( 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 .

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

(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 .

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

(B) Representative 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 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 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 .

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

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.


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

O O O O O O O O O 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 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

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


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

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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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


B - D . Examples of Min protein patterns emerging from nearly homogeneous initial conditions in E . coli cells of different sizes . Lateral dimensions ( in µm ) from top to bottom : 2x6 . 5 , 2x8 . 8 , and 5 . 2x8 . 8 respectively . The gray - scale images show cytosolic near - infrared fluorescence emitted by the protein eqFP670 at the first ( left ) and last ( right ) time points . The color montages show the sfGFP - MinD intensity ( indicated by the color scale at the bottom right ) over time . The scale bar in panel B corresponds to 5 μm . Red arrows show the oscillation mode at the respective time point .

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B-D. Examples of Min protein patterns emerging from nearly homogeneous initial conditions in E. coli cells of different sizes. Lateral dimensions (in µm) from top to bottom: 2x6.5, 2x8.8, and 5.2x8.8 respectively. The gray-scale images show cytosolic near-infrared fluorescence emitted by the protein eqFP670 at the first (left) and last (right) time points. The color montages show the sfGFP-MinD intensity (indicated by the color scale at the bottom right) over time. The scale bar in panel B corresponds to 5 μm. Red arrows show the oscillation mode at the respective time point.


E . Two early and two late frames depicting sfGFP - MinD patterns in a cell exhibiting stable transverse oscillations . The images share the scale bar in B .

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E. Two early and two late frames depicting sfGFP-MinD patterns in a cell exhibiting stable transverse oscillations. The images share the scale bar in B.


F . Difference in sfGFP - MinD intensity between the top half and bottom half of the cell plotted against time .

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F. Difference in sfGFP-MinD intensity between the top half and bottom half of the cell plotted against time.


Figure 3 . Computing stability in multistability regimes . A . Two plots that show the non - degeneracy of even and odd modes in an elliptical geometry for varying cell geometry and MinD recruitment rate . The degeneracy ( light blue area ) increases with the MinD recruitment rate .

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Figure 3. Computing stability in multistability regimes. A. Two plots that show the non-degeneracy of even and odd modes in an elliptical geometry for varying cell geometry and MinD recruitment rate. The degeneracy (light blue area) increases with the MinD recruitment rate.


Figure 4 . Basins of attraction predicted from systematic perturbations of patterns with shallow attachment gradients . A . Relative distribution of the final patterns ( indicated on the right ) observed after sampling all alignment angles of the MinD attachment template from 0 to 90 degrees . The MinD recruitment rate was set to a constant value kdD = 0 . 1 . The data shows the increase in the incidence of multistability as the cell size is increased beyond minimal values for cell length and cell width . B . Fractions of the final patterns in cells of 9 - and 10 - µm length after sampling all alignment angles of the MinD attachment template from 0 to 90 degrees . The data shows that increasing the MinD recruitment rate facilitates multistability .

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Figure 4. Basins of attraction predicted from systematic perturbations of patterns with shallow attachment gradients. A. Relative distribution of the final patterns (indicated on the right) observed after sampling all alignment angles of the MinD attachment template from 0 to 90 degrees. The MinD recruitment rate was set to a constant value kdD = 0.1. The data shows the increase in the incidence of multistability as the cell size is increased beyond minimal values for cell length and cell width. B. Fractions of the final patterns in cells of 9- and 10-µm length after sampling all alignment angles of the MinD attachment template from 0 to 90 degrees. The data shows that increasing the MinD recruitment rate facilitates multistability.


Figure 5 . The effect of cell - shape change during growth on the stability of Min protein patterns . A . Cytosolic fluorescence during growth of a cell from a small elliptical form into a large rectangular shape . Numbers in red indicate time in hours . Illustrations show the positions and orientations of the cell in the first and last time frames . Green and blue lines indicate the maximum and minimum Feret diameters , respectively . B . sfGFP - MinD patterns during the growth of the cell shown in A . Illustrations indicate the cell boundaries and oscillation angles observed in the first and last frames ( not to scale ) . C . Quantitative data obtained from the cell shown in A and B . The maximum and minimum Feret diameters ( green and blue ) , and the measured MinD oscillations ( red ) were expressed in terms of length ( top ) and angle ( bottom ) and plotted against time . The number of cells that fit this category was 41 / 97 . Arrows indicate the time when cell width reached the chamber width of 5 μm .

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Figure 5. The effect of cell-shape change during growth on the stability of Min protein patterns. A. Cytosolic fluorescence during growth of a cell from a small elliptical form into a large rectangular shape. Numbers in red indicate time in hours. Illustrations show the positions and orientations of the cell in the first and last time frames. Green and blue lines indicate the maximum and minimum Feret diameters, respectively. B. sfGFP-MinD patterns during the growth of the cell shown in A. Illustrations indicate the cell boundaries and oscillation angles observed in the first and last frames (not to scale). C. Quantitative data obtained from the cell shown in A and B. The maximum and minimum Feret diameters (green and blue), and the measured MinD oscillations (red) were expressed in terms of length (top) and angle (bottom) and plotted against time. The number of cells that fit this category was 41/97. Arrows indicate the time when cell width reached the chamber width of 5 μm.


D - F . Data are presented as in A - C for another cell that showed persistent oscillations along the horizontal axis throughout growth . The number of cells that fit this category was 28 / 97 .

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D-F. Data are presented as in A-C for another cell that showed persistent oscillations along the horizontal axis throughout growth. The number of cells that fit this category was 28/97.


G . Time - lapse images of sfGFP - MinD that reveal stochastic switching of patterns in a cell with an asymmetric shape and a low aspect ratio . White arrows indicate the oscillation axes .

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G. Time-lapse images of sfGFP-MinD that reveal stochastic switching of patterns in a cell with an asymmetric shape and a low aspect ratio. White arrows indicate the oscillation axes.


H . The angles of the maximum and minimum Feret diameters ( green and blue ) , and the measured MinD clusters ( red ) for the cell shown in panel G plotted against time . The number of cells that fit this category was 10 / 97 . All scale bars correspond to 5 μm .

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H. The angles of the maximum and minimum Feret diameters (green and blue), and the measured MinD clusters (red) for the cell shown in panel G plotted against time. The number of cells that fit this category was 10/97. All scale bars correspond to 5 μm.


Figure 6 . Transitions between various modes of Min protein patterns . A . Time - lapse images showing the transition from a longitudinal pole - to - pole mode to the transverse mode . Scale bar , 5 μm . B . Time - lapse images showing the transition from a transverse mode to a longitudinal pole - to - pole mode .

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Figure 6. Transitions between various modes of Min protein patterns. A. Time-lapse images showing the transition from a longitudinal pole-to-pole mode to the transverse mode. Scale bar, 5 μm. B. Time-lapse images showing the transition from a transverse mode to a longitudinal pole-to-pole mode.


C . Bar plot showing the distribution of the number of transitions . Inset : Data from experiments carried out under nutrient - poor conditions in which growth rates are reduced .

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C. Bar plot showing the distribution of the number of transitions. Inset: Data from experiments carried out under nutrient-poor conditions in which growth rates are reduced.


D . Representative time - course of a change in the mode of sfGFP - MinD oscillation . The black line is a sigmoidal fit . The dashed black lines indicate 15 ° and 75 ° and the dashed red line indicates 45 ° .

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D. Representative time-course of a change in the mode of sfGFP-MinD oscillation. The black line is a sigmoidal fit. The dashed black lines indicate 15° and 75° and the dashed red line indicates 45°.


E . Bar plot showing the time scale of the switch in the oscillations . Inset : Data from experiments carried out in nutrient - poor conditions .

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E. Bar plot showing the time scale of the switch in the oscillations. Inset: Data from experiments carried out in nutrient-poor conditions.


F . Bar plots showing the relative numbers of the indicated transitions that occur at different cell lengths . All cells have a width of 5 μm .

F. Bar plots showing the relative numbers of the indicated transitions that occur at different cell lengths. All cells have a width of 5 μm.


G . Distribution of final patterns in cells of the indicated widths as indicates , and lengths of 9 - 10 μm .

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G. Distribution of final patterns in cells of the indicated widths as indicates, and lengths of 9-10 μm.


H . Time - lapse images of various modes of transitions between patterns . Cell sizes from top to bottom are respectively 10x2x1 , 10x6x1 , 9x5x1 , 10x4x1 μm3 . Note that the cells are scaled differently . On the right is an illustration showing Min pattern transitions through intermediate states .

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H. Time-lapse images of various modes of transitions between patterns. Cell sizes from top to bottom are respectively 10x2x1, 10x6x1, 9x5x1, 10x4x1 μm3. Note that the cells are scaled differently. On the right is an illustration showing Min pattern transitions through intermediate states.


( B ) USH1C transcripts were consistently detected in all tested organs of WT and USH1C pigs , except in the heart and cerebellum . RT - PCR for GAPDH was used as control ( Ctrl . ) .

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(B) USH1C transcripts were consistently detected in all tested organs of WT and USH1C pigs, except in the heart and cerebellum. RT-PCR for GAPDH was used as control (Ctrl.).


( D ) Representative Western blot analysis proved lack of harmonin protein expression in retina and duodenum of USH1C pigs at the age of 3 weeks ( 3w ) ( 1 piglet , 1 retina , 1 duodenum ) and 1 year ( 1y ) ( 2 pigs , 1 retina each , 3 TRs ) . Arrow indicates the expected size of harmonin isoform a ( 72kDa ) .

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(D) Representative Western blot analysis proved lack of harmonin protein expression in retina and duodenum of USH1C pigs at the age of 3 weeks (3w) (1 piglet, 1 retina, 1 duodenum) and 1 year (1y) (2 pigs, 1 retina each, 3 TRs). Arrow indicates the expected size of harmonin isoform a (72kDa).


( E ) Representative immunofluorescence staining of harmonin ( green ) of longitudinal cryosection through WT and USH1C pig retinas ( 2 pigs , 1y , 1 retina each , 3 TRs , scale bar 10µm ) demonstrated the absence of harmonin staining in the outer segment ( OS ) of photoreceptor cells and in the other layers below the outer limiting membrane ( OLM , arrow ) of the USH1C pig retina .

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(E) Representative immunofluorescence staining of harmonin (green) of longitudinal cryosection through WT and USH1C pig retinas (2 pigs, 1y, 1 retina each, 3 TRs, scale bar 10µm) demonstrated the absence of harmonin staining in the outer segment (OS) of photoreceptor cells and in the other layers below the outer limiting membrane (OLM, arrow) of the USH1C pig retina.


( F ) Representative Western blot analysis of retinal protein extracts , indicating that the abundance of the USH proteins SANS ( USH1G ) , whirlin ( USH2D ) or myosin7a ( USH1B ) is unaffected ( 1 piglet , 3w , 1 retina each , 2 TRs ) .

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(F) Representative Western blot analysis of retinal protein extracts, indicating that the abundance of the USH proteins SANS (USH1G), whirlin (USH2D) or myosin7a (USH1B) is unaffected (1 piglet, 3w, 1 retina each, 2 TRs).