C , D . Scatter plots showing individual recordings of pairs of uninfected and infected neurons at − 60 mV ( AMPAR - mediated EPSCs ) in slices expressing EGFP - GSK3α ( C ) or EGFP - GSK3β ( D ) . Error bars represent SEM . Statistical significance was calculated according to the Wilcoxon signed - rank test . E , F . Scatter plots showing individual recordings of pairs of uninfected and infected neurons at + 40 mV ( NMDAR - mediated EPSCs ) in slices expressing EGFP - GSK3α ( E ) or EGFP - GSK3β ( F ) . Error bars represent SEM . Statistical significance was calculated according to the Wilcoxon signed - rank test .

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

C, D. Scatter plots showing individual recordings of pairs of uninfected and infected neurons at −60 mV (AMPAR-mediated EPSCs) in slices expressing EGFP-GSK3α (C) or EGFP-GSK3β (D). Error bars represent SEM. Statistical significance was calculated according to the Wilcoxon signed-rank test. E, F. Scatter plots showing individual recordings of pairs of uninfected and infected neurons at +40 mV (NMDAR-mediated EPSCs) in slices expressing EGFP-GSK3α (E) or EGFP-GSK3β (F). Error bars represent SEM. Statistical significance was calculated according to the Wilcoxon signed-rank test.


A . Representative confocal images from EGFP - GSK3α FRAP experiments . Upper left panel ( ' Baseline ' ) shows EGFP - GSK3α expression in a dendrite and dendritic spine of organotypic slices cultured from rat hippocampus . The spine was bleached ( ' Bleach ' ; yellow dashed circle ) and its fluorescence partially recovered 90 s later ( ' Recovery ' ) . Single dendritic spines were imaged once per second for 10 seconds prior to , and for 90 seconds following , spine photobleaching . Such single FRAP experiments were performed on different spines during basal conditions ( ' basal ' ; upper panels ) , immediately after cLTD induction with 20 µM NMDA for 5 minutes ( ' cLTD ' ; middle panels ) , and immediately following 10 minutes of washout ( ' washout ' ; lower panels ) . Scale bars = 1 µm . B . Time course of fluorescence recovery during FRAP experiments described in A . White circles ( ' basal ' ) show the average spine / dendrite fluorescence of FRAP on EGFP - GSK3α - expressing spines in slices prior to cLTD induction . Pink circles ( ' cLTD ' ) show fluorescence in spines in which cLTD had been induced using 20 µM NMDA . Green circles ( ' washout ' ) represent the average of spine fluorescence after NMDA washout for 10 minutes . Recovery trajectories for the three conditions are fitted with two - phase exponential curves ( red lines ) . C , D . As A , B but for spines expressing EGFP - GSK3β . Scale bars = 1 µm .

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

A. Representative confocal images from EGFP-GSK3α FRAP experiments. Upper left panel ('Baseline') shows EGFP-GSK3α expression in a dendrite and dendritic spine of organotypic slices cultured from rat hippocampus. The spine was bleached ('Bleach'; yellow dashed circle) and its fluorescence partially recovered 90 s later ('Recovery'). Single dendritic spines were imaged once per second for 10 seconds prior to, and for 90 seconds following, spine photobleaching. Such single FRAP experiments were performed on different spines during basal conditions ('basal'; upper panels), immediately after cLTD induction with 20 µM NMDA for 5 minutes ('cLTD'; middle panels), and immediately following 10 minutes of washout ('washout'; lower panels). Scale bars = 1 µm. B. Time course of fluorescence recovery during FRAP experiments described in A. White circles ('basal') show the average spine/dendrite fluorescence of FRAP on EGFP-GSK3α-expressing spines in slices prior to cLTD induction. Pink circles ('cLTD') show fluorescence in spines in which cLTD had been induced using 20 µM NMDA. Green circles ('washout') represent the average of spine fluorescence after NMDA washout for 10 minutes. Recovery trajectories for the three conditions are fitted with two-phase exponential curves (red lines). C, D. As A, B but for spines expressing EGFP-GSK3β. Scale bars = 1 µm.


E . Quantification of last 10 seconds of fluorescence recovery ( 90 - 100 seconds ) in B and D . Bars represent mean ± SEM . Statistical significance was calculated according to the Mann - Whitney U test . N = 12 , 11 and 11 spines for EGFP - GSK3α baseline , cLTD and washout groups and 20 , 11 and 9 spines for EGFP - GSK3β baseline , cLTD and washout groups , respectively .

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

E. Quantification of last 10 seconds of fluorescence recovery (90-100 seconds) in B and D. Bars represent mean ± SEM. Statistical significance was calculated according to the Mann-Whitney U test. N = 12, 11 and 11 spines for EGFP-GSK3α baseline, cLTD and washout groups and 20, 11 and 9 spines for EGFP-GSK3β baseline, cLTD and washout groups, respectively.


F . Spine / dendrite ratios showing enrichment of EGFP - GSK3α or EGFP - GSK3β in ' baseline ' , ' cLTD ' or ' recovery ' conditions . Bars represent mean ± SEM . Statistical significance was calculated according to the Mann - Whitney U test .

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

F. Spine/dendrite ratios showing enrichment of EGFP-GSK3α or EGFP-GSK3β in 'baseline', 'cLTD' or 'recovery' conditions. Bars represent mean ± SEM. Statistical significance was calculated according to the Mann-Whitney U test.


G . Representative western blots from whole extracts of organotypic slices incubated in ACSF ( untreated ; ' baseline ' ) , treated with 20 µM NMDA for 5 minutes ( ' cLTD ' ) or treated with 20 µM NMDA for 5 minutes and allowed to recover in plain ACSF for 10 minutes ( ' washout ' ) . Blots were probed for pSer21 / 9 GSK3α / β , total GSK3α / β , pSer845 GluA1 , and total GluA1 . H . Quantification of experiments as those shown in G . Error bars represent SEM and statistical significance was calculated according to the Mann - Whitney U test . N = 4 independent experiments for all conditions . Asterisks ( * * ) denote P < 0 . 01 .

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

G. Representative western blots from whole extracts of organotypic slices incubated in ACSF (untreated; 'baseline'), treated with 20 µM NMDA for 5 minutes ('cLTD') or treated with 20 µM NMDA for 5 minutes and allowed to recover in plain ACSF for 10 minutes ('washout'). Blots were probed for pSer21/9 GSK3α/β, total GSK3α/β, pSer845 GluA1, and total GluA1. H. Quantification of experiments as those shown in G. Error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test. N = 4 independent experiments for all conditions. Asterisks (**) denote P < 0.01.


A . Representative confocal images from EGFP - GSK3α FRAP experiments in organotypic slices cultured from C57 wild - type mice . Upper left panel ( ' Baseline ' ) shows EGFP - GSK3α expression in a dendrite and dendritic spine . The spine was bleached ( ' Bleach ' ; yellow dashed circle ) and its fluorescence partially recovered 90 s later ( ' Recovery ' ) . Single dendritic spines were imaged once per second for 10 seconds prior to , and for 90 seconds following , spine photobleaching . Such single FRAP experiments were performed on different spines during basal conditions ( ' basal ' ; upper panels ) , immediately after cLTD induction with 20 µM NMDA for 5 minutes ( ' cLTD ' ; middle panels ) , and immediately following 10 minutes of washout ( ' washout ' ; lower panels ) . Scale bars = 1 µm . B . Time course of fluorescence recovery during FRAP experiments described in A . White circles ( ' basal ' ) show the average spine / dendrite fluorescence of FRAP on EGFP - GSK3α - expressing spines in wild - type mouse slices prior to cLTD induction . Pink circles ( ' cLTD ' ) show fluorescence in spines in which cLTD had been induced using 20 µM NMDA . Green circles ( ' washout ' ) represent the average of spine fluorescence after NMDA washout for 10 minutes . Recovery trajectories for the three conditions are fitted with two - phase exponential curves ( red lines ) . C , D . As A , B but in slices cultured from C57 tauKO mice . Scale bars = 1 µm .

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

A. Representative confocal images from EGFP-GSK3α FRAP experiments in organotypic slices cultured from C57 wild-type mice. Upper left panel ('Baseline') shows EGFP-GSK3α expression in a dendrite and dendritic spine. The spine was bleached ('Bleach'; yellow dashed circle) and its fluorescence partially recovered 90 s later ('Recovery'). Single dendritic spines were imaged once per second for 10 seconds prior to, and for 90 seconds following, spine photobleaching. Such single FRAP experiments were performed on different spines during basal conditions ('basal'; upper panels), immediately after cLTD induction with 20 µM NMDA for 5 minutes ('cLTD'; middle panels), and immediately following 10 minutes of washout ('washout'; lower panels). Scale bars = 1 µm. B. Time course of fluorescence recovery during FRAP experiments described in A. White circles ('basal') show the average spine/dendrite fluorescence of FRAP on EGFP-GSK3α-expressing spines in wild-type mouse slices prior to cLTD induction. Pink circles ('cLTD') show fluorescence in spines in which cLTD had been induced using 20 µM NMDA. Green circles ('washout ') represent the average of spine fluorescence after NMDA washout for 10 minutes. Recovery trajectories for the three conditions are fitted with two-phase exponential curves (red lines). C, D. As A, B but in slices cultured from C57 tauKO mice. Scale bars = 1 µm.


E . Quantification of last 10 seconds of fluorescence recovery ( 90 - 100 seconds ) in B and D . Error bars represent SEM and statistical significance was calculated according to the Mann - Whitney U test . N = 13 , 11 and 12 spines for wt baseline , cLTD and recovery and 9 , 7 and 5 spines for tauKO baseline , cLTD and recovery groups , respectively .

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

E. Quantification of last 10 seconds of fluorescence recovery (90-100 seconds) in B and D. Error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test. N = 13, 11 and 12 spines for wt baseline, cLTD and recovery and 9, 7 and 5 spines for tauKO baseline, cLTD and recovery groups, respectively.


F . Spine / dendrite ratios showing enrichment of EGFP - GSK3α in wild - type or tauKO slices in ' baseline ' , ' cLTD ' or ' recovery ' conditions . Error bars represent SEM and statistical significance was calculated according to the Mann - Whitney U test .

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

F. Spine/dendrite ratios showing enrichment of EGFP-GSK3α in wild-type or tauKO slices in 'baseline', 'cLTD' or 'recovery' conditions. Error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test.


B . Representative traces for C - F , showing evoked AMPAR - and NMDAR - mediated EPSCs ( collected at − 60 mV and + 40 mV , respectively ) in paired uninfected ( black traces ) and EGFP - GSK3α - expressing neurons ( green traces ) in C57 wt or tauKO slices .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative traces for C-F, showing evoked AMPAR- and NMDAR-mediated EPSCs (collected at −60 mV and +40 mV, respectively) in paired uninfected (black traces) and EGFP-GSK3α-expressing neurons (green traces) in C57 wt or tauKO slices.


C - F . Scatter plots showing individual recordings of pairs of uninfected and infected neurons at − 60 mV ( AMPAR - mediated EPSCs ) in C57 wt ( C ) or tauKO slices ( D ) , or at + 40 mV ( NMDAR - mediated EPSCs ) in wt ( E ) or tauKO slices ( F ) . Red circles represent the averages of all pairs in a given plot , and n denotes the number of cell pairs . In all cases , error bars represent SEM and statistical significance was calculated according to the Mann - Whitney U test .

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

C-F. Scatter plots showing individual recordings of pairs of uninfected and infected neurons at −60 mV (AMPAR-mediated EPSCs) in C57 wt (C) or tauKO slices (D), or at +40 mV (NMDAR-mediated EPSCs) in wt (E) or tauKO slices (F). Red circles represent the averages of all pairs in a given plot, and n denotes the number of cell pairs. In all cases, error bars represent SEM and statistical significance was calculated according to the Mann-Whitney U test.


( C ) Confocal image of a constricting cell during ventral furrow formation showing an overlay between the myosin - II probe Sqh : : GFP and the plasma membrane marker GAP43 : : mCherry . Scale bar , 5

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

(C) Confocal image of a constricting cell during ventral furrow formation showing an overlay between the myosin-II probe Sqh::GFP and the plasma membrane marker GAP43::mCherry. Scale bar, 5 μm


( D ) Movie ( still frames ) of a gastrulating Drosophila embryo expressing endogenously tagged mVenus : : βH - spectrin imaged in a cross section using two - photon microscopy . βH - spectrin is enriched at the apical surface of ventral mesodermal cells during tissue invagination ( arrowheads ) . Scale bar , 50 μm .

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

(D) Movie (still frames) of a gastrulating Drosophila embryo expressing endogenously tagged mVenus::βH-spectrin imaged in a cross section using two-photon microscopy. βH-spectrin is enriched at the apical surface of ventral mesodermal cells during tissue invagination (arrowheads). Scale bar, 50 μm.


( E ) Z - Projections of confocal image stacks showing the apical cell surface of a Drosophila embryo expressing endogenously tagged mVenus : : βH - spectrin ( green ) and the plasma membrane marker GAP43 : : mCherry at 5 min ( top ) , 7 min ( middle ) and 9 min ( bottom ) after initial medio - apical accumulation of βH - spectrin . Scale bar , 20

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

(E) Z-Projections of confocal image stacks showing the apical cell surface of a Drosophila embryo expressing endogenously tagged mVenus::βH-spectrin (green) and the plasma membrane marker GAP43::mCherry at 5 min (top), 7 min (middle) and 9 min (bottom) after initial medio-apical accumulation of βH-spectrin. Scale bar, 20 μm


( F , G ) Immunostaining of βH - spectrin ( green ) visualized by STED nanoscopy revealed medio - apical βH - spectrin supra - cellular fibers in mesodermal cells ( F ) . In ectodermal cells , βH - spectrin localizes to apical cell junctions ( G ) . Note , in ( F ) the junctional βH - spectrin signal is shown in magenta as a proxy for cell membranes . Scale bars , 2 . 5 μm .

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

(F, G) Immunostaining of βH-spectrin (green) visualized by STED nanoscopy revealed medio-apical βH-spectrin supra-cellular fibers in mesodermal cells (F). In ectodermal cells, βH-spectrin localizes to apical cell junctions (G). Note, in (F) the junctional βH-spectrin signal is shown in magenta as a proxy for cell membranes. Scale bars, 2.5 μm.


( H - J ) Confocal images of the ventral surface of a Drosophila embryo expressing endogenously tagged mVenus : : βH - spectrin ( H ) , the myosin - II marker Sqh : : mCherry ( I ) and a merge of the two ( J ) with βH - spectrin in green and Sqh : : mCherry in magenta . Scale bars : 20 μm .

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

(H-J) Confocal images of the ventral surface of a Drosophila embryo expressing endogenously tagged mVenus::βH-spectrin (H), the myosin-II marker Sqh::mCherry (I) and a merge of the two (J) with βH-spectrin in green and Sqh::mCherry in magenta. Scale bars: 20 μm.


( K - L ) Co - staining of phalloidin ( F - actin reporter ; magenta ) and βH - spectrin ( green ) of the apical surface of a mesodermal cell at the onset of ventral furrow formation ( K ) and at a later time point ( L ) visualized by confocal microscopy . White dashed lines indicate the cell boundaries segmented based on the phalloidin - staining of sub - apical confocal sections . Scale bars : 5 μm .

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

(K-L) Co-staining of phalloidin (F-actin reporter; magenta) and βH-spectrin (green) of the apical surface of a mesodermal cell at the onset of ventral furrow formation (K) and at a later time point (L) visualized by confocal microscopy. White dashed lines indicate the cell boundaries segmented based on the phalloidin-staining of sub-apical confocal sections. Scale bars: 5 μm.


( A ) Z - Projection of confocal image stacks spanning 5 μm of a control embryo ( left ) co - expressing endogenously tagged mVenus : : βH - spectrin ( green ) and the plasma membrane marker GAP43 : : mCherry ( magenta ) and of an embryo additionally expressing a maternally driven anti - GFP nanobody KD module to knock - down βH - spectrin ( right ) . Co - expression of the anti - GFP nanobody KD system resulted in a drastic decrease of βH - spectrin protein levels as assessed by the fluorescent signal intensity which dropped below the detection limit .

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

(A) Z-Projection of confocal image stacks spanning 5 μm of a control embryo (left) co-expressing endogenously tagged mVenus::βH-spectrin (green) and the plasma membrane marker GAP43::mCherry (magenta) and of an embryo additionally expressing a maternally driven anti-GFP nanobody KD module to knock-down βH-spectrin (right). Co-expression of the anti-GFP nanobody KD system resulted in a drastic decrease of βH-spectrin protein levels as assessed by the fluorescent signal intensity which dropped below the detection limit.


( B ) Pie diagrams quantifying the percentage of control embryos ( top , n = 50 ) and βH - spectrin knock - down embryos ( bottom , n = 40 ) that formed ventral furrow either normally or with minor phenotypes ( green ) and the percentage of embryos that displayed severe abnormalities or did not at all undergo tissue invagination ( red ) . The assessment of phenotypes was done in an unbiased randomized approach .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Pie diagrams quantifying the percentage of control embryos (top, n=50) and βH-spectrin knock-down embryos (bottom, n=40) that formed ventral furrow either normally or with minor phenotypes (green) and the percentage of embryos that displayed severe abnormalities or did not at all undergo tissue invagination (red). The assessment of phenotypes was done in an unbiased randomized approach.


( C - J ) Confocal images of a control embryo ( C , D ) and βH - spectrin knock - down embryos with a minor phenotype ( E , F ) , with a severe phenotype that underwent tissue invagination ( G , H ) and with a severe phenotype that did not undergo tissue invagination ( I , J ) . Surface view is shown in ( C , E , G , I ) and the cross section through the tissue at two different time points in ( D , F , H , J ) . All embryos expressed the plasma membrane marker GAP43 : : mCherry . Scale bars , 20 μm .

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

(C-J) Confocal images of a control embryo (C,D) and βH-spectrin knock-down embryos with a minor phenotype (E,F), with a severe phenotype that underwent tissue invagination (G, H) and with a severe phenotype that did not undergo tissue invagination (I, J). Surface view is shown in (C,E,G,I) and the cross section through the tissue at two different time points in (D,F,H,J). All embryos expressed the plasma membrane marker GAP43::mCherry. Scale bars, 20 μm.


( K , L ) Quantification of apical surface area ( K ) and anisotropy ( L ) in control embryos ( green , nControl : 6 embryos ) and in βH - spectrin knock - down embryos ( red , nβH - spectrin KD : 7 embryos ) . Solid lines indicate the mean values and the semi - transparent regions the corresponding standard deviation .

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

(K, L) Quantification of apical surface area (K) and anisotropy (L) in control embryos (green, nControl: 6 embryos) and in βH-spectrin knock-down embryos (red, nβH-spectrin KD: 7 embryos). Solid lines indicate the mean values and the semi-transparent regions the corresponding standard deviation.


( D , E ) Still frames of a movie showing endogenously tagged mVenus : : βH - spectrin in the cross section of a gastrulating wild type ( D ) and twist mutant ( E ) embryo imaged using two - photon microscopy . The top frame corresponds to the time point where an initial bending of the apical surface was observed , the middle frame was taken 2 min and the lower frame 3 min afterwards . Scale bars , 30 µm .

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

(D, E) Still frames of a movie showing endogenously tagged mVenus::βH-spectrin in the cross section of a gastrulating wild type (D) and twist mutant (E) embryo imaged using two-photon microscopy. The top frame corresponds to the time point where an initial bending of the apical surface was observed, the middle frame was taken 2 min and the lower frame 3 min afterwards. Scale bars, 30 µm.


( F , G ) Confocal images showing apical βH - spectrin ( endogenously tagged mVenus : : βH - spectrin ) in a wild type ( F ) and twist mutant ( G ) embryo . Scale bars , 20 μm . Medio - apical βH - spectrin fibers observed in wild type embryos were absent in twist mutant embryos .

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

(F, G) Confocal images showing apical βH-spectrin (endogenously tagged mVenus::βH-spectrin) in a wild type (F) and twist mutant (G) embryo. Scale bars, 20 μm. Medio-apical βH-spectrin fibers observed in wild type embryos were absent in twist mutant embryos.


( H - Q ) Confocal or two - photon microscopy images of the apical surface of an embryo mounted with the dorsal side facing the objective and co - expressing endogenously tagged mVenus : : βH - spectrin ( green , H , K , M , P ) , the myosin - II marker Sqh : : mCherry ( magenta , I , N ) , and the optogenetic modules as explained in ( C ) . The apical surface is shown before photoactivation ( H - L ) and 1 min after photoactivation ( M - Q ) . Panels ( J , O ) show a merge of βH - spectrin and myosin - II and the dashed rectangle indicates regions shown in ( L and Q ) at higher magnification . Panels ( K , L ) and ( P , Q ) are zoom - ins into the region indicated in ( J ) an ( O ) , respectively . Scale bars , 20 μm ( H - J , M - O ) . 2 . 5 μm ( K , L , P , Q ) .

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

(H-Q) Confocal or two-photon microscopy images of the apical surface of an embryo mounted with the dorsal side facing the objective and co-expressing endogenously tagged mVenus::βH-spectrin (green, H, K, M, P), the myosin-II marker Sqh::mCherry (magenta, I, N), and the optogenetic modules as explained in (C). The apical surface is shown before photoactivation (H-L) and 1 min after photoactivation (M-Q). Panels (J, O) show a merge of βH-spectrin and myosin-II and the dashed rectangle indicates regions shown in (L and Q) at higher magnification. Panels (K, L) and (P, Q) are zoom-ins into the region indicated in (J) an (O), respectively. Scale bars, 20 μm (H-J, M-O). 2.5 μm (K, L, P, Q).


( R ) Quantification of apical βH - spectrin upon optogenetic stimulation of Rho signaling . The graph shows the mean intensity of βH - spectrin ( green ) at the apical surface over time as the fold change relative to the initial time point ( before photo - activation ) . The semi - transparent region indicates the standard deviation and dots indicate individual data points ( n = 5 embryos ) .

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

(R) Quantification of apical βH-spectrin upon optogenetic stimulation of Rho signaling. The graph shows the mean intensity of βH-spectrin (green) at the apical surface over time as the fold change relative to the initial time point (before photo-activation). The semi-transparent region indicates the standard deviation and dots indicate individual data points (n = 5 embryos).


( S - T ) Two - photon microscopy images of apical βH - spectrin ( green ) 1 . 5 min after optogenetic stimulation of Rho signaling in embryos that were previously injected with water ( S ) or the ROCK - inhibitor Y - 27634 ( T ) . Dashed boxes indicate photo - activated cells . Y - 27634 - injected embryos did not undergo apical constriction and did not accumulate βH - spectrin upon photo - activation , while water - injected embryos constricted and showed increased βH - spectrin levels . Scale bars : 10 µm .

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

(S-T) Two-photon microscopy images of apical βH-spectrin (green) 1.5 min after optogenetic stimulation of Rho signaling in embryos that were previously injected with water (S) or the ROCK-inhibitor Y-27634 (T). Dashed boxes indicate photo-activated cells. Y-27634-injected embryos did not undergo apical constriction and did not accumulate βH-spectrin upon photo-activation, while water-injected embryos constricted and showed increased βH-spectrin levels. Scale bars: 10 µm.


( A - C ) Confocal images of the apical cell surface of a βH - spectrin knock - down embryo showing the myosin - II marker Sqh : : mCherry ( green ) and E - cadherin : : mNeonGreen ( magenta ) during ventral furrow formation . ( A ) myosin - II accumulates normally at the apical surface and cells undergo an initial phase of constriction . The dashed rectangle indicates the region shown in ( B and C ) at higher magnification . Scale bar , 30 μm . Panels ( B , C ) show a sub - region within the ventral tissue at different time points . Initially a supracellular myosin - II network formed ( 0 s ) , before intercellular connections broke ( 13 s ) resulting in membrane tears ( dashed line ) with tissue retractions ( small arrow heads ) along the anterior - posterior axis . The intercellular network re - established ( 39 s , 65 s ) as new actomyosin fibers formed ( big arrow heads ) to reconnect neighboring cells . The intercellular network broke again causing tears ( dashed lines ) at different positions ( 104 s ) . Scale bars , 5 μm .

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

(A-C) Confocal images of the apical cell surface of a βH-spectrin knock-down embryo showing the myosin-II marker Sqh::mCherry (green) and E-cadherin::mNeonGreen (magenta) during ventral furrow formation. (A) myosin-II accumulates normally at the apical surface and cells undergo an initial phase of constriction. The dashed rectangle indicates the region shown in (B and C) at higher magnification. Scale bar, 30 μm. Panels (B,C) show a sub-region within the ventral tissue at different time points. Initially a supracellular myosin-II network formed (0 s), before intercellular connections broke (13 s) resulting in membrane tears (dashed line) with tissue retractions (small arrow heads) along the anterior-posterior axis. The intercellular network re-established (39 s, 65 s) as new actomyosin fibers formed (big arrow heads) to reconnect neighboring cells. The intercellular network broke again causing tears (dashed lines) at different positions (104 s). Scale bars, 5 μm.


( D , E ) Confocal images showing the myosin - II marker Sqh : : mCherry ( green ) , E - cadherin : : mNeonGreen ( magenta ) and the segmented membrane ( red ) of a single cell at different time points in a control embryo undergoing ratcheted constriction ( D ) and in a βH - spectrin knock - down embryo undergoing non - ratcheted pulsations ( E ) . Scale bars , 2 . 5 μm .

(D,E) Confocal images showing the myosin-II marker Sqh::mCherry (green), E-cadherin::mNeonGreen (magenta) and the segmented membrane (red) of a single cell at different time points in a control embryo undergoing ratcheted constriction (D) and in a βH-spectrin knock-down embryo undergoing non-ratcheted pulsations (E). Scale bars, 2.5 μm.


( F ) Heatmaps showing the apical surface of individual cells over time of control embryos ( left ) and βH - spectrin knock - down embryos ( right ) normalized to the initial time point ( nControl = 233 ; nβH - spectrinKD = 368 ; of 5 different embryos each ) . The color - coded scale bar is depicted on the left . Dark blue indicates a cell area bigger than the initial value ( > 1 ) and yellow indicates an area constricted to less than 1 / 5 of the initial area ( < 0 . 2 ) . Three different cell behaviors in βH - spectrin knock - down embryos were identified : cells that constricted , cells that showed impaired constriction and cells that expanded .

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

(F) Heatmaps showing the apical surface of individual cells over time of control embryos (left) and βH-spectrin knock-down embryos (right) normalized to the initial time point (nControl=233; nβH-spectrinKD=368; of 5 different embryos each). The color-coded scale bar is depicted on the left. Dark blue indicates a cell area bigger than the initial value (>1) and yellow indicates an area constricted to less than 1/5 of the initial area (<0.2). Three different cell behaviors in βH-spectrin knock-down embryos were identified: cells that constricted, cells that showed impaired constriction and cells that expanded.


( G ) Quantification of the percentage of cells that constricted ( to an area < 50 % , yellow ) , cells that showed impaired constriction ( to an area > 50 and < 100 % , green ) and cells that expanded ( to an area > 100 % , blue ) after 7 min in control embryos and in βH - spectrin knock - down embryos .

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

(G) Quantification of the percentage of cells that constricted (to an area < 50%, yellow), cells that showed impaired constriction (to an area >50 and <100%, green) and cells that expanded (to an area >100%, blue) after 7 min in control embryos and in βH-spectrin knock-down embryos.


( H , I ) Graph showing the average area over time of control cells ( H , n = 235 cells ) and cells in βH - spectrin knock - down embryos ( I ) that constricted ( yelow , n = 274 ) , of cells that showed impaired constriction ( green , n = 232 ) and of cells that expanded ( blue , n = 32 ) . The lines indicate the mean value and the semi - transparent region the standard deviation .

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

(H,I) Graph showing the average area over time of control cells (H, n=235 cells) and cells in βH-spectrin knock-down embryos (I) that constricted (yelow, n=274), of cells that showed impaired constriction (green, n=232) and of cells that expanded (blue, n=32). The lines indicate the mean value and the semi-transparent region the standard deviation.


( J ) Boxplots showing the distribution of apical cell area values 7 . 5 min after the onset of ventral furrow formation of ventral cells in control embryos , of ventral cells in βH - spectrin knock - down embryos that constricted , and ventral cells in βH - spectrin knock - down embryos that displayed impaired constriction and expanded . Sample numbers are equal to I . The values are presented as percentage of initial cell area . ANOVA result ( all data points ) : F ( 3 , 717 ) = 763 . 9 , p = 9 . 6e - 223 ; Cohen ' s d compared to the control : dconstricted = 0 . 1 , dimpaired = 1 . 5 , dexpanded = 3 . 7 . Due to a big effect size ( high sample number ) , statistical significance was assessed based on Cohen ' s d . A Cohen ' s d < 0 . 5 was considered not significant ( n . s . ) .

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

(J) Boxplots showing the distribution of apical cell area values 7.5 min after the onset of ventral furrow formation of ventral cells in control embryos, of ventral cells in βH-spectrin knock-down embryos that constricted, and ventral cells in βH-spectrin knock-down embryos that displayed impaired constriction and expanded. Sample numbers are equal to I. The values are presented as percentage of initial cell area. ANOVA result (all data points): F(3,717)=763.9, p=9.6e-223; Cohen's d compared to the control: dconstricted=0.1, dimpaired=1.5, dexpanded=3.7. Due to a big effect size (high sample number), statistical significance was assessed based on Cohen's d. A Cohen's d < 0.5 was considered not significant (n. s.).


( K ) Quantification of the peak constriction rate and expansion rate relative to control cells . Control cells and cells in βH - spectrin knock - down embryos that showed impaired constriction or expanded were compared . The mean value and standard deviation are shown . Please note the logarithmic scale . While in βH - spectrin knock - down cells the constriction rate was ~ 24 % lower ( mean value of 0 . 8 ) compared to control cells , the expansion rate increased to ~ 60 % ( mean value of 1 . 6 ) . The absolute change of the expansion rate was 2 . 5 times higher than the change of the constriction rate . ANOVA result ( all data points ) : F ( 3 , 717 ) = 349 . 2 , p = 9 . 7e - 140 ; pairwise comparison of βH - spectrin knock - down cells to control using Cohen ' s d > 1 . 4 .

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

(K) Quantification of the peak constriction rate and expansion rate relative to control cells. Control cells and cells in βH-spectrin knock-down embryos that showed impaired constriction or expanded were compared. The mean value and standard deviation are shown. Please note the logarithmic scale. While in βH-spectrin knock-down cells the constriction rate was ~24% lower (mean value of 0.8) compared to control cells, the expansion rate increased to ~60% (mean value of 1.6). The absolute change of the expansion rate was 2.5 times higher than the change of the constriction rate. ANOVA result (all data points): F(3,717)=349.2, p=9.7e-140; pairwise comparison of βH-spectrin knock-down cells to control using Cohen's d>1.4.


D Quantification of the frequency of cell clusters with a strong polarised PAR - 3 centre . Data information : Data are presented as means ± SEM in ( D ) n = 3 experiments in ( D ) Two - way ANOVA analysis in ( D ) P values were listed in the graphs .

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

D Quantification of the frequency of cell clusters with a strong polarised PAR-3 centre. Data information: Data are presented as means ± SEM in (D) n = 3 experiments in (D) Two-way ANOVA analysis in (D) P values were listed in the graphs.


F Line - scan profiles of PAR - 3 at the cell - cell interface of 2 - cell mESC doublets . Line scans were sectioned and fitted to each 5 % along the cell - cell interface length . Data information : ; individual line - scans and mean valued line - scans in ( F ) 15 - 32 cell clusters were analysed for each column in every experiment ; 15 cell doublets for each condition from one experiment in ( F )

F Line-scan profiles of PAR-3 at the cell-cell interface of 2-cell mESC doublets. Line scans were sectioned and fitted to each 5% along the cell-cell interface length. Data information: ; individual line-scans and mean valued line-scans in (F) 15-32 cell clusters were analysed for each column in every experiment; 15 cell doublets for each condition from one experiment in (F)


H , I Representative images of centralised ZO - 1 puncta and polarised Golgi apparatus , labelled by GM130 ( H ) and quantification of the frequency of cell doublets with a polarised ZO - 1 or Golgi apparatus centre ( I ) . Data information : Data are presented as means ± SEM in ; ( I ) ; n = 3 experiments in ( I ) student ' s t - test analysis in ( I ) P values were listed in the graphs . scale bars : 10 µm .

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

H, I Representative images of centralised ZO-1 puncta and polarised Golgi apparatus, labelled by GM130 (H) and quantification of the frequency of cell doublets with a polarised ZO-1 or Golgi apparatus centre (I). Data information: Data are presented as means ± SEM in ; (I); n = 3 experiments in (I) student's t-test analysis in (I) P values were listed in the graphs. scale bars: 10 µm.


J , K Representative images of polarised centrosomes ( J ) , labelled with γ - tubulin and distance between centrosomes ( K ) normalised to the length of the long axis of the doublets in 2 - cell clusters . Data information : individual values in dots and median values in bars in ( K ) . student ' s t - test analysis in ( K ) . P values were listed in the graphs . scale bars : 10 µm .

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

J, K Representative images of polarised centrosomes (J), labelled with γ-tubulin and distance between centrosomes (K) normalised to the length of the long axis of the doublets in 2-cell clusters. Data information: individual values in dots and median values in bars in (K). student's t-test analysis in (K). P values were listed in the graphs. scale bars: 10 µm.


quantification of the frequency of cell clusters with polarised mCherry - PAR - 6B ( C ) in control and mitomycin division - blocked mESC live cells after 24 hours in Matrigel . Data information : Data in ( C ) are presented as means ± SEM . n = 3 experiments . At least 20 clusters were analysed for each column in every experiment . Two - way ANOVA analysis ; P values were listed in the graphs .

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

quantification of the frequency of cell clusters with polarised mCherry-PAR-6B (C) in control and mitomycin division-blocked mESC live cells after 24 hours in Matrigel. Data information: Data in (C) are presented as means ± SEM. n = 3 experiments. At least 20 clusters were analysed for each column in every experiment. Two-way ANOVA analysis; P values were listed in the graphs.


A , B Immunofluorescence of PAR - 3 ( A ) and proportions of mESC clusters with a strong PAR - 3 centre ( B ) in wild - type ( W4 ) and E - cadherin knock - out ( Cdh1 KO ) mESCs at 24 hrs in Matrigel . Data information : Data are presented as means ± SEM in ( B ) n = 3 experiments in ( B ) Two - way ANOVA analysis in ( B ) , P values were listed in the graphs . scale bars : 10 µm .

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

A, B Immunofluorescence of PAR-3 (A) and proportions of mESC clusters with a strong PAR-3 centre (B) in wild-type (W4) and E-cadherin knock-out (Cdh1 KO) mESCs at 24 hrs in Matrigel. Data information: Data are presented as means ± SEM in (B) n = 3 experiments in (B) Two-way ANOVA analysis in (B), P values were listed in the graphs. scale bars: 10 µm.


C Line - scan profiles of PAR - 3 at the cell - cell interface in wild - type control , mitomycin C treated and Cdh1 KO control , mitomycin C treated 2 - cell doublets . D Ratio of PAR - 3 pixel intensity values at central and surrounding regions in > 2 - cell mESC clusters . Data information : individual and mean - valued line - scans in ( C ) individual cell cluster values ( small dots ) , mean experimental values ( large dots ) and means of 3 experiments ( bars ) ± SEM in ( D ) 17 - 50 cell clusters were analysed for each column in every experiment ; 15 doublets for each condition in ( C ) ; 17 - 30 cell clusters were analysed for each condition in every experiment in ( D ) ; Two - way ANOVA analysis in ( D ) , P values were listed in the graphs . scale bars : 10 µm .

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

C Line-scan profiles of PAR-3 at the cell-cell interface in wild-type control, mitomycin C treated and Cdh1 KO control, mitomycin C treated 2-cell doublets. D Ratio of PAR-3 pixel intensity values at central and surrounding regions in >2-cell mESC clusters. Data information: individual and mean-valued line-scans in (C) individual cell cluster values (small dots), mean experimental values (large dots) and means of 3 experiments (bars) ± SEM in (D) 17-50 cell clusters were analysed for each column in every experiment; 15 doublets for each condition in (C); 17-30 cell clusters were analysed for each condition in every experiment in (D); Two-way ANOVA analysis in (D), P values were listed in the graphs. scale bars: 10 µm.


E , F Representative images of PAR - 3 immunofluorescence ( E ) and line - scan profiles of PAR - 3 at the cell - cell interface ( F ) in WT homogeneous or WT ( ES - E14 ) / Cdh1 KO chimeric mESC 2 - cell doublets . * , WT mESCs . Data information : individual and mean - valued line - scans in ( F ) ; 15 doublets for each condition in ( F )

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

E, F Representative images of PAR-3 immunofluorescence (E) and line-scan profiles of PAR-3 at the cell-cell interface (F) in WT homogeneous or WT (ES-E14) /Cdh1 KO chimeric mESC 2-cell doublets. *, WT mESCs. Data information: individual and mean-valued line-scans in (F); 15 doublets for each condition in (F)


G , H Representative images of ZO - 1 puncta and Golgi apparatus ( G ) and proportions of mESC doublets with central ZO - 1 puncta or polarised Golgi apparatus ( H ) in WT and Cdh1 KO mESC doublets . Data information : Data are presented as means ± SEM in ( H ) ; student ' s t - test analysis in ( H ) ; P values were listed in the graphs . scale bars : 10 µm .

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

G, H Representative images of ZO-1 puncta and Golgi apparatus (G) and proportions of mESC doublets with central ZO-1 puncta or polarised Golgi apparatus (H) in WT and Cdh1 KO mESC doublets. Data information: Data are presented as means ± SEM in (H); student's t-test analysis in (H); P values were listed in the graphs. scale bars: 10 µm.


I Distance between centrosomes in cell doublets . The distance was normalised to the length of the long axis of the doubles . Data information : individual values in dots and median values in bars in ( I ) ; 35 - 40 doublets in each condition in ( I ) Two - way ANOVA analysis in ( I ) P values were listed in the graphs .

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

I Distance between centrosomes in cell doublets. The distance was normalised to the length of the long axis of the doubles. Data information: individual values in dots and median values in bars in (I); 35-40 doublets in each condition in (I) Two-way ANOVA analysis in (I) P values were listed in the graphs.


J Fluorescence recovery after photobleaching ( FRAP ) of E - cadherin - eGFP at the centre - most or side 2 µm regions of division - blocked mESC doublets ' cell - cell interfaces . K Mobile fraction of E - cadherin - eGFP calculated from the plotting of ( J ) . Data information : exponential association fitting curves ± SD in ( J ) ; means ± SD in ( K ) . 35 - 40 doublets in each condition in ( I ) ; 15 - 18 doublets for each condition in ( J ) & ( K ) . Two - way ANOVA analysis in ( K ) ; P values were listed in the graphs .

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

J Fluorescence recovery after photobleaching (FRAP) of E-cadherin-eGFP at the centre-most or side 2 µm regions of division-blocked mESC doublets' cell-cell interfaces. K Mobile fraction of E-cadherin-eGFP calculated from the plotting of (J). Data information: exponential association fitting curves ± SD in (J); means ± SD in (K). 35-40 doublets in each condition in (I); 15-18 doublets for each condition in (J) & (K). Two-way ANOVA analysis in (K); P values were listed in the graphs.


A - C Immunofluorescence of PAR - 3 and P - cadherin ( A ) , proportions of mESC clusters with a positive PAR - 3 centre ( B ) and line - scans of PAR - 3 at the cell - cell interface ( C ) in control , P - cadherin knock - down by RNAi , mitomycin treated and P - cadherin knock - down mitomycin treated W4 mESC doublets cultured for 24 hours in Matrigel . Data information : Data are presented as means ± SEM in ( B ) ; individual and mean - valued line - scans in ( C ) n = 3 experiments in ( B ) , 15 - 21 clusters were analysed for each column in every experiment ; 15 - 21 line - scans in ( C ) Two - way ANOVA analysis in ( B ) P values were listed in the graphs . scale bars : 5 µm .

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

A-C Immunofluorescence of PAR-3 and P-cadherin (A), proportions of mESC clusters with a positive PAR-3 centre (B) and line-scans of PAR-3 at the cell-cell interface (C) in control, P-cadherin knock-down by RNAi, mitomycin treated and P-cadherin knock-down mitomycin treated W4 mESC doublets cultured for 24 hours in Matrigel. Data information: Data are presented as means ± SEM in (B) ; individual and mean-valued line-scans in (C) n = 3 experiments in (B), 15-21 clusters were analysed for each column in every experiment; 15-21 line-scans in (C) Two-way ANOVA analysis in (B) P values were listed in the graphs. scale bars: 5 µm.


D - F Immunofluorescence of PAR - 3 and JAM - A ( D ) , proportions of mESC clusters with a positive PAR - 3 centre ( E ) and line - scans of PAR - 3 at the cell - cell interface ( F ) in control , JAM - A knock - down by RNAi , mitomycin treated and JAM - A knock - down mitomycin treated W4 mESC doublets cultured for 24 hours in Matrigel . Data information : Data are presented as means ± SEM in ( E ) , individual and mean - valued line - scans in ( F ) n = 3 experiments in ( E ) , 15 - 21 clusters were analysed for each column in every experiment ; 15 - 21 line - scans in ( F ) , Two - way ANOVA analysis in ( E ) , P values were listed in the graphs . scale bars : 5 µm .

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

D-F Immunofluorescence of PAR-3 and JAM-A (D), proportions of mESC clusters with a positive PAR-3 centre (E) and line-scans of PAR-3 at the cell-cell interface (F) in control, JAM-A knock-down by RNAi, mitomycin treated and JAM-A knock-down mitomycin treated W4 mESC doublets cultured for 24 hours in Matrigel. Data information: Data are presented as means ± SEM in (E), individual and mean-valued line-scans in (F) n = 3 experiments in (E), 15-21 clusters were analysed for each column in every experiment; 15-21 line-scans in (F), Two-way ANOVA analysis in (E), P values were listed in the graphs. scale bars: 5 µm.


G - I Immunofluorescence of PAR - 3 and Nectin - 2 ( G ) , proportions of mESC clusters with a positive PAR - 3 centre ( H ) and line - scans of PAR - 3 at the cell - cell interface ( I ) in control , Nectin - 2 knock - down by RNAi , mitomycin treated and Nectin - 2 knock - down mitomycin treated W4 mESC doublets cultured for 24 hours in Matrigel . Data information : Data are presented as means ± SEM in ( H ) ; individual and mean - valued line - scans in ( I ) . n = 3 experiments in ( H ) , 15 - 21 clusters were analysed for each column in every experiment ; 15 - 21 line - scans in ( I ) . Two - way ANOVA analysis in ( H ) ; P values were listed in the graphs . scale bars : 5 µm .

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

G-I Immunofluorescence of PAR-3 and Nectin-2 (G), proportions of mESC clusters with a positive PAR-3 centre (H) and line-scans of PAR-3 at the cell-cell interface (I) in control, Nectin-2 knock-down by RNAi, mitomycin treated and Nectin-2 knock-down mitomycin treated W4 mESC doublets cultured for 24 hours in Matrigel. Data information: Data are presented as means ± SEM in (H); individual and mean-valued line-scans in (I). n = 3 experiments in (H), 15-21 clusters were analysed for each column in every experiment; 15-21 line-scans in (I). Two-way ANOVA analysis in (H); P values were listed in the graphs. scale bars: 5 µm.


A , B Immunofluorescence of PAR - 3 ( A ) and proportions of mESC doublets with polarised PAR - 3 ( B ) in wild - type ( ES - E14 ) and E - cadherin knock - out ( Cdh1 KO ) cells at 30 hours in 0 . 5 % agarose . Data information : Data are presented as means ± SEM in ( B ) ; n = 3 experiments in ( B ) Two - way ANOVA analysis in ( B ) P values were listed in the graphs . scale bars : 5 µm .

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

A, B Immunofluorescence of PAR-3 (A) and proportions of mESC doublets with polarised PAR-3 (B) in wild-type (ES-E14) and E-cadherin knock-out (Cdh1 KO) cells at 30 hours in 0.5% agarose. Data information: Data are presented as means ± SEM in (B); n = 3 experiments in (B) Two-way ANOVA analysis in (B) P values were listed in the graphs. scale bars: 5 µm.


C Immunofluorescence of PAR - 3 in cell division blocked mESCs cultured on E - cadherin or Fibronectin coated glass topped with or without Matrigel for 24 hours . Data information : scale bars : 5 µm .

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

C Immunofluorescence of PAR-3 in cell division blocked mESCs cultured on E-cadherin or Fibronectin coated glass topped with or without Matrigel for 24 hours. Data information: scale bars: 5 µm.


D Heatmap of PAR - 3 in cells from one experiment of ( C ) . Squared frames were fitted to the main bodies of the cells . A pixel was the average value in each 10 % along the width of the frames . The heatmaps were stacks of 15 cells in one experiment . E PAR - 3 levels at the central 20 % of the heatmaps from three experiments of ( C ) . F Ratios between PAR - 3 in the 2 . 5 µm diameter central core and whole cell surface from three experiments of ( C ) . Data information : ; individual cell values ( small dots ) , mean experimental values ( large dots ) and means of experiments ± SEM ( bars ) in ( E ) & ( F ) . n = 3 experiments in ( E ) , ( F ) ; at least 15 clusters were analysed for each column in every experiment ; 15 - 20 cells for each column in every experiment of ( E ) & ( F ) . Two - way ANOVA analysis in ( E ) , ( F ) ; P values were listed in the graphs . scale bars : 5 µm .

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

D Heatmap of PAR-3 in cells from one experiment of (C). Squared frames were fitted to the main bodies of the cells. A pixel was the average value in each 10% along the width of the frames. The heatmaps were stacks of 15 cells in one experiment. E PAR-3 levels at the central 20% of the heatmaps from three experiments of (C). F Ratios between PAR-3 in the 2.5 µm diameter central core and whole cell surface from three experiments of (C). Data information: ; individual cell values (small dots), mean experimental values (large dots) and means of experiments ± SEM (bars) in (E) & (F). n = 3 experiments in (E), (F); at least 15 clusters were analysed for each column in every experiment; 15 - 20 cells for each column in every experiment of (E) & (F). Two-way ANOVA analysis in (E), (F); P values were listed in the graphs. scale bars: 5 µm.


D Percentage of cell clusters with different cavities relative to total cell clusters at different time points . The analysis was compared between the closed / rosette category among the conditions . E Percentage of cell clusters with closed cavities relative to total cell clusters with cavities calculated from ( D ) . Data information : Data are presented as means ± SEM in ( D ) & ( E ) . n = 20 ( 48 hours WT & Cdh1 KO ) , 30 ( 72 hours WT ) and 32 ( 72 hours Cdh1 KO ) images in ( A ) ; 3 experiments in ( D ) & ( E ) , at least 25 clusters were analysed for each column in every experiment . Two - way ANOVA analysis in ( D ) & ( E ) ; P values were listed in the graphs .

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

D Percentage of cell clusters with different cavities relative to total cell clusters at different time points. The analysis was compared between the closed/rosette category among the conditions. E Percentage of cell clusters with closed cavities relative to total cell clusters with cavities calculated from (D). Data information: Data are presented as means ± SEM in (D) & (E). n = 20 (48 hours WT & Cdh1 KO), 30 (72 hours WT) and 32 (72 hours Cdh1 KO) images in (A); 3 experiments in (D) & (E), at least 25 clusters were analysed for each column in every experiment. Two-way ANOVA analysis in (D) & (E); P values were listed in the graphs.


Immunoblot of whole cell lysates from multiple human lineage - committed cell - lines using a C - terminal anti - JARID2 antibody . The size of canonical form is 140kDa but an additional strong band was detected at around ~ 80kDa . The blot is divided according to cell - types , which are indicated in the figure .

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

Immunoblot of whole cell lysates from multiple human lineage-committed cell-lines using a C-terminal anti-JARID2 antibody. The size of canonical form is 140kDa but an additional strong band was detected at around ~80kDa. The blot is divided according to cell-types, which are indicated in the figure.


qPCR measurements of JARID2 mRNA levels in different cell lines . The RNA levels were measured relative to 18S rRNA . Means and SEs are calculated over at least 2 independent experiments and 3 technical replicates . The data is represented as mean ± SE .

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

qPCR measurements of JARID2 mRNA levels in different cell lines. The RNA levels were measured relative to 18S rRNA. Means and SEs are calculated over at least 2 independent experiments and 3 technical replicates. The data is represented as mean ± SE.


Immunoblot showing that the ~ 80kDa band disappears upon treatment of HEK293T and HaCaT cells with siRNAs against exon 3 and exon 15 of JARID2 RNA . Non - silencing control ( NSC ) was used as a transfection control .

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

Immunoblot showing that the ~80kDa band disappears upon treatment of HEK293T and HaCaT cells with siRNAs against exon 3 and exon 15 of JARID2 RNA. Non-silencing control (NSC) was used as a transfection control.


qPCR measurements of RNA levels ( n = 3 ) for the three isoforms of JARID2 in HaCaT cells . The RNA levels are plotted relative to 18S rRNA . Level of JARID2 isoform - 1 is significantly higher than that of isoform - 2 ( * * * P < 0 . 001 ) and isoform - 3 ( * * * * P < 0 . 0001 ) .

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

qPCR measurements of RNA levels (n=3) for the three isoforms of JARID2 in HaCaT cells. The RNA levels are plotted relative to 18S rRNA. Level of JARID2 isoform-1 is significantly higher than that of isoform-2 (***P<0.001) and isoform-3 (****P<0.0001).


Agarose gel showing RT - PCR products amplified using primers in exon3 and exon15 ( 2 . 9kb ) as well as RT - PCR product corresponding to JARID2 isoform - 1 ( 3 . 7kb ) amplified using the first ( exon 1 ) and the last exon ( exon18 ) . Only one product at the right size is observed indicating that it is unlikely that JARID2 might be a product of an alternatively spliced isoform of JARID2 .

Agarose gel showing RT-PCR products amplified using primers in exon3 and exon15 (2.9kb) as well as RT-PCR product corresponding to JARID2 isoform-1 (3.7kb) amplified using the first (exon 1) and the last exon (exon18). Only one product at the right size is observed indicating that it is unlikely that JARID2 might be a product of an alternatively spliced isoform of JARID2.


CRISPR - Cas9 knockout of JARID2 using a sgRNA guide designed to target main translation start site as seen in ( E ) . Immunoblot revealed that ~ 80kDa form was removed from JARID2 KO lines .

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

CRISPR-Cas9 knockout of JARID2 using a sgRNA guide designed to target main translation start site as seen in (E). Immunoblot revealed that ~80kDa form was removed from JARID2 KO lines.


Immunoblot after a full length JARID2 isoform - 1 was expressed from an exogenous vector in HaCaT cells showed an increase ( 2 - 3 fold ) in ~ 80kDa level .

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

Immunoblot after a full length JARID2 isoform-1 was expressed from an exogenous vector in HaCaT cells showed an increase (2-3 fold) in ~80kDa level.


Immunoprecipitation using anti - JARID2 antibody and followed by mass spectrometry identification of the ~ 80kDa band detected JARID2 peptides spanning only C - terminus of JARID2 . The identified peptides are shown as red arrows on schematic of JARID2 sequence .

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

Immunoprecipitation using anti-JARID2 antibody and followed by mass spectrometry identification of the ~80kDa band detected JARID2 peptides spanning only C-terminus of JARID2. The identified peptides are shown as red arrows on schematic of JARID2 sequence.


Immunoblot showing increase in expression levels of differentiation markers Involucrin ( IVL ) and Transglutaminase - 1 ( TGase - 1 ) as differentiation progressed indicating that keratinocyte differentiation protocol was successful . Protein levels on day 0 ( D0 ) , day 1 ( D1 ) , day 3 ( D3 ) and day 6 ( D6 ) of differentiation are shown . Immunoblot for JARID2 during D0 , D1 , D3 and D6 of differentiation ( as in B ) are shown .

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

Immunoblot showing increase in expression levels of differentiation markers Involucrin (IVL) and Transglutaminase-1 (TGase-1) as differentiation progressed indicating that keratinocyte differentiation protocol was successful. Protein levels on day 0 (D0), day 1 (D1), day 3 (D3) and day 6 (D6) of differentiation are shown. Immunoblot for JARID2 during D0, D1, D3 and D6 of differentiation (as in B) are shown.


Effect of JARID2 removal on levels of differentiation markers Involucrin ( IVL ) , Keratin - 1 ( KRT1 ) and Keratin - 10 ( KRT10 ) mRNAs as measured in qPCR experiment relative to 18S rRNA and the rescue using exogenous expression of ΔN - JARID2 . The effect of exogenous expression of full - length JARID2 ( FL - JARID2 ) in JARID2 knockout is also shown . Data from wildtype HaCaTs , two independent JARID2 knockout ( KO1 and KO2 ) HaCaT lines as well as KO cells exogenously expressing empty vector control , ΔN - JARID2 and FL - JARID2 are shown . Levels of Involucrin ( IVL ) , Keratin - 1 ( KRT1 ) and Keratin - 10 ( KRT10 ) mRNAs as above but measured on day 3 of differentiation .

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

Effect of JARID2 removal on levels of differentiation markers Involucrin (IVL), Keratin-1 (KRT1) and Keratin-10 (KRT10) mRNAs as measured in qPCR experiment relative to 18S rRNA and the rescue using exogenous expression of ΔN-JARID2. The effect of exogenous expression of full-length JARID2 (FL-JARID2) in JARID2 knockout is also shown. Data from wildtype HaCaTs, two independent JARID2 knockout (KO1 and KO2) HaCaT lines as well as KO cells exogenously expressing empty vector control, ΔN-JARID2 and FL-JARID2 are shown. Levels of Involucrin (IVL), Keratin-1 (KRT1) and Keratin-10 (KRT10) mRNAs as above but measured on day 3 of differentiation.


A metagene plot showing enrichment of full - length JARID2 in human induced pluripotent cells at down - regulated genes ( blue ) as compared to up - regulated genes ( grey ) in JARID2 KO cells . The plot is centered on Transcription Start Sites ( TSS ) of genes and distance from TSS is indicated on the x - axis .

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

A metagene plot showing enrichment of full-length JARID2 in human induced pluripotent cells at down-regulated genes (blue) as compared to up-regulated genes (grey) in JARID2 KO cells. The plot is centered on Transcription Start Sites (TSS) of genes and distance from TSS is indicated on the x-axis.


Metagene plots of average enrichment of EZH2 in ES cells and keratinocytes at down - regulated genes ( blue ) as compared to up - regulated genes ( grey ) in JARID2 KO cells . The plots are centered on Transcription Start Sites ( TSS ) of genes and distance from TSS is indicated on the x - axes .

Metagene plots of average enrichment of EZH2 in ES cells and keratinocytes at down-regulated genes (blue) as compared to up-regulated genes (grey) in JARID2 KO cells. The plots are centered on Transcription Start Sites (TSS) of genes and distance from TSS is indicated on the x-axes.


Levels of H3K27me3 in WT , JARID2 KO lines and JARID2 KO lines expressing ΔN - JARID2 . Histone H3 and Ponceau staining is used as a loading control .

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

Levels of H3K27me3 in WT, JARID2 KO lines and JARID2 KO lines expressing ΔN-JARID2. Histone H3 and Ponceau staining is used as a loading control.


A bar - plot showing percentage of down - and up - regulated genes among genes which lose H3K27me3 vs which do not lose H3K27me3 modification during differentiation .

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

A bar-plot showing percentage of down- and up-regulated genes among genes which lose H3K27me3 vs which do not lose H3K27me3 modification during differentiation.


B . Dot blot of tau levels in media from HEK293T cells overexpressing tau and FLAG - DnaJC5 compared to empty vector and tau . Intracellular protein levels are shown by Western blot , DnaJC5 was detected by FLAG antibody . Quantification of extracellular tau levels shown below ; data are mean ± SEM , n = 8 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Dot blot of tau levels in media from HEK293T cells overexpressing tau and FLAG-DnaJC5 compared to empty vector and tau. Intracellular protein levels are shown by Western blot, DnaJC5 was detected by FLAG antibody. Quantification of extracellular tau levels shown below; data are mean ± SEM, n=8, ***p<0.001.


C . Light / heavy ratio of tau peptides in label - free mass spectroscopy of media from HEK293T cells overexpressing tau or tau and DnaJC5 compared to 100 ng 15N labelled recombinant tau . Light peptides represent endogenous tau peptides . Data are mean ± SEM , n = 3 biological repeats , * * * p < 0 . 001 .

C. Light/heavy ratio of tau peptides in label-free mass spectroscopy of media from HEK293T cells overexpressing tau or tau and DnaJC5 compared to 100 ng 15N labelled recombinant tau. Light peptides represent endogenous tau peptides. Data are mean ± SEM, n=3 biological repeats, ***p<0.001.


A . Western blot of endogenous tau levels from M17 neuroblastoma cells overexpressing FLAG - DnaJC5 or empty vector . Media was concentrated prior to analysis . Intracellular DnaJC5 was detected with FLAG antibody . Quantification of extracellular tau levels are mean ± SEM , n = 3 , * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

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

A. Western blot of endogenous tau levels from M17 neuroblastoma cells overexpressing FLAG-DnaJC5 or empty vector. Media was concentrated prior to analysis. Intracellular DnaJC5 was detected with FLAG antibody. Quantification of extracellular tau levels are mean ± SEM, n=3, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


B . Western blottau levels from concentrated media from ex - vivo organotypic slice cultures of wildtype mice overexpressing GFP - AAV9 or FLAG - DnaJC5 - AAV9 . DnaJC5 is detected by FLAG antibody . Quantification of extracellular tau levels are mean ± SEM , n = 3 , * * p < 0 . 01 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

B. Western blottau levels from concentrated media from ex-vivo organotypic slice cultures of wildtype mice overexpressing GFP-AAV9 or FLAG-DnaJC5-AAV9. DnaJC5 is detected by FLAG antibody. Quantification of extracellular tau levels are mean ± SEM, n=3, **p<0.01 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


C . Western blottau levels from concentrated media from ex - vivo organotypic slice cultures of Cspa + / + or Cspa - / - mice . DnaJC5 was detected by DnaJC5 antibody ( Synaptic Systems ) . Quantification of extracellular tau levels are mean ± SEM , n = 3 , * * p < 0 . 01 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

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

C. Western blottau levels from concentrated media from ex-vivo organotypic slice cultures of Cspa+/+ or Cspa-/- mice. DnaJC5 was detected by DnaJC5 antibody (Synaptic Systems). Quantification of extracellular tau levels are mean ± SEM, n=3, **p<0.01 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


A . FLAG - DnaJC5 overexpression facilitates extracellular release of wildtype ( WT ) , P301L and R406W tau by dot blot in HEK293T cells . Intracellular protein levels are shown by Western blot ; DnaJC5 was detected by FLAG antibody . Quantification of extracellular tau levels shown below ; data are mean ± SEM , n = 3 , * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

A. FLAG-DnaJC5 overexpression facilitates extracellular release of wildtype (WT), P301L and R406W tau by dot blot in HEK293T cells. Intracellular protein levels are shown by Western blot; DnaJC5 was detected by FLAG antibody. Quantification of extracellular tau levels shown below; data are mean ± SEM, n=3, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


B . Overexpression of FLAG - DnaJC5 induces extracellular release of wildtype ( WT ) and A53T α - synuclein in HEK293T cells . Extracellular protein levels shown by dot blot ( quantification below , mean ± SEM , n = 3 * * * p < 0 . 001 , * * p < 0 . 01 by one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis ) and intracellular proteins levels were analyzed by Western blot , DnaJC5 was detected by FLAG antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Overexpression of FLAG-DnaJC5 induces extracellular release of wildtype (WT) and A53T α-synuclein in HEK293T cells. Extracellular protein levels shown by dot blot (quantification below, mean ±SEM, n=3 ***p<0.001, ** p<0.01 by one-way analysis of variance with Tukey's multiple comparison post-hoc analysis) and intracellular proteins levels were analyzed by Western blot, DnaJC5 was detected by FLAG antibody.


C . DnaJC5 overexpression induces extracellular release of GFP - tagged wildtype ( WT ) , A315T and Q343R TDP - 43 in HEK293T cells . Intracellular DnaJC5 was detected by FLAG antibody . Extracellular protein levels are analyzed by dot blot ( quantification below , mean ± SEM , n = 4 ) * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

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

C. DnaJC5 overexpression induces extracellular release of GFP-tagged wildtype (WT), A315T and Q343R TDP-43 in HEK293T cells. Intracellular DnaJC5 was detected by FLAG antibody. Extracellular protein levels are analyzed by dot blot (quantification below, mean ±SEM, n=4) ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


A . Dot blot of tau levels in media of cells overexpressing the phospho - deficient FLAG - DnaJC5 S10A compared to FLAG - DnaJC5 WT . Intracellular levels shown by Western blot , DnaJC5 was detected by FLAG antibody . Quantification of tau levels are mean ± SEM , n = 6 , * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

A. Dot blot of tau levels in media of cells overexpressing the phospho-deficient FLAG-DnaJC5 S10A compared to FLAG-DnaJC5 WT. Intracellular levels shown by Western blot, DnaJC5 was detected by FLAG antibody. Quantification of tau levels are mean ± SEM, n=6, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


B . Knockdown of SNAP23 by siRNA inhibits DnaJC5 - mediated tau release in HEK293T cells as shown by dot blot . Quantification of extracellular levels are mean ± SEM , n = 3 , * * p < 0 . 01 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis . DnaJC5 was detected by FLAG antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Knockdown of SNAP23 by siRNA inhibits DnaJC5-mediated tau release in HEK293T cells as shown by dot blot. Quantification of extracellular levels are mean ± SEM, n=3, **p<0.01 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. DnaJC5 was detected by FLAG antibody.


C . Knockdown of SNAP23 by siRNA inhibits DnaJC5 - mediated α - synuclein release in M17 cells as shown by dot blot . Quantification of extracellular levels are mean ± SEM , n = 3 , * * p < 0 . 01 by one - way ANOVA with a Tukey ' s post - hoc analysis . DnaJC5 is detected by FLAG antibody .

C. Knockdown of SNAP23 by siRNA inhibits DnaJC5-mediated α-synuclein release in M17 cells as shown by dot blot. Quantification of extracellular levels are mean ± SEM, n=3, **p<0.01 by one-way ANOVA with a Tukey's post-hoc analysis. DnaJC5 is detected by FLAG antibody.


D . Knockdown of SNAP23 by siRNA inhibits DnaJC5 - mediated TDP - 43 release in M17 cells as shown by dot blot . Quantification of extracellular levels are mean ± SEM , n = 3 , * * p < 0 . 01 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis . DnaJC5 was detected by FLAG antibody .

D. Knockdown of SNAP23 by siRNA inhibits DnaJC5-mediated TDP-43 release in M17 cells as shown by dot blot. Quantification of extracellular levels are mean ± SEM, n=3, **p<0.01 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. DnaJC5 was detected by FLAG antibody.


A . Confocal microscopy of tau ( red pseudocolor ) and Hsp70 ( green pseudocolor ) colocalization ( merged image ) levels in wild type primary neurons overexpressing GFP - AAV9 or FLAG - DnaJC5 - AAV9 . Boxed area are 5X digital zoom on a 60X lens shown below . Scale bars are 20 µm and 5 µm as indicated . Only neurons overexpressing virus were imaged . B . Quantification of tau - Hsp70 co - localization ( mean Pearson ' s coefficient ± SEM , n = 18 , * p < 0 . 05 , one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis ) .

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

A. Confocal microscopy of tau (red pseudocolor) and Hsp70 (green pseudocolor) colocalization (merged image) levels in wild type primary neurons overexpressing GFP-AAV9 or FLAG-DnaJC5-AAV9. Boxed area are 5X digital zoom on a 60X lens shown below. Scale bars are 20 µm and 5 µm as indicated. Only neurons overexpressing virus were imaged.B. Quantification of tau-Hsp70 co-localization (mean Pearson's coefficient ± SEM, n=18, *p <0.05, one-way analysis of variance with Tukey's multiple comparison post-hoc analysis).


C . Western blot of immunoprecipitatedtau from HEK293T reveals tau exists in an intracellular complex with DnaJC5 and Hsc70 . DnaJC5 was detected with FLAG antibody .

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

C. Western blot of immunoprecipitatedtau from HEK293T reveals tau exists in an intracellular complex with DnaJC5 and Hsc70. DnaJC5 was detected with FLAG antibody.


D . Western blot of HEK293T intracellular lysates show the DnaJC5 / Hsc70 / tau complex is disrupted by the addition of increasing amounts of YM - 01 ( 0 , 10 , 30 , 100 µM ) in a dose - dependent fashion . DnaJC5 was detected by FLAG antibody .

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

D. Western blot of HEK293T intracellular lysates show the DnaJC5/Hsc70/tau complex is disrupted by the addition of increasing amounts of YM-01 (0, 10, 30, 100 µM) in a dose-dependent fashion. DnaJC5 was detected by FLAG antibody.


E . The DnaJC5 / Hsc70 complex is disrupted when Hsc70 activity is inhibited by a dominant - negative point mutation , E175S . Western blot of immunoprecipitated DnaJC5 from HEK293T cells overexpressing WT or DN E175S Hsc70 . DnaJC5 is immunopreicipitated by DnaJC5 antibody ( Synaptic Systems ) . DnaJC5 was detected on Western blot by FLAG antibody .

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

E. The DnaJC5/Hsc70 complex is disrupted when Hsc70 activity is inhibited by a dominant-negative point mutation, E175S. Western blot of immunoprecipitated DnaJC5 from HEK293T cells overexpressing WT or DN E175S Hsc70. DnaJC5 is immunopreicipitated by DnaJC5 antibody (Synaptic Systems). DnaJC5 was detected on Western blot by FLAG antibody.


F . Dot blot of tau levels in media of M17 neuroblastoma cells treated with YM - 01 . Quantification of extracellular tau levels are mean ± SEM , n = 6 , * p < 0 . 05 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis . Intracellular levels shown by Western blot . DnaJC5 was detected on Western blot by FLAG antibody .

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

F. Dot blot of tau levels in media of M17 neuroblastoma cells treated with YM-01. Quantification of extracellular tau levels are mean ± SEM, n=6, *p<0.05 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. Intracellular levels shown by Western blot. DnaJC5 was detected on Western blot by FLAG antibody.


G . Dot blot of tau levels in media of primary neurons from transgenic overexpressing tau P301L mice treated with YM - 01 ( 10µM ) . FLAG - DnaJC5 was transduced into neurons using AAV9 . Quantification of extracellular tau levels are mean ± SEM , n = 6 , * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis . DnaJC5 was detected on Western blot by FLAG antibody .

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

G. Dot blot of tau levels in media of primary neurons from transgenic overexpressing tau P301L mice treated with YM-01 (10µM). FLAG-DnaJC5 was transduced into neurons using AAV9. Quantification of extracellular tau levels are mean ± SEM, n=6, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis. DnaJC5 was detected on Western blot by FLAG antibody.


A . Chemical inhibition of Hsc70 activity in HEK293T cells overexpressing FLAG - DnaJC5 reduces extracellular tau levels as shown by dot blot . Intracellular levels are shown by Western blot , DnaJC5 was detected by FLAG antibody . Quantification of extracellular tau levels is shown as average ± SEM , n = 3 , * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

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

A. Chemical inhibition of Hsc70 activity in HEK293T cells overexpressing FLAG-DnaJC5 reduces extracellular tau levels as shown by dot blot. Intracellular levels are shown by Western blot, DnaJC5 was detected by FLAG antibody. Quantification of extracellular tau levels is shown as average ± SEM, n=3, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


B . Chemical inhibition of Hsc70 activity in HEK293T cells overexpressing FLAG - DnaJC5 reduces extracellular α - synuclein levels as shown by dot blot . Intracellular levels are shown by Western blotDnaJC5 was detected by FLAG antibody . Quantification of extracellular synuclein levels is shown as average ± SEM , n = 3 , * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

B. Chemical inhibition of Hsc70 activity in HEK293T cells overexpressing FLAG-DnaJC5 reduces extracellular α-synuclein levels as shown by dot blot. Intracellular levels are shown by Western blotDnaJC5 was detected by FLAG antibody. Quantification of extracellular synuclein levels is shown as average ± SEM, n=3, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


C . Dominant - negative Hsc70 E175S blocks tau release when overexpressed in HEK293T cells as shown by dot blot . Intracellular levels are shown by Western blot . Quantification of extracellular tau levels is shown as average ± SEM , n = 4 , * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

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

C. Dominant-negative Hsc70 E175S blocks tau release when overexpressed in HEK293T cells as shown by dot blot. Intracellular levels are shown by Western blot. Quantification of extracellular tau levels is shown as average ± SEM, n=4, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


D . Knockdown of Hsc70 reduces extracellular tau release ( dot blot ) in HEK293T cells overexpressing FLAG - DnaJC5 . Intracellular levels are shown by Western blot , DnaJC5 was detected by FLAG antibody . Quantification of extracellular tau levels is shown as average ± SEM , n = 3 , * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

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

D. Knockdown of Hsc70 reduces extracellular tau release (dot blot) in HEK293T cells overexpressing FLAG-DnaJC5. Intracellular levels are shown by Western blot, DnaJC5 was detected by FLAG antibody. Quantification of extracellular tau levels is shown as average ± SEM, n=3, ***p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


E . A mutant tau that does not bind Hsc70 is not released when FLAG - DnaJC5 is overexpressed in HEK293T cells . Dot blot and quantification of extracellular tau levels are shown . Intracellular protein levels are shown with representative Western blot , DnaJC5 was detected by FLAG antibody . Data are mean ± SEM , n = 3 p < 0 . 001 . one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

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

E. A mutant tau that does not bind Hsc70 is not released when FLAG-DnaJC5 is overexpressed in HEK293T cells. Dot blot and quantification of extracellular tau levels are shown. Intracellular protein levels are shown with representative Western blot, DnaJC5 was detected by FLAG antibody. Data are mean ± SEM, n=3 p<0.001. one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


F . Dot blot of Hsp70 isoform overexpression effect on extracellular tau release in HEK293T cells . Intracellular protein levels are shown with representative Western blot . HSPA variants were detected with V5 antibody . Data are mean ± SEM , n = 3 p < 0 . 01 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

F. Dot blot of Hsp70 isoform overexpression effect on extracellular tau release in HEK293T cells. Intracellular protein levels are shown with representative Western blot. HSPA variants were detected with V5 antibody. Data are mean ± SEM, n=3 p<0.01 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


G . Dot blot of different FLAG - DnaJ overexpression effect on extracellular tau release in HEK293T cells . Intracellular protein levels are shown with representative Western blot . DnaJ levels were detected by FLAG antibody . Data are mean ± SEM , n = 3 ; * * * p < 0 . 001 one - way analysis of variance with Tukey ' s multiple comparison post - hoc analysis .

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

G. Dot blot of different FLAG-DnaJ overexpression effect on extracellular tau release in HEK293T cells. Intracellular protein levels are shown with representative Western blot. DnaJ levels were detected by FLAG antibody. Data are mean ± SEM, n=3; *** p<0.001 one-way analysis of variance with Tukey's multiple comparison post-hoc analysis.


H . Dot blot of increasing FLAG - DnaJC7 levels on FLAG - DnaJC5 overexpression - mediated extracellular tau release in HEK293T cells . Intracellular protein levels are shown with representative Western blot , DnaJ proteins were detected by FLAG antibody . Quantification of extracellular levels are mean ± SEM , n = 4 .

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

H. Dot blot of increasing FLAG-DnaJC7 levels on FLAG-DnaJC5 overexpression-mediated extracellular tau release in HEK293T cells. Intracellular protein levels are shown with representative Western blot, DnaJ proteins were detected by FLAG antibody. Quantification of extracellular levels are mean ± SEM, n=4.


( B ) The Western blot analysis of GFP , Spd2 and αTub ( loading control ) in the brain extracts from wild type ( Oregon R , OR ) , Spd2WT - OE and Spd2DK - OE third instar larva . Representative immune blot images are shown on the left . The signal intensities of Spd2 bands were quantified and normalised against αTub values . The values relative to control of each biological replicate ( n = 2 ) and their means are shown as dots and bars , respectively , in a scatted dot plot on the right . The numbers above the bars indicate means ± Range . GFP - Spd2DK proteins in Spd2DK - OE larval brain extracts showed approximately 2 . 04 - fold higher accumulation than GFP - Spd2WT proteins in Spd2WT - OE brain extracts . ( C ) The Western blot analysis of Spd2 , CycB and αTub in OR , fzrRNAi , Spd2WT - RES and Spd2DK - RES brain extracts . The signal intensities of Spd2 bands were quantified and normalised against αTub values . The values relative to control and their means are presented in a scatted dot plot on the right ( biological replicate n = 2 ) . Means ± Range are shown above the bars . RFP - Spd2DK proteins in Spd2DK - RES larval brain extracts showed approximately 2 . 08 - fold higher accumulation than RFP - Spd2WT proteins in Spd2WT - RES brain extracts .

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

(B) The Western blot analysis of GFP, Spd2 and αTub (loading control) in the brain extracts from wild type (Oregon R, OR), Spd2WT-OE and Spd2DK-OE third instar larva. Representative immune blot images are shown on the left. The signal intensities of Spd2 bands were quantified and normalised against αTub values. The values relative to control of each biological replicate (n = 2) and their means are shown as dots and bars, respectively, in a scatted dot plot on the right. The numbers above the bars indicate means ± Range. GFP-Spd2DK proteins in Spd2DK-OE larval brain extracts showed approximately 2.04-fold higher accumulation than GFP-Spd2WT proteins in Spd2WT-OE brain extracts. (C) The Western blot analysis of Spd2, CycB and αTub in OR, fzrRNAi, Spd2WT-RES and Spd2DK-RES brain extracts. The signal intensities of Spd2 bands were quantified and normalised against αTub values. The values relative to control and their means are presented in a scatted dot plot on the right (biological replicate n = 2). Means ± Range are shown above the bars. RFP-Spd2DK proteins in Spd2DK-RES larval brain extracts showed approximately 2.08-fold higher accumulation than RFP-Spd2WT proteins in Spd2WT-RES brain extracts.


( D ) Larval brains of OR , Spd2WT - RES , fzrRNAi and Spd2WT - OE flies were stained by an anti - Spd2 antibody . Representative images of NBs are shown on the top . Scale bar : 10 µm . The local signal intensities of Spd2 immunofluorescence at the centrosomes and in the cytoplasm of individual NBs in each brain were measured , and the normalised Spd2 signal intensities in individual NBs were presented as scattered dot plots ( see Materials and Methods for detail ) . The lines indicate means ± SD . The numbers of NBs ( n ) and the numbers of brains analysed ( N ) are control : n = 29 ( N = 6 ) , Spd2WT - RES : n = 30 ( N = 4 ) , fzrRNA : n = 30 ( N = 6 ) , and Spd2WT - OE : n = 27 ( N = 10 ) . P values were calculated using non - parametric two - tailed Mann - Whitney tests . Based on these results , the relative centrosomal Spd2 protein levels in NBs can be estimated as wildtype ( or control ) : Spd2WT - RES : fzrRNAi : Spd2DK - RES : Spd2WT - OE : Spd2DK - OE = 1 : 1 . 00 ~ 1 . 18 : 1 . 06 ~ 1 . 29 : 2 . 08 ~ 2 . 45 ( = 1 . 00 ~ 1 . 18 x 2 . 08 ) : 1 . 93 ~ 3 . 62 : 3 . 94 ~ 7 . 38 ( = 1 . 93 ~ 3 . 62 x 2 .

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

(D) Larval brains of OR, Spd2WT-RES, fzrRNAi and Spd2WT-OE flies were stained by an anti-Spd2 antibody. Representative images of NBs are shown on the top. Scale bar: 10 µm. The local signal intensities of Spd2 immunofluorescence at the centrosomes and in the cytoplasm of individual NBs in each brain were measured, and the normalised Spd2 signal intensities in individual NBs were presented as scattered dot plots (see Materials and Methods for detail). The lines indicate means ± SD. The numbers of NBs (n) and the numbers of brains analysed (N) are control: n= 29 (N = 6), Spd2WT-RES: n = 30 (N = 4), fzrRNA: n = 30 (N = 6), and Spd2WT-OE: n = 27 (N = 10). P values were calculated using non-parametric two-tailed Mann-Whitney tests. Based on these results, the relative centrosomal Spd2 protein levels in NBs can be estimated as wildtype (or control) : Spd2WT-RES: fzrRNAi: Spd2DK-RES: Spd2WT-OE: Spd2DK-OE = 1: 1.00~1.18: 1.06~1.29: 2.08~2.45 (= 1.00~1.18 x 2.08): 1.93~3.62: 3.94 ~ 7.38 (= 1.93~3.62 x 2.04


( C ) In time lapse movies , NBs that showed an active centrosome that was attached to the cortex ( " Attached " ) and NBs with both two centrosomes being inactivated and detached from the cortex ( " Detached " ) in interphase were counted in third instar larval brains of the indicated fly lines and their proportions are presented in bar graphs . The numbers of NBs analysed from at least four brains ( n ) are Control : 29 , fzrRNAi : 80 , Spd2WT - RES : 45 , Spd2DK - RES : 52 , Spd2WT - OE : 43 , Spd2DK - OE : 40 , Spd2WT - OE + fzrRNAi : 72 . p - values are fzrRNAi : 0 . 0069 , Spd2WT - RES : 0 . 2479 , Spd2DK - RES : 0 . 0116 , Spd2WT - OE : < 0 . 0001 , Spd2DK - OE : < 0 . 0001 , Spd2WT - OE + fzrRNAi : 0 . 0008 ( Pearson ' s chi - squared tests were performed between control and each condition ) .

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

(C) In time lapse movies, NBs that showed an active centrosome that was attached to the cortex ("Attached") and NBs with both two centrosomes being inactivated and detached from the cortex ("Detached") in interphase were counted in third instar larval brains of the indicated fly lines and their proportions are presented in bar graphs. The numbers of NBs analysed from at least four brains (n) are Control: 29, fzrRNAi: 80, Spd2WT-RES: 45, Spd2DK-RES: 52, Spd2WT-OE: 43, Spd2DK-OE: 40, Spd2WT-OE + fzrRNAi: 72. p-values are fzrRNAi: 0.0069, Spd2WT-RES: 0.2479, Spd2DK-RES: 0.0116, Spd2WT-OE: <0.0001, Spd2DK-OE: <0.0001, Spd2WT-OE + fzrRNAi: 0.0008 (Pearson's chi-squared tests were performed between control and each condition).