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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 .

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

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 concentration ( EC50 ) of the small molecular inhibitor guaiacol in A673 cells 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 ) 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 .

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

J. 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 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 .

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 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

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 ) 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 .

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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 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 ) .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 the five C3b molecules in the C3b - regulator complexes . Coloring and style as in B .

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

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 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 .

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

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 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 .

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 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 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 B-MEASURED_VAR 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 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 ) .

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 B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR 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 and FH domains CCP3 - 4 ( shown as blue and orange cartoons , resp . ) on the surface of C3b .

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

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 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 .

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

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

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 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 .

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

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 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 .

O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR 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 B-MEASURED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR 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 ) . 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 .

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

(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 the 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 the MCP ( CCP3 - 4 ) surface using data from Table 4 . Colors as in Fig . 4A .

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(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 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 .

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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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 assay is shown . Plotted are mean values from at least three independent experiments .

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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 ) 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 .

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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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 . 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 ) .

O B-MEASURED_VAR 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-MEASURED_VAR 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

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 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 .

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

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 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 .

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

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 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 .

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

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 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 .

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

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 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR 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 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 .

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 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 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

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 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 .

O O O O O O O B-CONTROLLED_VAR 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 B-MEASURED_VAR 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 B-MEASURED_VAR 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 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 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 .

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

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

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 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

(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 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 .

O O O O O 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 B-MEASURED_VAR 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 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 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 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 .

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

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 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 O O O O O O 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 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

(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 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 .

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

(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 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 .

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

(C) Representative immunofluorescence images 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 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 .

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 B-CONTROLLED_VAR 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

(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 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 .

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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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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

(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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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

(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 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 .

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

(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 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 .

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

(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 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 .

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

(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 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 .

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

(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 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 .

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 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

(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 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 .

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

(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 ) 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 .

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

(D) Quantification of the 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 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 .

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

(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.


( C ) Selected frames from live imaging analysis of SpindlyWT - EGFP behavior during KT alignment in Drosophila S2 cells expressing either PoloWT - mRFP or PoloT182D - mRFP . Insets show magnifications of the outlined regions that highlight KTs with delayed congression to the spindle equator ( yellow dashed line ) . Time 0 is the first shift in KT direction from poleward to anti - poleward movement . ( D ) Graph represents the variation in SpindlyWT - EGFP levels at KTs highlighted in ( C ) and its correlation with efficiency of alignment . Spindly - EGFP mean fluorescence intensity ( MFI ) levels were corrected for photobleaching during acquisition and normalized to the first frame of analysis to account for differences in expression levels . KTs were tracked during the process of congression ( using Polo - mRFP signal as a KT reference ) and the distance from the unaligned KT pair to the spindle equator was measured . All values were normalized to the last frame of analysis ( stable integration in metaphase plate ) . Time 0 is the first shift in KT direction from poleward to anti - poleward movement ( n ≥ 9 KTs from at least 5 cells for each condition , n ≥ 4 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 .

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 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 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 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

(C) Selected frames from live imaging analysis of SpindlyWT-EGFP behavior during KT alignment in Drosophila S2 cells expressing either PoloWT-mRFP or PoloT182D-mRFP. Insets show magnifications of the outlined regions that highlight KTs with delayed congression to the spindle equator (yellow dashed line). Time 0 is the first shift in KT direction from poleward to anti-poleward movement. (D) Graph represents the variation in SpindlyWT-EGFP levels at KTs highlighted in (C) and its correlation with efficiency of alignment. Spindly-EGFP mean fluorescence intensity (MFI) levels were corrected for photobleaching during acquisition and normalized to the first frame of analysis to account for differences in expression levels. KTs were tracked during the process of congression (using Polo-mRFP signal as a KT reference) and the distance from the unaligned KT pair to the spindle equator was measured. All values were normalized to the last frame of analysis (stable integration in metaphase plate). Time 0 is the first shift in KT direction from poleward to anti-poleward movement (n≥9 KTs from at least 5 cells for each condition, n≥4 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.


( E ) Representative immunofluorescence images of Spindly accumulation at aligned KTs in Drosophila S2 cells expressing either PoloWT - EGFP or PoloT182D - EGFP , in control or upon depletion of dynein heavy chain . Insets display magnifications of the outlined regions . CID was used as a KT reference . Data information : Scale bar : 5 μm .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_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

(E) Representative immunofluorescence images of Spindly accumulation at aligned KTs in Drosophila S2 cells expressing either PoloWT-EGFP or PoloT182D-EGFP, in control or upon depletion of dynein heavy chain. Insets display magnifications of the outlined regions. CID was used as a KT reference. Data information: Scale bar: 5 μm.


( F ) Representative immunofluorescence images of Spindly accumulation at unattached KTs in Drosophila S2 cells expressing either PoloWT - EGFP or PoloT182D - EGFP . Insets display magnifications of the outlined regions . Cells were treated with colchicine prior to fixation to generate unattached KTs . CID was used as a KT reference . Data information : Scale bar : 5 μm .

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

(F) Representative immunofluorescence images of Spindly accumulation at unattached KTs in Drosophila S2 cells expressing either PoloWT-EGFP or PoloT182D-EGFP. Insets display magnifications of the outlined regions. Cells were treated with colchicine prior to fixation to generate unattached KTs. CID was used as a KT reference. Data information: Scale bar: 5 μm.


( G ) Graph represents Spindly levels at aligned KTs for cells Spindly levels were determined relative to CID and all values were normalized to the mean fluorescence intensity quantified in control PoloWT - EGFP expressing cells , which was set to 100 % ( n ≥ 633 KTs from at least 35 cells for each condition , n = 2 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 .

O O O O O B-MEASURED_VAR 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 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

(G) Graph represents Spindly levels at aligned KTs for cells Spindly levels were determined relative to CID and all values were normalized to the mean fluorescence intensity quantified in control PoloWT-EGFP expressing cells, which was set to 100% (n≥633 KTs from at least 35 cells for each condition, n=2 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.


( H ) Graph represents Spindly levels at unattached KTs for cells Spindly levels were determined relative to CID and all values were normalized to the mean fluorescence intensity quantified in PoloWT - EGFP expressing cells , which was set to 100 % ( n ≥ 1282 KTs from at least 59 cells for each condition , n = 2 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 .

O O O O O B-MEASURED_VAR 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 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

(H) Graph represents Spindly levels at unattached KTs for cells Spindly levels were determined relative to CID and all values were normalized to the mean fluorescence intensity quantified in PoloWT-EGFP expressing cells, which was set to 100% (n≥1282 KTs from at least 59 cells for each condition, n=2 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.


( A ) In vitro kinase assays with the indicated recombinant protein in the presence of [ γ - 32P ] - ATP for 30 min . Phosphorylation was detected by autoradiography and levels of proteins were visualized by coomassie blue staining . Data information : 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

(A) In vitro kinase assays with the indicated recombinant protein in the presence of [γ-32P]-ATP for 30 min. Phosphorylation was detected by autoradiography and levels of proteins were visualized by coomassie blue staining. Data information: Scale bar: 5 μm.


( D ) Representative immunofluorescence images of Spindly - phospho ( ph ) Ser499 levels at unattached KTs in control Drosophila S2 cells and in Polo - depleted or BI2536 - treated cells . Insets display magnifications of the outlined regions . Cells were treated with colchicine prior to fixation to generate unattached KTs . CID was used as a KT reference . Data information : Scale bar : 5 μm .

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 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-MEASURED_VAR O O O O O O O O O O O O O O O O

(D) Representative immunofluorescence images of Spindly-phospho(ph)Ser499 levels at unattached KTs in control Drosophila S2 cells and in Polo-depleted or BI2536-treated cells. Insets display magnifications of the outlined regions. Cells were treated with colchicine prior to fixation to generate unattached KTs. CID was used as a KT reference. Data information: Scale bar: 5 μm.


( E ) Graph represents the levels of Spindly phS499 at unattached KTs for cells Spindly phS499 levels were determined relative to CID and all values were normalized to the control mean fluorescence intensity , which was set to 100 % ( n ≥ 1246 KTs from at least 63 cells for each condition , n = 2 independent experiments ) . Statistical analysis was calculated using a Kruskal - Wallis test for multiple comparisons . p values : * * * * , < 0 . 0001 Data information : Data are shown as mean ± SD . Scale bar : 5 μm .

O O O O O O O O B-MEASURED_VAR 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

(E) Graph represents the levels of Spindly phS499 at unattached KTs for cells Spindly phS499 levels were determined relative to CID and all values were normalized to the control mean fluorescence intensity, which was set to 100% (n≥1246 KTs from at least 63 cells for each condition, n=2 independent experiments). Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: ****, <0.0001 Data information: Data are shown as mean ± SD. Scale bar: 5 μm.


( F ) Representative immunofluorescence images of Spindly phS499 levels at unaligned and aligned KTs in a PoloWT - EGFP expressing Drosophila S2 cell . Insets show magnifications of the outlined regions , which highlight either a unaligned ( u ) KT or an aligned ( a ) KT . CID was used as a KT reference . Plotted profiles of signal intensities of phS499 and CID are shown for the highlighted KTs . Data information : Data are shown as mean ± SD . Scale bar : 5 μm .

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 B-MEASURED_VAR 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

(F) Representative immunofluorescence images of Spindly phS499 levels at unaligned and aligned KTs in a PoloWT-EGFP expressing Drosophila S2 cell. Insets show magnifications of the outlined regions, which highlight either a unaligned (u) KT or an aligned (a) KT. CID was used as a KT reference. Plotted profiles of signal intensities of phS499 and CID are shown for the highlighted KTs. Data information: Data are shown as mean ± SD. Scale bar: 5 μm.


( G ) Graph represents the levels of Spindly phS499 at unaligned KTs versus aligned KTs for cells Spindly phS499 levels were determined relative to CID and all values were normalized to the mean fluorescence intensity of unaligned KTs , which was set to 100 % ( n ≥ 34 KTs from 14 cells , n = 2 experiments ) . Statistical analysis was calculated using an unpaired t - test ( Mann - Whitney ) . p values : * * * * , < 0 . 0001 . Data information : Data are shown as mean ± SD . Scale bar : 5 μm .

O O O O O O O O B-MEASURED_VAR 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

(G) Graph represents the levels of Spindly phS499 at unaligned KTs versus aligned KTs for cells Spindly phS499 levels were determined relative to CID and all values were normalized to the mean fluorescence intensity of unaligned KTs, which was set to 100% (n≥34 KTs from 14 cells, n=2 experiments). Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: ****, <0.0001. Data information: Data are shown as mean ± SD. Scale bar: 5 μm.


( A ) Western blot analysis of MBP - Zwilch pull downs of full length ( FL ) his6x - SpindlyWT and phosphomimetic mutant his6x - SpindlyS499D . Immobilized MBP was used as negative control . B : Beads , FT : Flow - through . ( B ) Western blot analysis of MBP - Zwilch pull downs of his6x - SpindlyWT or his6x - SpindlyS499D fragments ( aa1 - 510 ) . Immobilized MBP was used as negative control . B : Beads , FT : Flow - through . ( C ) Graph represents the levels of his6x - Spindly relative to MBP - Zwilch from at least three independent pull down experiments . The values obtained for his6x - SpindlyWT were set to 1 . Data information : XXXX Data are shown as mean ± SD . Scale bar : 5 μm .

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

(A) Western blot analysis of MBP-Zwilch pull downs of full length (FL) his6x-SpindlyWT and phosphomimetic mutant his6x-SpindlyS499D. Immobilized MBP was used as negative control. B:Beads, FT: Flow-through. (B) Western blot analysis of MBP-Zwilch pull downs of his6x-SpindlyWT or his6x-SpindlyS499D fragments (aa1-510). Immobilized MBP was used as negative control. B:Beads, FT: Flow-through. (C) Graph represents the levels of his6x-Spindly relative to MBP-Zwilch from at least three independent pull down experiments. The values obtained for his6x-SpindlyWT were set to 1. Data information: XXXX Data are shown as mean ± SD. Scale bar: 5 μm.


( D ) Selected frames from live imaging analysis of SpindlyS499A - EGFP or SpindlyS499D - EGFP behavior during KT alignment in Drosophila S2 cells expressing PoloWT - mRFP . SpindlyWT - EGFP expressing cells were used as control . All Spindly - EGFP constructs were resistant to the RNAi targeted against endogenous Spindly . Insets show magnifications of the outlined regions that highlight KTs that delay in congressing to the spindle equator ( yellow dashed line ) . Time 0 is the first shift in KT direction from poleward to anti - poleward movement . ( E ) Graph represents the variation in SpindlyWT - EGFP , SpindlyS499A - EGFP and SpindlyS499D - EGFP levels at KTs highlighted in ( D ) and its correlation with efficiency of alignment . All values were determined as described in Fig . 6D . Time 0 is the first shift in KT direction from poleward to anti - poleward movement ( n ≥ 5 KTs from at least 4 cells for each condition , n ≥ 2 independent experiments ) . Data information : XXXX Data are shown as mean ± SD . Scale bar : 5 μm .

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 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 B-MEASURED_VAR 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 O O O O O O O O O O O O O O O O O O O O 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) Selected frames from live imaging analysis of SpindlyS499A-EGFP or SpindlyS499D-EGFP behavior during KT alignment in Drosophila S2 cells expressing PoloWT-mRFP. SpindlyWT-EGFP expressing cells were used as control. All Spindly-EGFP constructs were resistant to the RNAi targeted against endogenous Spindly. Insets show magnifications of the outlined regions that highlight KTs that delay in congressing to the spindle equator (yellow dashed line). Time 0 is the first shift in KT direction from poleward to anti-poleward movement. (E) Graph represents the variation in SpindlyWT-EGFP, SpindlyS499A-EGFP and SpindlyS499D-EGFP levels at KTs highlighted in (D) and its correlation with efficiency of alignment. All values were determined as described in Fig.6D. Time 0 is the first shift in KT direction from poleward to anti-poleward movement (n≥5 KTs from at least 4 cells for each condition, n≥2 independent experiments). Data information: XXXX Data are shown as mean ± SD. Scale bar: 5 μm.


( A ) Representative immunofluorescence images of ZW10 localization to unaligned KTs in Drosophila S2 cells expressing either SpindlyWT - , SpindlyS499A - or SpindlyS499D - EGFP . Insets display magnifications of the outlined regions , which highlight late congressing KTs . CID was used as a KT reference . ( B ) Graph represents the percentage of ZW10 levels at late congressing and non - oriented ( not oriented parallel to the spindle axis ) KTs for cells shown in ( A ) . ZW10 levels were determined relative to CID and all values were normalized to the control mean fluorescence intensity , which was set to 100 % ( n ≥ 72 KTs from at least 26 cells for each condition , n = 4 independent experiments ) . Data information : Statistical analysis was calculated using a Kruskal - Wallis test for multiple comparisons . p values : * , < 0 . 05 ; * * , < 0 . 01 ; * * * * , < 0 . 0001 . Data are shown as mean ± SD . Scale bar : 5 μm .

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

(A) Representative immunofluorescence images of ZW10 localization to unaligned KTs in Drosophila S2 cells expressing either SpindlyWT-, SpindlyS499A- or SpindlyS499D-EGFP. Insets display magnifications of the outlined regions, which highlight late congressing KTs. CID was used as a KT reference. (B) Graph represents the percentage of ZW10 levels at late congressing and non-oriented (not oriented parallel to the spindle axis) KTs for cells shown in (A). ZW10 levels were determined relative to CID and all values were normalized to the control mean fluorescence intensity, which was set to 100% (n≥72 KTs from at least 26 cells for each condition, n=4 independent experiments). Data information: Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: *, <0.05; **, <0.01; ****, <0.0001 . Data are shown as mean ±SD. Scale bar: 5 μm.


( C ) Selected frames from live imaging analysis of chromosome congression in Drosophila S2 cells expressing SpindlyWT - EGFP , SpindlyS499A - EGFP , SpindlyS499D - EGFP or SpindlyS499D - EGFP in a ZW10 - depleted background . The first frame shows EGFP - positive cells . Chromosome alignment was followed in vivo by direct visualization of KT - associated PoloWT - mRFP signal . Time 0 refers to nuclear envelope breakdown ( NEBD ) , metaphase refers to full KT alignment and Anaphase onset refers to onset of chromosome segregation . ( D ) Quantification of the time spent in prometaphase ( from NEBD until last KT alignment at the metaphase plate ) for cells shown in ( C ) ( n ≥ 10 cells for each condition , n ≥ 2 independent experiments ) . Data information : Statistical analysis was calculated using a Kruskal - Wallis test for multiple comparisons . p values : * , < 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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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 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 O O O O O

(C) Selected frames from live imaging analysis of chromosome congression in Drosophila S2 cells expressing SpindlyWT-EGFP, SpindlyS499A-EGFP, SpindlyS499D-EGFP or SpindlyS499D-EGFP in a ZW10-depleted background. The first frame shows EGFP-positive cells. Chromosome alignment was followed in vivo by direct visualization of KT-associated PoloWT-mRFP signal. Time 0 refers to nuclear envelope breakdown (NEBD), metaphase refers to full KT alignment and Anaphase onset refers to onset of chromosome segregation. (D) Quantification of the time spent in prometaphase (from NEBD until last KT alignment at the metaphase plate) for cells shown in (C) (n≥10 cells for each condition, n≥2 independent experiments). Data information: Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: *, <0.05; **, <0.01; ****, <0.0001 . Data are shown as mean ±SD. Scale bar: 5 μm.


( A ) Representative immunofluorescence images of ZW10 streaming from KTs in Drosophila S2 cells in metaphase expressing either SpindlyWT - , SpindlyS499A - or SpindlyS499D - EGFP . ZW10 streaming is also shown in S2 cells expressing SpindlyWT - or SpindlyS499A - EGFP in a dynein - depleted background . Insets display magnifications of the outlined regions , which highlight streaming robustness . ( B ) Graph represents the percentage of cells in metaphase showing different levels of ZW10 streaming ( n ≥ 35 cells for each condition , n = 2 independent experiments ) . ( C ) Graph represents the percentage of ZW10 levels at aligned KTs normalized to ZW10 levels at the spindle region . All values were normalized to the mean fluorescence intensity quantified in SpindlyWT - EGFP expressing cells , which was set to 100 % ( n ≥ 470 KTs from at least 35 cells for each condition , n = 2 independent experiments ) . Data information : Statistical analysis was calculated using a Kruskal - Wallis test for multiple comparisons . p values : * * * * , < 0 . 0001 . Data are shown as mean ± SD . Scale bar : 5 μm .

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

(A) Representative immunofluorescence images of ZW10 streaming from KTs in Drosophila S2 cells in metaphase expressing either SpindlyWT-, SpindlyS499A- or SpindlyS499D-EGFP. ZW10 streaming is also shown in S2 cells expressing SpindlyWT- or SpindlyS499A-EGFP in a dynein-depleted background. Insets display magnifications of the outlined regions, which highlight streaming robustness. (B) Graph represents the percentage of cells in metaphase showing different levels of ZW10 streaming (n≥35 cells for each condition, n=2 independent experiments). (C) Graph represents the percentage of ZW10 levels at aligned KTs normalized to ZW10 levels at the spindle region. All values were normalized to the mean fluorescence intensity quantified in SpindlyWT-EGFP expressing cells, which was set to 100% (n≥470 KTs from at least 35 cells for each condition, n=2 independent experiments). Data information: Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.


( D ) Representative immunofluorescence images of calcium - stable KT - MT attachments in metaphase S2 cells expressing SpindlyWT - , SpindlyS499A - , SpindlyS499D - or SpindlyS499D - EGFP in a ZW10 - depleted background . Insets display magnifications of the outlined regions which highlight different attachment configurations ( E - end on ; L - lateral ; M - merotelic ) . Cartoon depicts the attachment configuration of the respective KT pair . Asterisk highlights an aligned KT pair in which a sister KT appears to be laterally attached to the end of a MT fiber . Plotted profiles show the overlap between CENP - C and tubulin signals for the highlighted KT . CENP - C was used as a KT reference . ( E ) Graph represents the percentage of metaphase cells showing only end - on attachments or at least 1 KT with lateral or merotelic attachment , as shown in D ( n ≥ 44 cells for each condition , n ≥ 2 independent experiments ) . Data information : Statistical analysis was calculated using a Kruskal - Wallis test for multiple comparisons . p values : * * * * , < 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 B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 immunofluorescence images of calcium-stable KT-MT attachments in metaphase S2 cells expressing SpindlyWT-, SpindlyS499A-, SpindlyS499D- or SpindlyS499D-EGFP in a ZW10-depleted background. Insets display magnifications of the outlined regions which highlight different attachment configurations (E - end on; L - lateral; M - merotelic). Cartoon depicts the attachment configuration of the respective KT pair. Asterisk highlights an aligned KT pair in which a sister KT appears to be laterally attached to the end of a MT fiber. Plotted profiles show the overlap between CENP-C and tubulin signals for the highlighted KT. CENP-C was used as a KT reference. (E) Graph represents the percentage of metaphase cells showing only end-on attachments or at least 1 KT with lateral or merotelic attachment, as shown in D (n≥44 cells for each condition, n≥2 independent experiments). Data information: Statistical analysis was calculated using a Kruskal-Wallis test for multiple comparisons. p values: ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm.


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

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


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

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


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

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

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