F . Yeast strains containing Flag - tagged rbd2 - SHP , rbd2 - SHP * , rbd2 - G246R - SHP , rbd2 - G246R - SHP * were generated in rbd2Δ background by chromosomal integration of rbd2 fusion depicted in ( E ) . Indicated yeast strains were then assayed for Sre1 cleavage as in ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Yeast strains containing Flag-tagged rbd2-SHP, rbd2-SHP*, rbd2-G246R-SHP, and rbd2-G246R-SHP* were generated in rbd2Δ background by chromosomal integration of the rbd2 fusion proteins depicted in (E). Indicated yeast strains were then assayed for Sre1 cleavage as in (B).


G . Indicated strains were assayed for Sre2 cleavage as in ( C ) .

G. Indicated strains were assayed for Sre2 cleavage as in (C).


H . Wild - type , sre2Δ or strains containing chromosomal Flag - tagged rbd2 , rbd2 - G246R or rbd2 - S130S in rbd2Δ background ( lane 7 - 12 ) were treated with bortezomib ( Bz ) for 2 hr , phosphatase - treated , whole lysates were analyzed by western blot with anti - Sre2 serum or anti - Flag IgG .

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

H. Wild-type, sre2Δ or strains containing chromosomal Flag-tagged rbd2, rbd2-G246R or rbd2-S130S in rbd2Δ background (lane 7-12) were treated with bortezomib (Bz) for 2 hr, and phosphatase-treated, whole cell lysates were analyzed by western blot with anti-Sre2 serum or anti-Flag IgG.


A . Western blot of phosphatase - treated whole lysates from yeast strains carrying rbd2 - Flag - APEX2 ( rbd2 - F ) , rbd2 - G246R - Flag - APEX2 ( G246R ) or rbd2 - S130A - Flag - APEX2 ( S130A ) plasmid in rbd2Δ background sre1Δ yeast was probed with anti - Sre1 or anti - Flag IgG . P N denote Sre1 precursor forms , 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

A. Western blot of phosphatase-treated whole cell lysates from yeast strains carrying rbd2-Flag-APEX2 (rbd2-F), rbd2-G246R-Flag-APEX2 (G246R) or rbd2-S130A-Flag-APEX2 (S130A) plasmid in rbd2Δ background and sre1Δ yeast was probed with anti-Sre1 or anti-Flag IgG. P and N denote Sre1 precursor and cleaved nuclear forms, respectively.


B . Indicated yeast strains ( 200 ) containing chromosomal Flag - tagged rbd2 , rbd2 - G246R or rbd2 - S130A in rbd2Δ background ( lane 3 - 5 ) or yeast strains ( 200 ) carrying rbd2 - Flag - APEX2 , rbd2 - G246R - Flag - APEX2 or rbd2 - S130A - Flag - APEX2 plasmid in rbd2Δ background ( lane 6 - 8 ) were grown on rich medium in absence or presence of cobalt chloride ( CoCl2 ) .

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

B. Indicated yeast strains (200 cells) containing chromosomal Flag-tagged rbd2, rbd2-G246R or rbd2-S130A in rbd2Δ background (lane 3-5) or yeast strains (200 cells) carrying rbd2-Flag-APEX2, rbd2-G246R-Flag-APEX2 or rbd2-S130A-Flag-APEX2 plasmid in rbd2Δ background (lane 6-8) were grown on rich medium in the absence or presence of cobalt chloride (CoCl2).


C . Comparison of Rbd2 from overexpression system to endogenous expression . Western blot of whole lysates from yeast strains containing chromosomal Flag - tagged rbd2 , rbd2 - G246R or rbd2 - S130A in rbd2Δ background yeast strains carrying rbd2 - Flag - APEX2 , rbd2 - G246R - Flag - APEX2 or rbd2 - S130A - Flag - APEX2 plasmid in rbd2Δ background was probed with anti - FlagIgG . For , of Flag band in each lane was normalized to non - specific band ( asterisk ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Comparison of Rbd2 protein levels from overexpression system to endogenous expression. Western blot of whole cell lysates from yeast strains containing chromosomal Flag-tagged rbd2, rbd2-G246R or rbd2-S130A in rbd2Δ background and yeast strains carrying rbd2-Flag-APEX2, rbd2-G246R-Flag-APEX2 or rbd2-S130A-Flag-APEX2 plasmid in rbd2Δ background was probed with anti-FlagIgG. For quantification, the intensity of Flag band in each lane was normalized to the non-specific band (asterisk).


D . rbd2Δ expressing indicated plasmids of GFP - Anp1 or GFP - Sre2MS together with rbd2 - Flag - APEX2 , rbd2 - S130A - Flag - APEX2 or rbd2 - S130A / G246R - Flag - APEX2 were treated with Bz at a of 1 mM for 2 hr prior to biotin - labeling reaction . After labeling termination , were lysed denatured as described in Materials Methods . Biotinylated were then enriched using streptavidin magnetic beads . Lysates ( 1x input ) 100x - enriched eluates were analyzed by western blot with IRDye 800CW Streptavidin , anti - Flag IgG , anti - Cdc48 serum , anti - GFP IgG . of GFP band in enriched sample was normalized to that in corresponding input sample , then normalized to Flag in enriched sample . Relative intensities of biotinylated GFP level are shown in bar graph . Error bars denote standard deviation from three biological replicates .

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

D. rbd2Δ cells expressing indicated plasmids of GFP-Anp1 or GFP-Sre2MS together with rbd2-Flag-APEX2, rbd2-S130A-Flag-APEX2 or rbd2-S130A/G246R-Flag-APEX2 were treated with Bz at a concentration of 1 mM for 2 hr prior to biotin-labeling reaction. After labeling termination, cells were lysed and denatured as described in Materials and Methods. Biotinylated proteins were then enriched using streptavidin magnetic beads. Lysates (1x input) and 100x-enriched eluates were analyzed by western blot with IRDye 800CW Streptavidin, anti-Flag IgG, anti-Cdc48 serum, and anti-GFP IgG. Intensity of GFP band in enriched sample was normalized to that in the corresponding input sample, then normalized to the Flag intensity in enriched sample. Relative intensities of biotinylated GFP level are shown in bar graph. Error bars denote standard deviation from three biological replicates.


A , B Representative time - lapse ( A ) kymograph ( B ) of mouse zygotes expressing H2B - RFP ( magenta ) labelled with SiR - Tubulin ( green ) . Shown is an example of marked chromosomal displacement to cortex . C Changes in chromosome - to - centre distances ( d ) over time relative to anaphase - onset ( n = 50 blastomeres ) . Data information : In ( A , time is shown as h post - metaphase scale bar = 20 µm . In ( C , data are shown as mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative time-lapse images (A) and kymograph (B) of mouse zygotes expressing H2B-RFP (magenta) and labelled with SiR-Tubulin (green). Shown is an example of marked chromosomal displacement to the cortex. C Changes in chromosome-to-cell centre distances (d) over time relative to anaphase-onset (n = 50 blastomeres). Data information: In (A, time is shown as h post-metaphase and scale bar = 20 µm. In (C, data are shown as mean ± SEM.


B , C Representative time - lapse of in which DMSO ( B ) or nocodazole ( C ) was added after anaphase completion . D Changes in nucleus - to - centre distances ( d ) over time relative to time of drug addition following DMSO - ( n = 22 blastomeres ) or nocodazole - treatment ( n = 26 blastomeres ) at anaphase completion . Data information : In ( B , C , time is shown as h post - drug addition scale bar = 20 µm . In ( D , data are shown as mean ± . Statistical significance was assessed by an ordinary two - way ANOVA with Šidák ' s multiple comparisons ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , unremarked datapoints = not significant ) .

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 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 O O O O

B, C Representative time-lapse images of embryos in which DMSO (B) or nocodazole (C) was added after anaphase completion. D Changes in nucleus-to-cell centre distances (d) over time relative to time of drug addition following DMSO- (n = 22 blastomeres) or nocodazole-treatment (n = 26 blastomeres) at anaphase completion. Data information: In (B,C, time is shown as h post-drug addition and scale bar = 20 µm. In (D, data are shown as mean ± SEM. Statistical significance was assessed by an ordinary two-way ANOVA with Šidák's multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unremarked datapoints = not significant).


E - G Representative time - lapse of in which DMSO ( E ) , Nocodazole ( F ) or Cytochalasin D ( G ) was added when nuclei had travelled to cortex . H Changes in nucleus - to - centre distances ( d ) over time relative to time of drug addition DMSO - ( n = 20 blastomeres ) , nocodazole - ( n = 12 blastomeres ) or cytochalasin D - treatment ( n = 14 blastomeres ) after nuclei reached cortex . Data information : In E - G ) , time is shown as h post - drug addition scale bar = 20 µm . In H ) , data are shown as mean ± . Statistical significance was assessed by an ordinary two - way ANOVA with Šidák ' s multiple comparisons ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , unremarked datapoints = not significant ) .

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

E-G Representative time-lapse images of embryos in which DMSO (E), Nocodazole (F) or Cytochalasin D (G) was added when nuclei had travelled to the cortex. H Changes in nucleus-to-cell centre distances (d) over time relative to time of drug addition DMSO- (n = 20 blastomeres), nocodazole- (n = 12 blastomeres) or cytochalasin D-treatment (n = 14 blastomeres) after nuclei reached the cortex. Data information: In E - G), time is shown as h post-drug addition and scale bar = 20 µm. In H), data are shown as mean ± SEM. Statistical significance was assessed by an ordinary two-way ANOVA with Šidák's multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unremarked datapoints = not significant).


A , B Representative time - lapse of that have been washed out of either nocodazole after nuclei were prevented from travelling to cortex after anaphase ( A , n = 15 ) or cytochalasin D after nuclei had been trapped at cortex by disrupting actin ( B , n = 22 ) . C Graph comparing distribution of maximal d values in that developed to blastocyst stage or not . Each data point represents mean maximal d value of two blastomeres from same ( n = 45 ) . D Graph of time taken to develop to blastocyst stage in relation to maximal d values ( n = 27 ) . E Graph comparing distribution of times taken for nuclei to re - centre in that developed to blastocyst stage or not ( n = 45 ) . F Graph of time taken to develop to blastocyst stage in relation to times taken for nuclei to re - centre ( n = 27 ) . Data information : In ( A , B ) , time is shown as h post - drug washout scale bar = 20 µm . In ( C - F ) , data are shown as mean ± . In ( C , E ) , statistical significance was assessed by Welch ' s t - , ns : not significant . In ( D , F ) , strength of correlation ( r ) statistical significance were assessed using Pearson correlation , ns : not significant .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative time-lapse images of embryos that have been washed out of either nocodazole after nuclei were prevented from travelling to the cortex after anaphase (A, n = 15 embryos) or cytochalasin D after nuclei had been trapped at the cortex by disrupting actin (B, n = 22 embryos). C Graph comparing the distribution of maximal d values in embryos that developed to the blastocyst stage or not. Each data point represents the mean maximal d value of the two blastomeres from the same embryo (n = 45 embryos). D Graph of time taken to develop to the blastocyst stage in relation to maximal d values (n = 27 embryos). E Graph comparing the distribution of times taken for nuclei to re-centre in embryos that developed to the blastocyst stage or not (n = 45 embryos). F Graph of time taken to develop to the blastocyst stage in relation to times taken for nuclei to re-centre (n = 27 embryos). Data information: In (A, B), time is shown as h post-drug washout and scale bar = 20 µm. In (C - F), data are shown as mean ± SEM. In (C, E), statistical significance was assessed by Welch's t-test, ns: not significant. In (D, F), the strength of correlation (r) and statistical significance were assessed using the Pearson correlation test, ns: not significant.


A Representative time - lapse image of SiR - tubulin in DMSO - BFA - treated during first mitotic division subsequent interphase . Time is shown as h relative to first mitotic anaphase . B Changes in nucleus - to - centre distances ( d ) over time in BFA - treated , relative to maximal displacement to cortex ( n = 14 blastomeres ) . Data information : In ( A , scale bar = 20 µm .

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 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

A Representative time-lapse image of DNA and SiR-tubulin in DMSO- and BFA-treated embryos during the first mitotic division and subsequent interphase. Time is shown as h relative to the first mitotic anaphase. B Changes in nucleus-to-cell centre distances (d) over time in BFA-treated embryos, relative to maximal nuclear displacement to the cortex (n = 14 blastomeres). Data information: In (A, scale bar = 20 µm.


D Representative time - lapse of second mitotic division in DMSO - BFA - treated . Dashed lines highlight position of nuclei shortly before NEBD . Dashed yellow lines outline daughter - . Time is shown as h relative to second mitotic NEBD . E Dot plot of duration between first mitotic division anaphase - onset second mitotic division NEBD for DMSO - ( n = 47 blastomeres ) BFA - treated ( n = 47 blastomeres ) . F Plot of duration between NEBD anaphase of second mitotic division for DMSO - ( n = 44 blastomeres ) BFA - treated ( n = 43 blastomeres ) . G Plot of spindle - to - centre distances against pre - NEBD nucleus - to - centre ( n = 34 blastomeres ) . indicates mathematically regressed fit . Data information : In D ) , scale bar = 20 µm . In E - G , data are shown as mean ± . In ( E , F , statistical significance was assessed by Mann - Whitney , * * * * P < 0 . 0001 , ns : not significant . In ( G , strength of correlation ( r ) statistical significance were assessed using Pearson correlation . Two - p values are reported ( * * P < 0 . 01 ) .

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 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 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 O O 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 Representative time-lapse images of the second mitotic division in DMSO- and BFA-treated embryos. Dashed white lines highlight position of nuclei shortly before NEBD. Dashed yellow lines outline daughter-cells. Time is shown as h relative to the second mitotic NEBD. E Dot plot of the duration between first mitotic division anaphase-onset and second mitotic division NEBD for DMSO- (n = 47 blastomeres) and BFA-treated (n = 47 blastomeres) embryos. F Plot of the duration between NEBD and anaphase of the second mitotic division for DMSO- (n = 44 blastomeres) and BFA-treated (n = 43 blastomeres) embryos. G Plot of the spindle-to-cell centre distances against pre-NEBD nucleus-to-cell centre distance (n = 34 blastomeres). Line indicates mathematically regressed fit. Data information: In D), scale bar = 20 µm. In E - G, data are shown as mean ± SEM. In (E, F, statistical significance was assessed by Mann-Whitney test, ****P < 0.0001, ns: not significant. In (G, the strength of correlation (r) and statistical significance were assessed using the Pearson correlation test. Two-tailed p values are reported (**P < 0.01).


A Representative time - lapse of 2 - cultured with SiR - to label nuclei ( circles ) microinjected with oil droplets ( yellow circles ) . Time is shown as h : min relative to maximal displacement . B Plot of distances between oil droplets nuclei to - centre over time ( n = 14 oil - nucleus pairs ) . Data information : Scale bar = 20 µm in A , In ( B ) , data are shown as mean ± .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 time-lapse images of 2-cell embryos cultured with SiR-DNA to label nuclei (white circles) and microinjected with oil droplets (yellow circles). Time is shown as h:min relative to maximal nuclear displacement. B Plot of the distances between oil droplets and nuclei to the cell-centre over time (n = 14 oil-nucleus pairs). Data information: Scale bar = 20 µm in A, In (B), data are shown as mean ± SEM.


E Top panel : an example of bead distribution in a 2 - . Lower panel : an example of region imaged for bead tracking at high temporal resolution . F , G Trajectories of bead movement over a 20 s - duration in oocytes ( F , n = 217 beads from 23 oocytes ) 2 - ( n = 277 beads from 26 ) . Each coloured represents a single bead track . Data information : Scale bar = 20 µm in E , except 2 µm in lower panel of E .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Top panel: an example of fluorescent bead distribution in a 2-cell embryo. Lower panel: an example of region imaged for bead tracking at high temporal resolution. F, G Trajectories of bead movement over a 20 s-duration in oocytes (F, n = 217 beads from 23 oocytes) and 2-cell embryos (n = 277 beads from 26 embryos). Each coloured line represents a single bead track. Data information: Scale bar = 20 µm in E, except 2 µm in the lower panel of E.


B Shown is a representative 2 - immunostained for β - tubulin ( green ) ( magenta ) at anaphase completion ( n = 8 ) . C Dot plot of mean β - tubulin within regions that either lie ahead of nucleus ( cortex - facing aspect , dashed triangle in B ) or behind nucleus ( cleavage plane - facing aspect , dashed yellow triangle in B ) ( n = 14 ) . Data information : In ( B scale bar = 20 µm . In ( C , data are shown as mean ± . In ( C , statistical significance was assessed by Welch ' s t - , * * * * P < 0 . 0001 , ns = not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Shown is a representative 2-cell embryo immunostained for β-tubulin (green) and DNA (magenta) at anaphase completion (n = 8 embryos). C Dot plot of mean β-tubulin fluorescence intensity within cytoplasmic regions that either lie ahead of the nucleus (cortex-facing aspect, dashed white triangle in B) or behind the nucleus (cleavage plane-facing aspect, dashed yellow triangle in B) (n = 14 embryos). Data information: In (B scale bar = 20 µm. In (C, data are shown as mean ± SEM. In (C, statistical significance was assessed by Welch's t-test, ****P < 0.0001, ns = not significant.


D Representative time - lapse of expressing H2B - RFP labelled with SiR - tubulin following addition of dynarrestin at anaphase completion ( n = 14 ) . Data information : In ( D , time is shown as h post - drug addition . In D , scale bar = 20 µm .

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

D Representative time-lapse images of embryos expressing H2B-RFP and labelled with SiR-tubulin following the addition of dynarrestin at anaphase completion (n = 14 embryos). Data information: In (D, time is shown as h post-drug addition. In D, scale bar = 20 µm.


E Dot plot of maximal d values between DMSO - dynarrestin - treated ( n = 28 blastomeres per group ) . Data information : In E , data are shown as mean ± . In E ) , statistical significance was assessed by Welch ' s t - , * * * * P < 0 . 0001 , ns = not significant .

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

E Dot plot of maximal d values between DMSO- and dynarrestin-treated embryos (n = 28 blastomeres per group). Data information: In E, data are shown as mean ± SEM. In E), statistical significance was assessed by Welch's t-test, ****P < 0.0001, ns = not significant.


G Representative time - lapse of expressing H2B - RFP labelled with SiR - tubulin following addition of taxol at maximal displacement . Data information : In G , scale bar = 20 µm .

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

G Representative time-lapse images of embryos expressing H2B-RFP and labelled with SiR-tubulin following the addition of taxol at maximal nuclear displacement. Data information: In G, scale bar = 20 µm.


H Changes in nucleus - to - centre distances ( d ) over time relative to time of drug addition in DMSO - ( n = 19 ) taxol - ( n = 19 ) treated 2 - blastomeres ( mean ± ) . Data information : In H ) , data are shown as mean ± . In ( H ) , statistical significance was assessed by an ordinary two - way ANOVA with Šidák ' s multiple comparisons ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , unremarked datapoints = not significant ) .

O O O O 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

H Changes in nucleus-to-cell centre distances (d) over time relative to the time of drug addition in DMSO- (n = 19) and taxol- (n = 19) treated 2-cell blastomeres (mean ± SEM). Data information: In H), data are shown as mean ± SEM. In (H), statistical significance was assessed by an ordinary two-way ANOVA with Šidák's multiple comparisons test (*P < 0.05, **P < 0.01, ***P < 0.001, unremarked datapoints = not significant).


I Representative of immunostained for β - tubulin Hoechst following completion of live imaging ( shown in E ) in DMSO - taxol - treated ( n = 9 per group ) . Time is shown as h post - drug addition . Scale bar = 20 µm . Data information : In I ) , scale bar = 20 µm .

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

I Representative images of embryos immunostained for β-tubulin and Hoechst following the completion of live imaging (shown in E) in DMSO- and taxol-treated embryos (n = 9 embryos per group). Time is shown as h post-drug addition. Scale bar = 20 µm. Data information: In I), scale bar = 20 µm.


A . Hematoxylin eosin ( H & E ) - stained cross section of a human liver slice . Scale bar is representative of A - F . Image is also used in Fig . S3A . B . Spatial distribution relative abundance of glycogen ( represented by CL7 ) in an immediate adjacent tissue slice from A . image displays a gradient of from ( least abundant ) to red ( most abundant ) which is representative of B , D , F . C . H & E - stained cross section of a human lung slice . Image is also used in Fig . S3D . D . Spatial distribution relative abundance of glycogen ( represented by CL7 ) in an immediate adjacent tissue slice from C . E . H & E - stained cross section of frontal cortical region of a human brain tissue . Image is also used in Fig . S3G . F . Spatial distribution relative abundance of glycogen ( represented by CL7 ) in an immediate adjacent tissue slice from E . G . of relative abundance of total glycogen calculated from sum of CL3 - CL18 in human liver , lung , brain . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) . * 0 . 01 < P < 0 . 05 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 , analyzed by one - way ANOVA with Tukey ' s multiple comparison for total glycogen . H . Glycogen structure represented by normalized chain distribution ( to CL3 ) of each human liver , lung brain from G . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) .

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

A. Hematoxylin and eosin (H&E)-stained cross section of a human liver slice. Scale bar is representative of A-F. Image is also used in Fig. S3A. B. Spatial distribution and relative abundance of glycogen (represented by CL7) in an immediate adjacent tissue slice from A. The image displays a heatmap gradient of intensity from white (least abundant) to red (most abundant) which is representative of B, D, and F. C. H&E-stained cross section of a human lung slice. Image is also used in Fig. S3D. D. Spatial distribution and relative abundance of glycogen (represented by CL7) in an immediate adjacent tissue slice from C. E.H&E-stained cross section of frontal cortical region of a human brain tissue. Image is also used in Fig. S3G. F. Spatial distribution and relative abundance of glycogen (represented by CL7) in an immediate adjacent tissue slice from E. G. Quantification of relative abundance of total glycogen calculated from the sum of CL3-CL18 in human liver, lung, and brain. Values are presented as mean +/- standard error (n=3 technical replicates per region). *0.01 < P < 0.05; ***P < 0.001; ****P<0.0001, analyzed by one-way ANOVA with Tukey's multiple comparison for total glycogen. H. Glycogen structure represented by normalized chain length distribution (ratio to CL3) of each human liver, lung and brain from G. Values are presented as mean +/- standard error (n=3 technical replicates per region).


A . Hematoxylin eosin ( H & E ) - stained cross section of adenocarcinoma of prostate , lung squamous carcinoma ( LUSC ) , lung adenocarcinoma ( LUAD ) , Ewing sarcoma ( ES ) of abdomen . Annotated regions are T - , S - stroma , D / G - / glandular , A - adipose tissue . Scale bar is representative of A B . are also used in Fig . S4A , 5D S6G . B . Spatial distribution relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section shown in A . image displays a gradient of with ( least abundant ) to red ( most abundant ) . are also used in Fig . S4E , 5D , S6G . C . Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain ( CL ) distribution from regions shown in A . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) .

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

A. Hematoxylin and eosin (H&E)-stained cross section of adenocarcinoma of the prostate, lung squamous cell carcinoma (LUSC), lung adenocarcinoma (LUAD), and Ewing sarcoma (ES) of the abdomen tumor. Annotated regions are T-tumor, S-stroma, D/G-duct/glandular, and A-adipose tissue. Scale bar is representative of A and B. Images are also used in Fig. S4A, 5D and S6G. B. Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section shown in A. The image displays a heatmap gradient of intensity with white (least abundant) to red (most abundant). Images are also used in Fig. S4E, 5D, and S6G. C. Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length (CL) distribution from tumor regions shown in A. Values are presented as mean +/- standard error (n=3 technical replicates per region).


A . ( Left ) Hematoxylin eosin ( H & E ) - stained cross section of an Ewing sarcoma ( ES ) of shoulder . Annotated regions are annotated as T - M - muscle . ( Right ) Spatial distribution relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . image displays a gradient of from ( least abundant ) to red ( most abundant ) . Scale bar gradient below are representative of in A - E . B . ( Left ) H & E stained cross section of an ES of chest wall . Annotated regions are annotated as T - N - necrosis . ( Right ) Spatial distribution relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . C . ( Left ) H & E stained cross section of an ES of rib . Annotated regions are annotated as T - N - necrosis . ( Right ) Spatial distribution relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . D . ( Left ) H & E stained cross section of an ES of abdomen . Annotated regions are T - , S - stroma , A - adipose tissue . ( Right ) Spatial distribution relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . E . ( Left ) H & E stained cross section of an ES of bladder . Annotated regions are T - S - stroma . ( Right ) Spatial distribution relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . F . Magnified H & E cross section of ( top , left ) , ( top , right ) muscle tissue from ES rib with magnified image of CL7 ( m / z = 1175 ) of ( bottom , left ) , ( bottom , right ) muscle tissues . G . Magnified H & E cross section of ( top , left ) , ( top , middle ) stroma , ( top , right ) adipose tissues from ES abdomen with magnified image of CL7 ( m / z = 1175 ) of ( bottom , left ) , ( bottom , middle ) stroma ( bottom , right ) adipose tissues .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. (Left) Hematoxylin and eosin (H&E)-stained cross section of an Ewing sarcoma (ES) of the shoulder tumor. Annotated regions are annotated as T-tumor and M-muscle. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. The image displays a heatmap gradient of intensity from white (least abundant) to red (most abundant). Scale bar and intensity gradient below are representative of all images in A-E. B. (Left) H&E stained cross section of an ES of the chest wall tumor. Annotated regions are annotated as T-tumor and N-necrosis. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. C. (Left) H&E stained cross section of an ES of the rib tumor. Annotated regions are annotated as T-tumor and N-necrosis. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. D. (Left) H&E stained cross section of an ES of the abdomen tumor. Annotated regions are T-tumor, S-stroma, and A-adipose tissue. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. E. (Left) H&E stained cross section of an ES of the bladder tumor. Annotated regions are T-tumor and S-stroma. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. F. Magnified H&E cross section of (top, left) tumor, and (top, right) muscle tissue from ES rib with magnified image of CL7 (m/z=1175) of (bottom, left) tumor, and (bottom, right) muscle tissues. G. Magnified H&E cross section of (top, left) tumor, (top, middle) stroma, and (top, right) adipose tissues from ES abdomen with magnified image of CL7 (m/z=1175) of (bottom, left) tumor, (bottom, middle) stroma and (bottom, right) adipose tissues.


A . ( Left ) Hematoxylin eosin ( H & E ) stained cross section of a normal human tibia ( Top ) resected at same time as Ewing sarcoma ( ES ) of tibia ( Bottom ) . Annotated regions are CT - connective tissue , B - decalcified bone , BM - bone marrow , T - , S - stroma . ( Right ) Spatial distribution relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section shown in A . image displays a gradient of with ( least abundant ) to red ( most abundant ) . Scale bar gradient below are representative of in A . B . Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain ( CL ) distribution from normal tibia in A . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) . # 0 . 01 < P < 0 . 05 ; * * * * P < 0 . 0001 , analyzed by two - way ANOVA with Tukey ' s multiple comparison for each glycogen chain . C . Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain ( CL ) distribution from ES of tibia in A . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) . * * 0 . 001 < P < 0 . 01 ; * * * * P < 0 . 0001 , analyzed by two - t - for each glycogen chain .

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

A. (Left) Hematoxylin and eosin (H&E) stained cross section of a normal human tibia (Top) resected at the same time as the Ewing sarcoma (ES) of the tibia tumor (Bottom). Annotated regions are CT-connective tissue, B-decalcified bone, BM-bone marrow, T-tumor, and S-stroma. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section shown in A. The image displays a heatmap gradient of intensity with white (least abundant) to red (most abundant). Scale bar and intensity gradient below are representative of all images in A. B. Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length (CL) distribution from normal tibia in A. Values are presented as mean +/- standard error (n=3 technical replicates per region). #0.01 < P < 0.05; ****P<0.0001, analyzed by two-way ANOVA with Tukey's multiple comparison for each glycogen chain length.C. Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length (CL) distribution from the ES of the tibia tumor in A. Values are presented as mean +/- standard error (n=3 technical replicates per region). **0.001 < P < 0.01; ****P<0.0001, analyzed by two-tailed t-test for each glycogen chain length.


F . In vivo growth of A673 EV or GYS1 - KO injected in flanks of athymic nude mice . Values are presented as mean + / - standard error ( n = 8 biological replicates per group ) . * * * * P < 0 . 0001 , analyzed by two - t - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. In vivo tumor growth of A673 EV or GYS1-KO injected in the flanks of athymic nude mice. Values are presented as mean +/- standard error (n=8 biological replicates per group). ****P<0.0001, analyzed by two-tailed t-test.


G . Half maximal effective ( EC50 ) of small molecular inhibitor guaiacol in A673 in vitro .

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G. Half maximal effective concentration (EC50) of the small molecular inhibitor guaiacol in A673 cells in vitro.


J . Spatial distribution of phosphorylated CL5 of human tibia ( top ) tibial ES ( bottom ) . image displays a gradient of with black ( least abundant ) to yellow ( most abundant ) . K . Phosphorylated chain distribution in normal human tibia tibial ES in J . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) . * * * * P < 0 . 0001 , analyzed by two - t - for each glycogen chain .

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

J. Spatial distribution of phosphorylated CL5 of human tibia (top) and tibial ES tumor (bottom). The image displays a heatmap gradient of intensity with black (least abundant) to yellow (most abundant). K. Phosphorylated chain length distribution in normal human tibia and tibial ES tumor in J. Values are presented as mean +/- standard error (n=3 technical replicates per region). ****P<0.0001, analyzed by two-tailed t-test for each glycogen chain length.


N . Representatives image of immunohistochemical of laforin in ES samples with annotation between ( T ) stroma ( S ) tissues . Tissues were scanned digitally using Axio Scan . Z1 side scanner . Scale bar is shown below image . O . of immunohistochemical laforin between normal tissue ( n = 3 technical replicates per region ) , tibial ES ( n = 7 technical replicates per region ) , stroma of five ES tissues ( n = 7 ) . Values are presented as mean + / - standard error . * * 0 . 001 < P < 0 . 0 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 , analyzed by one - way ANOVA with Tukey ' s multiple comparison .

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

N. Representatives image of immunohistochemical staining of laforin in ES patient samples with annotation between tumor (T) and stroma (S) tissues. Tissues were scanned digitally using the Axio Scan.Z1 side scanner. Scale bar is shown below the image. O. Quantification of immunohistochemical laforin staining between normal tissue (n=3 technical replicates per region), tibial ES tumor (n=7 technical replicates per region), and stroma of five ES tissues (n=7). Values are presented as mean +/- standard error. **0.001 < P < 0.0; ***P < 0.001; ****P<0.0001, analyzed by one-way ANOVA with Tukey's multiple comparison.


P . ( Top ) Western blot showing confirmation of laforin overexpression in A673 xenograft tumors . ( Bottom ) growth of xenografts of A673 empty vector ( EV ) laforin overexpression ( LAOE ) . Values are presented as mean + / - standard error ( n = 8 biological replicates per group ) . * * * * P < 0 . 0001 , analyzed by two - t - .

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

P. (Top) Western blot showing confirmation of laforin overexpression in A673 xenograft tumors. (Bottom) Tumor growth of xenografts of A673 empty vector (EV) and laforin overexpression (LAOE). Values are presented as mean +/- standard error (n=8 biological replicates per group). ****P<0.0001, analyzed by two-tailed t-test.


A . From left to right , Surface representation of three - dimensional structures of C3b - MCP ( CCP1 - 4 ) ( blue ) , C3b - SPICE ( CCP1 - 4 ) ( green ) , C3b - FH ( CCP1 - 4 ) ( orange ) , C3b - CR1 ( CCP15 - 17 ) ( red ) , C3b - DAF ( CCP2 - 4 ) ( purple ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. From left to right, Surface representation of the three-dimensional structures of C3b-MCP (CCP1-4) (blue), C3b-SPICE (CCP1-4) (green), C3b-FH (CCP1-4) (orange), C3b-CR1 (CCP15-17) (dark red), C3b-DAF (CCP2-4) (purple).


B . Cartoon representation of five regulators . width of cartoon is proportional to B - factors observed in structural data . B - factor coloring ( from blue to red ) is in relative scale according to resolution of crystal structures .

O O O O O O O O O O O O O O O O O O O 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. Cartoon representation of the five regulators. The width of the cartoon is proportional to the B-factors observed in the structural data. The B-factor coloring (from blue to red) is in relative scale according to the resolution of the crystal structures.


C . Cartoon representation of five C3b molecules in C3b - regulator complexes . Coloring style as in B .

C. Cartoon representation of the five C3b molecules in the C3b-regulator complexes. Coloring and style as in B.


D . Highlight of electron density maps ( 2Fo - Fc , blue , contour level 1 . 2σ ; Fo - Fc , green red , contour level 3σ - 3σ , resp . ) for regions of C3b - regulator complexes showing flexibility . From left to right : putative site for MCP CCP2 , SPICE CCP4 , CR1 CCP15 , DAF CCP2 .

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D. Highlight of electron density maps (2Fo-Fc, blue, contour level 1.2σ; Fo-Fc, green and red, contour level 3σ and -3σ, resp.) for the regions of the C3b-regulator complexes showing flexibility. From left to right: the putative site for MCP CCP2, SPICE CCP4, CR1 CCP15, and DAF CCP2.


A . " Open - book " surface representation of C3b regulator molecules , highlighting their domain organizations using different colors as shown in neighboring schemes . Domains in complement regulators are labeled using domain numbering from CCPi to CCPiv on their contact sites on C3b . corresponding CCP domains in each regulator described in this work are shown in bottom - left scheme . Domains CCP1 - 2 of MCP are shown in shaded grey because they are not involved in C3b binding . table reports extent of regulator contact surface on C3b associated buried surface .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. "Open-book" surface representation of C3b and regulator molecules, highlighting their domain organizations using different colors as shown in the neighboring schemes. Domains in complement regulators are labeled using domain numbering from CCPi to CCPiv based on their contact sites on C3b. The corresponding CCP domains in each regulator described in this work are shown in the bottom-left scheme. Domains CCP1-2 of MCP are shown in shaded grey because they are not involved in C3b binding. The table reports the extent of the regulator contact surface on C3b and the associated buried surface area.


B . Opened view of molecular footprint of regulator - C3b interaction ( as shown in Fig . 2A ) , highlighting C3b domains on regulator surface ( top ) regulator domains on C3b ( bottom ) .

O O O O O O O O O O O O O O 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. Opened view of the molecular footprint of the regulator-C3b interaction (as shown in Fig. 2A), highlighting the C3b domains on the regulator surface (top) and the regulator domains on C3b (bottom).


A . Superposition of MCP FH domains CCP3 - 4 ( shown as blue orange cartoons , resp . ) on surface of C3b .

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A. Superposition of MCP and FH domains CCP3-4 (shown as blue and orange cartoons, resp.) on the surface of C3b.


B . Cartoon representation of superposition of domains CCP3 - 4 of MCP ( blue ) , FH ( orange ) SPICE ( green ) . Despite strong structural similarity conserved angle between domains CCP3 CCP4 , MCP SPICE show a rotation of approximately 100 ° in position of CCP4 compared to FH .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Cartoon representation of the superposition of domains CCP3-4 of MCP (blue), FH (orange) and SPICE (green). Despite the strong structural similarity and the conserved angle between domains CCP3 and CCP4, MCP and SPICE show a rotation of approximately 100° in the position of CCP4 compared to FH.


C . reported mutagenesis data on MCP CCP3 CCP4 ( data from Liszewki et al . ( Liszewski et al , 2000 ) ) are consistent with crystal structure . Shown are two orientations of surface of MCP CCP3 - 4 as in crystal structure of C3b - MCP , highlighting contact sites with C3b platform ( purple ) , MCP mutations identified as critical for C3b binding ( red ) those not affecting interaction ( blue ) . GB24 antibody epitope is shown as yellow contour on MCP surface .

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C. The reported mutagenesis data on MCP CCP3 and CCP4 (data from Liszewki et al. (Liszewski et al, 2000)) are consistent with the crystal structure. Shown are two orientations of the surface of MCP CCP3-4 as in the crystal structure of the C3b-MCP complex, highlighting the contact sites with the C3b platform (purple), the MCP mutations identified as critical for C3b binding (red) and those not affecting the interaction (blue). The GB24 antibody epitope is shown as yellow contour on the MCP surface.


D . Model of putative positioning of flexible CCP1 - 2 domains of MCP . model has been generated by superposing structure of CCP1 - 4 of MCP from PDB 3O8E ( Persson et al , 2010 ) onto structure of C3b - MCP ( CCP1 - 4 ) . CCP1 - 2 domains of MCP , not visible in C3b - MCP ( CCP1 - 4 ) structure , are shown in grey .

D. Model of the putative positioning of the flexible CCP1-2 domains of MCP. The model has been generated by superposing the structure of CCP1-4 of MCP from PDB 3O8E (Persson et al, 2010) onto the structure of the C3b-MCP (CCP1-4) complex. CCP1-2 domains of MCP, not visible in the C3b-MCP (CCP1-4) structure, are shown in grey.


E . Highlight of differences between SPICE VCP . 11 mutations distinguishing two viral regulators are shown on surface of SPICE in purple . contact surface of C3b on regulator in C3b - SPICE ( CCP1 - 4 ) is shown in green on right panel .

O O O O O O O O O O O O O O O O O O O O O O O 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. Highlight of the primary sequence differences between SPICE and VCP. The 11 mutations distinguishing the two viral regulators are shown on the surface of SPICE in purple. The contact surface of C3b on the regulator in the C3b-SPICE (CCP1-4) complex is shown in green on the right panel.


( A ) Mapping of known disease - related mutations on C3b ( Table 3 ) . high - resolution C3b three - dimensional structure is used as structural template , shown as surface . regulator contact surfaces ( on C3b - FH ( CCP1 - 4 ) ( Wu et al , 2009 ) C3d - FH ( CCP19 - 20 ) ( Kajander et al , 2011 ; Morgan et al , 2011 ) ) are shown as black contour . mutations reported in Table 3 are shown in blue on C3b surface . Disease - related mutants reported to affect regulator binding are shown in red labeled in right magnified panel .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mapping of known disease-related mutations on C3b (Table 3). The high-resolution C3b three-dimensional structure is used as structural template, shown as white surface. The regulator contact surfaces (based on C3b-FH (CCP1-4) (Wu et al, 2009) and C3d-FH (CCP19-20) (Kajander et al, 2011; Morgan et al, 2011)) are shown as black contour. All mutations reported in Table 3 are shown in blue on the C3b surface. Disease-related mutants reported to affect regulator binding are shown in red and labeled in the right magnified panel.


( B ) Mapping of disease - related mutations on FH ( CCP1 - 4 ) surface using data from Table 4 . Colors as in Fig . 4A .

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(B) Mapping of disease-related mutations on the FH (CCP1-4) surface using data from Table 4. Colors as in Fig. 4A.


( C ) Mapping of disease - related mutations on MCP ( CCP3 - 4 ) surface using data from Table 4 . Colors as in Fig . 4A .

(C) Mapping of disease-related mutations on the MCP (CCP3-4) surface using data from Table 4. Colors as in Fig. 4A.


B Negative stain image of 2D crystals containing FERM - kinase region of FAK bound to a PI ( 4 , 5 ) P2 monolayer . Top right insert : Fourier transform of 2D crystal image . Bottom right insert : 2 . 5x zoom of main image .

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

B Negative stain image of 2D crystals containing the FERM-kinase region of FAK bound to a PI(4,5)P2 monolayer. Top right insert: Fourier transform of the 2D crystal image. Bottom right insert: 2.5x zoom of the main image.


A Autophosphorylation as measured in a ELISA is shown for WT interaction mutants in presence of control vesicles ( PC ; phosphatidylcholine ) or PI ( 4 , 5 ) P2 containing vesicles . Plotted are mean values from two independent autophosphorylation reactions , each determined in duplicates ( n = 4 ) . Mutant nomenclature is as shown in Table 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-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

A Autophosphorylation as measured in a ELISA assay is shown for WT and interaction mutants in presence of control vesicles (PC; phosphatidylcholine) or PI(4,5)P2 containing vesicles. Plotted are mean values from two independent autophosphorylation reactions, each determined in duplicates (n=4). Mutant nomenclature is as shown in Table 2.


B Catalytic turnover activity as measured in a coupled kinase is shown . Plotted are mean values from at least three independent experiments .

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

B Catalytic turnover activity as measured in a coupled kinase assay is shown. Plotted are mean values from at least three independent experiments.


C To determine whether autophosphorylation occurs in cis or trans , autophosphorylation was measured for WT or a kinase dead form of FAK ( KD , contains K454R mutation ) in presence or absence of a substrate deficient FAK mutant ( Y397F ) / or PI ( 4 , 5 ) P2 vesicles . PI ( 4 , 5 ) P2 negative experiments contain control PC vesicles instead . Note that scales of two plots are different , i . e . autophosphorylation is increased in presence of PI ( 4 , 5 ) P2 . Plotted are mean values from two independent phosphorylation reactions , each determined in duplicates ( n = 4 ) . Left : Schematic of possible cis - trans - autophosphorylation ( red arrows ) in mixtures of FAK - WT / FAK - Y397F FAK - KD / FAK - Y397F .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 To determine whether autophosphorylation occurs in cis or trans, autophosphorylation was measured for WT or a kinase dead form of FAK (KD, contains K454R mutation) in presence or absence of a substrate deficient FAK mutant (Y397F) and/or PI(4,5)P2 vesicles. PI(4,5)P2 negative experiments contain control PC vesicles instead. Note that the scales of the two plots are different, i.e. autophosphorylation is increased in presence of PI(4,5)P2. Plotted are mean values from two independent phosphorylation reactions, each determined in duplicates (n=4). Left: Schematic of possible cis- and trans-autophosphorylation (red arrows) in mixtures of FAK-WT/FAK-Y397F and FAK-KD/FAK-Y397F.


D Src phosphorylation of Y577 in FAK in presence of PC or PI ( 4 , 5 ) P2 vesicles as determined by an ELISA method . plot on left is obtained in presence Src containing only kinase domain ( Src254 - 536 ) plot on right with Src containing additionally SH3 SH2 domains ( Src84 - 536 ) . Plotted are mean values from two independent phosphorylation reactions , each determined in duplicates ( n = 4 ) .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Src phosphorylation of Y577 in FAK in presence of PC or PI(4,5)P2 vesicles as determined by an ELISA method. The plot on the left is obtained in presence Src containing only the kinase domain (Src254-536) and the plot on the right with Src containing additionally the SH3 and SH2 domains (Src84-536). Plotted are mean values from two independent phosphorylation reactions, each determined in duplicates (n=4).


Interactions between WT or interface mutant FAK with a PI ( 4 , 5 ) P2 containing was probed using a pulldown with coated silica beads . Plotted are bound relative to binding for WT at highest PI ( 4 , 5 ) P2 . Mutant naming is as in Table 2 . For WT non - specific binding to a PC is shown for comparison . Plotted are mean values from at least three independent experiments . Error bars : s . d .

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

Interactions between WT or interface mutant FAK with a PI(4,5)P2 containing membrane was probed using a pulldown assay with membrane coated silica beads. Plotted are bound protein relative to the binding for WT at the highest PI(4,5)P2 concentration. Mutant naming is as in Table 2. For WT the non-specific binding to a PC membrane is shown for comparison. Plotted are mean values from at least three independent experiments. Error bars: s.d.


A FAK phosphorylation of WT or interface mutant FAK in SCC . Whole lysates were subjected to western blot with anti - pFAK Y397 , anti - pFAK Y576 / 577 , anti - pFAK Y861 , anti - pFAK Y925 anti - FAK . Anti - GAPDH served as a loading control . graph shows densitometric of relative pFAK / FAK ratios ( mean ) from three independent experiments . Student ' s t - was carried out to calculate statistical significance . Error bars : s . d . * = p < 0 . 01 , # = p < 0 . 05 . For full blots see source data .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 FAK phosphorylation of WT or interface mutant FAK in SCC cells. Whole cell lysates were subjected to western blot analysis with anti-pFAK Y397, anti-pFAK Y576/577, anti-pFAK Y861, anti-pFAK Y925 and anti-FAK. Anti-GAPDH served as a loading control. The graph shows densitometric analysis of relative pFAK/FAK ratios (mean) from three independent experiments. Student's t-test was carried out to calculate the statistical significance. Error bars: s.d. * = p < 0.01, # = p < 0.05. For full blots see source data.


B Downstream signaling in SCC expressing WT or interface mutant FAK . Whole lysates are analyzed by western blot with indicated antibodies . graph shows densitometric of relative phospho / total ratios ( mean ) from three independent experiments . Student ' s t - was carried out to calculate statistical significance . Error bars : s . d . * = p < 0 . 01 , # = p < 0 . 05 . For full blots see source data .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Downstream signaling in SCC cells expressing WT or interface mutant FAK. Whole cell lysates are analyzed by western blot analysis with the indicated antibodies. The graph shows densitometric analysis of relative phospho/total protein ratios (mean) from three independent experiments. Student's t-test was carried out to calculate the statistical significance. Error bars: s.d. * = p < 0.01, # = p < 0.05. For full blots see source data.


C Src phosphorylation interactions between FAK Src or pSrc Y416 in SCC . FAK , Src or pSrc Y416 were immunoprecipitated from whole SCC lysates using anti - pSrc Y416 , anti - FAK , anti - Src or non - specific IgG - agarose , followed by western blot with anti - FAK , anti - pSrc Y416 anti - Src . Anti - GAPDH served as a loading control . For full blots see source data .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Src phosphorylation and interactions between FAK and Src or pSrc Y416 in SCC cells . FAK, Src or pSrc Y416 were immunoprecipitated from whole cell SCC lysates using anti-pSrc Y416, anti-FAK, anti-Src or non-specific IgG-agarose, followed by western blot analysis with anti-FAK, anti-pSrc Y416 and anti-Src. Anti-GAPDH served as a loading control. For full blots see source data.


FAK - expressing SCC were grown on glass coverslips , fixed stained with anti - FAK anti - Paxillin DAPI Representative immunofluorescence are shown . 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

FAK-expressing SCC cells were grown on glass coverslips, fixed and stained with anti-FAK anti-Paxillin and DAPI Representative immunofluorescence images are shown. Scale bars, 20 μm.


B FAK - expressing SCC were grown on glass coverslips , fixed stained with anti - pSrc Y416 , anti - Paxillin DAPI Representative immunofluorescence are shown . Scale bars , 20 μm . graph ( B ) shows of internalized active Src from three independent experiments . Student ' s t - was carried out to calculate statistical significance . Error bars , s . d . * = 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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 FAK-expressing SCC cells were grown on glass coverslips, fixed and stained with anti-pSrc Y416 , anti-Paxillin and DAPI Representative immunofluorescence images are shown. Scale bars, 20 μm. The graph (B) shows the quantification of internalized active Src from three independent experiments. Student's t-test was carried out to calculate the statistical significance. Error bars, s.d. * = p < 0.01.


C Subcellular fractionation of SCC expressing FAK - WT or interface mutant FAK . Whole lysates ( WCL ) subcellular fractions were subjected to western blot with anti - FAK , anti - Tubulin ( marker for fraction ; C ) , anti - RCAS ( marker for perinuclear fraction ; PN ) anti - H4 ( marker for fraction ; N ) . graph shows densitometric of relative FAK amount of FAK mutants in different fractions from three independent experiments . Student ' s t - was carried out to calculate statistical significance . Error bars : s . d . * = p < 0 . 01 , # = p < 0 . 05 . For full blots see source data .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Subcellular fractionation of SCC cells expressing FAK-WT or interface mutant FAK. Whole cell lysates (WCL) and subcellular fractions were subjected to western blot analysis with anti-FAK, anti-Tubulin (marker for cytoplasmic fraction; C), anti-RCAS (marker for perinuclear fraction; PN) and anti-H4 (marker for nuclear fraction; N). The graph shows densitometric analysis of relative FAK amount of the FAK mutants in the different fractions from three independent experiments. Student's t-test was carried out to calculate the statistical significance. Error bars: s.d. * = p < 0.01, # = p < 0.05. For full blots see source data.


A FAK - WT , interface mutant FAK or FAK - / - SCC were resuspended in methylcellulose solution in growth medium on a layer of agarose observed for 3D sphere formation . were taken from 10 random fields after nine days ( representative are shown ) relative colony ( top graph ) as well as number of colonies ( bottom graph ) were assessed from three independent experiments . Scale bars , 200 μm . Student ' s t - was carried out to calculate statistical significance . Error bars s . d . * = p < 0 . 01 , # = p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 FAK-WT, interface mutant FAK or FAK -/- SCC cells were resuspended in methylcellulose solution in growth medium on a layer of agarose and observed for 3D sphere formation. Images were taken from 10 random fields after nine days (representative images are shown) and the relative colony size (top graph) as well as the number of colonies (bottom graph) were assessed from three independent experiments. Scale bars, 200 μm. Student's t-test was carried out to calculate the statistical significance. Error bars s.d. * = p < 0.01, # = p < 0.05.


B FAK - WT , interface mutant FAK or FAK - / - SCC were seeded on growth factor - reduced Matrigel in serum - free media . After 72 h , invasion towards a serum gradient was analyzed by with calcein taking at 10 μm increments through matrigel . Representative of invasion are shown . Scale bars , 200 μm . graph shows relative invasion from five independent experiments . Student ' s t - was carried out to calculate statistical significance . Error bars , s . e . m . * = p < 0 . 01 , # = p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 FAK-WT, interface mutant FAK or FAK -/- SCC cells were seeded on growth factor-reduced Matrigel in serum-free media. After 72 h, invasion towards a serum gradient was analyzed by staining the cells with calcein and taking images at 10 μm increments through the matrigel. Representative images of the invasion assay are shown. Scale bars, 200 μm. The graph shows relative invasion from five independent experiments. Student's t-test was carried out to calculate the statistical significance. Error bars, s.e.m. * = p < 0.01, # = p < 0.05.


( A ) Representative immunofluorescence of Polo - phospho ( ph ) - T182 at KTs of Drosophila neuroblasts throughout mitotic progression . Insets show magnifications of outlined regions showing kinetochores ( KTs ) in early prometaphase ( EP ) , late prometaphase ( LP ) KTs aligned KTs in metaphase ( M ) . Plotted profiles of signal intensities of phospho ( ph ) - T182 KT Spc105 are shown for highlighted KTs . ( B ) Graph represents of Polo phT182 at KTs for neuroblasts shown in ( A ) . phT182 signal was determined relative to Spc105 values normalized to mean value determined for EP , which was set to 100 % ( n ≥ 67 KTs from at least 19 neuroblasts for each condition , n = 4 independent experiments ) . Data information : Statistical was calculated using a Kruskal - Wallis for multiple comparisons . p values : ns , not significant ; * < 0 . 05 ; * * < 0 . 01 ; * * * * < 0 . 0001 . Data are shown as mean ± SD . Scale bar : 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

(A) Representative immunofluorescence images of Polo-phospho(ph)-T182 levels at KTs of Drosophila neuroblasts throughout mitotic progression. Insets show magnifications of the outlined regions showing kinetochores (KTs) in early prometaphase (EP), late prometaphase (LP) KTs and aligned KTs in metaphase (M). Plotted profiles of signal intensities of phospho(ph)-T182 and the KT protein Spc105 are shown for the highlighted KTs. (B) Graph represents the levels of Polo phT182 at KTs for neuroblasts shown in (A). phT182 signal was determined relative to Spc105 and all values normalized to the mean value determined for EP, which was set to 100% (n≥67 KTs from at least 19 neuroblasts for each condition, n=4 independent experiments).Data information: Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: ns, not significant; *<0.05; **<0.01; ****<0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.


( C ) Selected stills from live imaging of mitotic progression in neuroblasts expressing UASPoloWT or UASPoloT182D under control of inscuteable - Gal4 driver . Neuroblasts without Polo overexpression were used as control . Mitotic progression was followed in vivo by direct visualization of tubulin - RFP centromere marker CID - GFP . Time 0 refers to envelope breakdown ( NEBD ) . ( D ) of mitotic time ( from NEBD to anaphase onset ) for neuroblasts shown in ( C ) . NEBD was identified as time tubulin entered space anaphase onset as time sister KTs separated ( n ≥ 12 neuroblasts for each condition , n ≥ 3 independent experiments ) ( E ) of time spent in prometaphase ( from NEBD until last KT alignment at metaphase plate ) for neuroblasts shown in ( C ) ( n ≥ 11 neuroblasts for each condition , n ≥ 3 independent experiments ) . Data information : Statistical was calculated using a Kruskal - Wallis for multiple comparisons . p values : ns , not significant ; * < 0 . 05 ; * * < 0 . 01 ; * * * * < 0 . 0001 . Data are shown as mean ± SD . Scale bar : 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 O

(C) Selected stills from live imaging analysis of mitotic progression in neuroblasts expressing UASPoloWT or UASPoloT182D under the control of inscuteable-Gal4 driver. Neuroblasts without Polo overexpression were used as control. Mitotic progression was followed in vivo by direct visualization of tubulin-RFP and the centromere marker CID-GFP. Time 0 refers to nuclear envelope breakdown (NEBD). (D) Quantification of the mitotic time (from NEBD to anaphase onset) for neuroblasts shown in (C). NEBD was identified as the time tubulin entered the nuclear space and anaphase onset as the time sister KTs separated (n≥12 neuroblasts for each condition, n≥3 independent experiments) (E) Quantification of the time spent in prometaphase (from NEBD until last KT alignment at the metaphase plate) for neuroblasts shown in (C) (n≥11 neuroblasts for each condition, n≥3 independent experiments). Data information: Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: ns, not significant; *<0.05; **<0.01; ****<0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.


( F ) Measurement of inter - kinetochore during mitotic progression for neuroblasts shown in ( C ) . Only KT pairs within maximum of two consecutive z - planes were considered eligible for . were acquired every 20 sec . graph shows mean between two CID centroids in a KT pair over time ( t = 0 is NEBD ) . A linear regression was applied to data set . Vertical dashed lines highlight time at which overexpressing PoloWT ( gray ) or PoloT182D ( red ) reach average inter - kinetochore measured in metaphase without Polo overexpression ( black ) ( n ≥ 7 neuroblasts for each condition ) . Data information : Statistical was calculated using a Kruskal - Wallis for multiple comparisons . p values : ns , not significant ; * < 0 . 05 ; * * < 0 . 01 ; * * * * < 0 . 0001 . Data are shown as mean ± SD . Scale bar : 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

(F) Measurement of inter-kinetochore distance during mitotic progression for neuroblasts shown in (C). Only KT pairs within the maximum of two consecutive z-planes were considered eligible for quantification. Images were acquired every 20 sec. The graph shows the mean distance between two CID centroids in a KT pair over time (t=0 is NEBD). A linear regression was applied to the data set. Vertical dashed lines highlight the time at which cells overexpressing PoloWT (gray) or PoloT182D (red) reach the average inter-kinetochore distance measured in metaphase cells without Polo overexpression (black) (n≥7 neuroblasts for each condition). Data information: Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: ns, not significant; *<0.05; **<0.01; ****<0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.


( G ) Selected stills from live imaging of neuroblasts expressing Mad2 - GFP to follow KT - MT attachment status upon expression of PoloWT or PoloT182D . CID - RFP was used as KT reference . Insets show magnifications of outlined regions showing single KT pairs that take longer to align at metaphase plate . Asterisks indicate direction of chromosome segregation ( putative spindle pole positions ) . ( H ) Graph represents mean ( MFI ) for Mad2 - GFP at KTs measured from NEBD to anaphase onset for neuroblasts shown in ( G ) . Each represents average of KTs measured from a single neuroblast at each time point . Mad2 - GFP MFI was determined relative to CID - RFP MFI ( n ≥ 16 neuroblasts for each condition , n = 5 independent experiments ) . Data information : Statistical was calculated using a Kruskal - Wallis for multiple comparisons . p values : ns , not significant ; * < 0 . 05 ; * * < 0 . 01 ; * * * * < 0 . 0001 . Data are shown as mean ± SD . Scale bar : 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

(G) Selected stills from live imaging of neuroblasts expressing Mad2-GFP to follow KT-MT attachment status upon expression of PoloWT or PoloT182D. CID-RFP was used as KT reference. Insets show magnifications of the outlined regions showing single KT pairs that take longer to align at the metaphase plate. Asterisks indicate direction of chromosome segregation (putative spindle pole positions). (H) Graph represents the mean fluorescence intensity (MFI) for Mad2-GFP at KTs measured from NEBD to anaphase onset for neuroblasts shown in (G). Each line represents the average of all KTs measured from a single neuroblast at each time point. Mad2-GFP MFI was determined relative to CID-RFP MFI (n≥16 neuroblasts for each condition, n=5 independent experiments).Data information: Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: ns, not significant; *<0.05; **<0.01; ****<0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.


( A ) Representative immunofluorescence of KT alignment efficiency in mitotic S2 expressing PoloWT - EGFP , PoloT182D - EGFP or lacking expression of any Polo transgene . were treated with MG132 prior to fixation to prevent from exiting mitosis increase number of pre - anaphase figures . Insets display magnifications of outlined regions , which highlight both low - tension misaligned ( inset 1 2 ) high - tension aligned KTs ( inset 3 ) . ( B ) Graph represents of in each indicated mitotic state , as shown in ( A ) ( n ≥ 532 for each condition , n = 2 independent experiments ) . Data information : Data are shown as mean ± SD . Scale bar : 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

(A) Representative immunofluorescence images of KT alignment efficiency in mitotic S2 cells expressing PoloWT-EGFP, PoloT182D-EGFP or lacking expression of any Polo transgene. Cells were treated with MG132 prior to fixation to prevent cells from exiting mitosis and increase the number of pre-anaphase figures. Insets display magnifications of the outlined regions, which highlight both low-tension misaligned (inset 1 and 2) and high-tension aligned KTs (inset 3). (B) Graph represents the percentage of cells in each indicated mitotic state, as shown in (A) (n≥ 532 cells for each condition, n=2 independent experiments). Data information: Data are shown as mean ± SD. Scale bar: 5 μm.


( C ) Representative immunofluorescence of calcium - stable KT - MT attachments in metaphase S2 expressing either PoloWT - EGFP or PoloT182D - EGFP . Insets display magnifications of outlined regions . Asterisk highlights either an aligned KT pair attached to MTs in an end - on fashion ( PoloWT - EGFP ) or an aligned KT pair in which a sister KT is laterally attached to end of a MT fiber ( PoloT182D - EGFP ) . Plotted profiles show overlap between Polo - EGFP tubulin signals for highlighted KT . ( D ) Graph represents of metaphase showing at least 1 KT with a lateral interaction , as shown in ( C ) ( asterisk ) ( n ≥ 58 for each condition , n = 2 independent experiments ) . Data information : Data are shown as mean ± SD . Scale bar : 5 μm .

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(C) Representative immunofluorescence images of calcium-stable KT-MT attachments in metaphase S2 cells expressing either PoloWT-EGFP or PoloT182D-EGFP. Insets display magnifications of the outlined regions. Asterisk highlights either an aligned KT pair attached to MTs in an end-on fashion (PoloWT-EGFP) or an aligned KT pair in which a sister KT is laterally attached to the end of a MT fiber (PoloT182D-EGFP). Plotted profiles show the overlap between Polo-EGFP and tubulin signals for the highlighted KT. (D) Graph represents the percentage of metaphase cells showing at least 1 KT with a lateral interaction, as shown in (C) (asterisk) (n≥58 cells for each condition, n=2 independent experiments). Data information: Data are shown as mean ± SD. Scale bar: 5 μm.


( E ) Selected frames from live image of chromosome segregation fidelity in neuroblasts expressing either PoloWT or PoloT182D . Neuroblasts without Polo overexpression were used as control . Jupiter - GFP was imaged for direct visualization of mitotic spindle . Time 0 refers to anaphase onset .

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(E) Selected frames from live image analysis of chromosome segregation fidelity in neuroblasts expressing either PoloWT or PoloT182D. Neuroblasts without Polo overexpression were used as control. Jupiter-GFP was imaged for direct visualization of the mitotic spindle. Time 0 refers to anaphase onset.


( F ) Representative immunofluorescence of mitotic neuroblasts from squashed brains showing distinct chromosome content . Spc105 was used as reference for KTs . Chromosome content is shown for each neuroblast .

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(F) Representative immunofluorescence images of mitotic neuroblasts from squashed brains showing distinct chromosome content. Spc105 was used as reference for KTs. Chromosome content is shown for each neuroblast.


( G ) Table shows of of asynchronous migration ( n ≥ 23 neuroblasts for each condition , n ≥ 3 independent experiments ) of aneuploid respectively . For of aneuploidy , whole brain was analyzed prometaphase metaphase neuroblasts were scored for chromosome number . An aneuploid was considered when > 8 chromosomes ( chr ) were visualized ( n ≥ 360 neuroblasts from at least 7 larvae brains for each condition , n ≥ 3 independent experiments ) . ND , not determined .

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(G) Table shows the quantification of the percentage of asynchronous migration (n≥23 neuroblasts for each condition, n≥3 independent experiments) and of aneuploid cells respectively. For the quantification of aneuploidy, the whole brain was analyzed and prometaphase and metaphase neuroblasts were scored for chromosome number. An aneuploid cell was considered when >8 chromosomes (chr) were visualized (n≥360 neuroblasts from at least 7 larvae brains for each condition, n≥3 independent experiments). ND, not determined.


( B ) Representative of eye phenotype observed in adult flies that were expressing UASPoloT182D in eye imaginal disc during development at indicated temperatures . An example of an eye from an adult fly co - expressing UASPoloT182D with UASRodRNAi is also shown . Expression of UASlacZ transgene in eye imaginal disc was used as control for overexpression UAS dilution effect .

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(B) Representative images of the eye phenotype observed in adult flies that were expressing UASPoloT182D in the eye imaginal disc during development at indicated temperatures. An example of an eye from an adult fly co-expressing UASPoloT182D with UASRodRNAi is also shown. Expression of UASlacZ transgene in the eye imaginal disc was used as control for overexpression and UAS dilution effect.


( C ) Graph represents mean of born at 25 ° C either expressing eyGal4 - driven UASPoloT182D together with UASlacZ or in combination with UASRodRNAi ( n ≥ 3 independent crosses for each condition ) . number of with genotype of interest born in each cross was normalized to mean total number of that are born in a control cross ( eyGal4 > UASlacZ , n = 9 independent crosses ) . Data information : Data are shown as mean ± SD . Scale bar : 5 μm .

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(C) Graph represents the mean percentage of males born at 25°C either expressing eyGal4-driven UASPoloT182D together with UASlacZ or in combination with UASRodRNAi (n≥3 independent crosses for each condition). The number of males with the genotype of interest born in each cross was normalized to the mean total number of males that are born in a control cross (eyGal4>UASlacZ, n=9 independent crosses). Data information: Data are shown as mean ± SD. Scale bar: 5 μm.


( D ) Selected stills from live imaging of neuroblasts expressing either UASPoloT182D alone or in Rod - depleted background ( UASPoloT182D + UASRodRNAi ) . Neuroblasts without transgene expression were used as control neuroblasts expressing UASPoloT182D together with UASlacZ were used as control for UAS dilution effect . ( E ) of time spent in prometaphase ( from NEBD until last KT alignment at metaphase plate ) for neuroblasts shown in ( D ) ( n ≥ 17 neuroblasts for each condition , n ≥ 4 independent experiments ) . Statistical was calculated using an one - way ANOVA for multiple comparisons . p values : ns , not significant ; * * , < 0 . 01 . Data information : Data are shown as mean ± SD . Scale bar : 5 μm .

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(D) Selected stills from live cell imaging of neuroblasts expressing either UASPoloT182D alone or in Rod-depleted background (UASPoloT182D+UASRodRNAi). Neuroblasts without transgene expression were used as control and neuroblasts expressing UASPoloT182D together with UASlacZ were used as control for UAS dilution effect. (E) Quantification of the time spent in prometaphase (from NEBD until last KT alignment at the metaphase plate) for neuroblasts shown in (D) (n≥17 neuroblasts for each condition, n≥4 independent experiments). Statistical analysis was calculated using an one-way ANOVA test for multiple comparisons. p values: ns, not significant; **, <0.01. Data information: Data are shown as mean ± SD. Scale bar: 5 μm.


( F ) of of prometaphase / metaphase mitotic figures per brain showing abnormal chromosome number ( > 8 chromosomes ( chr ) ( n ≥ 360 neuroblasts from at least 7 larvae brains for each condition , n ≥ 3 independent experiments ) . Statistical was calculated using a Kruskal - Wallis for multiple comparisons . p values : ns , not significant ; * * , < 0 . 01 . Data information : Data are shown as mean ± SD . Scale bar : 5 μm .

(F) Quantification of the percentage of prometaphase/metaphase mitotic figures per brain showing abnormal chromosome number (>8 chromosomes (chr) (n≥360 neuroblasts from at least 7 larvae brains for each condition, n≥3 independent experiments). Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: ns, not significant; **, <0.01. Data information: Data are shown as mean ± SD. Scale bar: 5 μm.


( A ) Selected stills from live imaging of neuroblasts expressing UASPoloWT , UASPoloT182D , or depleted of dynein heavy chain ( UASDhcRNAi ) expressing Rod - GFP under control of its endogenous promoter . CID - RFP was used as a KT reference . Insets display magnifications of outlined regions , which highlight late congressing KTs . Asterisks indicate direction of chromosome segregation ( putative spindle pole positions ) . ( B ) Graph represents mean ( MFI ) for Rod - GFP determined relative to CID - RFP MFI for KT pairs highlighted in ( A ) . Time was measured from first frame a KT pair moved away from other congressing KTs , until metaphase alignment ( vertical dashed lines ) . Data information : Statistical was calculated using an unpaired t - ( Mann - Whitney ) . p values : * * , < 0 . 01 ; * * * * , < 0 . 0001 . Data are shown as mean ± SD . Scale bar : 5 μm .

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(A) Selected stills from live cell imaging of neuroblasts expressing UASPoloWT, UASPoloT182D, or depleted of dynein heavy chain (UASDhcRNAi) and expressing Rod-GFP under control of its endogenous promoter. CID-RFP was used as a KT reference. Insets display magnifications of the outlined regions, which highlight late congressing KTs. Asterisks indicate direction of chromosome segregation (putative spindle pole positions). (B) Graph represents the mean fluorescence intensity (MFI) for Rod-GFP determined relative to CID-RFP MFI for the KT pairs highlighted in (A). Time was measured from the first frame a KT pair moved away from the other congressing KTs, until metaphase alignment (vertical dashed lines). Data information: Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: **, <0.01; ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.


( C ) Graph represents of KT Rod - GFP determined relative to CID - RFP measured throughout mitotic progression for control neuroblasts ( UASPoloWT , n = 24 neuroblasts , n = 4 independent experiments ) . Data information : Statistical was calculated using an unpaired t - ( Mann - Whitney ) . p values : * * , < 0 . 01 ; * * * * , < 0 . 0001 . Data are shown as mean ± SD . Scale bar : 5 μm .

(C) Graph represents the levels of KT Rod-GFP determined relative to CID-RFP measured throughout mitotic progression for control neuroblasts (UASPoloWT, n= 24 neuroblasts, n=4 independent experiments). Data information: Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: **, <0.01; ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.


( D ) Graph represents of Rod - GFP determined relative to CID - RFP measured at late congressing KTs ( n ≥ 15 neuroblasts for each condition , n ≥ 3 independent experiments ) . Data information : Statistical was calculated using an unpaired t - ( Mann - Whitney ) . p values : * * , < 0 . 01 ; * * * * , < 0 . 0001 . Data are shown as mean ± SD . Scale bar : 5 μm .

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(D) Graph represents the levels of Rod-GFP determined relative to CID-RFP measured at late congressing KTs (n≥15 neuroblasts for each condition, n≥3 independent experiments). Data information: Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: **, <0.01; ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.


( E ) Selected stills from live imaging of Rod - GFP streaming from KTs in neuroblasts expressing either PoloWT or PoloT182D . Asterisks indicate direction of chromosome segregation ( putative spindle poles positions ) . Arrowheads highlight streaming of Rod - GFP from unaligned KTs . Representative kymographs are shown for Rod - GFP streaming from metaphase until anaphase onset ( AO ) . Frames were acquired every 5sec . Data information : Scale bar : 5 μm .

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(E) Selected stills from live imaging analysis of Rod-GFP streaming from KTs in neuroblasts expressing either PoloWT or PoloT182D. Asterisks indicate direction of chromosome segregation (putative spindle poles positions). Arrowheads highlight streaming of Rod-GFP from unaligned KTs. Representative kymographs are shown for Rod-GFP streaming from metaphase until anaphase onset (AO). Frames were acquired every 5sec. Data information: Scale bar: 5 μm.


( A ) Representative immunofluorescence of ZW10 at unaligned KTs in neuroblasts expressing UASPoloWT , UASPoloT182D or depleted of dynactin subunit p50 ( UASp50RNAi ) or Spindly ( UASSpindlyRNAi ) . Spc105 was used as a KT reference . Insets display magnifications of outlined regions which highlight both late congressing KT ( inset 1 ) sister KTs that are non - oriented along spindle axis ( inset 2 , except for UASPoloWT panel which displays congressed KTs ) . UASPoloWT neuroblasts were used as control . Data information : Scale bar : 5 μm .

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(A) Representative immunofluorescence images of ZW10 levels at unaligned KTs in neuroblasts expressing UASPoloWT, UASPoloT182D or depleted of dynactin subunit p50 (UASp50RNAi) or Spindly (UASSpindlyRNAi). Spc105 was used as a KT reference. Insets display magnifications of the outlined regions which highlight both late congressing KT (inset 1) and sister KTs that are non-oriented along the spindle axis (inset 2, except for the UASPoloWT panel which displays congressed KTs). UASPoloWT neuroblasts were used as control. Data information: Scale bar: 5 μm.


( B ) Graph represents ZW10 at late congressing non - oriented KTs for neuroblasts were selected when most KTs were congressed late congressing KTs were clearly identified . Similarly , non - oriented KTs were considered when KT - KT axis was not parallel to most congressed KTs ( or a metaphase plate ) . ZW10 signal was determined relative to Spc105 values were normalized to control mean , which was set to 100 % ( n ≥ 69 KTs from at least 19 neuroblasts for each condition , n ≥ 3 independent experiments ) . Data information : Statistical was calculated using an unpaired t - ( Mann - Whitney ) . p values : * , < 0 . 05 ; * * , < 0 . 01 ; * * * , < 0 . 001 ; * * * * , < 0 . 0001 . Data are shown as mean ± SD .

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(B) Graph represents ZW10 levels at late congressing and non-oriented KTs for neuroblasts Cells were selected when most KTs were congressed and late congressing KTs were clearly identified. Similarly, non-oriented KTs were considered when the KT-KT axis was not parallel to most congressed KTs (or a metaphase plate). ZW10 signal was determined relative to Spc105 and all values were normalized to the control mean fluorescence intensity, which was set to 100% (n≥69 KTs from at least 19 neuroblasts for each condition, n≥3 independent experiments). Data information: Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: *, <0.05; **, <0.01; ***, <0.001; ****, <0.0001. Data are shown as mean ± SD.


( C ) Selected stills from live imaging of KT alignment in neuroblasts expressing UASPoloT182D , either alone or in a Rod - depleted background ( UASPoloT182D + UASRodRNAi ) , depleted of dynactin ( UASp50RNAi ) , Spindly ( UASSpindlyRNAi ) , dynein ( UASDhcRNAi ) or Rod in a dynein - depleted background ( UASDhcRNAi + UASRodRNAi ) . Outlined regions show KTs that are non - oriented ( lateral attachment ) relative to spindle axis that were followed over time until stable biorientation ( arrowhead ) . Cartoon depicts behavior of respective KT pair over successive frames relative to spindle axis ( dashed lines ) . Neuroblasts without transgene expression were used as control . Data information : Scale bar : 5 μm .

(C) Selected stills from live imaging analysis of KT alignment in neuroblasts expressing UASPoloT182D, either alone or in a Rod-depleted background (UASPoloT182D+UASRodRNAi), depleted of dynactin (UASp50RNAi), Spindly (UASSpindlyRNAi), dynein (UASDhcRNAi) or Rod in a dynein-depleted background (UASDhcRNAi+UASRodRNAi). Outlined regions show KTs that are non-oriented (lateral attachment) relative to spindle axis and that were followed over time until stable biorientation (arrowhead). Cartoon depicts the behavior of the respective KT pair over successive frames relative to the spindle axis (dashed lines). Neuroblasts without transgene expression were used as control. Data information: Scale bar: 5 μm.


( D ) of time a KT pair takes from first contact with MTs until stable biorientation for neuroblasts ( n ≥ 12 KTs from at least 8 neuroblasts for each condition , n ≥ 5 independent experiments ) . Data information : Statistical was calculated using an unpaired t - ( Mann - Whitney ) . p values : * , < 0 . 05 ; * * , < 0 . 01 ; * * * , < 0 . 001 ; * * * * , < 0 . 0001 . Data are shown as mean ± SD .

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(D) Quantification of the time a KT pair takes from the first contact with MTs until stable biorientation for neuroblasts (n≥12 KTs from at least 8 neuroblasts for each condition, n≥5 independent experiments). Data information: Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: *, <0.05; **, <0.01; ***, <0.001; ****, <0.0001. Data are shown as mean ± SD.


( A ) Representative immunofluorescence of Spindly localization to unaligned KTs in Drosophila S2 expressing either PoloWT - EGFP or PoloT182D - EGFP . Insets display magnifications of outlined regions . were treated with MG132 prior to fixation to increase number of late prometaphase figures allow better identification of late congressing KTs . CID was used as a KT reference . ( B ) Graph represents of Spindly at late congressing non - oriented ( not oriented parallel to spindle axis ) KTs for shown in ( A ) . Spindly were determined relative to CID values were normalized to mean quantified in expressing PoloWT - EGFP , which was set to 100 % ( n ≥ 121 KTs from at least 39 for each condition , n = 3 independent experiments ) . Data information : Statistical was calculated using an unpaired t - ( Mann - Whitney ) . p values : * * * * , < 0 . 0001 . Data are shown as mean ± SD . Scale bar : 5 μm .

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(A) Representative immunofluorescence images of Spindly localization to unaligned KTs in Drosophila S2 cells expressing either PoloWT-EGFP or PoloT182D-EGFP. Insets display magnifications of the outlined regions. Cells were treated with MG132 prior to fixation to increase the number of late prometaphase figures and allow better identification of late congressing KTs. CID was used as a KT reference. (B) Graph represents the percentage of Spindly levels at late congressing and non-oriented (not oriented parallel to the spindle axis) KTs for cells shown in (A). Spindly levels were determined relative to CID and all values were normalized to the mean fluorescence intensity quantified in cells expressing PoloWT-EGFP, which was set to 100% (n≥121 KTs from at least 39 cells for each condition, n=3 independent experiments). Data information: Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.