Figure 3 ( A ) FACS profile of cells transfected with nondegradable KEN - box mutant sororin - FLAG ( sorKBM - FLAG ) after synchronization in G1 - phase and propidium iodide staining . ( B ) Immunofluorescence microscopy experiment to determine transfection efficiency . ( C ) Western blot of fractionated lysates to detect sororinKBM - FLAG . ( D ) Quantification of SMC3 - LAP residence time on chromatin after transfecting cells with sorKBM - FLAG in G1 - phase . Error bars denote s . e . m . ; n > 15 cells per condition . ( E - F ) Analysis of cell cycle distribution and transfection efficiency using cells synchronized in G2 - phase . ( G ) Quantification of stably chromatin - bound SMC3 - LAP from cells expressing nondegradable sororin in G2 - phase . Cells were treated with siRNA as indicated . ( H ) Western blot showing fractionated and nuclease digested chromatin from cells synchronized in G1 and G2 - phase after immunoprecipitation with FLAG antibodies .

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Figure 3(A) FACS profile of cells transfected with nondegradable KEN-box mutant sororin-FLAG (sorKBM-FLAG) after synchronization in G1-phase and propidium iodide staining.(B) Immunofluorescence microscopy experiment to determine transfection efficiency.(C) Western blot of fractionated lysates to detect sororinKBM-FLAG.(D) Quantification of SMC3-LAP residence time on chromatin after transfecting cells with sorKBM-FLAG in G1-phase. Error bars denote s.e.m.; n > 15 cells per condition.(E-F) Analysis of cell cycle distribution and transfection efficiency using cells synchronized in G2-phase.(G) Quantification of stably chromatin-bound SMC3-LAP from cells expressing nondegradable sororin in G2-phase. Cells were treated with siRNA as indicated.(H) Western blot showing fractionated and nuclease digested chromatin from cells synchronized in G1 and G2-phase after immunoprecipitation with FLAG antibodies.


Figure 4 ( A ) Examples of sororin ( dotted arrows ) and SMC3ChIP - seq and BrdU DIP - seq data from early S - phase as compared to G2 - phase using the Integrated Genome Browser ( IGB ; Nicol et al , 2009 ) . ( B ) Alignment of sororin , BrdU and BrdU - negative control sequencing bins in early S - phase to humanchromosome 18 ( GRCh37 / hg19 ) . Bars correspond to called peaks , curves to read enrichments after smoothing ( k = 25 kbp ) . ( C ) Quantification of the co - occupancy of BrdU incorporation and sororin localization in early S - phase and sororin in G2 - phase depicted as p - value distributions calculated with IntervalStats ( Chikina Troyanskaya , 2012 ) and compared to randomized controls . Sororin peaks were identified as overlapping peaks of 4 samples in early S - phase and 2 samples in G2 - phase , using only the common peaks . ( D ) Quantification of the co - occupancy of SMC3 in early S - phase and in G2 - phase with BrdU incorporation in early S - phase .

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Figure 4(A) Examples of sororin (dotted arrows) and SMC3ChIP-seq and BrdU DIP-seq data from early S-phase as compared to G2-phase using the Integrated Genome Browser (IGB; Nicol et al, 2009).(B) Alignment of sororin, BrdU and BrdU-negative control sequencing bins in early S-phase to humanchromosome 18 (GRCh37/hg19). Bars correspond to called peaks, curves to read enrichments after smoothing (k=25 kbp).(C) Quantification of the co-occupancy of BrdU incorporation and sororin localization in early S-phase and sororin in G2-phase depicted as p-value distributions calculated with IntervalStats (Chikina Troyanskaya, 2012) and compared to randomized controls. Sororin peaks were identified as overlapping peaks of 4 samples in early S-phase and 2 samples in G2-phase, using only the common peaks.(D) Quantification of the co-occupancy of SMC3 in early S-phase and in G2-phase with BrdU incorporation in early S-phase.


Figure 5 ( A ) Schematic representation of wild - type ( wt ) , targeted ( flx ) and deleted ( Δ ) Cdca5 alleles ( left ) and Southern blot of mouse genomic DNA ( right ) . Red triangles , loxP sites . ( B ) Observed and expected genotype distribution among offspring of heterozygous Cdca5 Δ / + mice ( newborn mice , n = 136 ; E9 . 5 , n = 54 ) . ( D ) Western blot of cell extracts to analyze depletion of sororin protein from fibroblasts after tamoxifen - induced Cre expression . Extracts were prepared either one or two days after release from the arrest as indicated . Chr , fractionated chromatin ; sol , soluble proteins . ( E ) Proliferation curve of cells with the indicated genotype after tamoxifen treatment . ( F ) Observed cell cycle distribution of fibroblasts treated with tamoxifen under starvation and subcultured in rich medium . ( G ) Quantification of mitotic cells with misaligned chromosomes after Cdca5 deletion and proliferation for 36 to 60 hours . Size bar , 10 µm ; error bars denote s . e . m . ; n > 500 cells per condition . ( H ) Phenotypic classification of nuclear morphology after Cdca5 deletion and proliferation for 5 to 7 days . Size bar , 10 µm ; error bars denote s . e . m . ; n > 500 cells per condition . ( I ) Western blot showing protein depletion efficiency . Cre expression was induced with tamoxifen for 24 hours in proliferating Smc3 and Cdca5 conditional knockout immortalized fibroblasts . ( J ) Analysis of chromosome spreads after Smc3 or Cdca5 deletion and nocodazole treatment . Representative images from every category are shown ; size bar , 10 µm ; error bars denote s . e . m . ; n > 500 per genotype .

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Figure 5(A) Schematic representation of wild-type (wt), targeted (flx) and deleted (Δ) Cdca5 alleles (left) and Southern blot of mouse genomic DNA (right). Red triangles, loxP sites.(B) Observed and expected genotype distribution among offspring of heterozygous Cdca5 Δ/+ mice (newborn mice, n = 136; E9.5, n = 54).(D) Western blot of cell extracts to analyze depletion of sororin protein from fibroblasts after tamoxifen-induced Cre expression. Extracts were prepared either one or two days after release from the arrest as indicated. Chr, fractionated chromatin; sol, soluble proteins.(E) Proliferation curve of cells with the indicated genotype after tamoxifen treatment.(F) Observed cell cycle distribution of fibroblasts treated with tamoxifen under starvation and subcultured in rich medium.(G) Quantification of mitotic cells with misaligned chromosomes after Cdca5 deletion and proliferation for 36 to 60 hours. Size bar, 10 µm; error bars denote s.e.m.; n > 500 cells per condition.(H) Phenotypic classification of nuclear morphology after Cdca5 deletion and proliferation for 5 to 7 days. Size bar, 10 µm; error bars denote s.e.m.; n > 500 cells per condition.(I) Western blot showing protein depletion efficiency. Cre expression was induced with tamoxifen for 24 hours in proliferating Smc3 and Cdca5 conditional knockout immortalized fibroblasts.(J) Analysis of chromosome spreads after Smc3 or Cdca5 deletion and nocodazole treatment. Representative images from every category are shown; size bar, 10 µm; error bars denote s.e.m.; n > 500 per genotype.


Figure 6 ( A ) Schematic illustration of the LAP - AID - tag integrated into the bacterial artificial chromosome containing the mouse Cdca5 locus . ( B ) Immunofluorescence staining of cells expressing sor - LAP - AID and the F - box helper protein after treatment with auxin . Antibodies against GFP mark the fusion protein and Aurora B antibody stains cells in G2 / M - phase . Scale bar , 10 µm . ( C ) Quantification of immunofluorescence after treating cells with auxin . Error bars denote s . e . m . ; a . u . , arbitrary units . ( D ) Time course quantification of GFP immunofluorescence signal decrease after auxin addition . ( E ) Analysis of chromosome spreads after auxin treatment . ( F ) Western blot showing cell extracts after treatment with auxin .

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Figure 6(A) Schematic illustration of the LAP-AID-tag integrated into the bacterial artificial chromosome containing the mouse Cdca5 locus.(B) Immunofluorescence staining of cells expressing sor-LAP-AID and the F-box helper protein after treatment with auxin. Antibodies against GFP mark the fusion protein and Aurora B antibody stains cells in G2/M-phase. Scale bar, 10 µm.(C) Quantification of immunofluorescence after treating cells with auxin. Error bars denote s.e.m.; a.u., arbitrary units.(D) Time course quantification of GFP immunofluorescence signal decrease after auxin addition.(E) Analysis of chromosome spreads after auxin treatment.(F) Western blot showing cell extracts after treatment with auxin.


Figure 7 ( A ) Cell cycle distribution of immortalized Cdca5 Δ / Δ sor - LAP - AID fibroblasts after 24 - hour incubation with aphidicolin and release . ( B ) Western blot showing cell extracts after incubation with aphidicolin and release . ( C ) Still images of a fluorescence recovery after photobleaching experiment . Size bar , 5 μm . Bleach area radius , 2 µm . ( D ) Graph depicting the normalized intensity after photobleaching to quantify turnover of sororin ( n = 15 ) . ( E ) Quantification of the relative abundance of sororin - LAP - AID on chromatin . Error bars denote s . e . m . ( F ) Quantification of the chromatin residence time of sor - LAP - AID at different times . Error bars denote s . e . m .

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Figure 7(A) Cell cycle distribution of immortalized Cdca5 Δ/Δ sor-LAP-AID fibroblasts after 24-hour incubation with aphidicolin and release.(B) Western blot showing cell extracts after incubation with aphidicolin and release.(C) Still images of a fluorescence recovery after photobleaching experiment. Size bar, 5 μm. Bleach area radius, 2 µm.(D) Graph depicting the normalized intensity after photobleaching to quantify turnover of sororin (n = 15).(E) Quantification of the relative abundance of sororin-LAP-AID on chromatin. Error bars denote s.e.m.(F) Quantification of the chromatin residence time of sor-LAP-AID at different times. Error bars denote s.e.m.


Figure 8 ( B ) Quantification of EdU - labeled prometaphase cells after treating cells with auxin and EdU at indicated time points . Error bars denote s . e . m . ; n > 200 cells per condition . ( C ) Analysis of chromosome spreads after auxin and EdU treatment and Giemsa staining . ( D ) Representative picture of the most prominent phenotype class upon auxin and EdU treatment and immunofluorescence microscopy . Scale bar , 10 µm . ( E ) Analysis of chromosome spreads after auxin treatment and EdU and DAPI labeling . Error bars denote s . e . m . ; n > 200 cells per condition .

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Figure 8(B) Quantification of EdU-labeled prometaphase cells after treating cells with auxin and EdU at indicated time points. Error bars denote s.e.m.; n > 200 cells per condition.(C) Analysis of chromosome spreads after auxin and EdU treatment and Giemsa staining.(D) Representative picture of the most prominent phenotype class upon auxin and EdU treatment and immunofluorescence microscopy. Scale bar, 10 µm.(E) Analysis of chromosome spreads after auxin treatment and EdU and DAPI labeling. Error bars denote s.e.m.; n > 200 cells per condition.


Figure 1 ( a ) Widefield immunofluorescence images of PFA fixed HeLa cells showing endogenous FLCN and LAMP1 staining in ( top ) normal growth or ( bottom ) starvation conditions . Orange arrows highlight association of FLCN with LAMP1 positive membranes in the peri - nuclear region . White dotted line shows cell periphery in starved condition . Scale bar = 10μm . ( b ) ( left ) Quantification of FLCN / LAMP1 association in growth and starvation conditions . A cell is scored as positive if 5 or more discrete FLCN / LAMP1 puncta were observed at 100x magnification using a widefield microscope . Data represents 60 cells from 3 independent experiments , error bars show S . E . M , * * * = p < 0 . 001 . ( right ) Western blot shows levels of phosphorylated of S6K and 4EBP in whole cell extracts under the same conditions . ( c ) Western blot showing typical efficiency of FLCN knockdown in HeLa cells using a pool consisting of 4 oligonucleotides and 2 independent oligonucleotides from the pool . Graph shows relative FLCN expression ( n = 3 , error bars show S . E . M . ) ( d ) Representative confocal fluorescence microscopy images showing LAMP1 ( magenta ) distribution in HeLa cells under normal growth or starvation conditions in cells depleted of FLCN or control cells transfected with a non - targeting siRNA . The location of the Golgi ( green ) is highlighted by giantin staining . ( e ) Schematic illustrating application of the cumulative intensity distribution method for quantification of lysosome distribution . ( f ) Graph showing cumulative distribution of LAMP1 intensity in normal growth and starvation conditions in HeLa cells . P - value is determined by the extra sum of F - squares test following non - linear regression and curve fitting . Error bars show S . E . M . from 30 cells in 3 replicates . ( g ) Graph showing cumulative distribution of LAMP1 intensity in starvation conditions for control cells transfected with non - targeting siRNA or cells depleted of FLCN . Error bars show S . E . M . from 30 cells in 3 replicates .

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Figure 1(a) Widefield immunofluorescence images of PFA fixed HeLa cells showing endogenous FLCN and LAMP1 staining in (top) normal growth or (bottom) starvation conditions. Orange arrows highlight association of FLCN with LAMP1 positive membranes in the peri-nuclear region. White dotted line shows cell periphery in starved condition. Scale bar = 10μm.(b) (left) Quantification of FLCN/LAMP1 association in growth and starvation conditions. A cell is scored as positive if 5 or more discrete FLCN/LAMP1 puncta were observed at 100x magnification using a widefield microscope. Data represents 60 cells from 3 independent experiments, error bars show S.E.M, *** = p<0.001. (right) Western blot shows levels of phosphorylated of S6K and 4EBP in whole cell extracts under the same conditions.(c) Western blot showing typical efficiency of FLCN knockdown in HeLa cells using a pool consisting of 4 oligonucleotides and 2 independent oligonucleotides from the pool. Graph shows relative FLCN expression (n=3, error bars show S.E.M.)(d) Representative confocal fluorescence microscopy images showing LAMP1 (magenta) distribution in HeLa cells under normal growth or starvation conditions in cells depleted of FLCN or control cells transfected with a non-targeting siRNA. The location of the Golgi (green) is highlighted by giantin staining.(e) Schematic illustrating application of the cumulative intensity distribution method for quantification of lysosome distribution.(f) Graph showing cumulative distribution of LAMP1 intensity in normal growth and starvation conditions in HeLa cells. P-value is determined by the extra sum of F-squares test following non-linear regression and curve fitting. Error bars show S.E.M. from 30 cells in 3 replicates.(g) Graph showing cumulative distribution of LAMP1 intensity in starvation conditions for control cells transfected with non-targeting siRNA or cells depleted of FLCN. Error bars show S.E.M. from 30 cells in 3 replicates.


Figure 2 ( a ) Representative spinning disk confocal image showing FLCN - GFP and lysosomes ( Lysotracker - Red ) in a live FLCN - GFP / HA - FNIP2 transfected HeLa cell ( from Movie EV1 ) . Top panels show the first frame of a 180 frame ( 1fps ) image series . Bottom panels show a maximum intensity projection of the image series to highlight mobile and immobile components . Boxed and magnified regions show typical peri - nuclear ( i ) and peripheral ( ii ) regions in the cell as well as a FLCN - GFP tubule ( iii ) Scale bar = 5μm . ( b ) Spinning disk confocal image series of a region live FLCN - GFP / HA - FNIP2 transfected HeLa cell highlighting dynamic FLCN - GFP tubules ( Movie EV2 ) . First merge panel shows FLCN - GFP and lysotracker - Red at T = 0 Subsequent panels show only FLCN - GFP at 30 second intervals . c ) Confocal image of a region of a methanol fixed FLCN - GFP / HA - FNIP2 transfected HeLa cell showing Rab7 ( red ) and LAMP1 ( blue ) association with FLCN - GFP tubules . Scale bar = 1μm .

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Figure 2(a) Representative spinning disk confocal image showing FLCN-GFP and lysosomes (Lysotracker-Red) in a live FLCN-GFP/HA-FNIP2 transfected HeLa cell (from Movie EV1). Top panels show the first frame of a 180 frame (1fps) image series. Bottom panels show a maximum intensity projection of the image series to highlight mobile and immobile components. Boxed and magnified regions show typical peri-nuclear (i) and peripheral (ii) regions in the cell as well as a FLCN-GFP tubule (iii) Scale bar = 5μm.(b) Spinning disk confocal image series of a region live FLCN-GFP/HA-FNIP2 transfected HeLa cell highlighting dynamic FLCN-GFP tubules (Movie EV2). First merge panel shows FLCN-GFP and lysotracker-Red at T=0 Subsequent panels show only FLCN-GFP at 30 second intervals.c) Confocal image of a region of a methanol fixed FLCN-GFP/HA-FNIP2 transfected HeLa cell showing Rab7 (red) and LAMP1 (blue) association with FLCN-GFP tubules. Scale bar = 1μm.


Figure 3 ( a ) Graphs showing cumulative distribution of LAMP1 intensity in control GFP ( black ) , GFP - Rab34 ( green ) or GFP - Rab7 ( blue ) transfected cells . Note black line and symbols partially obscure blue . ( b ) GST - RILP pull - down analysis from HeLa cells in normal and starved conditions showing an enhanced capacity of endogenous Rab34 to bind RILP in starvation conditions . Quantification is mean of 3 independent experiments . Error is S . E . M . ( c ) Representative confocal fluorescence microscopy images of FLCN depleted or control cells , transfected with GFP - Rab34 . Scale bar = 10μm . ( d ) ( left ) Graphs showing mean GFP - Rab34 expression ( grey values ) and cumulative distribution of LAMP1 intensity ( right ) in FLCN depleted or control siRNA cells transfected HeLa cells expressing GFP - Rab34 . Error bars show S . E . M . . from 30 cells in 3 replicates . Scale bar = 10μm . ( e ) Western blot and graph showing typical efficiency of Rab34 siRNA knockdown after 48 hours in HeLa cells . Error bars show S . E . M from 3 experiments , * * * = p < 0 . 001 . ( f ) Representative confocal fluorescence microscopy images showing LAMP1 ( magenta ) distribution in HeLa cells under normal growth or starvation conditions in cells depleted of Rab34 or control cells transfected with a non - targeting siRNA . Giantin is shown in green . Scale bar = 10μm . ( g ) Graphs showing cumulative distribution of LAMP1 intensity in normal growth or starvation conditions for control cells or cells depleted of Rab34 using siRNA . ( h ) Graph showing percentage of LAMP1 in cis - medial Golgi - region ( giantin overlap ) in the indicated conditions . Error bars show S . E . M from 30 cells in 3 replicates , * * * = p < 0 . 001 .

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Figure 3(a) Graphs showing cumulative distribution of LAMP1 intensity in control GFP (black), GFP-Rab34 (green) or GFP-Rab7 (blue) transfected cells. Note black line and symbols partially obscure blue.(b) GST-RILP pull-down analysis from HeLa cells in normal and starved conditions showing an enhanced capacity of endogenous Rab34 to bind RILP in starvation conditions. Quantification is mean of 3 independent experiments. Error is S.E.M.(c) Representative confocal fluorescence microscopy images of FLCN depleted or control cells, transfected with GFP-Rab34. Scale bar = 10μm.(d) (left) Graphs showing mean GFP-Rab34 expression (grey values) and cumulative distribution of LAMP1 intensity (right) in FLCN depleted or control siRNA cells transfected HeLa cells expressing GFP-Rab34. Error bars show S.E.M.. from 30 cells in 3 replicates. Scale bar = 10μm.(e) Western blot and graph showing typical efficiency of Rab34 siRNA knockdown after 48 hours in HeLa cells. Error bars show S.E.M from 3 experiments, *** = p<0.001.(f) Representative confocal fluorescence microscopy images showing LAMP1 (magenta) distribution in HeLa cells under normal growth or starvation conditions in cells depleted of Rab34 or control cells transfected with a non-targeting siRNA. Giantin is shown in green. Scale bar = 10μm.(g) Graphs showing cumulative distribution of LAMP1 intensity in normal growth or starvation conditions for control cells or cells depleted of Rab34 using siRNA.(h) Graph showing percentage of LAMP1 in cis-medial Golgi-region (giantin overlap) in the indicated conditions. Error bars show S.E.M from 30 cells in 3 replicates, *** = p<0.001.


Figure 4 ( a ) ( left ) Representative confocal fluorescence microscopy image showing methanol fixed HeLa cell transfected with GFP - Rab34 and stained for LAMP1 . Linescan analysis shows relative intensity of GFP and LAMP1 signals . Scale bar = 10μm . ( b ) Maximum intensity projection images from a confocal Z - stack showing LAMP1 and endogenous Rab34 in P - M fixed HeLa cells from normal and starved conditions . Whole cells and fields from which these panels are derived are shown in Figure EV4A . Boxes highlight regions shown in Figure 4C . Scale bar = 2μm . ( c ) Single confocal sections from the Z - stack in figure 4B . Orange arrows show close LAMP1 / Rab34 association . ( d ) Analysis of Lysotracker - Red dynamics using PCC decay in HeLa cells transfected with either GFP or GFP - Rab34 . Data is from 10 cells per condition . Error bars show S . E . M . ( e ) Live - cell spinning disk confocal microscopy image series at 10 sec intervals ( from Movie EV3 ) showing GFP - Rab34 positive compartment making dynamic contacts with lysosomes . Scale bar = 1μm ( f ) Live - cell spinning disk confocal microscopy image series at 10 sec intervals ( from Movie EV4 ) of FLCN - GFP and mCherry - Rab34 in the peri - nuclear region of a FLCN - GFP / HA - FNIP2 / mCherry - Rab34 transfected HeLa cell . Blue arrow highlights FLCN - Rab34 association moving on a linear trajectory . Yellow arrow highlights shorter saltatory movements . White arrow highlights dynamic associations between distinct FLCN - GFP and Rab34 positive structures . Scale bar = 1μm ( g ) Confocal image showing localisation of endogenous FLCN and Rab34 in starved HeLa cells .

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Figure 4(a) (left) Representative confocal fluorescence microscopy image showing methanol fixed HeLa cell transfected with GFP-Rab34 and stained for LAMP1. Linescan analysis shows relative intensity of GFP and LAMP1 signals. Scale bar = 10μm.(b) Maximum intensity projection images from a confocal Z-stack showing LAMP1 and endogenous Rab34 in P-M fixed HeLa cells from normal and starved conditions. Whole cells and fields from which these panels are derived are shown in Figure EV4A. Boxes highlight regions shown in Figure 4C. Scale bar = 2μm.(c) Single confocal sections from the Z-stack in figure 4B. Orange arrows show close LAMP1/Rab34 association.(d) Analysis of Lysotracker-Red dynamics using PCC decay in HeLa cells transfected with either GFP or GFP-Rab34. Data is from 10 cells per condition. Error bars show S.E.M.(e) Live-cell spinning disk confocal microscopy image series at 10 sec intervals (from Movie EV3) showing GFP-Rab34 positive compartment making dynamic contacts with lysosomes. Scale bar = 1μm(f) Live-cell spinning disk confocal microscopy image series at 10 sec intervals (from Movie EV4) of FLCN-GFP and mCherry-Rab34 in the peri-nuclear region of a FLCN-GFP/HA-FNIP2/mCherry-Rab34 transfected HeLa cell. Blue arrow highlights FLCN-Rab34 association moving on a linear trajectory. Yellow arrow highlights shorter saltatory movements. White arrow highlights dynamic associations between distinct FLCN-GFP and Rab34 positive structures. Scale bar = 1μm(g) Confocal image showing localisation of endogenous FLCN and Rab34 in starved HeLa cells.


Figure 5Images from fixed GFP - Rab34 transfected cells showing contacts between GFP - Rab34 positive peri - nuclear membranes and lysosomes . ( a ) Spinning disk confocal microscopy images acquired at 160x magnification . Left panels show a maximum intensity projection imaged of a Z - stack , sampled according to the Nyquist criterion and deconvolved . Right panels shows a 3D volume rendering of the same data . Images from fixed GFP - Rab34 transfected cells showing contacts between GFP - Rab34 positive peri - nuclear membranes and lysosomes . ( b ) A projection of 4 planes from a structured illumination microscopy super - resolution Z - stack of the same cells . Images from fixed GFP - Rab34 transfected cells showing contacts between GFP - Rab34 positive peri - nuclear membranes and lysosomes . ( c ) A thin section electron microscopy image of the peri - nuclear region of a GFP - Rab34 transfected HeLa cell . * indicates lysosomes . Images from fixed GFP - Rab34 transfected cells showing contacts between GFP - Rab34 positive peri - nuclear membranes and lysosomes . ( d ) A cryo - immuno - EM image of a GFP - Rab34 transfected HeLa cell . GFP - Rab34 ( dark black dots ) is identified by immunostaining for GFP and detection with gold conjugated protein A . The lysosome is intimately associated with Rab34 positive membranes . Sites of contact are highlighted by arrows .

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

Figure 5Images from fixed GFP-Rab34 transfected cells showing contacts between GFP-Rab34 positive peri-nuclear membranes and lysosomes.(a) Spinning disk confocal microscopy images acquired at 160x magnification. Left panels show a maximum intensity projection imaged of a Z-stack, sampled according to the Nyquist criterion and deconvolved. Right panels shows a 3D volume rendering of the same data.Images from fixed GFP-Rab34 transfected cells showing contacts between GFP-Rab34 positive peri-nuclear membranes and lysosomes.(b) A projection of 4 planes from a structured illumination microscopy super-resolution Z-stack of the same cells.Images from fixed GFP-Rab34 transfected cells showing contacts between GFP-Rab34 positive peri-nuclear membranes and lysosomes.(c) A thin section electron microscopy image of the peri-nuclear region of a GFP-Rab34 transfected HeLa cell. * indicates lysosomes.Images from fixed GFP-Rab34 transfected cells showing contacts between GFP-Rab34 positive peri-nuclear membranes and lysosomes.(d) A cryo-immuno-EM image of a GFP-Rab34 transfected HeLa cell. GFP-Rab34 (dark black dots) is identified by immunostaining for GFP and detection with gold conjugated protein A. The lysosome is intimately associated with Rab34 positive membranes. Sites of contact are highlighted by arrows.


Figure 6 ( a ) Confocal immunofluorescence microscopy images of HeLa cells transfected with Rab34 - dsRED - Mito , FLCN - GFP and HA - FNIP2 . White arrows highlight FLCN - GFP / HA - FNIP2 co - localisation distinct from Rab34 - mitochondria / FLCN - GFP localisation . Scale bar = 10μm . ( b ) Confocal immunofluorescence microscopy images of Rab34 - dsRED - Mito ( WT ) and FLCN - GFP DENN only or ΔDENN transfected HeLa cells showing DENN domain dependent recruitment to mitochondria . Scale bar = 10μm . ( c ) Western blot analysis of GST - RILP pull down showing association of endogenous FLCN , Rab34 and Rab7 with RILP . Input lane shows 1 % input cell extract . ( d ) Western blot analysis of GST - RILP pull down from UOK257 cells showing DENN domain dependent association of FLCN with RILP N - terminal , Rab binding domain ( RBD ) and C - terminal fragments . ( e ) Western blot showing binding of His - tagged FLCN - DENN domain in E . coli extract to GST - RILP . ( f ) Western blot analysis of GST - RILP pull down showing direct interaction between RILP and GTP hydrolysis deficient ( Q111L ) Rab34 ( at 200nM , in the presence of 1mM GTPγS ) and FLCN - DENN domain ( 1μM ) , but not low nucleotide affinity ( T66N ) Rab34 . FLCN - DENN enhances association of RILP with Q111L Rab34 . Graph shows quantification of relative RILP / Rab34 Q111L binding . Error bars show SEM . * * = p < 0 . 01 . ( g ) Western blot analysis of GST - RILP / Rab34 Q111L pull down ( Rab34 at 200nM ) showing that enhanced association of with RILP is achieved with low concentrations of FLCN . ( h ) Western blot analysis of GST - RILPpull down experiments with GTPγS loaded ( in the presence of 1mM GTPγS ) and GDP loaded ( in the presence of 1mM GDP ) wildtype Rab34 , showing FLCN - DENN ( 500nM ) induced interaction with RILP . ( i ) Western blot analysis of a GFP - Trap immunoprecipitation experiment from HeLa cells showing that expression of HA - RILP promotes the association of GFP - Rab34 and HA - Rab7 .

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

Figure 6(a) Confocal immunofluorescence microscopy images of HeLa cells transfected with Rab34-dsRED-Mito, FLCN-GFP and HA-FNIP2. White arrows highlight FLCN-GFP/HA-FNIP2 co-localisation distinct from Rab34-mitochondria/FLCN-GFP localisation. Scale bar = 10μm.(b) Confocal immunofluorescence microscopy images of Rab34-dsRED-Mito (WT) and FLCN-GFP DENN only or ΔDENN transfected HeLa cells showing DENN domain dependent recruitment to mitochondria. Scale bar = 10μm.(c) Western blot analysis of GST-RILP pull down showing association of endogenous FLCN, Rab34 and Rab7 with RILP. Input lane shows 1% input cell extract.(d) Western blot analysis of GST-RILP pull down from UOK257 cells showing DENN domain dependent association of FLCN with RILP N-terminal, Rab binding domain (RBD) and C-terminal fragments.(e) Western blot showing binding of His-tagged FLCN-DENN domain in E.coli extract to GST-RILP.(f) Western blot analysis of GST-RILP pull down showing direct interaction between RILP and GTP hydrolysis deficient (Q111L) Rab34 (at 200nM, in the presence of 1mM GTPγS) and FLCN-DENN domain (1μM), but not low nucleotide affinity (T66N) Rab34. FLCN-DENN enhances association of RILP with Q111L Rab34. Graph shows quantification of relative RILP / Rab34 Q111L binding. Error bars show SEM. ** = p<0.01.(g) Western blot analysis of GST-RILP / Rab34 Q111L pull down (Rab34 at 200nM) showing that enhanced association of with RILP is achieved with low concentrations of FLCN.(h) Western blot analysis of GST-RILPpull down experiments with GTPγS loaded (in the presence of 1mM GTPγS) and GDP loaded (in the presence of 1mM GDP) wildtype Rab34, showing FLCN-DENN (500nM) induced interaction with RILP.(i) Western blot analysis of a GFP-Trap immunoprecipitation experiment from HeLa cells showing that expression of HA-RILP promotes the association of GFP-Rab34 and HA-Rab7.


Figure 7 ( a ) Western blot showing endogenous Rab7 and Rab34 expression in UOK257 and UOK257 - 2 cell extracts and relative levels of the active forms as measured by GST - RILP pull down . HSC70 is used as a loading control . Graphs show quantification of bound Rab7 / Rab34 from 3 independent experiments , error bars show S . E . M . , * = p < 0 . 05 . ( b ) ( top ) Representative confocal immunofluorescence microscopy images showing intracellular distribution of LAMP1 in UOK257 and UOK257 - 2 cells . Dashed line highlights cell periphery in UOK257 - 2 cells . Scale bar = 10μm . ( bottom left ) Graph showing cumulative distribution of LAMP1 intensity in UOK257 vs UOK257 - 2 cells . Error bars show S . E . M . from 30 cells in 3 replicates . ( bottom right ) Graph showing Lysotracker - Red PCC decay with time from confocal movies of UOK257 and UOK257 - 2 cells . Mean values from 5 cells . Error bars show S . E . M . ( c ) Widefield immunofluorescence images showing early endosomes ( EEA1 ) and Golgi ( golgin - 97 ) in UOK257 and UOK257 - 2 cells . Scale bar = 10μm . ( d ) Western blot showing FLCN and FLCNΔDENN expression in stable UOK257 derived cell lines . ( e ) Representative widefield fluorescence microscopy images showing intracellular distribution of LAMP1 in UOK257 , UOK257 - FLCN and UOK257 - FLCNΔDENN cells . Scale bar = 10μm . Graphs showing cumulative distribution of LAMP1 intensity in UOK257 , UOK257 - FLCN and UOK257 - FLCNΔDENN cells . Error bars show S . E . M from 30 cells in 3 replicates . ( f ) ( left ) Western blot showing endogenous Rab7 and Rab34 expression in UOK257 , UOK257 - FLCN and UOK257 - FLCNΔDENN cells and relative levels of activity as measured by GST - RILP pull down . HSC70 is used as a loading control . ( right ) Graphs show quantification of bound Rab7 / Rab34 from 3 independent experiments ( p < 0 . 05 , * * * = p < 0 . 001 ) . ( g ) Western blot showing endogenous RILP expression in the indicated cell lines . Graph shows quantification of RILP expression from 3 independent experiments ( * = p < 0 . 05 , * * * = p < 0 . 001 ) .

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

Figure 7(a) Western blot showing endogenous Rab7 and Rab34 expression in UOK257 and UOK257-2 cell extracts and relative levels of the active forms as measured by GST-RILP pull down. HSC70 is used as a loading control. Graphs show quantification of bound Rab7/Rab34 from 3 independent experiments, error bars show S.E.M., * = p<0.05.(b) (top) Representative confocal immunofluorescence microscopy images showing intracellular distribution of LAMP1 in UOK257 and UOK257-2 cells. Dashed line highlights cell periphery in UOK257-2 cells. Scale bar = 10μm. (bottom left) Graph showing cumulative distribution of LAMP1 intensity in UOK257 vs UOK257-2 cells. Error bars show S.E.M. from 30 cells in 3 replicates. (bottom right) Graph showing Lysotracker-Red PCC decay with time from confocal movies of UOK257 and UOK257-2 cells. Mean values from 5 cells. Error bars show S.E.M.(c) Widefield immunofluorescence images showing early endosomes (EEA1) and Golgi (golgin-97) in UOK257 and UOK257-2 cells. Scale bar = 10μm.(d) Western blot showing FLCN and FLCNΔDENN expression in stable UOK257 derived cell lines.(e) Representative widefield fluorescence microscopy images showing intracellular distribution of LAMP1 in UOK257, UOK257-FLCN and UOK257-FLCNΔDENN cells. Scale bar = 10μm. Graphs showing cumulative distribution of LAMP1 intensity in UOK257, UOK257-FLCN and UOK257-FLCNΔDENN cells. Error bars show S.E.M from 30 cells in 3 replicates.(f) (left) Western blot showing endogenous Rab7 and Rab34 expression in UOK257, UOK257-FLCN and UOK257-FLCNΔDENN cells and relative levels of activity as measured by GST-RILP pull down. HSC70 is used as a loading control. (right) Graphs show quantification of bound Rab7/Rab34 from 3 independent experiments (p<0.05, *** = p<0.001).(g) Western blot showing endogenous RILP expression in the indicated cell lines. Graph shows quantification of RILP expression from 3 independent experiments (* = p<0.05, *** = p<0.001).


Figure 1A . Representative plots of splenocytes from CD45 . 1 mice adoptively transferred with CD45 . 2 OT - II WT or CCR5 - / - lymph node cell suspensions , five weeks after infection with rVACV - OVA virus . The gating strategy used to identify the memory CD4 + T cell subtypes is shown ( n = 5 ) . B . Absolute number of OT - II cells recovered in spleens of mice as in A ( n = 5 ) . C , Percentage of CD4 + TEM ( C ) in the OT - II WT and CCR5 - / - populations ( n = 5 ) . D . Percentage of TCM ( D ) in the OT - II WT and CCR5 - / - populations ( n = 5 ) . E . IFNγ - producing OT - II WT and CCR5 - / - memory cells isolated from mice as in A and restimulated ex vivo with OVA323 - 339 ( 1 μM ) ( n = 4 ) . G - I . Representative plots ( G ) and quantification of the frequency ( H ) and absolute number ( I ) of Tfh cells ( CD4 + CD44 + PD - 1 + CXCR5 + ) in the spleen after primary immunization ( day 7 ) with NIP - OVA ( n = 7 ) . J , K . ELISA analysis of high - ( J ) and low - affinity ( K ) isotype - specific anti - NIP antibodies in sera from OVA / OVA - and OVA / KLH - immunized mice ( day 15 post - challenge ; n = 5 mice / group ) . Data representative of one experiment of two .

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

Figure 1A. Representative plots of splenocytes from CD45.1 mice adoptively transferred with CD45.2 OT-II WT or CCR5-/- lymph node cell suspensions, five weeks after infection with rVACV-OVA virus. The gating strategy used to identify the memory CD4+ T cell subtypes is shown (n = 5).B. Absolute number of OT-II cells recovered in spleens of mice as in A (n = 5).C, Percentage of CD4+ TEM (C) in the OT-II WT and CCR5-/- populations (n = 5).D. Percentage of TCM (D) in the OT-II WT and CCR5-/- populations (n = 5).E. IFNγ-producing OT-II WT and CCR5-/- memory cells isolated from mice as in A and restimulated ex vivo with OVA323-339 (1 μM) (n = 4).G-I. Representative plots (G) and quantification of the frequency (H) and absolute number (I) of Tfh cells (CD4+CD44+PD-1+CXCR5+) in the spleen after primary immunization (day 7) with NIP-OVA (n = 7).J, K. ELISA analysis of high- (J) and low-affinity (K) isotype-specific anti-NIP antibodies in sera from OVA/OVA- and OVA/KLH-immunized mice (day 15 post-challenge; n = 5 mice/group). Data representative of one experiment of two.


Figure 2A , Representative histograms and quantification of mean fluorescence intensity ( MFI ; A ) in OT - II WT and CCR5 ‑ / ‑ lymphoblasts expanded in IL - 2 or IL - 15 , as specified . Data shown as mean ± SEM ( n ≥ 3 ) . B . the percentage of cells positive for the indicated memory markers ( B ) in OT - II WT and CCR5 ‑ / ‑ lymphoblasts expanded in IL - 2 or IL - 15 , as specified . Data shown as mean ± SEM ( n ≥ 3 ) . C - F . IL - 2 - ( C , D ) and IL - 15 - expanded lymphoblasts ( E , F ) were restimulated with indicated concentrations of OVA323 - 339 ; cell proliferation ( thymidine incorporation into DNA ; C , E ) and IL - 2 production ( by ELISA ; D , F ) were measured after 72 h . Data are presented as mean ± SEM ( n = 5 ) .

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Figure 2A, Representative histograms and quantification of mean fluorescence intensity (MFI; A) in OT-II WT and CCR5‑/‑ lymphoblasts expanded in IL-2 or IL-15, as specified. Data shown as mean ± SEM (n ≥3).B. the percentage of cells positive for the indicated memory markers (B) in OT-II WT and CCR5‑/‑ lymphoblasts expanded in IL-2 or IL-15, as specified. Data shown as mean ± SEM (n ≥3).C-F. IL-2- (C, D) and IL-15-expanded lymphoblasts (E, F) were restimulated with indicated concentrations of OVA323-339; cell proliferation (thymidine incorporation into DNA; C, E) and IL-2 production (by ELISA; D, F) were measured after 72 h. Data are presented as mean ± SEM (n = 5).


Figure 3A - C . Analysis of TCR nanoclustering by EM in OT - II WT and CCR5 - / - naïve cells ( A ; n = 6 cells / genotype ; WT : 3427 , CCR5 - / - : 3528 particles ) , and IL - 2 - ( B ; WT , n = 8 cells , 15419 particles ; CCR5 - / - , n = 6 cells , 5410 particles ) or IL - 15 - expanded lymphoblasts ( C ; WT , n = 8 cells , 27518 particles ; CCR5 - / - , n = 7 cells , 22696 particles ) . A representative small field image at the top of each panel shows gold particle distribution in the cell surface replicas of anti - CD3ε - labeled cells ; at bottom , quantification ( mean ± SEM ) of gold particles in clusters of indicated size in WT ( gray bars ) and CCR5 - / - cells ( red ) . Insets show the distribution of clusters of one , two , three , four , or more than four particles , and statistical analysis . D , E . Posterior distribution in naïve ( D ) and IL - 2 - expanded lymphoblasts ( E ) of the clustering parameter b for WT ( gray ) and CCR5 - / - cells ( red ) ; randomly generated distributions of receptors are shown in blue . The mean value of the b parameter is indicated for each condition . The probability of a chance distribution similar to that determined in cells is nearly 0 % by the ROPE . F . Comparison of TCR oligomer size using BN - PAGE and anti - CD3ζ immunoblotting in day 10 , IL - 2 - expanded WT and CCR5 - / - OT - II lymphoblasts lysed in buffer containing digitonin or Brij - 96 . The marker protein is ferritin ( f1 , 440 and f2 , 880 kDa forms ) . The ratio of TCR nanoclusters to monomeric TCR in each lysis condition was quantified by densitometry ( right ; n = 5 ) . G . Top , representative small field EM images showing gold particle distribution in the cell surface replicas of CD4 + T cells isolated from OVA / OVA - immunized WT and CCR5 - / - mice . Bottom , quantification ( mean ± SEM ) of gold particles in clusters of the indicated size ( WT , gray bars ; n = 5 cells , 14680 particles ; CCR5 - / - , red ; n = 7 cells , 15374 particles ) . Insets show the distribution between clusters of one , two , three , four or more than four particles , and statistical analysis .

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

Figure 3A-C. Analysis of TCR nanoclustering by EM in OT-II WT and CCR5-/- naïve cells (A; n = 6 cells/genotype; WT: 3427, CCR5-/-: 3528 particles), and IL-2- (B; WT, n = 8 cells, 15419 particles; CCR5-/-, n = 6 cells, 5410 particles) or IL-15-expanded lymphoblasts (C; WT, n = 8 cells, 27518 particles; CCR5-/-, n = 7 cells, 22696 particles). A representative small field image at the top of each panel shows gold particle distribution in the cell surface replicas of anti-CD3ε-labeled cells; at bottom, quantification (mean ± SEM) of gold particles in clusters of indicated size in WT (gray bars) and CCR5-/- cells (red). Insets show the distribution of clusters of one, two, three, four, or more than four particles, and statistical analysis.D, E. Posterior distribution in naïve (D) and IL-2-expanded lymphoblasts (E) of the clustering parameter b for WT (gray) and CCR5-/- cells (red); randomly generated distributions of receptors are shown in blue. The mean value of the b parameter is indicated for each condition. The probability of a chance distribution similar to that determined in cells is nearly 0% by the ROPE.F. Comparison of TCR oligomer size using BN-PAGE and anti-CD3ζ immunoblotting in day 10, IL-2-expanded WT and CCR5-/- OT-II lymphoblasts lysed in buffer containing digitonin or Brij-96. The marker protein is ferritin (f1, 440 and f2, 880 kDa forms). The ratio of TCR nanoclusters to monomeric TCR in each lysis condition was quantified by densitometry (right; n = 5).G. Top, representative small field EM images showing gold particle distribution in the cell surface replicas of CD4+ T cells isolated from OVA/OVA-immunized WT and CCR5-/- mice. Bottom, quantification (mean ± SEM) of gold particles in clusters of the indicated size (WT, gray bars; n = 5 cells, 14680 particles; CCR5-/-, red; n = 7 cells, 15374 particles). Insets show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis.


Figure 4A . OT - II WT cells were activated with OVA323 - 339 , alone or with TAK - 779 . After three days , antigen and TAK - 779 were removed and lymphoblasts expanded in IL - 2 - containing medium . TCR nanoclustering was analyzed in anti - CD3ε - labeled surface replicas of day 10 lymphoblasts . Top , representative small field EM images showing gold particle distribution in the cell surface replicas of WT CD4 + T cells alone or with TAK - 779 . Bottom , quantification of gold particles in clusters of the indicated size . Inset , distribution of gold particles between clusters of one , two , three , four or more than four particles in vehicle - ( gray bars ; n = 5 cells , 11266 particles ) and TAK - 779 - treated cells ( black ; n = 6 cells , 5138 particles ) . B . OT - II WT cells were activated with OVA323 - 339 , and TAK - 779 was added at days 3 , 5 , and 7 after antigen removal . Analysis as above , untreated ( gray bars ; n = 5 cells , 6400 particles ) and TAK - 779 - treated cells ( black ; n = 6 cells , 7153 particles ) . Inset show the distribution between clusters of one , two , three , four or more than four particles , and statistical analysis . C . OT - II WT naïve cells were activated with antigen in the presence or not of the CXCR4 inhibitor AMD3100 . Left , representative EM images showing gold particle distribution in the cell surface replicas . Right , analysis of gold particles in clusters as above , vehicle - ( gray bars ; n = 6 cells , 12339 particles ) and AMD3100 - treated cells ( black ; n = 7 cells , 17059 particles ) . Inset show the distribution between clusters of one , two , three , four or more than four particles , and statistical analysis .

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Figure 4A. OT-II WT cells were activated with OVA323-339, alone or with TAK-779. After three days, antigen and TAK-779 were removed and lymphoblasts expanded in IL-2-containing medium. TCR nanoclustering was analyzed in anti-CD3ε-labeled surface replicas of day 10 lymphoblasts. Top, representative small field EM images showing gold particle distribution in the cell surface replicas of WT CD4+ T cells alone or with TAK-779. Bottom, quantification of gold particles in clusters of the indicated size. Inset, distribution of gold particles between clusters of one, two, three, four or more than four particles in vehicle- (gray bars; n = 5 cells, 11266 particles) and TAK-779-treated cells (black; n = 6 cells, 5138 particles). B. OT-II WT cells were activated with OVA323-339, and TAK-779 was added at days 3, 5, and 7 after antigen removal. Analysis as above, untreated (gray bars; n = 5 cells, 6400 particles) and TAK-779-treated cells (black; n = 6 cells, 7153 particles). Inset show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis.C. OT-II WT naïve cells were activated with antigen in the presence or not of the CXCR4 inhibitor AMD3100. Left, representative EM images showing gold particle distribution in the cell surface replicas. Right, analysis of gold particles in clusters as above, vehicle- (gray bars; n = 6 cells, 12339 particles) and AMD3100-treated cells (black; n = 7 cells, 17059 particles). Inset show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis.


Figure 5A . Total Chol levels in WT and CCR5 - / - OT - II lymphoblasts ( day 10 , IL - 2 - expanded ) as determined by a fluorometric assay ( n = 6 ) . B - D . SM ( B ) , Cer ( C ) and dhCer ( D ) levels in WT and CCR5 - / - OT - II 10 - day lymphoblasts , as determined by UPLC - TOF MS . Values , after normalization with C17 standards and cell number in each sample , are the mean of two independent experiments ( n = 6 ) . E . RT - qPCR determination of CerS mRNA levels in naïve and IL - 2 - expanded WT and CCR5 ‑ / - OT - II 10 - day lymphoblasts ( n = 3 - 5 ) . F . Representative immunoblot showing CerS2 protein levels in naïve and WT and CCR5 - / - OT - II 10 - day lymphoblasts , and densitometric quantification of blots as above ( n = 10 ) . G . ChIP analysis of the CerS2 promoter using an anti - H3K9Ac antibody . Scheme of the CerS2 promoter showing CpG islands and primers used for amplification . Relative ChIP of the CerS2 promoter in WT and CCR5 - / - OT - II 10 - day lymphoblasts ( n = 3 ) . H . Relative CerS2 mRNA level in CD4 T cells treated with PTx ( n = 3 ) . L . Representative immunofluorescence images showing pSer142 - GATA - 1 staining ( green ) of OT - II WT and CCR5 - / - lymphoblasts . The green channel ( top ) and the merge with nuclear DAPI staining ( blue ; bottom ) are shown . Scale bar , 10 μm . M . Quantification of nuclear staining of the cells plotted as integrated density fluorescence intensity in DAPI - stained area ; n ≥ 50 cells / condition ) . N . Top , basic scheme of the CerS2 promoter , indicating the putative GATA - 1 binding site ( blue ) and location of the primers used for amplification in ChIP assays ( black arrows ) . Bottom , relative anti - GATA - 1 ChIP levels in OT - II WT and CCR5 - / - lymphoblasts ( n = 5 ) .

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

Figure 5A. Total Chol levels in WT and CCR5-/- OT-II lymphoblasts (day 10, IL-2-expanded) as determined by a fluorometric assay (n = 6).B-D. SM (B), Cer (C) and dhCer (D) levels in WT and CCR5-/- OT-II 10-day lymphoblasts, as determined by UPLC-TOF MS. Values, after normalization with C17 standards and cell number in each sample, are the mean of two independent experiments (n = 6).E. RT-qPCR determination of CerS mRNA levels in naïve and IL-2-expanded WT and CCR5‑/- OT-II 10-day lymphoblasts (n = 3-5).F. Representative immunoblot showing CerS2 protein levels in naïve and WT and CCR5-/- OT-II 10-day lymphoblasts, and densitometric quantification of blots as above (n = 10).G. ChIP analysis of the CerS2 promoter using an anti-H3K9Ac antibody. Scheme of the CerS2 promoter showing CpG islands and primers used for amplification. Relative ChIP of the CerS2 promoter in WT and CCR5-/- OT-II 10-day lymphoblasts (n = 3).H. Relative CerS2 mRNA level in CD4 T cells treated with PTx (n = 3).L. Representative immunofluorescence images showing pSer142-GATA-1 staining (green) of OT-II WT and CCR5-/- lymphoblasts. The green channel (top) and the merge with nuclear DAPI staining (blue; bottom) are shown. Scale bar, 10 μm. M. Quantification of nuclear staining of the cells plotted as integrated density fluorescence intensity in DAPI-stained area; n ≥50 cells/condition).N. Top, basic scheme of the CerS2 promoter, indicating the putative GATA-1 binding site (blue) and location of the primers used for amplification in ChIP assays (black arrows). Bottom, relative anti-GATA-1 ChIP levels in OT-II WT and CCR5-/- lymphoblasts (n = 5).


Figure 6A . Scheme of the strategy used to form TCR proteoliposomes , and size of LUV generated at the indicated lipid molar ratio . Polydispersity index values are shown as black squares for each condition ( n = 3 ) . SBP , streptavidin - binding - peptide - tagged TCR . B . Representative immunoblots comparing TCR nanocluster sizes via BN - PAGE and anti - CD3ζ immunoblotting in TCR proteoliposomes lysed in the presence of Brij - 96 or digitonin . The marker protein is ferritin ( f1 , 440 and f2 , 880 kDa forms ) . C . The ratio of the nanocluster and monomeric TCR in each lysis condition was quantified by densitometry from immunoblots as in B ( n ≥ 4 ) . D . Cer levels in OT ‑ II 10 - day lymphoblasts , untreated or treated with SMase ( n = 4 ) . E . Representative small field images showing gold particle distribution , and quantification ( mean ± SEM ) of gold particles in clusters of the indicated size in cell surface replicas from untreated ( gray bars ; n = 5 cells , 8126 particles ) and SMase - treated ( 1h ) OT - II lymphoblasts ( black cyan ; n = 6 cells , 8457 particles ) after CD3ε labeling , as determined by EM . The inset shows distribution between clusters of one , two , three , four or more than four particles . F . GFP expression in shCtrl - ( black ) and shCerS2 ( orange ) - transduced 2B4 cells after puromycin selection , as determined by FACS . G . Relative CerS2 mRNA levels in TAK - 779 - treated 2B4 cells as in F . Values were normalized to those obtained in untransduced TAK - 779 - treated 2B4 cells ( n = 3 ) . H . Representative immunoblot with anti - CerS2 antibody to determine CerS2 protein levels in shCtrl and ShCerS2 - transduced 2B4 cells as in G . Filters were rehybridized with β - actin as loading control . I . TCR nanoclustering of shCtrl - and shCerS2 transduced 2B4 cells in the presence of TAK - 779 as determined by EM . Representative small field images and quantification ( mean ± SEM ) of gold particles in clusters of indicated sizes in cell surface replicas shCtrl ( gray black bars ; n = 6 cells , 12337 particles ) and shCerS2 2B4 lymphoblasts ( orange ; n = 7 cells , 13456 particles ) . Inset , distribution between clusters of indicated size and statistical analysis . J . Percentage of CD69 + shCtrl ( black ) and shCerS2 ( orange ) 2B4 cells restimulated with plate - bound anti - CD3ε antibody in the presence of TAK - 779 ( n = 3 ) .

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

Figure 6A. Scheme of the strategy used to form TCR proteoliposomes, and size of LUV generated at the indicated lipid molar ratio. Polydispersity index values are shown as black squares for each condition (n = 3). SBP, streptavidin-binding-peptide-tagged TCR.B. Representative immunoblots comparing TCR nanocluster sizes via BN-PAGE and anti-CD3ζ immunoblotting in TCR proteoliposomes lysed in the presence of Brij-96 or digitonin. The marker protein is ferritin (f1, 440 and f2, 880 kDa forms).C. The ratio of the nanocluster and monomeric TCR in each lysis condition was quantified by densitometry from immunoblots as in B (n ≥ 4).D. Cer levels in OT‑II 10-day lymphoblasts, untreated or treated with SMase (n = 4).E. Representative small field images showing gold particle distribution, and quantification (mean ± SEM) of gold particles in clusters of the indicated size in cell surface replicas from untreated (gray bars; n = 5 cells, 8126 particles) and SMase-treated (1h) OT-II lymphoblasts (black cyan; n = 6 cells, 8457 particles) after CD3ε labeling, as determined by EM. The inset shows distribution between clusters of one, two, three, four or more than four particles.F. GFP expression in shCtrl- (black) and shCerS2 (orange)-transduced 2B4 cells after puromycin selection, as determined by FACS.G. Relative CerS2 mRNA levels in TAK-779-treated 2B4 cells as in F. Values were normalized to those obtained in untransduced TAK-779-treated 2B4 cells (n = 3).H. Representative immunoblot with anti-CerS2 antibody to determine CerS2 protein levels in shCtrl and ShCerS2-transduced 2B4 cells as in G. Filters were rehybridized with β-actin as loading control.I. TCR nanoclustering of shCtrl- and shCerS2 transduced 2B4 cells in the presence of TAK-779 as determined by EM. Representative small field images and quantification (mean ± SEM) of gold particles in clusters of indicated sizes in cell surface replicas shCtrl (gray black bars; n = 6 cells, 12337 particles) and shCerS2 2B4 lymphoblasts (orange; n = 7 cells, 13456 particles). Inset, distribution between clusters of indicated size and statistical analysis.J. Percentage of CD69+ shCtrl (black) and shCerS2 (orange) 2B4 cells restimulated with plate-bound anti-CD3ε antibody in the presence of TAK-779 (n = 3).


Figure 7A . Analysis of TCR nanoclustering in lymphoblasts from healthy WT and ccr5Δ32 homozygous donors by EM . Top , representative small field image showing gold particle distribution in cell surface replicas of anti - CD3ε - labeled cells ; bottom , quantification ( mean ± SEM ) of gold particles in clusters of the indicated size in the WT ( gray bars ; n = 5 cells , 17689 particles ) and Δ32 / Δ32 cells ( light red ; n = 4 cells , 16938 particles ) . Insets show the distribution between clusters of one , two , three , four or more than four particles , and statistical analysis . B . Normalized SM , Cer and dhCer levels in lymphoblasts obtained as in A . A representative experiment is shown ( n = 3 donors / genotype ; n = 2 independent experiments ) C . Relative CerS2 mRNA levels in day 8 WT and ccr5Δ32 lymphoblasts . Each data point is the average of a technical triplicate from 3 donors in two independent experiments ( n = 6 )

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

Figure 7A. Analysis of TCR nanoclustering in lymphoblasts from healthy WT and ccr5Δ32 homozygous donors by EM. Top, representative small field image showing gold particle distribution in cell surface replicas of anti-CD3ε-labeled cells; bottom, quantification (mean ± SEM) of gold particles in clusters of the indicated size in the WT (gray bars; n = 5 cells, 17689 particles) and Δ32/Δ32 cells (light red; n = 4 cells, 16938 particles). Insets show the distribution between clusters of one, two, three, four or more than four particles, and statistical analysis.B. Normalized SM, Cer and dhCer levels in lymphoblasts obtained as in A. A representative experiment is shown (n = 3 donors/genotype; n = 2 independent experiments)C. Relative CerS2 mRNA levels in day 8 WT and ccr5Δ32 lymphoblasts. Each data point is the average of a technical triplicate from 3 donors in two independent experiments (n = 6)


Figure 1D , E Detection of FLAIL and asFLAIL gene expression in Col - 0 and flail mutants by strand - specific RT - qPCR . Transcript levels were normalized to UBQ expression levels . Y - axis showed relative values compared to the expression level of Col - 0 . G Violin graph showed number of rosette leaves after appearance of the first flower bud in Col - 0 . Data represented the mean of six independent experiments .

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Figure 1D, E Detection of FLAIL and asFLAIL gene expression in Col-0 and flail mutants by strand-specific RT-qPCR. Transcript levels were normalized to UBQ expression levels. Y-axis showed relative values compared to the expression level of Col-0.G Violin graph showed number of rosette leaves after appearance of the first flower bud in Col-0. Data represented the mean of six independent experiments.


Figure 2B , C Detection of sense FLAIL and asFLAIL transcript expression in Col - 0 , flail3 mutant and complementation lines by strand - specific RT - qPCR . pFLAIL : gFLAIL18 / 88 and pasFLAIL : gasFLAIL18 / 39 represented sense and antisense FLAIL complementation lines , respectively with flail3 35S : GUS transgenic line as a control . Positions of primers used for strand - specific RT - qPCR analyses were the same as those in Figure 1A . Transcript levels were normalized to UBQ expression levels . Y - axis showed relative values compared to the expression level of Col - 0 . D Representative morphological phenotypes of 4 - week - old plants of Col - 0 , flail3 mutant , transgenic lines . Scale bar : 2 cm . E Violin graph showed number of rosette leaves after appearance of the first flower bud in Col - 0 .

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Figure 2B, C Detection of sense FLAIL and asFLAIL transcript expression in Col-0, flail3 mutant and complementation lines by strand-specific RT-qPCR. pFLAIL:gFLAIL18/88 and pasFLAIL:gasFLAIL18/39 represented sense and antisense FLAIL complementation lines, respectively with flail3 35S:GUS transgenic line as a control. Positions of primers used for strand-specific RT-qPCR analyses were the same as those in Figure 1A. Transcript levels were normalized to UBQ expression levels. Y-axis showed relative values compared to the expression level of Col-0.D Representative morphological phenotypes of 4-week-old plants of Col-0, flail3 mutant, transgenic lines. Scale bar: 2 cm. E Violin graph showed number of rosette leaves after appearance of the first flower bud in Col-0.


Figure 3A Heatmap of 13 flowering genes and sense FLAIL was cropped with systematic gene names illustrating that the expression of dysregulated flowering genes in flail3 was fully ( AGL24 , CO , COOLAIR , CIR1 , FBH4 , LAC8 , PLL25 , PRE1 ) or partially ( AGL20 , DDF1 , MAF4 , NMT3 , PHYA ) rescued in complementation line . DEGs analyzed by DESeq2 with | log2 fold change | > 1 and adjusted p value < 0 . 05 were considered significant differential expression . Three biological replicates for Col - 0 , flail3 , and two for flail3 pFLAIL : gFLAIL18 ( complement ) . Flowering genes were highlighted in red . The color scale reflected the log 2 - fold change in gene expression , ranging from down - regulated ( blue ) to up - regulated ( red ) . Genome browser screenshots illustrating the expression of sense FLAIL and dysregulated flowering genes in flail3 were rescued in complementation line ( top panel ) . Normalized read counts ( TPM from RNA - seq ) for differentially expressed flowering genes in Col - 0 , flail3 , and flail3 pFLAIL : gFLAIL18 plants ( bottom panel ) .

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

Figure 3A Heatmap of 13 flowering genes and sense FLAIL was cropped with systematic gene names illustrating that the expression of dysregulated flowering genes in flail3 was fully (AGL24, CO, COOLAIR, CIR1, FBH4, LAC8, PLL25, PRE1) or partially (AGL20, DDF1, MAF4, NMT3, PHYA) rescued in complementation line. DEGs analyzed by DESeq2 with |log2 fold change| > 1 and adjusted p value < 0.05 were considered significant differential expression. Three biological replicates for Col-0, flail3, and two for flail3 pFLAIL:gFLAIL18 (complement). Flowering genes were highlighted in red. The color scale reflected the log 2-fold change in gene expression, ranging from down-regulated (blue) to up-regulated (red).Genome browser screenshots illustrating the expression of sense FLAIL and dysregulated flowering genes in flail3 were rescued in complementation line (top panel). Normalized read counts (TPM from RNA-seq) for differentially expressed flowering genes in Col-0, flail3, and flail3 pFLAIL:gFLAIL18 plants (bottom panel).


Figure 4E , F Genome browser screenshots illustrating two of FLAIL bound targets CIR1 ( E ) and LAC8 ( F ) in ChIRP - seq .

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Figure 4E, F Genome browser screenshots illustrating two of FLAIL bound targets CIR1 (E) and LAC8 (F) in ChIRP-seq.


Figure 5FLAIL affects AS of LAC8 . Light blue arrows indicate probes used for gel electrophoresis in C ; LAC8 isoforms were detected by RT - PCR with black arrow indicating LAC8 . 1 , grey arrow indicating LAC8 . 2 , and brown arrow indicating LAC8 . 3 . - RT , negative control without adding reverse transcriptase . Isoform bands were quantified using Image Lab software relative to the respective UBQ band of each sample . F - H ChIRP - qPCR using probe pools FLAIL - asDNA ( Odd and Even ) identified FLAIL binding snRNAs U1 , U2 and U5 . Luc - asDNA probes retrieved RNAs as a negative control . Retrieved FLAIL transcript was served as a positive control ; UBQ and a non - coding , unspliced , conserved snoRNA lncCOBRA1 ( AT1G05913 ) as negative controlsI Morphological phenotypes of 4 - week - old plants of Col - 0 , flail3 , lac8 - 52 - 34 , lac8 flail3 - 55 - 1 mutants . Scale bar : 2 cm . J Violin graph showed number of rosette leaves after appearance of the first flower bud in Col - 0 . Data represented the mean of at least seven independent experiments in Col - 0 , flail3 , lac8 - 52 - 34 , lac8 flail3 - 55 - 1 .

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Figure 5FLAIL affects AS of LAC8. Light blue arrows indicate probes used for gel electrophoresis in C; LAC8 isoforms were detected by RT-PCR with black arrow indicating LAC8.1, grey arrow indicating LAC8.2, and brown arrow indicating LAC8.3. -RT, negative control without adding reverse transcriptase. Isoform bands were quantified using Image Lab software relative to the respective UBQ band of each sample.F-H ChIRP-qPCR using probe pools FLAIL-asDNA (Odd and Even) identified FLAIL binding snRNAs U1, U2 and U5. Luc-asDNA probes retrieved RNAs as a negative control. Retrieved FLAIL transcript was served as a positive control; UBQ and a non-coding, unspliced, conserved snoRNA lncCOBRA1 (AT1G05913) as negative controlsI Morphological phenotypes of 4-week-old plants of Col-0, flail3, lac8-52-34, lac8 flail3-55-1 mutants. Scale bar: 2 cm. J Violin graph showed number of rosette leaves after appearance of the first flower bud in Col-0. Data represented the mean of at least seven independent experiments in Col-0, flail3, lac8-52-34, lac8 flail3-55-1.


Figure 1 ( A ) Left quadriceps muscle biopsy performed at age 10 months ( i - iii ) demonstrating a dystrophic process with variation in fiber size ( black arrow ) , increased connective tissue ( blue arrow ) and rare internalized nuclei ( white arrow ) on hematoxylin and eosin ( H & E ) staining ( i ) and Gömöritrichrome staining ( ii ) and type 1 fiber atrophy on nicotinamide adenine dinucleotide ( NADH ) staining ( iii ) . Muscle biopsy in an unaffected ( control ) individual ( iv - vi ) . Scale bar : 100μm . ( B ) Western blot data ( n = 1 ) with quantification demonstrated reduced α - Dystroglycan ( α - DG ) in P1 ' s muscle compared to control . ( C ) Western blot data with quantification demonstrated reduced α - Dystroglycan in fibroblasts from P1 and P2 compared to control ( c ) , although not to the extent of the reduction observed in fibroblasts from a patient with a confirmed α - dystroglycanopathy ( α - DG pt ) due to biallelic pathogenic variants in LARGE ( n = 1 ) . ( D ) Brain MRI findings in P1 at 28 months of age ( top ) revealing normal appearing cortex , cerebellum and pons on T1 - weighted images and T2 - weighted image . There is evidence of mild thinning of the corpus callosum ( arrow ) . Brain MRI in an unaffected ( control ) individual at 30 months of age ( bottom ) demonstrating normal sized corpus callosum .

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Figure 1(A) Left quadriceps muscle biopsy performed at age 10 months (i-iii) demonstrating a dystrophic process with variation in fiber size (black arrow), increased connective tissue (blue arrow) and rare internalized nuclei (white arrow) on hematoxylin and eosin (H&E) staining (i) and Gömöritrichrome staining (ii) and type 1 fiber atrophy on nicotinamide adenine dinucleotide (NADH) staining (iii). Muscle biopsy in an unaffected (control) individual (iv-vi). Scale bar: 100μm.(B) Western blot data (n=1) with quantification demonstrated reduced α-Dystroglycan (α-DG) in P1's muscle compared to control. (C) Western blot data with quantification demonstrated reduced α-Dystroglycan in fibroblasts from P1 and P2 compared to control (c), although not to the extent of the reduction observed in fibroblasts from a patient with a confirmed α-dystroglycanopathy (α-DG pt) due to biallelic pathogenic variants in LARGE (n=1).(D) Brain MRI findings in P1 at 28 months of age (top) revealing normal appearing cortex, cerebellum and pons on T1-weighted images and T2-weighted image. There is evidence of mild thinning of the corpus callosum (arrow). Brain MRI in an unaffected (control) individual at 30 months of age (bottom) demonstrating normal sized corpus callosum.


Figure 2 ( B ) Sashimi plots comparing muscle RNA sequencing reads in P1 ( red ) and two control muscle samples ( blue and green ) at the exons 3 - 5 of BET1 .

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Figure 2(B) Sashimi plots comparing muscle RNA sequencing reads in P1 (red) and two control muscle samples (blue and green) at the exons 3-5 of BET1.


Figure 3 ( A ) Immunoblot of patient fibroblast cell lines ( P1 and P2 ) and three independent controls ( C1 - 3 ) . Immunoblots were probed with the indicated primary antibodies . Quantification of BET1 protein levels normalized to GAPDH . Data represented are mean ± SD of three independent biological experiments . Significant differences of P1 BET1 - levels were checked compared to each control ( * * * = p < 0 . 001 , one - way - ANOVA followed by Tukey ` s post - hoc test ) . ( B ) Subcellular localization of BET1 in control and patient fibroblasts by indirect immunofluorescent confocal microscopy . Representative immunofluorescence images of BET1 ( red ) and cis - Golgi marker GM130 ( green ) . Scale bar : 20µm . Colocalization of BET1 with GM130 was determined using Pearson ' s correlation coefficient ( PCC ) measured per cell ( right panel ; C1 : n = 63 , C2 : n = 50 , C3 : n = 49 and P2 : n = 33 ) ( * * * = p ≤ 0 . 001 , one - way ANOVA was followed by Tukey ' s post - hoc test ) . Means are shown ± SEM . ( C ) Representative immunofluorescence images of BET1 ( red ) and ER marker PDI ( green ) . Scale bar : 20 µm . Right panel shows PCCs for BET1 versus PDI ( C1 : n = 61 , C2 : n = 42 , C3 : n = 59 and P2 : n = 32 of two biological replicates ) . ( * * * = p ≤ 0 . 001 , one - way ANOVA was followed by Tukey ' s post - hoc test ) . Means are shown ± SEM . ( D ) HeLa - cells stably expressing the pC4 - reporter were transfected with control siRNA ( ctrl ) or two independent siRNAs ( # 1 and # 2 ) directed against BET1 ( siRNA # 1 is shown in figure EV 2B ) . 2d post - transfection the pC4 - secretion assay was performed for 0 and 10 min post induction of secretion . Representative images are shown ( upper panel ctrl and lower panel # 2 siRNA , respectively . Scale bar : 20 µm . Right panels represent magnifications of dashed areas .

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Figure 3(A) Immunoblot of patient fibroblast cell lines (P1 and P2) and three independent controls (C1-3). Immunoblots were probed with the indicated primary antibodies. Quantification of BET1 protein levels normalized to GAPDH. Data represented are mean ± SD of three independent biological experiments. Significant differences of P1 BET1-levels were checked compared to each control (***=p<0.001, one-way-ANOVA followed by Tukey`s post-hoc test).(B) Subcellular localization of BET1 in control and patient fibroblasts by indirect immunofluorescent confocal microscopy. Representative immunofluorescence images of BET1 (red) and cis-Golgi marker GM130 (green). Scale bar: 20µm.Colocalization of BET1 with GM130 was determined using Pearson's correlation coefficient (PCC) measured per cell (right panel; C1: n=63, C2: n=50, C3: n=49 and P2: n=33) (***=p≤0.001, one-way ANOVA was followed by Tukey's post-hoc test). Means are shown ± SEM. (C) Representative immunofluorescence images of BET1 (red) and ER marker PDI (green). Scale bar: 20 µm. Right panel shows PCCs for BET1 versus PDI (C1: n=61, C2: n=42, C3: n=59 and P2: n=32 of two biological replicates). (***=p≤0.001, one-way ANOVA was followed by Tukey's post-hoc test). Means are shown ± SEM.(D) HeLa-cells stably expressing the pC4-reporter were transfected with control siRNA (ctrl) or two independent siRNAs (#1 and #2) directed against BET1 (siRNA #1 is shown in figure EV 2B). 2d post-transfection the pC4-secretion assay was performed for 0 and 10 min post induction of secretion. Representative images are shown (upper panel ctrl and lower panel #2 siRNA, respectively. Scale bar: 20 µm. Right panels represent magnifications of dashed areas.


Figure 4 ( B ) Stability of hydrogen bonds between BET1Asp68 and BET1Asp72 respectively was estimated using molecular dynamics simulation in three replicas . Parameter occupancy could be more than 100 % if several stable hydrogen bonds could be established by residue . Bars represent average fraction of the simulated time the residue is involved in a hydrogen bond ( s ) . Data represented are mean ± SD .

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Figure 4(B) Stability of hydrogen bonds between BET1Asp68 and BET1Asp72 respectively was estimated using molecular dynamics simulation in three replicas. Parameter occupancy could be more than 100% if several stable hydrogen bonds could be established by residue. Bars represent average fraction of the simulated time the residue is involved in a hydrogen bond(s). Data represented are mean ± SD.


Figure 5 ( A ) Volcano plot of AP - MS data using HA - BET1 or Ile51Ser . ( B ) Co - IPs using HA - tagged constructs expressed in HEK293 cells . As baits HA - tagged proteins were immunoprecipitated with anti - HA beads . IP samples and cell lysates were subjected to immunoblotting with α - HA , α - ERGIC - 53 , α - BET1 , α - SEC22b , α - GOSR2 and α - GAPDH antibodies , respectively . ( E ) Subcellular localization of ERGIC - 53 in fibroblasts by indirect immunofluorescent confocal microscopy . Representative immunofluorescence images of ERGIC - 53 ( green ) and GM130 ( red ) in fibroblasts from P1 , P2 and a control cell line . Scale bar : 20 µm . PCC ( right panel ) for GM130 versus ERGIC - 53 ( C1 : n = 74 , C2 : n = 95 , C3 : n = 59 , P1 : n = 72 and P2 : n = 76 ) . ( * * * = p ≤ 0 . 001 , one - way ANOVA was followed by Tukey ' s post - hoc test ) . Shown are means ± SEM . Immunofluorescence images shown are one example of a total of two biological replicates . ( F ) Colocalization of ERGIC - 53 and BET1 in control and P2 - derived fibroblasts . Representative immunofluorescence images of ERGIC - 53 ( green ) and BET1 ( red ) in fibroblasts from P2 and a control cell line . Scale bar : 20 µm . PCC ( right panel ) for BET1 versus ERGIC - 53 ( C1 : n = 72 , C2 : n = 46 , C3 : n = 56 and P2 : n = 37 ) . ( * * * = p ≤ 0 . 001 , one - way ANOVA was followed by Tukey ' s post - hoc test ) . Data represented are mean ± SEM of two independent experiments . ( H ) The number of cells with intact Golgi was divided by the number of total cells per image and plotted against time ( three biological replicates ) . Dots are means ± SD . For quantification , a sigmoidal fit was performed with the software GraphPad Prism 7 . Dashed lines indicate extrapolated plateaus . LogIC50 ± 95 % confidence interval : C1 = 59 . 38 ± 2 . 31 min , P1 = 72 . 89 ± 1 . 65 min , P2 = 68 . 57 ± 1 . 929 min . Significant differences were assumed when 95 % confidence interval do not overlap .

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Figure 5(A) Volcano plot of AP-MS data using HA-BET1 or Ile51Ser.(B) Co-IPs using HA-tagged constructs expressed in HEK293 cells. As baits HA-tagged proteins were immunoprecipitated with anti-HA beads. IP samples and cell lysates were subjected to immunoblotting with α-HA, α-ERGIC-53, α-BET1, α-SEC22b, α-GOSR2 and α-GAPDH antibodies, respectively.(E) Subcellular localization of ERGIC-53 in fibroblasts by indirect immunofluorescent confocal microscopy. Representative immunofluorescence images of ERGIC-53 (green) and GM130 (red) in fibroblasts from P1, P2 and a control cell line. Scale bar: 20 µm. PCC (right panel) for GM130 versus ERGIC-53 (C1: n=74, C2: n=95, C3: n=59, P1: n=72 and P2: n=76). (*** = p≤ 0.001, one-way ANOVA was followed by Tukey's post-hoc test). Shown are means ± SEM. Immunofluorescence images shown are one example of a total of two biological replicates.(F) Colocalization of ERGIC-53 and BET1 in control and P2-derived fibroblasts. Representative immunofluorescence images of ERGIC-53 (green) and BET1 (red) in fibroblasts from P2 and a control cell line. Scale bar: 20 µm. PCC (right panel) for BET1 versus ERGIC-53 (C1: n=72, C2: n=46, C3: n=56 and P2: n=37). (*** = p≤ 0.001, one-way ANOVA was followed by Tukey's post-hoc test). Data represented are mean ± SEM of two independent experiments.(H) The number of cells with intact Golgi was divided by the number of total cells per image and plotted against time (three biological replicates). Dots are means ±SD. For quantification, a sigmoidal fit was performed with the software GraphPad Prism 7. Dashed lines indicate extrapolated plateaus. LogIC50 ±95% confidence interval: C1 = 59.38±2.31 min, P1 = 72.89±1.65 min, P2 = 68.57±1.929 min. Significant differences were assumed when 95% confidence interval do not overlap.


Figure 2 ( B ) HA - ATG9A - BirA * was expressed in HCT - 116 ATG13 WT , ATG13 KO or ATG13 KO cells reconstituted with WT ATG13 or ATG13 ∆ HORMA . Cells were grown in full DMEM media , treated with 50 µM biotin for 12 hrs , followed by detergent lysis and incubation with streptavidin resin . The graph on right shows quantification of normalized ATG101 infrared signal . Mean ± SEM , n = 3 ( biological replicates ) . Significance measured using RM one - way ANOVA test followed by Fisher ' s LSD tests . ( C ) Cells were treated as in panel B but included reconstitution with ATG13 ∆ 2AA mutant . The graph on right shows quantification of normalized ATG101 infrared signal . Mean ± SEM , n = 3 ( biological replicates ) . Significance measured using RM one - way ANOVA test followed by Fisher ' s LSD tests . ( right ) . ( D ) HA - ATG9A - BirA * was overexpressed in WT and ULK1 / 2 Double KO MEFs . Cells were subjected to streptavidin pulldown and immunoblotting with indicated antibodies . The graph on right shows quantification of normalized ATG13 infrared signal . Mean ± SEM . n = 3 ( biological replicates ) . Significance measured using one - sample t - test compared to hypothetical mean of 1 ( right ) . ns = p > 0 . 05 , * = p ≤ 0 . 05 , * * = p ≤ 0 . 01 , * * * = p ≤ 0 . 001 , * * * * = p ≤ 0 . 0001 .

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Figure 2(B) HA-ATG9A-BirA* was expressed in HCT-116 ATG13 WT, ATG13 KO or ATG13 KO cells reconstituted with WT ATG13 or ATG13 ∆HORMA. Cells were grown in full DMEM media, treated with 50 µM biotin for 12 hrs, followed by detergent lysis and incubation with streptavidin resin. The graph on right shows quantification of normalized ATG101 infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests.(C) Cells were treated as in panel B but included reconstitution with ATG13 ∆2AA mutant. The graph on right shows quantification of normalized ATG101 infrared signal. Mean ± SEM, n=3 (biological replicates). Significance measured using RM one-way ANOVA test followed by Fisher's LSD tests. (right).(D) HA-ATG9A-BirA* was overexpressed in WT and ULK1/2 Double KO MEFs. Cells were subjected to streptavidin pulldown and immunoblotting with indicated antibodies. The graph on right shows quantification of normalized ATG13 infrared signal. Mean ± SEM. n=3 (biological replicates). Significance measured using one-sample t-test compared to hypothetical mean of 1 (right). ns=p>0.05, *=p≤0.05, **= p≤0.01, ***=p≤0.001, ****= p≤0.0001.


Figure 3 ( A ) - ( G ) Representative images of ATG9A colocalization with different organelle and autophagy markers . HCT - 116 ATG9A - HA KI - ATG13 WT or ATG13 KO cells were grown in full DMEM media , fixed and labeled with antibodies for HA and indicated organelle / cellular markers and imaged ( Scaler bar = 10 µm ) . ( H ) Quantification of ATG9A colocalization with indicated organelle markers of golgi ( GOLGIN97 ) , mitochondria ( TUFM ) , ER ( PDIA3 ) , endosomal system ( VPS26A ) , early endosome ( EEA1 ) , lysosome ( LAMP1 ) , autophagy adaptor ( p62 ) , and autophagosome markers ( ATG16L1 , WIPI4 , ATG2A , LC3II , WIPI2 ) respectively . Mean ± SEM , n = 1 independent experiment with 30 technical replicates . Images for p62 colocalization ( G ) are from the same single independent

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Figure 3(A)-(G) Representative images of ATG9A colocalization with different organelle and autophagy markers. HCT-116 ATG9A-HA KI-ATG13 WT or ATG13 KO cells were grown in full DMEM media, fixed and labeled with antibodies for HA and indicated organelle/cellular markers and imaged (Scaler bar=10 µm). (H) Quantification of ATG9A colocalization with indicated organelle markers of golgi (GOLGIN97), mitochondria (TUFM), ER (PDIA3), endosomal system (VPS26A), early endosome (EEA1), lysosome (LAMP1), autophagy adaptor (p62), and autophagosome markers (ATG16L1, WIPI4, ATG2A, LC3II, WIPI2) respectively. Mean ± SEM, n=1 independent experiment with 30 technical replicates. Images for p62 colocalization (G) are from the same single independent experiment