( B ) Full coverage plot of chromosome 17 centromeric array ( chr17 : 22 , 500 , 000 - 26 , 900 , 000 of the hg38 assembly ) showing enrichment of CENP - A by CUT & RUN - seq in the untreated ( NT ) and IAA wash out ( WO ) sample . CENP - A is reloaded to cen17 _ 1 ( D17Z1 ) . A shift of CENP - A occupancy to cen17 _ 2 ( D17Z1B ) is not detected .

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

(B) Full coverage plot of chromosome 17 centromeric array (chr17:22,500,000-26,900,000 of the hg38 assembly) showing enrichment of CENP-A by CUT&RUN-seq in the untreated (NT) and IAA wash out (WO) sample. CENP-A is reloaded to cen17_1 (D17Z1). A shift of CENP-A occupancy to cen17_2 (D17Z1B) is not detected.


( F ) Representative images showing ectopic CENP - A on a chromosome spread after induction of CENP - A overexpression in DLD - 1 cells . Centromere position is marked using immunofluorescence staining for CENP - B . Yellow dashed lines contour representative chromosomes . Scale bar , 2µm . Schematic to the right illustrate the observed CENP - AmRFP - DHFR localization pattern on a chromosome . Right panel : Comparison of CENP - ADHFR level per pixel at the centromere and on chromosome arms following IAA and DOX / TMP treatment . Each dot represents one centromere or a region on the chromosome arms .

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(F) Representative images showing ectopic CENP-A on a chromosome spread after induction of CENP-A overexpression in DLD-1 cells. Centromere position is marked using immunofluorescence staining for CENP-B. Yellow dashed lines contour representative chromosomes. Scale bar, 2µm. Schematic to the right illustrate the observed CENP-AmRFP-DHFR localization pattern on a chromosome. Right panel: Comparison of CENP-ADHFR level per pixel at the centromere and on chromosome arms following IAA and DOX/TMP treatment. Each dot represents one centromere or a region on the chromosome arms.


( G ) Schematic illustration for the experiments shown in H , I . ( H ) Representative images of chromosome spreads after re - expression of endogenous CENP - AEA into a CENP - AmRFP - DHFR overexpression background and subsequent removal of the overexpression as illustrated in ( G ) . Antibody against CENP - C was used to score for the presence of the functional centromere . Scale bar , 5µm . ( I ) Quantification of the indicated events after one CENP - AOFF / ON cycle compared to treatment shown in ( G ) .

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(G) Schematic illustration for the experiments shown in H, I. (H) Representative images of chromosome spreads after re-expression of endogenous CENP-AEA into a CENP-AmRFP-DHFR overexpression background and subsequent removal of the overexpression as illustrated in (G). Antibody against CENP-C was used to score for the presence of the functional centromere. Scale bar, 5µm. (I) Quantification of the indicated events after one CENP-AOFF/ON cycle compared to treatment shown in (G).


( A ) Schematic illustration of the CENP - AOFF / ON cycle performed in the experiments shown in B , C . ( B ) Representative images of de novo CENP - A reloading in CENP - B wild - type ( + / + ) and CENP - B knock - out ( - / - ) cells . Cells with centromeric CENP - A are marked with a red dashed contour line while a yellow contour lines mark cell without centromeric CENP - A . Scale bar , 5 µm . ( C ) Quantification of relative number of RPE - 1 cells with centromeric CENP - A in the indicated conditions . Each dot represents one experiment with at least 20 cells per condition . Error bars represent standard error of the mean ( SEM ) from 3 independent experiments .

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(A) Schematic illustration of the CENP-AOFF/ON cycle performed in the experiments shown in B, C. (B) Representative images of de novo CENP-A reloading in CENP-B wild-type (+/+) and CENP-B knock-out (-/-) cells. Cells with centromeric CENP-A are marked with a red dashed contour line while a yellow contour lines mark cell without centromeric CENP-A. Scale bar, 5 µm. (C) Quantification of relative number of RPE-1 cells with centromeric CENP-A in the indicated conditions. Each dot represents one experiment with at least 20 cells per condition. Error bars represent standard error of the mean (SEM) from 3 independent experiments.


( E ) Representative images of DLD - 1 CENP - B ( + / + ) or ( - / - ) cells in late M phase following one CENP - AOFF / ON cycle . EdU staining was used to confirm successful wash - out of Palbociclib and cell progressing through S - phase . Yellow dashed lines contour nucleus of cells in late M phase . Scale bar 5 µm . ( F ) Quantification of indicated events observed in late M phase cells in the indicated cell lines . Error bars show SEM from 4 independent experiments .

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(E) Representative images of DLD-1 CENP-B (+/+) or (-/-) cells in late M phase following one CENP-AOFF/ON cycle. EdU staining was used to confirm successful wash-out of Palbociclib and cell progressing through S-phase. Yellow dashed lines contour nucleus of cells in late M phase. Scale bar 5 µm. (F) Quantification of indicated events observed in late M phase cells in the indicated cell lines. Error bars show SEM from 4 independent experiments.


( C ) Representative IF - FISH images of mitotic spreads in non - treated cell ( NT ) and following a CENP - AOFF / ON cycle ( IAA WO ) . Chromosomes are contoured by a green ( X ) , magenta ( Y ) or white ( other autosomes ) dashed line . The centromeres of chromosome X / Y are highlighted by a green / magenta arrow . Scale bar , 2 µm .

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(C) Representative IF-FISH images of mitotic spreads in non-treated cell (NT) and following a CENP-AOFF/ON cycle (IAA WO). Chromosomes are contoured by a green (X), magenta (Y) or white (other autosomes) dashed line. The centromeres of chromosome X/Y are highlighted by a green/magenta arrow. Scale bar, 2 µm.


( D ) Quantification of CENP - C ( as read - out of CENP - A ) presence at the X ( green ) or Y ( magenta ) centromere in the indicated cell lines . Each dot represents one experiment . Error bars represent SD from 3 independent experiments . Unpaired t test , * * p = 0 . 0042 .

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(D) Quantification of CENP-C (as read-out of CENP-A) presence at the X (green) or Y (magenta) centromere in the indicated cell lines. Each dot represents one experiment. Error bars represent SD from 3 independent experiments. Unpaired t test, **p=0.0042.


( G ) Representative IF - FISH images showing CENP - C localization on the Y chromosome in cell growing under selective pressure ( G418 ) following a CENP - AOFF / ON cycle . Chromosomes are highlighted by a white dashed contour line . CENP - C was found at the native centromere position ( top panel , blue ) , the Y chromosome fused to another chromosome indicated by the increased size of the chromosome and the absence of Chr - Y painting probe staining ( second panel , orange ) or CENP - C was found on a different location on the Y chromosome and did not overlap with Cen - Y DNA ( lower panels , red ) . Scale bar , 2 µm . ( H ) Quantification of indicated events depicted in G . Each dot represents one experiment ( > 20 spreads for experiment ) . Error bars represent SEM from 3 independent experiments . Unpaired t - test , * p = 0 . 0137 and 0 . 039 .

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

(G) Representative IF-FISH images showing CENP-C localization on the Y chromosome in cell growing under selective pressure (G418) following a CENP-AOFF/ON cycle. Chromosomes are highlighted by a white dashed contour line. CENP-C was found at the native centromere position (top panel, blue), the Y chromosome fused to another chromosome indicated by the increased size of the chromosome and the absence of Chr-Y painting probe staining (second panel, orange) or CENP-C was found on a different location on the Y chromosome and did not overlap with Cen-Y DNA (lower panels, red). Scale bar, 2 µm. (H) Quantification of indicated events depicted in G. Each dot represents one experiment (>20 spreads for experiment). Error bars represent SEM from 3 independent experiments. Unpaired t-test, *p=0.0137 and 0.039.


( B ) Representative immunofluorescence images showing CENP - C recruitment at the LacO array in the indicated conditions . LacO array is displayed in the insets .

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(B) Representative immunofluorescence images showing CENP-C recruitment at the LacO array in the indicated conditions. LacO array is displayed in the insets.


( C ) Bar plot showing frequency of CENP - C ( orange ) or CENP - A ( green ) recruitment to LacO array in the indicated conditions . Each dot represents one independent experiment ( > 20 cell analyzed for experiment ) .

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(C) Bar plot showing frequency of CENP-C (orange) or CENP-A (green) recruitment to LacO array in the indicated conditions. Each dot represents one independent experiment (>20 cell analyzed for experiment).


( D ) Quantification of CENP - C ( orange ) or CENP - A ( green ) protein levels at the LacO array in the indicated conditions normalized to protein intensities in non - treated conditions at the mCherry - LacI - CENP - B - LacO array using CENP - A or CENP - C antibody . Each dot represents one independent experiment ( > 20 cell analyzed for experiment ) . Error bars represent SEM from 3 independent experiments .

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(D) Quantification of CENP-C (orange) or CENP-A (green) protein levels at the LacO array in the indicated conditions normalized to protein intensities in non-treated conditions at the mCherry-LacI-CENP-B-LacO array using CENP-A or CENP-C antibody. Each dot represents one independent experiment (>20 cell analyzed for experiment). Error bars represent SEM from 3 independent experiments.


( E ) Representative immunofluorescence images showing CENP - A recruitment to ectopic mCherry - LacI - CENP - B in the indicated conditions . LacO array is shown in the inset .

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(E) Representative immunofluorescence images showing CENP-A recruitment to ectopic mCherry-LacI-CENP-B in the indicated conditions. LacO array is shown in the inset.


( F ) Quantification of the frequency of CENP - C recruitment to LacO arrays by different LacI constructs . Error bars represent SEM from 5 independent experiments .

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(F) Quantification of the frequency of CENP-C recruitment to LacO arrays by different LacI constructs. Error bars represent SEM from 5 independent experiments.


( G ) CENP - B immunoblot following GST pulldown experiments using GST tagged CENP - C ( 1 - 727 ) as bait and with the indicated proteins as preys .

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(G) CENP-B immunoblot following GST pulldown experiments using GST tagged CENP-C (1-727) as bait and with the indicated proteins as preys.


( C ) Representative immunofluorescence images showing CENP - A and CENP - C reloading in the indicated conditions . Cells with centromeric CENP - A / CENP - C are marked with a red dashed contour line while a yellow contour lines mark cell without centromeric CENP - A . The centromere position is marked using immunofluorescence staining for CENP - B . White dashed lines contour DAPI stained nucleus of representative cells .

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

(C) Representative immunofluorescence images showing CENP-A and CENP-C reloading in the indicated conditions. Cells with centromeric CENP-A/CENP-C are marked with a red dashed contour line while a yellow contour lines mark cell without centromeric CENP-A. The centromere position is marked using immunofluorescence staining for CENP-B. White dashed lines contour DAPI stained nucleus of representative cells.


( D ) Bar plot showing the percentage of cells with CENP - A and CENP - C co - localization after a CENP - A / COFF / ON cycle in the indicated conditions . Error bars show SD from 5 independent experiments .

(D) Bar plot showing the percentage of cells with CENP-A and CENP-C co-localization after a CENP-A/COFF/ON cycle in the indicated conditions. Error bars show SD from 5 independent experiments.


( E ) Box plot of CUT & RUN qPCR for CENP - A ( green ) or CENP - C ( orange ) . Box plot shows median , 25th and 75th percentiles , whiskers show minimum and maximum values . Results from 3 independent experiments including combined data from qPCR reactions using alpha - satellite primers and primers binding at the centromere of chromosome 4 . Enrichment is measured relative to the IgG control and normalized to Alu repeats .

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(E) Box plot of CUT&RUN qPCR for CENP-A (green) or CENP-C (orange). Box plot shows median, 25th and 75th percentiles, whiskers show minimum and maximum values. Results from 3 independent experiments including combined data from qPCR reactions using alpha-satellite primers and primers binding at the centromere of chromosome 4. Enrichment is measured relative to the IgG control and normalized to Alu repeats.


( C ) Representative immunofluorescence images showing exogenous CENP - AmRFP - DHFR reloading in the presence ( control ) , but not in the absence ( sample ) of endogenous CENP - A and CENP - C . Cells with centromeric CENP - A are marked with a red dashed contour line while a yellow contour line marks cell without centromeric CENP - A . ( D ) Quantification of relative number of cells showing centromeric eCENP - AmRFP - DHFR in the indicated conditions . Error bars represent SEM from 3 independent experiments .

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(C) Representative immunofluorescence images showing exogenous CENP-AmRFP-DHFR reloading in the presence (control), but not in the absence (sample) of endogenous CENP-A and CENP-C. Cells with centromeric CENP-A are marked with a red dashed contour line while a yellow contour line marks cell without centromeric CENP-A. (D) Quantification of relative number of cells showing centromeric eCENP-AmRFP-DHFR in the indicated conditions. Error bars represent SEM from 3 independent experiments.


( E ) Bar plot of CUT & RUN qPCR quantification using CENP - A antibody and primers binding at the centromere of chromosome 4 . Enrichment is measured relative to the IgG control and normalized to Alu repeats .

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(E) Bar plot of CUT&RUN qPCR quantification using CENP-A antibody and primers binding at the centromere of chromosome 4. Enrichment is measured relative to the IgG control and normalized to Alu repeats.


( F ) Representative immunofluorescence chromosome spreads showing reloading of exogenous eCENP - CmRFP - DHFR in the presence ( control ) and in the absence ( sample ) of endogenous CENP - A and CENP - C . Cells were arrested with Colcemid for 3 h prior to spread . Cells with centromeric CENP - C are marked with a red dashed contour line while a yellow contour line marks cell without centromeric CENP - C . Chromosomes in inset are highlighted using a white dashed line . ( G ) Quantification of relative number of cells with eCENP - CmRFP - DHFR in the indicated conditions . Each dot represents one independent experiment with at least 30 cells per condition .

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(F) Representative immunofluorescence chromosome spreads showing reloading of exogenous eCENP-CmRFP-DHFR in the presence (control) and in the absence (sample) of endogenous CENP-A and CENP-C. Cells were arrested with Colcemid for 3 h prior to spread. Cells with centromeric CENP-C are marked with a red dashed contour line while a yellow contour line marks cell without centromeric CENP-C. Chromosomes in inset are highlighted using a white dashed line. (G) Quantification of relative number of cells with eCENP-CmRFP-DHFR in the indicated conditions. Each dot represents one independent experiment with at least 30 cells per condition.


( H ) Bar plot of CUT & RUN qPCR results using CENP - C antibody and primers binding at the centromere of chromosome 4 . Enrichment is measured relative to the IgG control and normalized to Alu repeats . Each dot represents one independent experiment .

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(H) Bar plot of CUT&RUN qPCR results using CENP-C antibody and primers binding at the centromere of chromosome 4. Enrichment is measured relative to the IgG control and normalized to Alu repeats. Each dot represents one independent experiment.


( A ) Gating strategy for the sorting of freshly purified resting human CD4 + T cells based on CENP - A expression . Dead cells were excluded ( first plot ) using Fixable Viability Dye eFluor 780 , CENP - Ahigh and CENP - Alow cells ( both CENP - B + ) were sorted ( second plot ) . Post - sort analysis of each population is then shown ( third and fourth plots ) .

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(A) Gating strategy for the sorting of freshly purified resting human CD4+ T cells based on CENP-A expression. Dead cells were excluded (first plot) using Fixable Viability Dye eFluor 780, CENP-Ahigh and CENP-Alow cells (both CENP-B+) were sorted (second plot). Post-sort analysis of each population is then shown (third and fourth plots).


( B ) Representative images of sorted CD4 + T cells with high or low CENP - A level . The nucleus is contoured by a white dashed line . Scale bar , 5 µm .

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(B) Representative images of sorted CD4+ T cells with high or low CENP-A level. The nucleus is contoured by a white dashed line. Scale bar, 5 µm.


( D ) Tracking of cell division by CFSE dilution . The black line separates cells that have not divided ( nº of div = 0 , CFSE - high ) from those that have undergone at least one division ( nº of div ≥ 1 , CFSE - low ) . ( E ) CENP - A expression and CFSE dilution . Black lines were set based on CENP - A isotype control and CFSE maximal staining at day 0 ( cells in shaded quadrants are CFSE - high / CENP - A - low ) . One representative donor is depicted . ( F ) Absolute number of CFSE - high / CENP - A - low CD4 + T cells ( shaded gate in E ) . Symbols represent individual donors . Error bars represent SEM from 6 independent donors . One - way ANOVA , multiple comparisons , n = 6 . * p < 0 . 05 ; * * p < 0 . 01 . ( G ) Frequency of cells without centromeric CENP - A determined by immunofluorescence microscopy before and post activation . Symbols represent individual donors , n = 4 . Error bars represent SEM from 6 independent donors .

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(D) Tracking of cell division by CFSE dilution. The black line separates cells that have not divided (nº of div=0, CFSE-high) from those that have undergone at least one division (nº of div≥1, CFSE-low). (E) CENP-A expression and CFSE dilution. Black lines were set based on CENP-A isotype control and CFSE maximal staining at day 0 (cells in shaded quadrants are CFSE-high/CENP-A-low). One representative donor is depicted. (F) Absolute number of CFSE-high/CENP-A-low CD4+ T cells (shaded gate in E). Symbols represent individual donors. Error bars represent SEM from 6 independent donors. One-way ANOVA, multiple comparisons, n=6. *p < 0.05; **p < 0.01. (G) Frequency of cells without centromeric CENP-A determined by immunofluorescence microscopy before and post activation. Symbols represent individual donors, n=4. Error bars represent SEM from 6 independent donors.


Representative Western blots showing GAPDH association with mitochondria - enriched fractions in different HD models . The levels of GAPDH were normalized to mitochondrial matrix protein , aconitase . n = 3 . * P = 0 . 0005 ; * * P = 0 . 019 ; # P = 0 . 042 .

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Representative Western blots showing GAPDH association with mitochondria-enriched fractions in different HD models. The levels of GAPDH were normalized to mitochondrial matrix protein, aconitase. n = 3. *P = 0.0005; **P = 0.019; #P = 0.042.


Measurement of GAPDH activity in the mitochondria - enriched fraction isolated from PC12 cells . n = 3 . * P = 0 . 0005 .

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Measurement of GAPDH activity in the mitochondria-enriched fraction isolated from PC12 cells. n = 3. *P = 0.0005.


Western blots showing oxidized GAPDH in the total lysates of PC12 cells and isolated mitochondria . The levels of oxidized GAPDH were normalized to beta - actin ( total lysate ) and aconitase ( mitochondrial fraction ) , respectively . n = 2 . * P = 0 . 023 ; * * P = 0 . 039 .

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Western blots showing oxidized GAPDH in the total lysates of PC12 cells and isolated mitochondria. The levels of oxidized GAPDH were normalized to beta-actin (total lysate) and aconitase (mitochondrial fraction), respectively. n = 2. *P = 0.023; **P = 0.039.


Western blot showing co - immunoprecipitation of GAPDH from the total lysates of PC12 cells in RIPA buffer without detergent ( Triton X - 100 ) . Immunoprecipitated samples were immunoblotted with antibodies against mitochondrial ( VDAC and Tom20 ) , endoplasmic reticulum ( calnexin ) , and lysosomal markers ( LAMP1 ) . ctr : control . n = 2 .

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Western blot showing co-immunoprecipitation of GAPDH from the total lysates of PC12 cells in RIPA buffer without detergent (Triton X-100). Immunoprecipitated samples were immunoblotted with antibodies against mitochondrial (VDAC and Tom20), endoplasmic reticulum (calnexin), and lysosomal markers (LAMP1). ctr: control. n = 2.


Representative electron microscopy showing mitochondrial morphology in PC12 cells expressing Q23 ( top panel , left ) and Q74 ( bottom panel , left ) and in brain tissues from wild - type ( WT ) ( top panel , right ) and HD transgenic mice ( bottom panel , right ) . An indicated portion of each image ( in yellow ) is expanded to illustrate the mitochondrial morphology . Scale bars : 1 μm .

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Representative electron microscopy showing mitochondrial morphology in PC12 cells expressing Q23 (top panel, left) and Q74 (bottom panel, left) and in brain tissues from wild-type (WT) (top panel, right) and HD transgenic mice (bottom panel, right). An indicated portion of each image (in yellow) is expanded to illustrate the mitochondrial morphology. Scale bars: 1 μm.


Measurement of mitochondrial ROS production ( using Mitosox ) in different HD models . Results are presented as percent of control [ Q23 , normal fibroblast ( Nor ) , Q7 ] . n = 3 . * P = 0 . 001 ; * * P = 0 . 0001 ; * * * P = 0 . 0001 ; # P = 0 . 0045 .

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Measurement of mitochondrial ROS production (using Mitosox) in different HD models. Results are presented as percent of control [Q23, normal fibroblast (Nor), Q7]. n = 3. *P = 0.001; **P = 0.0001; ***P = 0.0001; #P = 0.0045.


Mitochondrial membrane potential measured by using JC - 1 dye in models of HD and control cells ( MMP , ΔΨm ) . n = 3 . * P = 0 . 008 ; * * P = 0 . 044 ; * * * P = 0 . 025 ; # P = 0 . 005 .

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Mitochondrial membrane potential measured by using JC-1 dye in models of HD and control cells (MMP, ΔΨm). n = 3. *P = 0.008; **P = 0.044; ***P = 0.025; #P = 0.005.


Mitochondrial respiratory function in PC12 cells , represented by respiratory control ratio ( RCR ; [ state3 ] / [ state4 ] ) . n = 2 . * P = 0 . 028 .

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Mitochondrial respiratory function in PC12 cells, represented by respiratory control ratio (RCR; [state3]/[state4]). n = 2. *P = 0.028.


ATP levels in HD patient - derived fibroblast cells . n = 2 . * P = 0 . 003 ; * * P = 0 . 004 .

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ATP levels in HD patient-derived fibroblast cells. n = 2. *P = 0.003; **P = 0.004.


Western blots showing cytochrome c release from mitochondria . n = 2 . * P = 0 . 003 ; * * P = 0 . 003 ; * * * P = 0 . 01 ; # P = 0 . 008 .

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Western blots showing cytochrome c release from mitochondria. n = 2. *P = 0.003; **P = 0.003; ***P = 0.01; #P = 0.008.


Cell viability determined by colorimetric assays utilizing WST8 conversion to WST8 formazan in different HD models . n = 3 . * P , * * P , * * * P , # P < 0 . 005 .

Cell viability determined by colorimetric assays utilizing WST8 conversion to WST8 formazan in different HD models. n = 3. *P, **P, ***P, #P < 0.005.


Representative immunofluorescence of normal and HD patient - derived fibroblasts stained for GAPDH and mitochondrial ROS with Mitosox . Three independent images per condition were analyzed to calculate Mitosox intensity and the Pearson ' s correlation coefficient for quantification of colocalization between GAPDH and mitochondrial ROS . # P = 0 . 03 ; # # P = 0 . 02 ; * P = 0 . 006 ; * * P = 0 . 005 . Images were acquired at 63 × magnification , and brightness and contrast of images were adjusted by 50 % .

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

Representative immunofluorescence of normal and HD patient-derived fibroblasts stained for GAPDH and mitochondrial ROS with Mitosox. Three independent images per condition were analyzed to calculate Mitosox intensity and the Pearson's correlation coefficient for quantification of colocalization between GAPDH and mitochondrial ROS. #P = 0.03; ##P = 0.02; *P = 0.006; **P = 0.005. Images were acquired at 63× magnification, and brightness and contrast of images were adjusted by 50%.


A Representative Western blots showing separation of lysosome - and mitochondria - enriched fractions by a density gradient . Total extracts from different HD models were fractionated by the density gradient . Eight fractions were collected from top ( # 1 ) to the bottom ( # 8 ) . The amount of mitochondria in the lysosomal fractions was presented as a percentage of VDAC present in the entire gradient . n = 3 .

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

A Representative Western blots showing separation of lysosome- and mitochondria-enriched fractions by a density gradient. Total extracts from different HD models were fractionated by the density gradient. Eight fractions were collected from top (#1) to the bottom (#8). The amount of mitochondria in the lysosomal fractions was presented as a percentage of VDAC present in the entire gradient. n = 3.


B , C Representative electron microscopy of PC12 cells expressing Q23 or Q74 and brain tissues from wild - type ( WT ) and HD transgenic R6 / 2 mice showing mitochondrial ( as indicated by yellow arrows ) association with lysosomal vacuoles ( shown by red arrows ) . The vacuoles containing mitochondrial remnants are presented as a percentage of the total vacuoles counted . Analysis ( of six independent images ) was carried out by an observer blinded to the experimental conditions . * P < 0 . 023 ; * * P < 0 . 035 . Scale bar : 1 μm .

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B, C Representative electron microscopy of PC12 cells expressing Q23 or Q74 and brain tissues from wild-type (WT) and HD transgenic R6/2 mice showing mitochondrial (as indicated by yellow arrows) association with lysosomal vacuoles (shown by red arrows). The vacuoles containing mitochondrial remnants are presented as a percentage of the total vacuoles counted. Analysis (of six independent images) was carried out by an observer blinded to the experimental conditions. *P < 0.023; **P < 0.035. Scale bar: 1 μm.


D Western blot showing association of mitochondrial membrane with lysosomal membrane . The presence of membranes of each organelle was assessed using anti - MFN1 , VDAC ( as mitochondria markers ) , and LAMP1 ( as a lysosomal marker ) . n = 3 .

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D Western blot showing association of mitochondrial membrane with lysosomal membrane. The presence of membranes of each organelle was assessed using anti-MFN1, VDAC (as mitochondria markers), and LAMP1 (as a lysosomal marker). n = 3.


E Representative Western blot showing mitochondrial contents ( VDAC , Tom20 , and aconitase ) in the total lysates of PC12 cells with Q23 or Q74 . Mitochondrial protein levels were normalized to beta - actin . n = 3 . * P = 0 . 024 ; * * P = 0 . 0008 ; * * * P = 0 . 03 .

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E Representative Western blot showing mitochondrial contents (VDAC, Tom20, and aconitase) in the total lysates of PC12 cells with Q23 or Q74. Mitochondrial protein levels were normalized to beta-actin. n = 3. *P = 0.024; **P = 0.0008; ***P = 0.03.


F Representative Western blot showing mitochondrial contents ( VDAC , Tom20 , and aconitase ) in the total lysates of normal fibroblast cells and HD patient - derived fibroblasts . Mitochondrial protein levels were normalized to the cytosolic protein , enolase . n = 3 . * P = 0 . 008 ; * * P = 0 . 002 ; # P = 0 . 0008 .

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F Representative Western blot showing mitochondrial contents (VDAC, Tom20, and aconitase) in the total lysates of normal fibroblast cells and HD patient-derived fibroblasts. Mitochondrial protein levels were normalized to the cytosolic protein, enolase. n = 3. *P = 0.008; **P = 0.002; #P = 0.0008.


G Representative Western blot showing mitochondrial contents ( VDAC , Tom20 , and aconitase ) in the total lysates of mice striatal cells with Q7 and Q111 . n = 3 . * P = 0 . 0008 ; * * P = 0 . 0007 ; # P = 0 . 001 .

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G Representative Western blot showing mitochondrial contents (VDAC, Tom20, and aconitase) in the total lysates of mice striatal cells with Q7 and Q111. n = 3. *P = 0.0008; **P = 0.0007; #P = 0.001.


H Western blot showing mitochondrial contents ( Tom20 and aconitase ) in the total lysates of brain tissues from wild - type ( WT ) and HD transgenic mice . n = 2 . * P = 0 . 02 ; * * P = 0 . 03 * § P = 0 . 01 ; * * § P < 0 . 04 .

H Western blot showing mitochondrial contents (Tom20 and aconitase) in the total lysates of brain tissues from wild-type (WT) and HD transgenic mice. n = 2. *P = 0.02; **P = 0.03 *§P = 0.01; **§P < 0.04.


Western blots showing co - immunoprecipitation of GAPDH from the total lysates and isolated mitochondria of PC12 cells with anti - GAPDH antibody . Anti - EGFP antibody detects the presence of polyglutamine repeats . Bottom panel shows co - immunoprecipitation of GAPDH in the total lysate of PC12 cells treated with formaldehyde to cross - link proteins . Ctr * = control . n = 3 .

Western blots showing co-immunoprecipitation of GAPDH from the total lysates and isolated mitochondria of PC12 cells with anti-GAPDH antibody. Anti-EGFP antibody detects the presence of polyglutamine repeats. Bottom panel shows co-immunoprecipitation of GAPDH in the total lysate of PC12 cells treated with formaldehyde to cross-link proteins. Ctr* = control. n = 3.


Western blots showing co - immunoprecipitation of GAPDH from the total lysate ( left ) and isolated mitochondria ( right ) of brain tissues of wild - type ( WT ) and transgenic R6 / 2 ( HD ) mice with anti - GAPDH antibody . The presence of huntingtin was examined with anti - huntingtin antibody and anti - polyglutamine antibody , respectively . Co - immunoprecipitated huntingtin or polyglutamine ( polyQ ) was normalized to GAPDH . Left : n = 2 . * P = 0 . 03 ; * * P = 0 . 02 . Right : One representative experiment from two repeats .

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Western blots showing co-immunoprecipitation of GAPDH from the total lysate (left) and isolated mitochondria (right) of brain tissues of wild-type (WT) and transgenic R6/2 (HD) mice with anti-GAPDH antibody. The presence of huntingtin was examined with anti-huntingtin antibody and anti-polyglutamine antibody, respectively. Co-immunoprecipitated huntingtin or polyglutamine (polyQ) was normalized to GAPDH. Left: n = 2. *P = 0.03; **P = 0.02. Right: One representative experiment from two repeats.


Sub - mitochondrial localization of GAPDH and expanded polyglutamine repeats . Isolated mitochondria were left untreated or subjected to the proteinase K treatment in the presence or absence of detergent ( Triton X - 100 ) . n = 3 .

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

Sub-mitochondrial localization of GAPDH and expanded polyglutamine repeats. Isolated mitochondria were left untreated or subjected to the proteinase K treatment in the presence or absence of detergent (Triton X-100). n = 3.


Representative Western blot showing levels of mitochondrial proteins , as indicated by the presence of aconitase , VDAC , and Tom20 , in the total lysates of PC12 cells with Q23 or Q74 before ( represented as NT : no treatment ) and after overexpression of inactive GAPDH ( represented as G ) . n = 3 . * P < 0 . 035 ; * * P = 0 . 001 ; # P = 0 . 001 .

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Representative Western blot showing levels of mitochondrial proteins, as indicated by the presence of aconitase, VDAC, and Tom20, in the total lysates of PC12 cells with Q23 or Q74 before (represented as NT: no treatment) and after overexpression of inactive GAPDH (represented as G). n = 3. *P < 0.035; **P = 0.001; #P = 0.001.


Representative Western blot showing levels of mitochondrial proteins in the total lysates of mice striatal cells with Q7 or Q111 before ( NT ) and after overexpression of inactive GAPDH ( G ) . n = 2 . * P = 0 . 006 ; * * P = 0 . 007 ; # P = 0 . 001 .

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Representative Western blot showing levels of mitochondrial proteins in the total lysates of mice striatal cells with Q7 or Q111 before (NT) and after overexpression of inactive GAPDH (G). n = 2. *P = 0.006; **P = 0.007; #P = 0.001.


Determination of mitochondrial ROS production in different HD models . Results are shown as percent of control . n = 3 . * P = 0 . 008 ; # P = 0 . 054 ; * * P = 0 . 03 .

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Determination of mitochondrial ROS production in different HD models. Results are shown as percent of control. n = 3. *P = 0.008; #P = 0.054; **P = 0.03.


Determination of mitochondrial membrane potential using JC - 1 in different HD models and control cells ( MMP , ΔΨm ) . The difference in membrane potential between NT ( no inactive GAPDH overexpression ) and G ( inactive GAPDH overexpression ) in cells with expanded polyglutamine repeats ( PC12 cells with Q74 and HD patient - derived fibroblasts ) was not statistically significant . n = 3 .

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Determination of mitochondrial membrane potential using JC-1 in different HD models and control cells (MMP, ΔΨm). The difference in membrane potential between NT (no inactive GAPDH overexpression) and G (inactive GAPDH overexpression) in cells with expanded polyglutamine repeats (PC12 cells with Q74 and HD patient-derived fibroblasts) was not statistically significant. n = 3.


ATP levels in normal and HD patient - derived fibroblasts . The difference in ATP levels between NT ( no inactive GAPDH overexpression ) and G ( inactive GAPDH overexpression ) in HD patient - derived fibroblasts was not statistically significant . n = 2 .

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ATP levels in normal and HD patient-derived fibroblasts. The difference in ATP levels between NT (no inactive GAPDH overexpression) and G (inactive GAPDH overexpression) in HD patient-derived fibroblasts was not statistically significant. n = 2.


Cell viability determined by colorimetric assays in different HD models and control cells . n = 3 . * P = 0 . 02 ; * * P = 0 . 02 ; # # P = 0 . 01 .

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Cell viability determined by colorimetric assays in different HD models and control cells. n = 3. *P = 0.02; **P = 0.02; ##P = 0.01.


Immunofluorescence of normal and HD patient - derived fibroblasts , with and without overexpression of inactive GAPDH , stained for late endosome with anti - Rab7 antibody and mitochondria with anti - Tom20 antibody . Images were acquired at 63 × magnification , and brightness and contrast of all images were adjusted by 30 % . Correlation coefficient ( juvenile ) : * * P = 0 . 007 ; * # P = 0 . 005 . Mitochondrial interconnectivity ( juvenile ) : # # P = 0 . 001 ; # # * P = 0 . 02 . Correlation coefficient ( adult ) : § § P = 0 . 001 ; * § P = 0 . 005 . Mitochondrial interconnectivity ( adult ) : ≠ ≠ P = 0 . 043 ; ≠ ≠ * P = 0 . 015 .

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Immunofluorescence of normal and HD patient-derived fibroblasts, with and without overexpression of inactive GAPDH, stained for late endosome with anti-Rab7 antibody and mitochondria with anti-Tom20 antibody. Images were acquired at 63× magnification, and brightness and contrast of all images were adjusted by 30%. Correlation coefficient (juvenile): **P = 0.007; *#P = 0.005. Mitochondrial interconnectivity (juvenile): ##P = 0.001; ##*P = 0.02. Correlation coefficient (adult): §§P = 0.001; *§P = 0.005. Mitochondrial interconnectivity (adult): ≠≠P = 0.043; ≠≠*P = 0.015.


Representative Western blot showing levels of mitochondrial proteins , as indicated by the presence of aconitase , VDAC , and Tom20 , in the total lysates of PC12 cells with Q23 or Q74 in an in vitro reconstituted micro - mitophagic system . NT : no inactive GAPDH treatment ; G : inactive GAPDH treatment . n = 3 . * P = 0 . 0007 ; * * P = 0 . 001 ; # P = 0 . 008 .

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Representative Western blot showing levels of mitochondrial proteins, as indicated by the presence of aconitase, VDAC, and Tom20, in the total lysates of PC12 cells with Q23 or Q74 in an in vitro reconstituted micro-mitophagic system. NT: no inactive GAPDH treatment; G: inactive GAPDH treatment. n = 3. *P = 0.0007; **P = 0.001; #P = 0.008.


Representative Western blots showing levels of mitochondrial proteins ( aconitase and Tom20 ) in the total lysates of fibroblast cells ( juvenile and adult ) and mice striatal cells with Q7 or Q111 in an in vitro reconstituted mitophagic system . n = 2 . § P = 0 . 01 ; § § P = 0 . 02 ; * P = 0 . 04 ; * * P = 0 . 02 ; # P = 0 . 001 ; # # P = 0 . 024 .

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Representative Western blots showing levels of mitochondrial proteins (aconitase and Tom20) in the total lysates of fibroblast cells (juvenile and adult) and mice striatal cells with Q7 or Q111 in an in vitro reconstituted mitophagic system. n = 2. §P = 0.01; §§P = 0.02; *P = 0.04; **P = 0.02; #P = 0.001; ##P = 0.024.


Representative Western blot showing levels of mitochondrial proteins ( aconitase and Tom20 ) in the total lysates of PC12 cells . The cells were treated with 1 mM of 3 - methyladenine ( 3MA ) for 5 h prior to the micro - mitophagy reconstitution assay . n = 2 . * P = 0 . 001 ; * * P = 0 . 01 ; * # P = 0 . 003 ; * * # P = 0 . 01 ; # P = 0 . 02 ; § P = 0 . 049 ; # § P = 0 . 004 ; # # § P = 0 . 007 .

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

Representative Western blot showing levels of mitochondrial proteins (aconitase and Tom20) in the total lysates of PC12 cells. The cells were treated with 1 mM of 3-methyladenine (3MA) for 5 h prior to the micro-mitophagy reconstitution assay. n = 2. *P = 0.001; **P = 0.01; *#P = 0.003; **#P = 0.01; #P = 0.02; §P = 0.049; #§P = 0.004; ##§P = 0.007.


Western blot showing the level of Atg5 expression in the Atg5 + / + ( WT ) and Atg5 − / − ( KO ) MEF cells .

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Western blot showing the level of Atg5 expression in the Atg5+/+ (WT) and Atg5−/− (KO) MEF cells.


Western blot showing levels of GAPDH association with mitochondria in Atg5 KO MEF cells . n = 2 . * P = 0 . 04 .

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Western blot showing levels of GAPDH association with mitochondria in Atg5 KO MEF cells. n = 2. *P = 0.04.


Western blot showing relative mitochondrial mass in the total lysates of Atg5 KO MEF cells . n = 2 . * * P = 0 . 03 ; * * * P = 0 . 02 . Expression levels of Q23 or Q73 were detected with anti - His antibody .

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Western blot showing relative mitochondrial mass in the total lysates of Atg5 KO MEF cells. n = 2. **P = 0.03; ***P = 0.02. Expression levels of Q23 or Q73 were detected with anti-His antibody.


Western blot showing relative mitochondrial mass in the total lysates of Atg5 KO MEF cells without and with recombinant inactive GAPDH treatment ( represented as NT and G , respectively ) . n = 2 . * P = 0 . 02 ; * * P = 0 . 03 ; * # P = 0 . 03 ; # P = 0 . 005 .

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Western blot showing relative mitochondrial mass in the total lysates of Atg5 KO MEF cells without and with recombinant inactive GAPDH treatment (represented as NT and G, respectively). n = 2. *P = 0.02; **P = 0.03; *#P = 0.03; #P = 0.005.


( C ) Representative traces obtained with ADP • BeFx . The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor ( green ) and acceptor ( red ) fluorescence . The lowest trace was obtained by exciting the acceptor fluorophore directly . The arrow indicates a bleaching event . ( D ) Distribution of FRET values determined from 97 traces as in ( C ) fit with a Gaussian model ( black curve ) .

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(C) Representative traces obtained with ADP•BeFx. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event. (D) Distribution of FRET values determined from 97 traces as in (C) fit with a Gaussian model (black curve).


( E ) As in ( C ) , but in the presence of ATP . Periods in which a fluorescently labeled SecA molecule is bound are indicated by grey shading . ( F ) As in ( D ) , but with ATP ( 257 traces ) .

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(E) As in (C), but in the presence of ATP. Periods in which a fluorescently labeled SecA molecule is bound are indicated by grey shading. (F) As in (D), but with ATP (257 traces).


( A ) A representative FRET trace ( blue line ) was obtained as in Fig 1 , and fit with a hidden Markov model ( black dashed line ) .

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(A) A representative FRET trace (blue line) was obtained as in Fig 1, and fit with a hidden Markov model (black dashed line).


( B ) Traces as in ( A ) obtained in the presence of different nucleotides , were used to determine the number of states best fit by the Markov model . ( C ) Transition density plot of idealized ATP FRET states obtained in ( B ) .

(B) Traces as in (A) obtained in the presence of different nucleotides, were used to determine the number of states best fit by the Markov model. (C) Transition density plot of idealized ATP FRET states obtained in (B).


( D ) The distributions of dwell times of the low FRET states observed in ATP were fit with a single exponential ( 1500 low FRET states ) . The inset shows average dwell time and error , defined as the standard error based on the number of traces . ( E ) As in ( D ) , but with high FRET ( 1656 high FRET states ) .

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(D) The distributions of dwell times of the low FRET states observed in ATP were fit with a single exponential (1500 low FRET states). The inset shows average dwell time and error, defined as the standard error based on the number of traces. (E) As in (D), but with high FRET (1656 high FRET states).


( F ) Representative traces obtained with ATPγS . The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor ( green ) and acceptor ( red ) fluorescence . The lowest trace was obtained by exciting the acceptor fluorophore directly . The arrow indicates a bleaching event . ( G ) Distribution of FRET values determined from 168 traces as in ( D ) fit with a Gaussian model ( black curve ) .

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(F) Representative traces obtained with ATPγS. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event. (G) Distribution of FRET values determined from 168 traces as in (D) fit with a Gaussian model (black curve).


( C ) Representative traces obtained with ADP • BeFx . The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor ( green ) and acceptor ( red ) fluorescence . The lowest trace was obtained by exciting the acceptor fluorophore directly . The arrow indicates a bleaching event . ( D ) Distribution of FRET values determined from 197 traces as in ( B ) fit with a Gaussian model ( black curve ) .

(C) Representative traces obtained with ADP•BeFx. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event. (D) Distribution of FRET values determined from 197 traces as in (B) fit with a Gaussian model (black curve).


( E ) As in ( C ) , but with ATP . Periods in which a fluorescently labeled SecA molecule is bound are indicated by grey shading . ( F ) As in ( D ) , but with ATP ( 228 traces ) .

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(E) As in (C), but with ATP. Periods in which a fluorescently labeled SecA molecule is bound are indicated by grey shading. (F) As in (D), but with ATP (228 traces).


( A ) FRET traces of clamp movements in the presence of different nucleotides were used to determine the number of states best fit by a Markov model . ( B ) Transition density plot of idealized ATP FRET states obtained in ( A ) .

(A) FRET traces of clamp movements in the presence of different nucleotides were used to determine the number of states best fit by a Markov model. (B) Transition density plot of idealized ATP FRET states obtained in (A).


( C ) Representative traces obtained with ATPγS . The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor ( green ) and acceptor ( red ) fluorescence . The lowest trace was obtained by exciting the acceptor fluorophore directly . The arrow indicates a bleaching event . ( D ) Distribution of FRET values determined from 315 traces as in ( C ) fit with a Gaussian model ( black curve ) .

(C) Representative traces obtained with ATPγS. The upper FRET trace was calculated from the middle traces obtained by exciting the donor fluorophore and measuring both donor (green) and acceptor (red) fluorescence. The lowest trace was obtained by exciting the acceptor fluorophore directly. The arrow indicates a bleaching event. (D) Distribution of FRET values determined from 315 traces as in (C) fit with a Gaussian model (black curve).


( E ) Comparison of high and low FRET state occupancy in ADP • Pi and ADP • Vi for the clamp and THF .

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(E) Comparison of high and low FRET state occupancy in ADP•Pi and ADP•Vi for the clamp and THF.


( F ) The distributions of dwell times of the low FRET states observed in ATP were fit with a single exponential ( 1539 low FRET states ) . The inset shows average dwell time and error , defined as the standard error based on the number of traces . ( G ) As in ( F ) , but with high FRET ( 1773 high FRET states ) .

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

(F) The distributions of dwell times of the low FRET states observed in ATP were fit with a single exponential (1539 low FRET states). The inset shows average dwell time and error, defined as the standard error based on the number of traces. (G) As in (F), but with high FRET (1773 high FRET states).


( H ) Comparison of dwell times of the high FRET states for the two - helix finger ( THF ) and clamp for different fluorophore positions . Errors as in ( G ) with significance based on two sample Kolmogorov - Smirnov tests with a 1 % threshold . n . s . P = 0 . 012 ( left ) , n . s . P = 0 . 681 ( right ) ; * P < 1 * 10 - 35 ( between each pair of THF / clamp mutants ) . 809C / 394C : 1656 high FRET states ; 809C / 103C : 1349 states ; 233C / 103C : 1773 states ; 233C / 336C : 1778 states .

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

(H) Comparison of dwell times of the high FRET states for the two-helix finger (THF) and clamp for different fluorophore positions. Errors as in (G) with significance based on two sample Kolmogorov-Smirnov tests with a 1% threshold. n.s.P = 0.012 (left), n.s.P = 0.681 (right); *P < 1*10-35 (between each pair of THF/clamp mutants). 809C/394C: 1656 high FRET states; 809C/103C: 1349 states; 233C/103C: 1773 states; 233C/336C: 1778 states.


A . Confocal microscopy analysis showing the localization of endogenous Rab22a ( green ) and different endocytic markers ( red ) : early endosomal marker EEA1 , late endosome / lysosomal marker Lamp1 and the recycling compartment marker TfR in BMDCs at steady state . The means ± SEM of the Pearson correlation coefficients ( Pcc ) from Rab22a and EEA1 ( 0 . 4317 ± 0 . 05101 ) , Lamp1 ( 0 . 8412 ± 0 . 03562 ) and TfR ( 0 . 6409 ± 0 . 02999 ) were estimated from 15 images analyzed for each marker . Scale bars : 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Confocal microscopy analysis showing the localization of endogenous Rab22a (green) and different endocytic markers (red): early endosomal marker EEA1, late endosome/lysosomal marker Lamp1 and the recycling compartment marker TfR in BMDCs at steady state. The means ± SEM of the Pearson correlation coefficients (Pcc) from Rab22a and EEA1 (0.4317 ± 0.05101), Lamp1 (0.8412 ± 0.03562) and TfR (0.6409 ± 0.02999) were estimated from 15 images analyzed for each marker. Scale bars: 5 µm.


B . Colocalization of endogenous Rab22a ( red ) and EEA1 ( magenta ) around endosomes containing fluorescent soluble OVA ( OVA - FITC , green ) after 30 min of internalization by BMDCs . More than 60 % of the endosomes were positive for Rab22a and EEA1 . The indicated boxes are shown at higher magnification in the insets . Scale bars : 5 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Colocalization of endogenous Rab22a (red) and EEA1 (magenta) around endosomes containing fluorescent soluble OVA (OVA-FITC, green) after 30 min of internalization by BMDCs. More than 60% of the endosomes were positive for Rab22a and EEA1. The indicated boxes are shown at higher magnification in the insets. Scale bars: 5 µm.


C . IF detection of endogenous Rab22a ( green ) and Lamp1 ( red ) after 1 h of phagocytosis of 3 µm latex beads ( LB ) in BMDCs . Around 50 % of the phagosomes were double positive for Rab22a and Lamp1 . Asterisks indicate the LB . Scale bars : 5 µm .

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

C. IF detection of endogenous Rab22a (green) and Lamp1 (red) after 1 h of phagocytosis of 3 µm latex beads (LB) in BMDCs. Around 50% of the phagosomes were double positive for Rab22a and Lamp1. Asterisks indicate the LB. Scale bars: 5 µm.


D . BMDCs were incubated with 3 μm magnetic beads for 15 min at 37 ° C and chased for 0 or 45 min . The panel shows immunoblotting of purified phagosomes and the total cell lysates ( TCL ) analyzed for Lamp1 and Rab22a . A total protein amount of 10 µg and 50 µg was loaded for purified phagosomes and TCL , respectively . The blot is representative of three independent experiments .

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

D. BMDCs were incubated with 3 μm magnetic beads for 15 min at 37°C and chased for 0 or 45 min. The panel shows immunoblotting of purified phagosomes and the total cell lysates (TCL) analyzed for Lamp1 and Rab22a. A total protein amount of 10 µg and 50 µg was loaded for purified phagosomes and TCL, respectively. The blot is representative of three independent experiments.


A . Immunoblotting and densitometry quantification of Rab22a in JAWS - IIDCs infected with lentiviruses encoding a random sequence ( Scramble ) and a shRNA specific for silencing Rab22a ( Rab22a KD ) . Data are representative of at least three independent experiments .

O O O O O O O O O O O O O O O O O O O O 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. Immunoblotting and densitometry quantification of Rab22a in JAWS-IIDCs infected with lentiviruses encoding a random sequence (Scramble) and a shRNA specific for silencing Rab22a (Rab22a KD). Data are representative of at least three independent experiments.


B . RNA extraction and qPCR quantification of Rab22a in the same cells analyzed in A . Data show mean ± SEM of triplicate values and are representative of two independent experiments . * * P = 0 . 0047 .

O O O O O O O O O O O O O O O O O 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. RNA extraction and qPCR quantification of Rab22a in the same cells analyzed in A. Data show mean ± SEM of triplicate values and are representative of two independent experiments. ** P = 0.0047.


C . IF labeling and confocal microscopy analysis showing the distribution of MHC - I molecules ( H - 2Kb , green ) and Rab22a ( red ) in Scramble and Rab22a KD JAWS - II DCs . Nuclei stained with DAPI and DIC images are shown on the left . Overlay is shown in the right panels . Scale bars : 5 µm . Data are representative of at least 30 images analyzed for each experimental condition from three independent experiments .

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

C. IF labeling and confocal microscopy analysis showing the distribution of MHC-I molecules (H-2Kb, green) and Rab22a (red) in Scramble and Rab22a KD JAWS-II DCs. Nuclei stained with DAPI and DIC images are shown on the left. Overlay is shown in the right panels. Scale bars: 5 µm. Data are representative of at least 30 images analyzed for each experimental condition from three independent experiments.


D . FACS analysis of MHC - I labeled in intact ( cell surface ) and permeabilized ( total ) Scramble and Rab22a KD JAWS - II cells . Data represent mean ± SEM of triplicate values and are representative of three independent experiments . P = 0 . 1082 ( ns ) and * * * P = 0 . 0003 .

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

D. FACS analysis of MHC-I labeled in intact (cell surface) and permeabilized (total) Scramble and Rab22a KD JAWS-II cells. Data represent mean ± SEM of triplicate values and are representative of three independent experiments. P = 0.1082 (ns) and ***P = 0.0003.


E . MHC - I staining on isolated phagosomes was measured by FACS at the indicated time periods after 3 µm LB internalization by Scramble and Rab22a KD JAWS - II DCs . Data represent mean ± SEM of three independent experiments . * * * P < 0 . 001 at 3 h and * P < 0 . 05 at 5 h between both DC types .

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

E. MHC-I staining on isolated phagosomes was measured by FACS at the indicated time periods after 3 µm LB internalization by Scramble and Rab22a KD JAWS-II DCs. Data represent mean ± SEM of three independent experiments. ***P < 0.001 at 3 h and *P < 0.05 at 5 h between both DC types.


F . MHC - I molecules recycling ability was measured by FACS at the indicated time periods by Scramble and Rab22a KD JAWS - II DCs . Data represent mean ± SEM of three independent experiments . * P < 0 . 05 at 10 min and * * P < 0 . 01 at 20 and 40 min .

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

F. MHC-I molecules recycling ability was measured by FACS at the indicated time periods by Scramble and Rab22a KD JAWS-II DCs. Data represent mean ± SEM of three independent experiments. *P < 0.05 at 10 min and **P < 0.01 at 20 and 40 min.


G . The transferrin ( Tfn ) recycling ability was measured by FACS at the indicated time periods by Scramble and Rab22a KD JAWS - II DCs . Data represent mean ± SEM of three independent experiments . P > 0 . 05 ( ns ) at 10 , 20 and 40 min .

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

G. The transferrin (Tfn) recycling ability was measured by FACS at the indicated time periods by Scramble and Rab22a KD JAWS-II DCs. Data represent mean ± SEM of three independent experiments. P > 0.05 (ns) at 10, 20 and 40 min.


A - C . The cross - presentation ability after incubation with A ) soluble OVA , B ) OVA / BSA - coated beads and C ) the SIINFEKL control peptide at the indicated concentrations by Scramble and Rab22a KD JAWS - II DCs was evaluated with the B3Z hybridoma . Data represent mean ± SEM of triplicate values and are representative of three independent experiments . A ) * * * P = 0 . 0001 and B ) P = 0 . 1432 ( ns ) ; * * P = 0 . 0044 . The two - tailed Student ' s unpaired t test was performed .

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

A-C. The cross-presentation ability after incubation with A) soluble OVA, B) OVA/BSA-coated beads and C) the SIINFEKL control peptide at the indicated concentrations by Scramble and Rab22a KD JAWS-II DCs was evaluated with the B3Z hybridoma. Data represent mean ± SEM of triplicate values and are representative of three independent experiments. A) ***P = 0.0001 and B) P = 0.1432 (ns); **P = 0.0044. The two-tailed Student's unpaired t test was performed.


D - E . Evaluation of endocytosis and phagocytosis in Scramble and Rab22a KD JAWS - II DCs . D ) The endocytosis of fluorescent OVA after 1 h of internalization and E ) the phagocytosis of 3 µm fluorescent LB at different times of internalization were assessed by FACS analysis . The antigen internalization was conducted at 37 ° C for effective uptake and at 4 ° C as negative control . Data represent mean ± SEM of triplicate values and are representative of three independent experiments .

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

D-E. Evaluation of endocytosis and phagocytosis in Scramble and Rab22a KD JAWS-II DCs. D) The endocytosis of fluorescent OVA after 1 h of internalization and E) the phagocytosis of 3 µm fluorescent LB at different times of internalization were assessed by FACS analysis. The antigen internalization was conducted at 37°C for effective uptake and at 4°C as negative control. Data represent mean ± SEM of triplicate values and are representative of three independent experiments.


F . The kinetics of OVA degradation , as percentage of proteases inhibitors , in isolated phagosomes at the indicated time periods post - internalization from Scramble and Rab22a KD JAWS - II DCs was assessed by FACS analysis . Data represent mean ± SEM of three independent experiments .

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

F. The kinetics of OVA degradation, as percentage of proteases inhibitors, in isolated phagosomes at the indicated time periods post-internalization from Scramble and Rab22a KD JAWS-II DCs was assessed by FACS analysis. Data represent mean ± SEM of three independent experiments.


G . Immunoblotting of Rab22a and Actin in BMDCs infected with lentiviruses encoding a random sequence ( Scramble ) and two shRNA specific for silencing Rab22a ( Rab22a KD # 1 and # 2 ) .

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

G. Immunoblotting of Rab22a and Actin in BMDCs infected with lentiviruses encoding a random sequence (Scramble) and two shRNA specific for silencing Rab22a (Rab22a KD #1 and #2).


H - I . The cross - presentation capacity after the incubation with H ) soluble OVA at the indicated concentrations by Scramble , Rab22a KD # 1 and Rab22a KD # 2 BMDCs was evaluated as described before for JAWS - II DCs . Data represent mean ± SEM of triplicate values and are representative of two independent experiments . * * * P = 0 . 0001 . The two - tailed Student ' s unpaired t test was performed .

H-I. The cross-presentation capacity after the incubation with H) soluble OVA at the indicated concentrations by Scramble, Rab22a KD #1 and Rab22a KD #2 BMDCs was evaluated as described before for JAWS-II DCs. Data represent mean ± SEM of triplicate values and are representative of two independent experiments. ***P = 0.0001. The two-tailed Student's unpaired t test was performed.


H - I . The cross - presentation capacity after the incubation with I ) the SIINFEKL control peptide at the indicated concentrations by Scramble , Rab22a KD # 1 and Rab22a KD # 2 BMDCs was evaluated as described before for JAWS - II DCs . Data represent mean ± SEM of triplicate values and are representative of two independent experiments . * * * P = 0 . 0001 . The two - tailed Student ' s unpaired t test was performed .

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

H-I. The cross-presentation capacity after the incubation with I) the SIINFEKL control peptide at the indicated concentrations by Scramble, Rab22a KD #1 and Rab22a KD #2 BMDCs was evaluated as described before for JAWS-II DCs. Data represent mean ± SEM of triplicate values and are representative of two independent experiments. ***P = 0.0001. The two-tailed Student's unpaired t test was performed.


J . Soluble OVA was electroporated into the cytosol of Scramble and Rab22a KD JAWS - II DCs and T cell activation was determined 2 h later with the B3Z hibridoma . To control endogenous MHC - I antigen presentation specificity , DCs were also treated with brefreldin A ( BFA ) . The use of this drug markedly reduced CD8 + T cell response to similar levels obtained by DCs without any antigen ( ∅ ) . Data represent mean ± SEM of triplicate values and are representative of three independent experiments .

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

J. Soluble OVA was electroporated into the cytosol of Scramble and Rab22a KD JAWS-II DCs and T cell activation was determined 2 h later with the B3Z hibridoma. To control endogenous MHC-I antigen presentation specificity, DCs were also treated with brefreldin A (BFA). The use of this drug markedly reduced CD8+ T cell response to similar levels obtained by DCs without any antigen (∅). Data represent mean ± SEM of triplicate values and are representative of three independent experiments.


K . Immunoblotting showing the amount of OVA incorporated by Scramble ( a ) and Rab22a KD ( b ) JAWS - II DCs after electroporation and BFA treatment .

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

K. Immunoblotting showing the amount of OVA incorporated by Scramble (a) and Rab22a KD (b) JAWS-II DCs after electroporation and BFA treatment.


A . Scramble and Rab22a KD JAWS - II DCs were infected with OVA - YFP - expressing T . gondii ( TgRHYFPSAG1 - OVA ) for 8 h and confocal images detecting the parasite ( green ) , endogenous Rab22a ( red ) and GRA6 ( magenta ) were taken . Top panels : Scramble cells ; bottom panels : Rab22a KD cells . White boxes are shown at higher magnification in the insets . The nuclear marker DAPI ( blue ) and DIC images are shown in the left panels . Overlays are shown in the right panels . Scale bars : 5 µm . Images are representative of at least 30 analyzed from three independent experiments .

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A. Scramble and Rab22a KD JAWS-II DCs were infected with OVA-YFP-expressing T. gondii (TgRHYFPSAG1-OVA) for 8 h and confocal images detecting the parasite (green), endogenous Rab22a (red) and GRA6 (magenta) were taken. Top panels: Scramble cells; bottom panels: Rab22a KD cells. White boxes are shown at higher magnification in the insets. The nuclear marker DAPI (blue) and DIC images are shown in the left panels. Overlays are shown in the right panels. Scale bars: 5 µm. Images are representative of at least 30 analyzed from three independent experiments.


B . The cross - presentation of OVA secreted by T . gondii ( TgRHYFPSAG1 - OVA ) after 8 h of infection at the indicated MOI was evaluated by B3Z T cell activation . Data represent mean ± SEM of triplicate values and are representative of three independent experiments . * * * P < 0 . 001 . A two - way ANOVA and the Bonferroni post - test were performed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The cross-presentation of OVA secreted by T. gondii (TgRHYFPSAG1-OVA) after 8 h of infection at the indicated MOI was evaluated by B3Z T cell activation. Data represent mean ± SEM of triplicate values and are representative of three independent experiments. ***P < 0.001. A two-way ANOVA and the Bonferroni post-test were performed.


C . The efficiency of infection ( 8 h ) of TgRHYFPSAG1 - OVA in Scramble and Rab22a KD JAWS - II DCs was measured by FACS analysis at the indicated MOI . Data represent mean ± SEM of duplicate values and are representative of three independent experiments .

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

C. The efficiency of infection (8 h) of TgRHYFPSAG1-OVA in Scramble and Rab22a KD JAWS-II DCs was measured by FACS analysis at the indicated MOI. Data represent mean ± SEM of duplicate values and are representative of three independent experiments.


D . Immunoblotting showing the total amount of OVA in Scramble and Rab22a KD JAWS - II DCs after 90 min and 7 h of infection with TgRHYFPSAG1 - OVA parasite at MOI 2 and 6 . Fifty µg of total cell lysates were loaded onto each lane . Data are representative of two independent experiments .

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

D. Immunoblotting showing the total amount of OVA in Scramble and Rab22a KD JAWS-II DCs after 90 min and 7 h of infection with TgRHYFPSAG1-OVA parasite at MOI 2 and 6. Fifty µg of total cell lysates were loaded onto each lane. Data are representative of two independent experiments.


( C ) Still frames from time - lapse recordings of an embryo expressing a myosin - II probe ( Sqh : : GFP ) . Embryos were mounted vertically to image the transverse cross - section using two - photon microscopy . At the onset of gastrulation , myosin - II localized to ring structures representing the leading edge of the cellularization front ( lower arrow ) . During ventral furrow formation ( torques open rectangle ) , myosin - II accumulated at the apical side ( upper arrow ) of the cells that invaginate and the basal pool was progressively depleted . Scale bar , 40 µm

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(C) Still frames from time-lapse recordings of an embryo expressing a myosin-II probe (Sqh::GFP). Embryos were mounted vertically to image the transverse cross-section using two-photon microscopy. At the onset of gastrulation, myosin-II localized to ring structures representing the leading edge of the cellularization front (lower arrow). During ventral furrow formation (torques open rectangle), myosin-II accumulated at the apical side (upper arrow) of the cells that invaginate and the basal pool was progressively depleted. Scale bar, 40 µm


( D ) Quantification of basal myosin - II levels ( N = 3 embryos ) in ectodermal and mesodermal cells . In the mesoderm , basal myosin - II levels decreasd 5 - fold over the course of internalization , while in the ectoderm only ~ 3 - fold . Additionally , myosin - II depletion appeared to be accelerated in the mesodermal cells as indicated by the steeper slope of the polynomial fit ( 3rd degree with r2Ectoderm : 0 . 98 and r2Mesoderm : 0 . 87 ) . Note the logarithmic y - axis

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

(D) Quantification of basal myosin-II levels (N=3 embryos) in ectodermal and mesodermal cells. In the mesoderm, basal myosin-II levels decreasd 5-fold over the course of internalization, while in the ectoderm only ~3-fold. Additionally, myosin-II depletion appeared to be accelerated in the mesodermal cells as indicated by the steeper slope of the polynomial fit (3rd degree with r2Ectoderm: 0.98 and r2Mesoderm: 0.87). Note the logarithmic y-axis


( E - H ) Embryos at the end of cellularization co - expressing RhoGEF2 - CRY2 ; CIBN : : pmGFP , and Sqh : : mCherry were mounted with the dorsal epithelium facing the objective and a region of interest ( red dashed line ) was defined to specifically illuminate the cell base using two - photon microscopy . A representative embryo ( N = 3 ) showing basal myosin - II from top view ( sum - of - slice projection of 6 focal planes ) at the initial stage of the experiment ( E ) and 6 min after activation ( F ) . Z - projection of the same embryo showing the cross section of the activated region ( AR ) ( G ) and non - activated region ( NR ) ( H ) with the superimposed plasma membrane signal ( CIBN : : GFPpm in magenta ) recorded immediately after the final Sqh : : mCherry acquisition ( displayed in white ) . The contrast has been adjusted to better visualize the distribution of myosin - II along the apicobasal axis in the respective area of the tissue . Scale bars , 20 µm

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(E-H) Embryos at the end of cellularization co-expressing RhoGEF2-CRY2; CIBN::pmGFP, and Sqh::mCherry were mounted with the dorsal epithelium facing the objective and a region of interest (red dashed line) was defined to specifically illuminate the cell base using two-photon microscopy. A representative embryo (N=3) showing basal myosin-II from top view (sum-of-slice projection of 6 focal planes) at the initial stage of the experiment (E) and 6 min after activation (F). Z-projection of the same embryo showing the cross section of the activated region (AR) (G) and non-activated region (NR) (H) with the superimposed plasma membrane signal (CIBN::GFPpm in magenta) recorded immediately after the final Sqh::mCherry acquisition (displayed in white). The contrast has been adjusted to better visualize the distribution of myosin-II along the apicobasal axis in the respective area of the tissue. Scale bars, 20 µm


( I ) Quantification of myosin - II levels ( N = 3 ) within the region of activation ( AR , red ) relative to the non - activated region ( NR , green ) over the course of 6 min . The y - axis represents the fold - change of myosin - II signal intensity in the activated region versus non - activated region . A polynomial function ( 2nd degree , r2AR = 0 . 88 ) was fitted to the data ( straight line ) indicating the level of saturation that is reached at about 1 . 6 - fold change of myosin - II in the activated area after 6 min

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

(I) Quantification of myosin-II levels (N=3) within the region of activation (AR, red) relative to the non-activated region (NR, green) over the course of 6 min. The y-axis represents the fold-change of myosin-II signal intensity in the activated region versus non-activated region. A polynomial function (2nd degree, r2AR = 0.88) was fitted to the data (straight line) indicating the level of saturation that is reached at about 1.6-fold change of myosin-II in the activated area after 6 min


( J Analysis of the extent of signal spreading in the z - dimension ( apico - basla ) of basal myosin - II . Myosin - II signal intensity was plotted along the apicobasal axis and a Gaussian curve fitted to the basal peak . The width of the Gaussian fit was used as a parameter to estimate myosin - II signal spreading in the z - dimension , which was limited to 4 µm ( N = 3 ) . In each box plot , the central mark , the bottom , and the top edge of each box indicates the median , the 25th and 75th percentile , respectively . Whiskers extent to the most extreme data

(J Analysis of the extent of signal spreading in the z-dimension (apico-basla) of basal myosin-II. Myosin-II signal intensity was plotted along the apicobasal axis and a Gaussian curve fitted to the basal peak. The width of the Gaussian fit was used as a parameter to estimate myosin-II signal spreading in the z-dimension, which was limited to 4 µm (N=3). In each box plot, the central mark, the bottom, and the top edge of each box indicates the median, the 25th and 75th percentile, respectively. Whiskers extent to the most extreme data point


( K ) Quantification of the diameter of basal actomyosin rings in the non - activated region ( NR , green ) and activated region ( AR , red ) showing increased speed ( 4 . 8 - fold change ) and extent of ring constrictions in the photo - activated region ( n = > 41 cells ) . The ring diameter was normalized to the mean value of the first time point . The constriction speed was estimated by the slope of a fitted linear function to the data . ( L ) Light activation of the cell base resulted in a compaction of the tissue in the activated ( AR ) compared to the non - activated region ( NR ) . Actomyosin rings were counted and normalized to the analyzed area to score ring density , which increased 1 . 35 times in the activated region after 5 min of photo - activation , showing that cells were still interconnected at the base and that photo - activation did not affect tissue integrity ( N = 3 embryos ; nNR : 431 , nAR : 302 ) . In each box plot , the central mark , the bottom , and the top edge of each box indicates the median , the 25th and 75th percentile , respectively . Whiskers extent to the most extreme data point

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

(K) Quantification of the diameter of basal actomyosin rings in the non-activated region (NR, green) and activated region (AR, red) showing increased speed (4.8-fold change) and extent of ring constrictions in the photo-activated region (n= >41 cells). The ring diameter was normalized to the mean value of the first time point. The constriction speed was estimated by the slope of a fitted linear function to the data. (L) Light activation of the cell base resulted in a compaction of the tissue in the activated (AR) compared to the non-activated region (NR). Actomyosin rings were counted and normalized to the analyzed area to score ring density, which increased 1.35 times in the activated region after 5 min of photo-activation, showing that cells were still interconnected at the base and that photo-activation did not affect tissue integrity (N = 3 embryos; nNR: 431, nAR: 302). In each box plot, the central mark, the bottom, and the top edge of each box indicates the median, the 25th and 75th percentile, respectively. Whiskers extent to the most extreme data point


Ventral furrow formation was inhibited upon photo - stabilization of myosin - II levels at the cell base . Embryos co - expressing the optogenetic module and the myosin - II probe Sqh : : mCh were mounted with the ventral tissue facing the objective . A region of photo - activation ( two - photon λ = 950 nm ) was defined at the cell base in the anterior half of the embryo , and whole - cell myosin - II signal was recorded ( confocal setup ; λ = 561 nm ) in an alternating fashion . Scale bars , 20 µm . ( A ) Top view showing apical myosin - II 10 min after the initial activation . While cells in the non - activated region have internalized and disappeared from the apical focal planes ( right ) , cells in the activated region failed to internalize despite showing myosin - II accumulation at the apical surface ( left ) . Red and Blue dotted lines indicate the location of the cross - section panels displayed in ( C ) and ( D ) . ( B ) Quantification of apical myosin - II levels demonstrate that the defects in tissue invagination were not due to impaired apical myosin - II accumulation as the average apical myosin - II level per cell did not significantly differ between the non - activated ( NR ) and activated region ( AR ) . The intensity values were normalized using the median value of the combined cell population in the NR and AR . ( N = 3 embryos ; nNR : 94 , nAR : 78 ) . Significances ( by two - sample t - Test ) : n . s . indicates no statistically significant differences . In each box plot , the central mark , the bottom , and the top edge of each box indicates the median , the 25th and 75th percentile , respectively . Whiskers extent to the most extreme data point and the ' + ' symbol indicates an outlier . ( C

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

Ventral furrow formation was inhibited upon photo-stabilization of myosin-II levels at the cell base. Embryos co-expressing the optogenetic module and the myosin-II probe Sqh::mCh were mounted with the ventral tissue facing the objective. A region of photo-activation (two-photon λ=950 nm) was defined at the cell base in the anterior half of the embryo, and whole-cell myosin-II signal was recorded (confocal setup; λ=561 nm) in an alternating fashion. Scale bars, 20 µm. (A) Top view showing apical myosin-II 10 min after the initial activation. While cells in the non-activated region have internalized and disappeared from the apical focal planes (right), cells in the activated region failed to internalize despite showing myosin-II accumulation at the apical surface (left). Red and Blue dotted lines indicate the location of the cross-section panels displayed in (C) and (D). (B) Quantification of apical myosin-II levels demonstrate that the defects in tissue invagination were not due to impaired apical myosin-II accumulation as the average apical myosin-II level per cell did not significantly differ between the non-activated (NR) and activated region (AR). The intensity values were normalized using the median value of the combined cell population in the NR and AR. (N = 3 embryos; nNR: 94, nAR: 78). Significances (by two-sample t-Test): n.s. indicates no statistically significant differences. In each box plot, the central mark, the bottom, and the top edge of each box indicates the median, the 25th and 75th percentile, respectively. Whiskers extent to the most extreme data point and the '+' symbol indicates an outlier. (C


Ventral furrow formation was inhibited upon photo - stabilization of myosin - II levels at the cell base . Embryos co - expressing the optogenetic module and the myosin - II probe Sqh : : mCh were mounted with the ventral tissue facing the objective . A region of photo - activation ( two - photon λ = 950 nm ) was defined at the cell base in the anterior half of the embryo , and whole - cell myosin - II signal was recorded ( confocal setup ; λ = 561 nm ) in an alternating fashion . Scale bars , 20 µm ( C , D ) z - projections ( transverse cross section ) of myosin - II time - lapse recordings of the non - activated ( C ) and activated region ( D ) . While cells in the non - activated region internalized , resulting in a complete closure of the furrow ( C ) , stabilization of basal myosin - II levels prevented invagination ( D ) . Scale bars , 20

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

Ventral furrow formation was inhibited upon photo-stabilization of myosin-II levels at the cell base. Embryos co-expressing the optogenetic module and the myosin-II probe Sqh::mCh were mounted with the ventral tissue facing the objective. A region of photo-activation (two-photon λ=950 nm) was defined at the cell base in the anterior half of the embryo, and whole-cell myosin-II signal was recorded (confocal setup; λ=561 nm) in an alternating fashion. Scale bars, 20 µm (C, D) z-projections (transverse cross section) of myosin-II time-lapse recordings of the non-activated (C) and activated region (D). While cells in the non-activated region internalized, resulting in a complete closure of the furrow (C), stabilization of basal myosin-II levels prevented invagination (D). Scale bars, 20 µm


( A - C ) Embryos co - expressing the optogenetic module and Sqh : : mCherry mounted with the ventral epithelium facing the objective were subjected to cycles of photo - activation of the cell base in the anterior ( left ) half of the embryo and confocal recording of the whole embryo . The experiment was started at the end of cellularization when initial accumulation of apical myosin - II was detected and terminated at different time points to record the plasma membrane signal ( CIBN : : GFP - pm ) in the whole embryo using two - photon illumination . Still frames visualizing the plasma membrane ( CIBN : : GFP - pm ) at a sub - apical plane at the initial time point of the experiment ( A ) , 3 min ( B ) and 10 min ( C ) after initial photo - activation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Embryos co-expressing the optogenetic module and Sqh::mCherry mounted with the ventral epithelium facing the objective were subjected to cycles of photo-activation of the cell base in the anterior (left) half of the embryo and confocal recording of the whole embryo. The experiment was started at the end of cellularization when initial accumulation of apical myosin-II was detected and terminated at different time points to record the plasma membrane signal (CIBN::GFP-pm) in the whole embryo using two-photon illumination. Still frames visualizing the plasma membrane (CIBN::GFP-pm) at a sub-apical plane at the initial time point of the experiment (A), 3 min (B) and 10 min (C) after initial photo-activation.


Embryos expressing RhoGEF2 - CRY2 / CIBN : : GFPpm and the membrane marker GAP43 : : mCherry were mounted with the ventral epithelium facing the objective . To analyze apical dynamics at each activation cycle , the photo - activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm - sized apical image stack resulting in a total time resolution of 35 s . The basal surface of the anterior ( left ) half of the embryo was photo - activated . ( A ) Still frames of the time lapse membrane ( GAP43 : : mCherry ) recording are shown at the initial time point ( top ) , after 6 . 5 min ( middle ) , and after 13 min ( bottom ) of photo - activation . Cells in the non - activated region ( green ) underwent normal apical constriction . In the activated region , two populations of cells were observed , one that constricted ( purple ) and one that did not constrict ( red ) . Cells in the activated region that by the end of the experiment constricted to an area smaller than half of their initial area are considered as constricting cells . Cells that constricted less were considered to be defective in apical constriction and are referred to as non - constricting . Scale bar 20 µm .

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

Embryos expressing RhoGEF2-CRY2/CIBN::GFPpm and the membrane marker GAP43::mCherry were mounted with the ventral epithelium facing the objective. To analyze apical dynamics at each activation cycle, the photo-activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm-sized apical image stack resulting in a total time resolution of 35 s. The basal surface of the anterior (left) half of the embryo was photo-activated. (A) Still frames of the time lapse membrane (GAP43::mCherry) recording are shown at the initial time point (top), after 6.5 min (middle), and after 13 min (bottom) of photo-activation. Cells in the non-activated region (green) underwent normal apical constriction. In the activated region, two populations of cells were observed, one that constricted (purple) and one that did not constrict (red). Cells in the activated region that by the end of the experiment constricted to an area smaller than half of their initial area are considered as constricting cells. Cells that constricted less were considered to be defective in apical constriction and are referred to as non-constricting. Scale bar 20 µm.


Embryos expressing RhoGEF2 - CRY2 / CIBN : : GFPpm and the membrane marker GAP43 : : mCherry were mounted with the ventral epithelium facing the objective . To analyze apical dynamics at each activation cycle , the photo - activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm - sized apical image stack resulting in a total time resolution of 35 s . The basal surface of the anterior ( left ) half of the embryo was photo - activated . ( B ) All cells show pulsatile behavior . Graphs illustrate the area of three representative cells in each category ( non - activated region : top ; activated region with constricting cells : middle ; activated region with non - constricting cells : bottom ) over the course of one experiment . Yellow circles indicate the position of automatically identified local maxima used for further analyses .

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

Embryos expressing RhoGEF2-CRY2/CIBN::GFPpm and the membrane marker GAP43::mCherry were mounted with the ventral epithelium facing the objective. To analyze apical dynamics at each activation cycle, the photo-activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm-sized apical image stack resulting in a total time resolution of 35 s. The basal surface of the anterior (left) half of the embryo was photo-activated. (B) All cells show pulsatile behavior. Graphs illustrate the area of three representative cells in each category (non-activated region: top; activated region with constricting cells: middle; activated region with non-constricting cells: bottom) over the course of one experiment. Yellow circles indicate the position of automatically identified local maxima used for further analyses.