Atg18 variants to liposomes . SUVs were made of EPC with 5 % of the indicated phosphoinositides , or of EPC and the indicated fraction of PS . Cholesterol had been added to 20 mol % of the total phospholipid amount . Liposomes were incubated with the purified proteins for 30 min at room temperature ( 25 ° C ) , centrifuged and the supernatants ( S ) and pellets ( P ) analyzed by SDS - PAGE and Coomassie staining . Binding is shown for b , Atg18FGGG ;

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Binding of recombinant Atg18 variants to liposomes. SUVs were made of EPC with 5% of the indicated phosphoinositides, or of EPC and the indicated fraction of PS. Cholesterol had been added to 20 mol% of the total phospholipid amount. Liposomes were incubated with the purified proteins for 30 min at room temperature (25°C), centrifuged and the supernatants (S) and pellets (P) analyzed by SDS-PAGE and Coomassie staining. Binding is shown for b, Atg18FGGG;


Binding of recombinant Atg18 variants to liposomes . SUVs were made of EPC with 5 % of the indicated phosphoinositides , or of EPC and the indicated fraction of PS . Cholesterol had been added to 20 mol % of the total phospholipid amount . Liposomes were incubated with the purified proteins for 30 min at room temperature ( 25 ° C ) , centrifuged and the supernatants ( S ) and pellets ( P ) analyzed by SDS - PAGE and Coomassie staining . Binding is shown for c , Atg18DLoop ;

Binding of recombinant Atg18 variants to liposomes. SUVs were made of EPC with 5% of the indicated phosphoinositides, or of EPC and the indicated fraction of PS. Cholesterol had been added to 20 mol% of the total phospholipid amount. Liposomes were incubated with the purified proteins for 30 min at room temperature (25°C), centrifuged and the supernatants (S) and pellets (P) analyzed by SDS-PAGE and Coomassie staining. Binding is shown for c, Atg18DLoop;


Binding of recombinant Atg18 variants to liposomes . SUVs were made of EPC with 5 % of the indicated phosphoinositides , or of EPC and the indicated fraction of PS . Cholesterol had been added to 20 mol % of the total phospholipid amount . Liposomes were incubated with the purified proteins for 30 min at room temperature ( 25 ° C ) , centrifuged and the supernatants ( S ) and pellets ( P ) analyzed by SDS - PAGE and Coomassie staining . Binding is shown for d , Atg18SLoop .

Binding of recombinant Atg18 variants to liposomes. SUVs were made of EPC with 5% of the indicated phosphoinositides, or of EPC and the indicated fraction of PS. Cholesterol had been added to 20 mol% of the total phospholipid amount. Liposomes were incubated with the purified proteins for 30 min at room temperature (25°C), centrifuged and the supernatants (S) and pellets (P) analyzed by SDS-PAGE and Coomassie staining. Binding is shown for d, Atg18SLoop.


e , The bands from a - d were quantified from 3 independent experiments . The mean with SEM is shown .

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e, The bands from a-d were quantified from 3 independent experiments. The mean with SEM is shown.


a . Autophagy measured with the pho8 ∆ 60 assay in atg18 cells complemented with wt , FGGG , SLoop or DLoop alleles of ATG18 .

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a. Autophagy measured with the pho8∆60 assay in atg18 cells complemented with wt, FGGG, SLoop or DLoop alleles of ATG18.


b . Same assay as in a , but in atg18 atg21 cells complemented with wt or SLoop versions of ATG18 and ATG21 . Atg21SLoop contains K426F and F429K substitutions . Helical wheel projections for both Atg21 helices are given . The arrows indicate the magnitude of the hydrophobic moment .

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b. Same assay as in a, but in atg18 atg21 cells complemented with wt or SLoop versions of ATG18 and ATG21. Atg21SLoop contains K426F and F429K substitutions. Helical wheel projections for both Atg21 helices are given. The arrows indicate the magnitude of the hydrophobic moment.


c . Vacuole fragmentation in atg2 cells , assayed 15 min after a moderate hypertonic shock as in Fig . 1 .

c. Vacuole fragmentation in atg2 cells, assayed 15 min after a moderate hypertonic shock as in Fig. 1.


d . Number of vacuoles per cell in c , assayed as in Figure 1b . n = 3 .

d. Number of vacuoles per cell in c, assayed as in Figure 1b. n=3.


a , Time lapse analysis of Atg18 added to liposomes . 10 µL of 10 µM purified recombinant Atg18wt was added to 100 µL buffer containing GUVs with 5 % PI3P and 1 % PI ( 3 , 5 ) P2 . The sample was imaged on a spinning disc confocal microscope . Most of the tubules underwent scission into small vesicles ( See movie EV6 ) . Scale bar 5 µm . Areas in boxes 1 and 2 of c are shown at higher magnification in b and c . Scale bars : 2 . 5 µm .

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a, Time lapse analysis of Atg18 added to liposomes. 10 µL of 10 µM purified recombinant Atg18wt was added to 100 µL buffer containing GUVs with 5% PI3P and 1% PI(3,5)P2. The sample was imaged on a spinning disc confocal microscope. Most of the tubules underwent scission into small vesicles (See movie EV6). Scale bar 5 µm. Areas in boxes 1 and 2 of c are shown at higher magnification in b and c. Scale bars: 2.5 µm.


d , Quantification of fission activity . For each movie , the whole frame was divided into squares of 10 per 10 µm and the number of small vesicles per 100 µm2 counted . The mean of three independent experiments is shown with SEM .

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d, Quantification of fission activity. For each movie, the whole frame was divided into squares of 10 per 10 µm and the number of small vesicles per 100 µm2 counted. The mean of three independent experiments is shown with SEM.


a , 1µM Atg18 was added to wells containing sedimented GUVs with 10 % PI3P or PI ( 3 , 5 ) P2 while imaging a single plane by spinning disc microscopy . Membrane tension exerted by Atg18 binding suffices to induce liposome collapse , which allows to better judge the mobility of the tubular or clustered membranes that remain . Images are extracted from movies EV2 to EV5 . Scale bar 5 µm .

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a, 1µM Atg18 was added to wells containing sedimented GUVs with 10% PI3P or PI(3,5)P2 while imaging a single plane by spinning disc microscopy. Membrane tension exerted by Atg18 binding suffices to induce liposome collapse, which allows to better judge the mobility of the tubular or clustered membranes that remain. Images are extracted from movies EV2 to EV5. Scale bar 5 µm.


b , Atg18 was covalently labeled with the green fluorescent dye Alexa488 TFP ( ThermoFisher ) and used in experiments as in a , with rhodamine - phosphatidylethanolamine - labeled ( Rh - PE ) GUVs containing the indicated phosphoinositides . Scale bar 5

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b, Atg18 was covalently labeled with the green fluorescent dye Alexa488 TFP (ThermoFisher) and used in experiments as in a, with rhodamine-phosphatidylethanolamine-labeled (Rh-PE) GUVs containing the indicated phosphoinositides. Scale bar 5 µm


c , Effect of 1µM of purified recombinant Atg18 variants on GUVs containing 10mol % of the indicated phosphoinositides . Vesicles are shown after 1 to 3 min of incubation .

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c, Effect of 1µM of purified recombinant Atg18 variants on GUVs containing 10mol% of the indicated phosphoinositides. Vesicles are shown after 1 to 3 min of incubation.


a , Cross - linking of Atg18 . Purified recombinant Atg18wt or Atg18FGGG ( 1 . 5 µM ) was incubated with liposomes containing 5 % PI3P , PI ( 3 , 5 ) P2 , PI ( 4 , 5 ) P2 or 15 % PS as a control for negative charge . After addition of a cleavable cross - linker or buffer only , liposomes were pelleted , dissolved in sample buffer , incubated in the presence or absence of 100 mM dithiothreitol ( DTT ) for 5 min at room temperature and analysed by SDS - PAGE and Coomassie staining . Cross - link products migrate at the upper limit of the separating gel .

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

a, Cross-linking of Atg18. Purified recombinant Atg18wt or Atg18FGGG (1.5 µM) was incubated with liposomes containing 5% PI3P, PI(3,5)P2, PI(4,5)P2 or 15% PS as a control for negative charge. After addition of a cleavable cross-linker or buffer only, liposomes were pelleted, dissolved in sample buffer, incubated in the presence or absence of 100 mM dithiothreitol (DTT) for 5 min at room temperature and analysed by SDS-PAGE and Coomassie staining. Cross-link products migrate at the upper limit of the separating gel.


b , Cross - linking experiment with recombinant Atg18wt as in a , performed in the presence of increasing concentrations of liposomes containing 5 % PI ( 3 , 5 ) P2 .

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

b, Cross-linking experiment with recombinant Atg18wt as in a, performed in the presence of increasing concentrations of liposomes containing 5% PI(3,5)P2.


B : brain MRI images of II - 1 ( FLAIR ) . A : sagittal interemispheric sequence showing profound cerebellar atrophy ( arrow ) ; B : transverse sequence showing involvement of the dentate nuclei of cerebellum ; C : coronal sequence revealing cortical atrophy , a wide third ventricle , and high signal changes in the thalami , particularly on the left ( arrow ) , D : the thalamic lesion is indicated by an arrow .

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B: brain MRI images of II-1 (FLAIR). A: sagittal interemispheric sequence showing profound cerebellar atrophy (arrow); B: transverse sequence showing involvement of the dentate nuclei of cerebellum; C: coronal sequence revealing cortical atrophy, a wide third ventricle, and high signal changes in the thalami, particularly on the left (arrow), D: the thalamic lesion is indicated by an arrow.


C : histochemical staining of skeletal muscle from II - 2 . Combined COX / SDH staining shows scattered COX negative ( blue ) fibres ( arrows ) . The black bar corresponds to 100 µm .

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C: histochemical staining of skeletal muscle from II-2. Combined COX/SDH staining shows scattered COX negative (blue) fibres (arrows). The black bar corresponds to 100 µm.


D : respiratory chain complex activities in skeletal muscle . Specific activities ( nmol min - 1 mg - 1 ) of complex I , II and IV are reduced in II - 2 ( red ) compared to the controls mean ( blue ) ; CS activity is also low ( < 50 % of controls ) suggesting a reduction of mitochondrial mass ( see main text for further details ) . Each activity was measured in triplicate .

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D: respiratory chain complex activities in skeletal muscle. Specific activities (nmol min-1 mg-1) of complex I, II and IV are reduced in II-2 (red) compared to the controls mean (blue); CS activity is also low (<50% of controls) suggesting a reduction of mitochondrial mass (see main text for further details). Each activity was measured in triplicate.


E : Western blot analysis of PITRM1 in primary fibroblasts ( left ) and skeletal muscle ( right ) of controls ( CTR ) and subject II - 2 . Densitometric quantification using the Genetools software is shown below the blots . In blue is PITRM1WT and in red PITRM1R183Q .

E: Western blot analysis of PITRM1 in primary fibroblasts (left) and skeletal muscle (right) of controls (CTR) and subject II-2. Densitometric quantification using the Genetools software is shown below the blots. In blue is PITRM1WT and in red PITRM1R183Q.


F : proteolytic activity on two fluorescent oligopeptides ( AnaSpec ) , reported at the bottom of each histogram , by 6 - His tagged PITRM1WT and PITRM1R183Q proteins expressed in E . coli and affinity - purified by Ni - agarose chromatography . Values are expressed as arbitrary units per sec per µg of protein ( a . u . s - 1µg - 1 ) . Experiments were performed in duplicate .

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F: proteolytic activity on two fluorescent oligopeptides (AnaSpec), reported at the bottom of each histogram, by 6-His tagged PITRM1WT and PITRM1R183Q proteins expressed in E. coli and affinity-purified by Ni-agarose chromatography. Values are expressed as arbitrary units per sec per µg of protein (a.u. s-1µg-1). Experiments were performed in duplicate.


G : degradation rate of Aβ1 - 42 by purified 6 - His tagged PITRM1WT and PITRM1R183Q proteins . Quantification of these experiments is displayed below the blots . Experiments were performed in duplicate .

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G: degradation rate of Aβ1-42 by purified 6-His tagged PITRM1WT and PITRM1R183Q proteins. Quantification of these experiments is displayed below the blots. Experiments were performed in duplicate.


A : cell growth in glucose ( GLU ) and galactose ( GAL ) . C29V , C47V , TpLV : immortalized fibroblast cell lines from control individuals ; PITRM1V : immortalized fibroblasts from subject II - 2 , carrying the PITRM1R183Q mutation . pLKO . 1 : empty vector ; sh38 : shRNA38 ; sh41 : shRNA41 . Each cell line was measured six times . Error bars indicate ± SD . Statistic analysis was by two - way ANOVA post - hoc Bonferroni test * * * p < 0 . 001 .

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A: cell growth in glucose (GLU) and galactose (GAL). C29V, C47V, TpLV: immortalized fibroblast cell lines from control individuals; PITRM1V: immortalized fibroblasts from subject II-2, carrying the PITRM1R183Q mutation. pLKO.1: empty vector; sh38: shRNA38; sh41: shRNA41. Each cell line was measured six times. Error bars indicate ±SD. Statistic analysis was by two-way ANOVA post-hoc Bonferroni test ***p<0.001.


B : Western - blot analysis of PITRM1 in C29V cells transduced with the empty vector plKO . 1 and the two shRNA , sh38 and sh41 . SDHB and b - Actin are used as loading controls .

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B: Western-blot analysis of PITRM1 in C29V cells transduced with the empty vector plKO.1 and the two shRNA, sh38 and sh41. SDHB and b-Actin are used as loading controls.


C : co - localization of PITRM1 ( red ) with TOM20 ( T20 , green ) in human fibroblast cells from a control ( CTR ) and subject II - 2 . Note that the intensity of PITRM1 immunofluorescence is much lower in II - 2 cells than in CTR cells ( see main text for further details ) . Nuclei are stained in blue by DAPI . A white bar corresponds to 10 µM .

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C: co-localization of PITRM1 (red) with TOM20 (T20, green) in human fibroblast cells from a control (CTR) and subject II-2. Note that the intensity of PITRM1 immunofluorescence is much lower in II-2 cells than in CTR cells (see main text for further details). Nuclei are stained in blue by DAPI. A white bar corresponds to 10 µM.


D : mitochondrial membrane potential ( ΔP ) . In healthy cells with high mitochondrial ΔP , JC - 1 forms complexes known as J - aggregates that show an intense red fluorescence . On the contrary in unhealthy cells with low ΔP , JC - 1 remains in the monomeric form , showing only green fluorescence . Values are referred to as % of those of the control means , taken as 100 % . The results are the mean of 3 independent experiments . Error bars indicate ± SD . Statistic analysis was by two - way ANOVA * * p < 0 . 01 ; * * * * p < 0 . 0001 .

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D: mitochondrial membrane potential (ΔP). In healthy cells with high mitochondrial ΔP, JC-1 forms complexes known as J-aggregates that show an intense red fluorescence. On the contrary in unhealthy cells with low ΔP, JC-1 remains in the monomeric form, showing only green fluorescence. Values are referred to as % of those of the control means, taken as 100%. The results are the mean of 3 independent experiments. Error bars indicate ±SD. Statistic analysis was by two-way ANOVA **p <0.01; ****p<0.0001.


A : oxidative growth . W303 - 1Bcym1Δ strains harbouring the wild - type CYM1 allele ( CYM1wt ) , the cym1R163Q mutant allele or the empty vector were serially diluted from 107 to 104 cells / ml . Five microliters of each dilution were spotted on SC agar plates without uracil , supplemented with 2 % glucose , 2 % glycerol or 2 % ethanol . Plates were incubated at 37 ° C for 3 - 7 days .

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A: oxidative growth. W303-1Bcym1Δ strains harbouring the wild-type CYM1 allele (CYM1wt), the cym1R163Q mutant allele or the empty vector were serially diluted from 107 to 104 cells/ml. Five microliters of each dilution were spotted on SC agar plates without uracil, supplemented with 2% glucose, 2% glycerol or 2% ethanol. Plates were incubated at 37°C for 3-7 days.


B : oxygen consumption rate ( OCR ) . Cells grown at 37 ° C SC medium without uracil were supplemented with 0 . 5 % glucose . Values were normalized to the OCR of the CYM1wt strain ( 49 nmol O2 min − 1 mg − 1 ) and represented as the mean of at least three values ± SD . Statistical analysis was by unpaired , two - tail Student ' s t test : * * * p < 0 . 001 .

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B: oxygen consumption rate (OCR). Cells grown at 37°C SC medium without uracil were supplemented with 0.5% glucose. Values were normalized to the OCR of the CYM1wt strain (49 nmol O2 min−1 mg−1) and represented as the mean of at least three values ± SD. Statistical analysis was by unpaired, two-tail Student's t test: ***p<0.001.


C : reduced versus oxidized cytochrome spectra . Peaks at 550 , 560 and 602 nm correspond to cytochromes c , b and aa3 , respectively . The height of each peak relative to the baseline is an index of cytochrome content .

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C: reduced versus oxidized cytochrome spectra. Peaks at 550, 560 and 602 nm correspond to cytochromes c, b and aa3, respectively. The height of each peak relative to the baseline is an index of cytochrome content.


D : respiratory chain complex activities . Biochemical activities of Succinate Quinone DCPIP Reductase , SQDR ( CII ) , NADH - cytochrome c oxidoreductase activity NCCR ( CIII ) , and cytochrome c oxidase ( CIV ) were measured on a mitochondrial - enriched fraction from cells grown at 37 ° C as in panel B . Values were normalized to that of CYM1wt strain and represented as the mean of three independent experiments ± SD . Statistical analysis was by unpaired , two - tail Student ' s t test : * * p < 0 . 01 ; * * * p < 0 . 001 .

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D: respiratory chain complex activities. Biochemical activities of Succinate Quinone DCPIP Reductase, SQDR (CII), NADH-cytochrome c oxidoreductase activity NCCR (CIII), and cytochrome c oxidase (CIV) were measured on a mitochondrial-enriched fraction from cells grown at 37°C as in panel B. Values were normalized to that of CYM1wt strain and represented as the mean of three independent experiments ± SD. Statistical analysis was by unpaired, two-tail Student's t test: **p<0.01; ***p<0.001.


E : Western - blot using an anti - HA monoclonal antibody visualizing the CYM1wt and cym1R163Q recombinant proteins both fused in frame with the HA epitope on the C - terminus . Western - blot analysis on total protein extract . Total protein extracts were obtained by strains expressing HA - tagged CYM1wt and cym1R163Q . PGK was used as a loading control and signals were normalized to the wt . The quantification was performed on 5 independent blots . F : prolonged exposure of a Western - blot containing the CYM1wt and cym1R163Q recombinant proteins reveals the presence of a band corresponding to a degradation product in the cym1R163Q lanes ( arrow ) .

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E: Western-blot using an anti-HA monoclonal antibody visualizing the CYM1wt and cym1R163Q recombinant proteins both fused in frame with the HA epitope on the C-terminus. Western-blot analysis on total protein extract. Total protein extracts were obtained by strains expressing HA-tagged CYM1wt and cym1R163Q. PGK was used as a loading control and signals were normalized to the wt. The quantification was performed on 5 independent blots.F: prolonged exposure of a Western-blot containing the CYM1wt and cym1R163Q recombinant proteins reveals the presence of a band corresponding to a degradation product in the cym1R163Q lanes (arrow).


G : Western blot of Aβ1 - 42myc monomer and dimer incubated with purified mitochondrial extracts from cells grown at 37 ° C in SC medium supplemented with 0 . 15 % glucose and 2 % galactose . VDAC is used as a loading control . Each experiment was performed in triplicate .

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G: Western blot of Aβ1-42myc monomer and dimer incubated with purified mitochondrial extracts from cells grown at 37°C in SC medium supplemented with 0.15% glucose and 2% galactose. VDAC is used as a loading control. Each experiment was performed in triplicate.


A : Western - blot analysis of Pitrm1 protein in brain , muscle and liver of two 4 - mo male Pitrm1 + / - mice and two Pitrm1 + / + littermates . Densitometric analysis is reported in the histograms below the blots . Pitrm1 + / + is in blue , Pitrm1 + / - in red . Error bars indicate ±

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A: Western-blot analysis of Pitrm1 protein in brain, muscle and liver of two 4-mo male Pitrm1+/- mice and two Pitrm1+/+ littermates. Densitometric analysis is reported in the histograms below the blots. Pitrm1+/+ is in blue, Pitrm1+/- in red. Error bars indicate ±SD


B : representative hindlimb - clasping phenomenon is shown in a 4 mo Pitrm1 + / - male mouse , consisting in strong adduction of the hindlimbs when the animal is suspended by the tail ; a littermate Pitrm1 + / + control displays the normal reflex , consisting in wide abduction of the limbs . All examined Pitrm1 + / - animals displayed this abnormal reflex from 2 months of age .

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B: representative hindlimb-clasping phenomenon is shown in a 4 mo Pitrm1+/- male mouse, consisting in strong adduction of the hindlimbs when the animal is suspended by the tail; a littermate Pitrm1+/+ control displays the normal reflex, consisting in wide abduction of the limbs. All examined Pitrm1+/- animals displayed this abnormal reflex from 2 months of age.


C : rotarod test . Blue and red lines refer to Pitrm1 + / + ( n = 9 ) and Pitrm1 + / - ( n = 7 ) 4 mo animals , respectively . Error bars indicate ± SD . Statistical analysis was by unpaired , two - tail Student ' s t test . * p < 0 . 05 ; * * p < 0 . 01 .

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C: rotarod test. Blue and red lines refer to Pitrm1+/+ (n=9) and Pitrm1+/- (n=7) 4 mo animals, respectively. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student's t test. *p<0.05; ** p<0.01.


D : negative geotaxis test . The experiment was carried out on the same group of animals as in panel B . Error bars indicate ± SD . Statistical analysis was by unpaired , two - tail Student ' s t test . * p < 0 . 05 .

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D: negative geotaxis test. The experiment was carried out on the same group of animals as in panel B. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student's t test. * p<0.05.


E : cylinder test . The experiment was carried out on the same group of animals as in panel b . Error bars indicate ± SD . Statistical analysis was by unpaired , two - tail Student ' s t test . * * * p < 0 . 001 .

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E: cylinder test. The experiment was carried out on the same group of animals as in panel b. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student's t test. *** p<0.001.


F : pole test . The experiment was carried out on the same group of animals as in panel B . Error bars indicate ± SD . Statistical analysis was by unpaired , two - tail Student ' s t test . * * * p < 0 . 001 .

F: pole test. The experiment was carried out on the same group of animals as in panel B. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student's t test. ***p<0.001.


A : Western - blot analysis of amyloid - precursor protein ( APP ) in 6 mo male mice . GAPDH was used as a loading control . Densitometric analysis on a total of 5 independent samples is shown in the histogram on the right .

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A: Western-blot analysis of amyloid-precursor protein (APP) in 6 mo male mice. GAPDH was used as a loading control. Densitometric analysis on a total of 5 independent samples is shown in the histogram on the right.


B : morphological analysis of an AD subject and of Pitrm1 + / - and Pitrm1 + / + mouse brains . TF : Thioflavin T ( thalamus ) ; CR : Congo - red ( brain cortex ) ; PL : polarised light ( same sections as those stained with CR ) ; Aβ1 - 42 immunostaining ( pons ) ; Ub : ubiquitin immunohistochemistry shown as brownish staining ( brain cortex ) . The white bar indicates 20 µm .

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B: morphological analysis of an AD subject and of Pitrm1+/- and Pitrm1+/+ mouse brains. TF: Thioflavin T (thalamus); CR: Congo-red (brain cortex); PL: polarised light (same sections as those stained with CR); Aβ1-42 immunostaining (pons); Ub: ubiquitin immunohistochemistry shown as brownish staining (brain cortex). The white bar indicates 20 µm.


A : upper panel : import of Aβ1 - 42 into mitochondria ; lower panel : relative quantification from two independent experiments . Values of Aβ1 - 42 signal were normalized to HSP60 signal and the resulting value at 5 min without trypsin was arbitrarily chosen as 1 . Error bars indicate ± SD . Statistical analysis was by unpaired , two - tail Student ' s t test . * p < 0 . 05 .

A: upper panel: import of Aβ1-42 into mitochondria; lower panel: relative quantification from two independent experiments. Values of Aβ1-42 signal were normalized to HSP60 signal and the resulting value at 5 min without trypsin was arbitrarily chosen as 1. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student's t test. *p<0.05.


B : upper panel : pulse and chase experiment to assess clearance of Aβ1 - 42 ; lower panel : quantification from three independent experiments . Values of Aβ1 - 42 signal were normalized to HSP60 signal and the pulse value was arbitrarily chosen as 1 . Error bars indicate ± SD . Statistical analysis was by unpaired , two - tail Student ' s t test . * p < 0 . 05 , * * p < 0 . 01 .

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B: upper panel: pulse and chase experiment to assess clearance of Aβ1-42; lower panel: quantification from three independent experiments. Values of Aβ1-42 signal were normalized to HSP60 signal and the pulse value was arbitrarily chosen as 1. Error bars indicate ±SD. Statistical analysis was by unpaired, two-tail Student's t test. *p<0.05, **p<0.01.


A : MEFs from Pitrm1 + / + , Pitrm1 + / - , Mfn1 - / - , Mfn2 - / - , as well as h - PITRM1 R183Q and h - PITRM1 WT were exposed to Aβ1 - 40 peptide for 18h . Note that Aβ1 - 40 signal was still evident in Pitrm1 + / - and h - PITRM1 R183Q cells . B : quantification of three independent experiments similar to those shown in panel A . Statistical analysis was by unpaired , two - tail Student ' s t test . * * p < 0 . 01 , * * * p < 0 . 001 .

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A: MEFs from Pitrm1+/+, Pitrm1+/-, Mfn1-/-, Mfn2-/-, as well as h-PITRM1 R183Q and h- PITRM1 WT were exposed to Aβ1-40 peptide for 18h. Note that Aβ1-40 signal was still evident in Pitrm1+/- and h-PITRM1 R183Q cells.B: quantification of three independent experiments similar to those shown in panel A. Statistical analysis was by unpaired, two-tail Student's t test. **p<0.01, ***p<0.001.


( A ) A titration of anti - MHV polyclonal serum was added either directly to media ( extracellular ) or electroporated into L929 cells ( intracellular ) , then cells were infected with MHV - A59 . The kinetics of cell growth was monitored by measuring total cell area . Data information : Data was analysed using a Students t - test . Error bars depict the mean + / - SEM . All data represents at least two independent replicates .

(A) A titration of anti-MHV polyclonal serum was added either directly to media (extracellular) or electroporated into L929 cells (intracellular), then cells were infected with MHV-A59. The kinetics of cell growth was monitored by measuring total cell area. Data information: Data was analysed using a Students t-test. Error bars depict the mean +/- SEM. All data represents at least two independent replicates.


( D ) Kinetics of cell growth following MHV - A59 infection in the presence of a titration of electroporated anti - N antibody . Data information : Data was analysed using a Students t - test . Error bars depict the mean + / - SEM . All data represents at least two independent replicates .

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(D) Kinetics of cell growth following MHV-A59 infection in the presence of a titration of electroporated anti-N antibody. Data information: Data was analysed using a Students t-test. Error bars depict the mean +/- SEM. All data represents at least two independent replicates.


( G & H ) WT or TRIM21 KO L929 cells were infected with MHV - A59 in the presence of electroporated polyclonal antiserum ( G ) or anti - N antibody ( H ) and quantified by cell area 48h later . Co - electroporation of Protein A / G with anti - N antibody into WT cells mimics the TRIM21 KO phenotype . Data information : Data was analysed using a Students t - test . Error bars depict the mean + / - SEM . All data represents at least two independent replicates .

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(G&H) WT or TRIM21 KO L929 cells were infected with MHV-A59 in the presence of electroporated polyclonal antiserum (G) or anti-N antibody (H) and quantified by cell area 48h later. Co-electroporation of Protein A/G with anti-N antibody into WT cells mimics the TRIM21 KO phenotype. Data information: Data was analysed using a Students t-test. Error bars depict the mean +/- SEM. All data represents at least two independent replicates.


( I ) L929 Cells were infected with MHV - A59 for 4h , then cyclohexamide ( CHX ) was added to block further viral protein synthesis . After 5h , cells were electroporated with anti - N or anti - GFP antibodies and left for a further 3h before being western blotted for cellular N protein levels ( * denotes a non - specific band ) . Data information : Data was analysed using a Students t - test . Error bars depict the mean + / - SEM . All data represents at least two independent replicates .

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(I) L929 Cells were infected with MHV-A59 for 4h, then cyclohexamide (CHX) was added to block further viral protein synthesis. After 5h, cells were electroporated with anti-N or anti-GFP antibodies and left for a further 3h before being western blotted for cellular N protein levels (*denotes a non-specific band). Data information: Data was analysed using a Students t-test. Error bars depict the mean +/- SEM. All data represents at least two independent replicates.


( A & B ) Vero cells OE ACE2 and TMPRSS2 were infected with SARS - CoV - 2 in the presence of IgG or anti - N antibodies added directly into media or electroporated into cells . Viral replication was then determined by RT - qPCR ( A ) or plaque assay ( B ) . Electroporation of anti - N antibodies significantly inhibits SARS - CoV - 2 replication ( * * * = p < 0 . 0002 ) . Data information : All data represents at least three independent replicates . Error bars depict the mean + / - SEM . Statistical comparisons were performed using a one - way ANOVA .

(A&B) Vero cells OE ACE2 and TMPRSS2 were infected with SARS-CoV-2 in the presence of IgG or anti-N antibodies added directly into media or electroporated into cells. Viral replication was then determined by RT-qPCR (A) or plaque assay (B). Electroporation of anti-N antibodies significantly inhibits SARS-CoV-2 replication (*** = p < 0.0002). Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM. Statistical comparisons were performed using a one-way ANOVA.


( C & D ) As with A & B , except with a titration of electroporated anti - N antibodies . Data information : All data represents at least three independent replicates . Error bars depict the mean + / - SEM . Statistical comparisons were performed using a one - way ANOVA .

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(C&D) As with A&B, except with a titration of electroporated anti-N antibodies. Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM. Statistical comparisons were performed using a one-way ANOVA.


( F & G ) 293T ACE2 OE / TRIM21 KO cells reconstituted with EV or TRIM21 , electroporated with IgG or anti - N antibodies , and infected with SARS - CoV - 2 . Viral replication was then determined by RT - qPCR ( F ) or plaque assay ( G ) . Electroporation of anti - N antibodies significantly inhibits SARS - CoV - 2 replication only in TRIM21 - reconstituted cells ( * = p < 0 . 05 ) . Data information : All data represents at least three independent replicates . Error bars depict the mean + / - SEM . Statistical comparisons were performed using two - way ANOVA .

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(F&G) 293T ACE2 OE/TRIM21 KO cells reconstituted with EV or TRIM21, electroporated with IgG or anti-N antibodies, and infected with SARS-CoV-2. Viral replication was then determined by RT-qPCR (F) or plaque assay (G). Electroporation of anti-N antibodies significantly inhibits SARS-CoV-2 replication only in TRIM21-reconstituted cells (* = p < 0.05). Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM. Statistical comparisons were performed using two-way ANOVA.


( A ) Capillary - based protein detection ( Jess ) of antibodies against SARS - CoV - 2 antigens in convalescent sera .

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(A) Capillary-based protein detection (Jess) of antibodies against SARS-CoV-2 antigens in convalescent sera.


( B ) Quantification of antigen - specific antibodies in convalescent sera . Data information : All data represents at least three independent replicates . Error bars depict the mean + / - SEM .

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(B) Quantification of antigen-specific antibodies in convalescent sera. Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM.


( C ) 293T cells OE ACE2 infected with SARS - CoV - 2 in the presence of electroporated convalescent sera . There are statistically significant differences in the ability of serum from different individuals to inhibit viral replication intracellularly ( * * = p < 0 . 005 , * = p < 0 . 05 ) . ( D ) 293T ACE2 OE / TRIM21 KOs reconstituted with either empty vector ( EV ) or TRIM21 - expressing vector ( TRIM21 ) infected with SARS - CoV - 2 in the presence of electroporated convalescent sera . Intracellular neutralization of viral replication is only observed in cells reconstituted with TRIM21 ( * * = p < 0 . 005 ) . Data information : All data represents at least three independent replicates . Error bars depict the mean + / - SEM . Statistical comparisons were performed using a one - way ( C ) or two - way ( D ) ANOVA .

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(C) 293T cells OE ACE2 infected with SARS-CoV-2 in the presence of electroporated convalescent sera. There are statistically significant differences in the ability of serum from different individuals to inhibit viral replication intracellularly (** = p< 0.005, * = p< 0.05). (D) 293T ACE2 OE/TRIM21 KOs reconstituted with either empty vector (EV) or TRIM21-expressing vector (TRIM21) infected with SARS-CoV-2 in the presence of electroporated convalescent sera. Intracellular neutralization of viral replication is only observed in cells reconstituted with TRIM21 (** = p< 0.005). Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM. Statistical comparisons were performed using a one-way (C) or two-way (D) ANOVA.


( C ) Correlation between N - antibody titre , as calculated by Jess , and intracellular neutralization ( fold decrease in SARS - CoV - 2 replication ) , as measured by EDNA , in convalescent sera from multiple individuals . ( D ) Intracellular neutralization of SARS - CoV - 2 by titrated serum from two strongly N - antibody positive individuals . ( Data information : All data represents at least three independent replicates . Error bars depict the mean + / - SEM .

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(C) Correlation between N-antibody titre, as calculated by Jess, and intracellular neutralization (fold decrease in SARS-CoV-2 replication), as measured by EDNA, in convalescent sera from multiple individuals. (D) Intracellular neutralization of SARS-CoV-2 by titrated serum from two strongly N-antibody positive individuals. ( Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM.


( E ) ELISpot measuring IFNγ production in T cells from SARS - CoV - 2 convalescents upon stimulation with a peptide library covering N - protein . Data information : All data represents at least three independent replicates . Error bars depict the mean + / - SEM .

(E) ELISpot measuring IFNγ production in T cells from SARS-CoV-2 convalescents upon stimulation with a peptide library covering N-protein. Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM.


A Cytokine release by PBMCs challenged with heat inactivated S . aureus ( hiSa ) , and / or RNase A ( RA ) treated solution , or Sa19 ( TLR13 activating ORN ) upon TLR2 blockade ( T2 . 5 ; n . p . , not performed ; * * , p ≤ 0 . 01 ; n = 3 ) .

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A Cytokine release by PBMCs challenged with heat inactivated S. aureus (hiSa), and/or RNase A (RA) treated solution, or Sa19 (TLR13 activating ORN) upon TLR2 blockade (T2.5; n.p., not performed; **, p ≤ 0.01; n = 3).


C Cytokine release of PBMCs transfected with ORNs including TLR7 / 8 ligand RNA40 ( n = 3 ) .

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C Cytokine release of PBMCs transfected with ORNs including TLR7/8 ligand RNA40 (n = 3).


D , E Activity of PBMCs upon bacterial RNA challenge , pretreatment with chloroquine in ( D ) only ( S , Svedberg ; r , ribosomal ; tot , total ; n = 3 ) .

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D, E Activity of PBMCs upon bacterial RNA challenge, pretreatment with chloroquine in (D) only (S, Svedberg; r, ribosomal; tot, total; n = 3).


F Activity of PBMCs upon mitochondrial ( mt ) RNA challenge ( Hs , human ; triangle , increasing doses ; LFA , Lipofectamine 2000 ; n = 3 ) .

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F Activity of PBMCs upon mitochondrial (mt) RNA challenge (Hs, human; triangle, increasing doses; LFA, Lipofectamine 2000; n = 3).


G Activity of PBMCs challenged with ORNs and total S . aureus ( Sa ) RNA ( Myd88d / d , mutant MyD88 expression not impairing LPS driven IL - 8 production ; n = 2 ) .

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G Activity of PBMCs challenged with ORNs and total S. aureus (Sa) RNA (Myd88d/d, mutant MyD88 expression not impairing LPS driven IL-8 production; n = 2).


H Responsiveness of undifferentiated ( undiff ) and 3 - or 8 days PMA differentiated ( ddi ) THP1 cells to Sa19 challenge ( n = 3 ) .

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H Responsiveness of undifferentiated (undiff) and 3- or 8 days PMA differentiated (ddi) THP1 cells to Sa19 challenge (n = 3).


I Activity of parental and Unc93b1 - / - - 3ddiTHP - 1 cells challenged with ORNs ( n = 3 ) .

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I Activity of parental and Unc93b1-/--3ddiTHP-1 cells challenged with ORNs (n = 3).


A Cytokine release of 3 days differentiated ( ddi ) THP1 cells toward stimulation with TLR ligands and bacterial RNAs ( S , Svedberg ; r , ribosomal ; tot , total ; n = 3 ) .

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A Cytokine release of 3 days differentiated (ddi) THP1 cells toward stimulation with TLR ligands and bacterial RNAs (S, Svedberg; r, ribosomal; tot, total; n = 3).


B , C Responsiveness of Unc93b1 - / - - 3ddiTHP1 cells treated with T2 . 5 and challenged with heat inactivated ( hi ) bacteria ( triangle , decreasing doses ; n = 3 ) .

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B, C Responsiveness of Unc93b1-/--3ddiTHP1 cells treated with T2.5 and challenged with heat inactivated (hi) bacteria (triangle, decreasing doses; n=3).


D Ratios of constitutive mRNA amounts in 3 - versus 8ddi ( grey bar ) and of each of both versus undifferentiated THP1 cells ( black or white bar , respectively ) according to a transcriptome profiling result ( n = 1 ) .

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D Ratios of constitutive mRNA amounts in 3- versus 8ddi (grey bar) and of each of both versus undifferentiated THP1 cells (black or white bar, respectively) according to a transcriptome profiling result (n = 1).


E , F NF - κB driven relative luciferase activity ( Rel . luc . act . ) of hTLR8 overexpressing HEK293 and murine RAW264 . 7 cells , respectively , upon challenges ( vector , empty plasmid ; n = 3 ) .

E, F NF-κB driven relative luciferase activity (Rel. luc. act.) of hTLR8 overexpressing HEK293 and murine RAW264.7 cells, respectively, upon challenges (vector, empty plasmid; n = 3).


G Transfection of TLR8 siRNA impairs 3ddiTHP1 cell responsiveness ( n = 3 ) .

G Transfection of TLR8 siRNA impairs 3ddiTHP1 cell responsiveness (n = 3).


A , B Cytokine release of 3 days differentiated ( ddi ) THP1 cells challenged with ( A ) ORNs or ( B ) bacterial RNA fractions ( tot , total ; S , Svedberg ; r , ribosomal ) .

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A, B Cytokine release of 3 days differentiated (ddi) THP1 cells challenged with (A) ORNs or (B) bacterial RNA fractions (tot, total; S, Svedberg; r, ribosomal).


C Responsiveness of Tlr8 - / - - 3ddiTHP1 cells challenged with heat inactivated ( hi ) bacteria ( triangle , decreasing doses ; * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ) upon TLR2 blockade ( T2 . 5 ) .

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C Responsiveness of Tlr8-/--3ddiTHP1 cells challenged with heat inactivated (hi) bacteria (triangle, decreasing doses; *, p ≤ 0.05; **, p ≤ 0.01) upon TLR2 blockade (T2.5).


D Responsiveness of 3ddiTHP1 cells upon challenge with human ( Hs ) mitochondrial ( mt ) RNA ( triangle , increasing doses ) .

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D Responsiveness of 3ddiTHP1 cells upon challenge with human (Hs) mitochondrial (mt) RNA (triangle, increasing doses).


E - H NF - B driven relative luciferase activity ( Rel . luc . act . ) of hTLR8 + HEK293 cells or cytokine release by PBMCs all transfected with RNAs indicated and additional uridine ( U ) if indicated ( n . p . , not performed ; vector , empty plasmid ) .

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E-H NF-B driven relative luciferase activity (Rel. luc. act.) of hTLR8+ HEK293 cells or cytokine release by PBMCs all transfected with RNAs indicated and additional uridine (U) if indicated (n.p., not performed; vector, empty plasmid).


A Cytokine release of 3 days differentiated THP - 1 cells of indicated genotypes upon TLR2 blockade ( T2 . 5 ) and challenge with TLR ligands , heat inactivated ( hi ) or viable bacteria .

A Cytokine release of 3 days differentiated THP-1 cells of indicated genotypes upon TLR2 blockade (T2.5) and challenge with TLR ligands, heat inactivated (hi) or viable bacteria.


B PBMC activity upon endosome function inhibition ( chloroquine ) and TLR2 blockade ( T2 . 5 ) followed by TLR ligand challenge or bacterial infection .

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B PBMC activity upon endosome function inhibition (chloroquine) and TLR2 blockade (T2.5) followed by TLR ligand challenge or bacterial infection.