Figure 4Western 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 . 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 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 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 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

Figure 4Western 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.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.


Figure 5Representative 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 . 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 . 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 . 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 .

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 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 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 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 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

Figure 5Representative 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.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.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.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.


Figure 6Representative 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 . 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 . 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 . 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 .

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 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 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 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 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

Figure 6Representative 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.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.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.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.


Figure 1 ( 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 ) .

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

Figure 1(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).


Figure 2 ( 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 ) . ( 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 ) .

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 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 O O O O O O O O O O O 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

Figure 2(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).(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).


Figure 3 ( 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 ) .

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

Figure 3(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).


Figure 4 ( 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 ) . ( 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 ) . ( 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 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 O O O O O O O O O O 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 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

Figure 4(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).(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).(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.


Figure 1A . 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 . 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 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 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 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 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

Figure 1A. 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.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.


Figure 2A . 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 . 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 . 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 . 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 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 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 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 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 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

Figure 2A. 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.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.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.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.


Figure 3A - 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 . 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 ) . 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 . 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 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 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 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 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 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 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

Figure 3A-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.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).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.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.


Figure 4A . 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 . 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 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 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 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

Figure 4A. 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.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.


Figure 1 ( 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 ( 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 ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1 (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  (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  (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


Figure 2Ventral 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2Ventral 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 µm


Figure 3 ( 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 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

Figure 3(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.


Figure 4Embryos 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 . 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 . ( C , D ) ( C ) Diagram showing the extent of ratchet - like constrictions . The mean difference in apical area between adjacent peaks ( relaxed cell surface ) is indicated by a red dot and reflects the extent to which cells shrink in a single pulse . Cells in the non - activated region ( NR ) constricted on average more than 5 µm2 , which is characteristic for ratchet - like constriction . Non - constricting cells in the activated region ( AR , non - con . ) displayed a mean difference in apical area per pulse of about 0 , reflecting non - ratchet constrictions meaning that pulsing cells constricted but relaxed subsequently back to their initial size . Constricting cells in the activated region ( AR , con . ) show an intermediate behavior with less efficient constrictions than in the NR . Data points are significantly different as determined by ANOVA ( F ( 2 , 539 ) = 72 . 04 , p = 1 . 9e - 28 ) and post - hoc Tukey test . ( D ) The pulsation period ( T ) . All cells pulse with a period of about 90 s . There were no statistically significant differences between group means as determined by one - way ANOVA ( F ( 2 , 540 ) = 1 . 438 , p = 0 . 24 ) . ( C and D ) Red dots indicate the mean values and the error bars represent the standard deviation . More than 500 pulses ( nNR = 119 , nAR , non - con . = 128 , nAR , con = 295 ) of more than 100 cells were analyzed . Data information : In all panels , * * * * indicates p ≤ 0 . 0001 , and n . s . indicates no statistically significant differences according to two - sample t - Test . 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 . ( E ) Diagram showing the prevalence of cells in the photo - activated region that constricted ( con . ) and that did not constrict ( Non - con . ) . More than 4 / 5 of the cells did not constrict to half of the initial area ( N = 5 embryos ; n : 407 cells ; Cohen ' s D : d = 10 ) . 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 . Data information : In all panels , * * * * indicates p ≤ 0 . 0001 , and n . s . indicates no statistically significant differences according to two - sample t - Test . 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 . ( F , G ) Graphs showing the average apical area ( F ) and anisotropy ( G ) of cells in the non - activated region ( green ) , in the activated region that constricted ( purple ) and that did not constrict ( red ) . The straight line indicates the mean value at a given time - point , the semi - transparent colored area indicated the respective standard deviation . Cells in the non - activated region constricted efficiently and progressively acquired anisotropy . Constricting cells in the activated region constricted slower in an isotropic manner . Non - constricting cells in the activated region did not show net decrease in apical area and gain of anisotropy ( nNR = 34 , nAR , non - con . = 20 , nAR , con = 59 ) .

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 O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4Embryos 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.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. (C, D) (C) Diagram showing the extent of ratchet-like constrictions. The mean difference in apical area between adjacent peaks (relaxed cell surface) is indicated by a red dot and reflects the extent to which cells shrink in a single pulse. Cells in the non-activated region (NR) constricted on average more than 5 µm2, which is characteristic for ratchet-like constriction. Non-constricting cells in the activated region (AR, non-con.) displayed a mean difference in apical area per pulse of about 0, reflecting non-ratchet constrictions meaning that pulsing cells constricted but relaxed subsequently back to their initial size. Constricting cells in the activated region (AR, con.) show an intermediate behavior with less efficient constrictions than in the NR. Data points are significantly different as determined by ANOVA (F(2,539)=72.04, p=1.9e-28) and post-hoc Tukey test. (D) The pulsation period (T). All cells pulse with a period of about 90 s. There were no statistically significant differences between group means as determined by one-way ANOVA (F(2,540)=1.438, p=0.24). (C and D) Red dots indicate the mean values and the error bars represent the standard deviation. More than 500 pulses (nNR = 119, nAR, non-con. = 128, nAR, con = 295) of more than 100 cells were analyzed. Data information: In all panels, **** indicates p ≤ 0.0001, and n.s. indicates no statistically significant differences according to two-sample t-Test.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. (E) Diagram showing the prevalence of cells in the photo-activated region that constricted (con.) and that did not constrict (Non-con.). More than 4/5 of the cells did not constrict to half of the initial area (N = 5 embryos; n: 407 cells; Cohen's D: d = 10). 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. Data information: In all panels, **** indicates p ≤ 0.0001, and n.s. indicates no statistically significant differences according to two-sample t-Test.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. (F,G) Graphs showing the average apical area (F) and anisotropy (G) of cells in the non-activated region (green), in the activated region that constricted (purple) and that did not constrict (red). The straight line indicates the mean value at a given time-point, the semi-transparent colored area indicated the respective standard deviation. Cells in the non-activated region constricted efficiently and progressively acquired anisotropy. Constricting cells in the activated region constricted slower in an isotropic manner. Non-constricting cells in the activated region did not show net decrease in apical area and gain of anisotropy (nNR = 34, nAR, non-con. = 20, nAR, con = 59).


Figure 5 ( C - E ) Embryos expressing either of the optogenetic anchor proteins and RhoGEF2 - CRY2 : : mCherry were imaged during late cellularization . ( C ) z - projections of confocal image stacks show the localization of the respective GFP - tagged optogenetic anchor . Please note that the construct PatJ - CIBN ( indicated by asterisk ) lacks a fluorescent marker and its localization was thus inferred from the recruited RhoGEF2 - CRY2 : : mCh . All anchor proteins showed predominantly basal localization during cellularization and at the onset of ventral furrow formation . Scale bars , 20 μm . ( D ) A region of interest ( right side of the dashed line ) in a 10 µm - sized stack was photo - activated using two - photon illumination for 30 s before acquiring the RhoGEF2 - CRY2 : : mCherry signal using confocal microscopy . All anchor proteins were able to recruit RhoGEF2 - CRY2 : : mCherry specifically to the activated region . Scale bars , 10 μm . ( E ) Although all basal anchors were effective at recruiting RhoGEF2 - CRY2 : : mCherry to the cell base , only PatJ - CIBN : : GFP - pm resulted in effective myosin - II recruitment and actomyosin ring constriction

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5 (C-E) Embryos expressing either of the optogenetic anchor proteins and RhoGEF2-CRY2::mCherry were imaged during late cellularization. (C) z-projections of confocal image stacks show the localization of the respective GFP-tagged optogenetic anchor. Please note that the construct PatJ-CIBN (indicated by asterisk) lacks a fluorescent marker and its localization was thus inferred from the recruited RhoGEF2-CRY2::mCh. All anchor proteins showed predominantly basal localization during cellularization and at the onset of ventral furrow formation. Scale bars, 20 μm. (D) A region of interest (right side of the dashed line) in a 10 µm-sized stack was photo-activated using two-photon illumination for 30 s before acquiring the RhoGEF2-CRY2::mCherry signal using confocal microscopy. All anchor proteins were able to recruit RhoGEF2-CRY2::mCherry specifically to the activated region. Scale bars, 10 μm. (E) Although all basal anchors were effective at recruiting RhoGEF2-CRY2::mCherry to the cell base, only PatJ-CIBN::GFP-pm resulted in effective myosin-II recruitment and actomyosin ring constriction


Figure 6 ( A ) Localization of the basal - specific optogenetic anchor CIBN - PatJ : : GFP - pm during ventral furrow formation . Embryos expressing CIBN - PatJ : : GFP - pm were imaged using two - photon excitation during ventral furrow formation . Transverse cross section ( z - projection ) at a stage when cells were already apically constricted and elongated , and the ventral tissue already internalized . Embryos co - expressing CIBN - PatJ : : GFP - pm , RhoGEF2 - CRY2 and Sqh : : mCherry ( B , C were mounted with the ventral tissue facing the objective . After cells underwent apical constriction and cell elongation the basal volume of the ventral cells was photo - activated using two - photon illumination and the myosin - II signal was recorded in alternation . ( B ) myosin - II distribution in a cross section ( z - projection ) 3 min after photo - activation . myosin - II was locally recruited to the base as indicated by the red arrowheads in the activated region ( red line ) . ( C ) Top view ( sum of slice of 4 µm - sized image stack ) showing basal myosin - II recruitment in the photo - activated region ( red dashed square ) Data information : Scale bars , 20 µmEmbryos co - expressing CIBN - PatJ : : GFP - pm , RhoGEF2 - CRY an the membrane marker GAP43 : : mCherry ( D - I ) were mounted with the ventral tissue facing the objective . After cells underwent apical constriction and cell elongation the basal volume of the ventral cells was photo - activated using two - photon illumination and the myosin - II signal was recorded in alternation ( D ) Cross section ( z - projection ) showing the plasma membrane and ( E ) top view showing the apical plasma membrane at the initial time point before photo - activation . At this stage , the central mesodermal tissue has started to internalize and cells were constricted at the apical surface . In an alternating fashion , the base of the cells in the anterior half of the embryo ( left ) was photo - activated for 100 sec and the GAP43 : : mCherry signal in the entire field of view was recorded . The furrow further ingressed and closed completely 3 min ( F ) and 6 min ( G ) after initial activation , respectively . In the activated region , the tissue failed to internalize and to form a closed furrow both at 3 min ( H ) and 6 min ( I ) after initial photo - activation . The width of the central tissue forming the ventral furrow at the initial point of the experiment is 40 µm ( D ) . In the non - activated region 6 min after initial activation the width of the tissue shrank to about 25 µm ( G ) , whereas it remained constant at 40 µm in the activated region ( I ) . Data information : Scale bars , 20

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Localization of the basal-specific optogenetic anchor CIBN-PatJ::GFP-pm during ventral furrow formation. Embryos expressing CIBN-PatJ::GFP-pm were imaged using two-photon excitation during ventral furrow formation. Transverse cross section (z-projection) at a stage when cells were already apically constricted and elongated, and the ventral tissue already internalized.Embryos co-expressing CIBN-PatJ::GFP-pm, RhoGEF2-CRY2 and Sqh::mCherry (B,C were mounted with the ventral tissue facing the objective. After cells underwent apical constriction and cell elongation the basal volume of the ventral cells was photo-activated using two-photon illumination and the myosin-II signal was recorded in alternation. (B) myosin-II distribution in a cross section (z-projection) 3 min after photo-activation. myosin-II was locally recruited to the base as indicated by the red arrowheads in the activated region (red line). (C) Top view (sum of slice of 4 µm-sized image stack) showing basal myosin-II recruitment in the photo-activated region (red dashed square) Data information: Scale bars, 20 µmEmbryos co-expressing CIBN-PatJ::GFP-pm, RhoGEF2-CRY an the membrane marker GAP43::mCherry (D-I) were mounted with the ventral tissue facing the objective. After cells underwent apical constriction and cell elongation the basal volume of the ventral cells was photo-activated using two-photon illumination and the myosin-II signal was recorded in alternation (D) Cross section (z-projection) showing the plasma membrane and (E) top view showing the apical plasma membrane at the initial time point before photo-activation. At this stage, the central mesodermal tissue has started to internalize and cells were constricted at the apical surface. In an alternating fashion, the base of the cells in the anterior half of the embryo (left) was photo-activated for 100 sec and the GAP43::mCherry signal in the entire field of view was recorded. The furrow further ingressed and closed completely 3 min (F) and 6 min (G) after initial activation, respectively. In the activated region, the tissue failed to internalize and to form a closed furrow both at 3 min (H) and 6 min (I) after initial photo-activation. The width of the central tissue forming the ventral furrow at the initial point of the experiment is 40 µm (D). In the non-activated region 6 min after initial activation the width of the tissue shrank to about 25 µm (G), whereas it remained constant at 40 µm in the activated region (I). Data information: Scale bars, 20 µm


Figure 1 ( A ) Immunoblot showing the activation of PI3K / AKT pathway ( by assessing the levels of p - AKT Ser473 ) of ECs 24h after induction of Pik3caH1047R expression . ( B ) Volcano plot of genes analysed by RNAseq in ECs upon Pik3caH1047R expression . Differentially expressed genes ( DEGs ) ( p adj < 0 . 05 ) in Pik3caH1047R over Pik3caWT ECs at 24h ( grey , unchanged ; orange , downregulated ; purple , upregulated ) . n = 4 biological replicates per genotype . ( D ) Immunoblot showing the activation of PI3K / AKT pathway of Pik3caWT and Pik3caH1047R ECs after 2h starvation ( - ) and 30 min stimulation with 20 % fetal bovine serum ( FBS ) ( + ) . ( E , F ) ( E ) Cell cycle analysis by flow cytometry of Pik3caWT and Pik3caH1047R ECs after cultured either without serum ( - ) or 20 % serum ( + ) for 24h . ( F ) Percentage of proliferative ECs ( in S and G2M phases ) from Fig 1E . Error bars are s . e . m . n ≥ 3 biological replicates per genotype . Statistical analysis was performed by nonparametric Mann - Whitney test . * * p < 0 . 01 and * * * * p < 0 . 0001 were considered statistically significant .

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 O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O 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 1(A) Immunoblot showing the activation of PI3K/AKT pathway (by assessing the levels of p-AKT Ser473) of ECs 24h after induction of Pik3caH1047R expression.(B) Volcano plot of genes analysed by RNAseq in ECs upon Pik3caH1047R expression. Differentially expressed genes (DEGs) (p adj < 0.05) in Pik3caH1047R over Pik3caWT ECs at 24h (grey, unchanged; orange, downregulated; purple, upregulated). n=4 biological replicates per genotype.(D) Immunoblot showing the activation of PI3K/AKT pathway of Pik3caWT and Pik3caH1047R ECs after 2h starvation (-) and 30 min stimulation with 20% fetal bovine serum (FBS) (+).(E, F) (E) Cell cycle analysis by flow cytometry of Pik3caWT and Pik3caH1047R ECs after cultured either without serum (-) or 20% serum (+) for 24h. (F) Percentage of proliferative ECs (in S and G2M phases) from Fig 1E. Error bars are s.e.m. n ≥ 3 biological replicates per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. **p < 0.01 and ****p < 0.0001 were considered statistically significant.


Figure 2 ( B ) Representative images of control and EC - Pik3caH1047R / WT mouse retinas isolated at indicated time points stained with IB4 for blood vessels . Scale bars : 150 μm . ( D ) Quantification of IB4 - positive areas per retina . Error bars are s . e . m . n ≥ 3 retinas per genotype . ( E ) Representative images of EC - mTmG mouse retinas isolated at indicated time points stained with IB4 and GFP . Scale bars : 150 μm . ( F ) Quantification of GFP / IB4 ratio per retina . Error bars are s . e . m . n ≥ 3 retinas per genotype . Data information : Statistical analysis was performed by nonparametric Mann - Whitney test . * p < 0 . 05 was considered statistically significant .

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 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 2(B) Representative images of control and EC-Pik3caH1047R/WT mouse retinas isolated at indicated time points stained with IB4 for blood vessels. Scale bars: 150 μm.(D) Quantification of IB4-positive areas per retina. Error bars are s.e.m. n ≥ 3 retinas per genotype.(E) Representative images of EC-mTmG mouse retinas isolated at indicated time points stained with IB4 and GFP. Scale bars: 150 μm. (F) Quantification of GFP/IB4 ratio per retina. Error bars are s.e.m. n ≥ 3 retinas per genotype. Data information: Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 was considered statistically significant.


Figure 3 ( A , B ) Representative images of EC - Pik3caH1047R / WT ( A ) and EC - mTmG ( B ) P6 retinas from mice treated with decreasing doses of 4 - OHT on P1 . Retinas were stained for blood vessels ( IB4 ) and GFP as indicated . Scale bars : 150 μm . ( C ) Quantification of GFP / IB4 ratio of EC - mTmG retinas . Error bars are s . e . m . n ≥ 3 retinas per genotype . Data information : Statistical analysis was performed by nonparametric Mann - Whitney test . * p < 0 . 05 was considered statistically significant . ( D ) Quantification of IB4 - positive area per retina in Pik3caH1047R / WT retinas . Data presented as a percentage of the control for each 4 - OHT dose . Error bars are s . e . m . n = 4 retinas per genotype . Data information : Statistical analysis was performed by nonparametric Mann - Whitney test . * p < 0 . 05 was considered statistically significant .

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 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

Figure 3(A, B) Representative images of EC-Pik3caH1047R/WT (A) and EC-mTmG (B) P6 retinas from mice treated with decreasing doses of 4-OHT on P1. Retinas were stained for blood vessels (IB4) and GFP as indicated. Scale bars: 150 μm.(C) Quantification of GFP/IB4 ratio of EC-mTmG retinas. Error bars are s.e.m. n ≥ 3 retinas per genotype. Data information: Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 was considered statistically significant.(D) Quantification of IB4-positive area per retina in Pik3caH1047R/WT retinas. Data presented as a percentage of the control for each 4-OHT dose. Error bars are s.e.m. n = 4 retinas per genotype. Data information: Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 was considered statistically significant.


Figure 4 ( A ) Representative images of EC - Pik3caH1047R / WT P6 retinas isolated from mice treated with 0 . 125 mg / kg of 4 - OHT on P1 . Retinas were immunostained for blood vessels ( IB4 ) . Red asterisks show arteries and arterioles and yellow arrowheads veins . Scale bars : 150 μm ( left panel ) and 30 μm ( right , high magnification panels ) . ( B ) Representative images of P6 retinas from control and EC - Pik3caH1047R / WT mice treated with 0 . 125 mg / kg 4 - OHT on P1 , following immunostaining for p - S6 ( S235 / 236 ) and blood vessels ( IB4 ) . Scale bars : 150 μm ( left panels ) and 30 μm ( right panels , high magnification ) . ( C ) Representative control and EC - Pik3caH1047R / WT P6 retinas immunostained for blood vessels ( IB4 ) , EC nuclei ( Erg ) and EdU . Scale bars : 150 μm ( left panels ) and 30 μm ( right panels , high magnification ) . ( D - G ) Quantification of ( D ) p - S6 ( S235 / 236 ) intensity ( presented as a fold change of vehicle - treated control ) , ( E ) EC proliferation by EdU staining , ( F ) EC number by Erg positive cells and ( G ) retinal vascularity by IB4 - positive area in control and EC - Pik3caH1047R / WT retinas . Error bars are s . e . m . n > 5 retinas per genotype . Statistical analysis was performed by nonparametric Mann - Whitney test . * * p < 0 . 01 was considered statistically significant .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4(A) Representative images of EC-Pik3caH1047R/WT P6 retinas isolated from mice treated with 0.125 mg/kg of 4-OHT on P1. Retinas were immunostained for blood vessels (IB4). Red asterisks show arteries and arterioles and yellow arrowheads veins. Scale bars: 150 μm (left panel) and 30 μm (right, high magnification panels). (B) Representative images of P6 retinas from control and EC-Pik3caH1047R/WT mice treated with 0.125 mg/kg 4-OHT on P1, following immunostaining for p-S6 (S235/236) and blood vessels (IB4). Scale bars: 150 μm (left panels) and 30 μm (right panels, high magnification). (C) Representative control and EC-Pik3caH1047R/WT P6 retinas immunostained for blood vessels (IB4), EC nuclei (Erg) and EdU. Scale bars: 150 μm (left panels) and 30 μm (right panels, high magnification). (D-G) Quantification of (D) p-S6 (S235/236) intensity (presented as a fold change of vehicle-treated control), (E) EC proliferation by EdU staining, (F) EC number by Erg positive cells and (G) retinal vascularity by IB4-positive area in control and EC-Pik3caH1047R/WT retinas. Error bars are s.e.m. n > 5 retinas per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. **p < 0.01 was considered statistically significant.


Figure 5 ( B , C ) Representative images of P6 retinas isolated from control and EC - Pik3caH1047R / WT mouse littermates . Blood vessels were stained with IB4 . Lower panels showing high magnification images of the representative areas showing ( B ) blood vessels ( IB4 ) , EC nuclei ( Erg ) , EdU incorporation and ( C ) pS6 ( S235 / 236 ) . Scale bars : 150 μm ( upper panel ) and 30 μm ( lower panels ) . ( D - G ) Quantification of ( D ) retinal vascularity by IB4 staining , ( E ) EC number by Erg immunostaining , ( F ) EC proliferation by EdU staining , and ( G ) pS6 ( S235 / 236 ) intensity ( presented as a fold change of vehicle - treated control ) . Bars represent the mean ± s . e . m . n ≥ 4 retinas per genotype . Statistical analysis was performed by nonparametric Mann - Whitney test . * p < 0 . 05 and * * p < 0 . 01 were considered statistically significant .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5(B, C) Representative images of P6 retinas isolated from control and EC-Pik3caH1047R/WT mouse littermates. Blood vessels were stained with IB4. Lower panels showing high magnification images of the representative areas showing (B) blood vessels (IB4), EC nuclei (Erg), EdU incorporation and (C) pS6 (S235/236). Scale bars: 150 μm (upper panel) and 30 μm (lower panels). (D-G) Quantification of (D) retinal vascularity by IB4 staining, (E) EC number by Erg immunostaining, (F) EC proliferation by EdU staining, and (G) pS6 (S235/236) intensity (presented as a fold change of vehicle-treated control). Bars represent the mean ± s.e.m. n ≥ 4 retinas per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 and **p < 0.01 were considered statistically significant.


Figure 6 ( B , C ) Representative confocal images of P6 retinas isolated from control and EC - Pik3caH1047R / WT mouse littermates . Blood vessels were stained with IB4 . Lower panels showing high magnification images of the representative areas showing ( B ) blood vessels ( IB4 ) , EC nuclei ( Erg ) , EdU incorporation and ( C ) pS6 ( S235 / 236 ) . Scale bars : 150 μm ( upper panels ) and 30 μm ( lower panels ) . ( D - G ) Quantification of ( D ) retinal vascularity by IB4 staining , ( E ) EC number by Erg immunostaining , ( F ) EC proliferation by EdU staining , and ( G ) pS6 ( S235 / 236 ) intensity ( presented as a fold change of vehicle - treated control ) . Bars represent the mean ± s . e . m . n ≥ 3 retinas per genotype . Statistical analysis was performed by nonparametric Mann - Whitney test . * p < 0 . 05 and * * p < 0 . 01 were considered statistically significant .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(B, C) Representative confocal images of P6 retinas isolated from control and EC-Pik3caH1047R/WT mouse littermates. Blood vessels were stained with IB4. Lower panels showing high magnification images of the representative areas showing (B) blood vessels (IB4), EC nuclei (Erg), EdU incorporation and (C) pS6 (S235/236). Scale bars: 150 μm (upper panels) and 30 μm (lower panels). (D-G) Quantification of (D) retinal vascularity by IB4 staining, (E) EC number by Erg immunostaining, (F) EC proliferation by EdU staining, and (G) pS6 (S235/236) intensity (presented as a fold change of vehicle-treated control). Bars represent the mean ± s.e.m. n ≥ 3 retinas per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 and **p < 0.01 were considered statistically significant.


Figure 7 ( B , C ) Representative confocal images of P6 retinas isolated from control and EC - Pik3caH1047R / WT mouse littermates . Blood vessels were stained with IB4 . Lower panels showing high magnification images of the representative areas showing ( B ) blood vessels ( IB4 ) , EC nuclei ( Erg ) , EdU incorporation and ( C ) pS6 ( S235 / 236 ) . Scale bars : 150 μm ( upper panels ) and 30 μm ( lower panels ) . ( D - G ) Quantification of ( D ) retinal vascularity by IB4 staining , ( E ) EC number by Erg immunostaining , ( F ) EC proliferation by EdU staining , and ( G ) pS6 ( S235 / 236 ) intensity ( presented as a fold change of vehicle - treated control ) . Bars represent the mean ± s . e . m . n ≥ 4 retinas per genotype . Statistical analysis was performed by nonparametric Mann - Whitney test . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 were considered statistically significant .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(B, C) Representative confocal images of P6 retinas isolated from control and EC-Pik3caH1047R/WT mouse littermates. Blood vessels were stained with IB4. Lower panels showing high magnification images of the representative areas showing (B) blood vessels (IB4), EC nuclei (Erg), EdU incorporation and (C) pS6 (S235/236). Scale bars: 150 μm (upper panels) and 30 μm (lower panels). (D-G) Quantification of (D) retinal vascularity by IB4 staining, (E) EC number by Erg immunostaining, (F) EC proliferation by EdU staining, and (G) pS6 (S235/236) intensity (presented as a fold change of vehicle-treated control). Bars represent the mean ± s.e.m. n ≥ 4 retinas per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05, **p < 0.01 and ***p < 0.001 were considered statistically significant.