( 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR B-EXP_ASSAY O B-ORGANISM B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O B-ORGANISM B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR 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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O B-ORGANISM B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-SUBCELLULAR 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-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY 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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE B-EXP_ASSAY O O B-SUBCELLULAR O O B-ORGANISM B-CELL_LINE O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-SUBCELLULAR 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 B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O B-ORGANISM B-CELL_LINE O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O O O B-SUBCELLULAR 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 B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O O B-SUBCELLULAR O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR B-EXP_ASSAY O B-ORGANISM B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O B-ORGANISM B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY O B-ORGANISM B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-SUBCELLULAR O B-ORGANISM B-CELL_LINE O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-CELL_LINE O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD O 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-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O O B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-SUBCELLULAR O B-SUBCELLULAR B-EXP_ASSAY O O B-CELL_LINE O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-EXP_ASSAY O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-SUBCELLULAR O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O O 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-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O 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 B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

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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 .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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


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 .

O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O 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-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O 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-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 .

O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O B-GENEPROD O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O 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-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O

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 .

O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O 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. 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 .

O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O

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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O 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. 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 ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O B-TISSUE O B-GENEPROD B-ORGANISM O O O O O O O O O O O B-ORGANISM O O B-TISSUE O O B-TISSUE O O O O O O O O O B-ORGANISM O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O O O O O O

(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 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-GENEPROD B-ORGANISM B-TISSUE O O B-ORGANISM O O O O B-TISSUE O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O B-TISSUE I-TISSUE O B-TISSUE O O B-CELL_TYPE I-CELL_TYPE O O O O O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O O B-GENEPROD B-ORGANISM B-TISSUE O

(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 ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O B-ORGANISM O O O O B-TISSUE O O O O O O

(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).


( A , B ) Upregulation of GFAP in the retina of USH1C pigs at an age of 3 weeks ( 3w ) . ( A ) Representative immunofluorescence staining of GFAP in Müller glia cells which extend throughout almost the entire retina from the OLM ( arrow ) to the ganglion cell layer ( GCL ) of the retina . The consistent increase of GFAP expression in the Müller glia of USH1C pigs indicates Müller cell activation and gliosis . IS , inner segment ; ONL , outer nuclear layer ; OPL , outer plexiform layer ; INL , inner nuclear layer ; IPL , inner plexiform layer ; scale bar : 20 µm . ( B ) Left panel : Western blot analysis of GFAP protein expression in 3w old USH1C piglets and age matched controls . Anti - actin Western blot was used as loading control . Right panel : Quantification of Western blot bands in 4 gels by the LI - COR Odyssey system revealed a strong increase GFAP expression in 3w USH1C piglets when compared to age - matched WT controls ( 2 piglets , 3w , 2 retinas each , 2 TRs , error bars represent standard deviation ( SD ) of the mean , two - tailed Student ' s t tests , * p < 0 . 05 ) .

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(A, B) Upregulation of GFAP in the retina of USH1C pigs at an age of 3 weeks (3w). (A) Representative immunofluorescence staining of GFAP in Müller glia cells which extend throughout almost the entire retina from the OLM (arrow) to the ganglion cell layer (GCL) of the retina. The consistent increase of GFAP expression in the Müller glia of USH1C pigs indicates Müller cell activation and gliosis. IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; scale bar: 20 µm. (B) Left panel: Western blot analysis of GFAP protein expression in 3w old USH1C piglets and age matched controls. Anti-actin Western blot was used as loading control. Right panel: Quantification of Western blot bands in 4 gels by the LI-COR Odyssey system revealed a strong increase GFAP expression in 3w USH1C piglets when compared to age-matched WT controls (2 piglets, 3w, 2 retinas each, 2 TRs, error bars represent standard deviation (SD) of the mean, two-tailed Student's t tests, *p<0.05).


( C , D ) Reduced synaptic width in USH1C pigs at an age of 1 year ( 1y ) . ( C ) Fluorescent microscopic analysis of the cone synapse phenotype . Left panel : Representative images of longitudinal sections through WT and USH1C pig retinae stained for the pre - synaptic marker PSD - 95 ( green ) and by fluorescent peanut agglutinin ( PNA , red ) for cone synaptic pedicles ( white arrows ) and counter - stained by DAPI for nuclear DNA ( blue ) . Scale bar 10µm . Middle panel : Higher magnification of a PNA - stained cone synaptic pedicle . Synapse width was determined as the maximum extension of consistent PNA signals . Scale bar 1µm . Right panel : Measuring cone synaptic pedicle width in WT and USH1C pigs by applying a Fiji script to PNA - stained sections indicated reduced synaptic width ( 2 pigs , 1y , 1 retina each , number of examined synapses indicated , error bars represent SD , two - tailed Student ' s t tests , * * * p < 0 . 001 ) . ( D ) Determining cone synaptic pedicles width by TEM . Left panel : Representative images of retinal cross sections . Scale bar 500nm . Right panel : Quantification of synaptic width confirmed the significantly reduced width of cone synaptic pedicles in USH1C pigs . ( 1 pig , 1y , 1 retina each , number of examined synapses indicated ,

O O O O O O B-SUBCELLULAR O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE B-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD B-ORGANISM B-TISSUE B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD B-ORGANISM O O O O O O B-GENEPROD O B-EXP_ASSAY O O O B-SUBCELLULAR O O O B-ORGANISM O O O O B-TISSUE O O O O O B-SUBCELLULAR O O O O O O 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-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O B-TISSUE O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD B-ORGANISM O O O B-ORGANISM O O O O B-TISSUE O O O O O B-SUBCELLULAR O O

(C, D) Reduced synaptic width in USH1C pigs at an age of 1 year (1y). (C) Fluorescent microscopic analysis of the cone synapse phenotype. Left panel: Representative images of longitudinal sections through WT and USH1C pig retinae stained for the pre-synaptic marker PSD-95 (green) and by fluorescent peanut agglutinin (PNA, red) for cone synaptic pedicles (white arrows) and counter-stained by DAPI for nuclear DNA (blue). Scale bar 10µm. Middle panel: Higher magnification of a PNA-stained cone synaptic pedicle. Synapse width was determined as the maximum extension of consistent PNA signals. Scale bar 1µm. Right panel: Measuring cone synaptic pedicle width in WT and USH1C pigs by applying a Fiji script to PNA-stained sections indicated reduced synaptic width (2 pigs, 1y, 1 retina each, number of examined synapses indicated, error bars represent SD, two-tailed Student's t tests, ***p<0.001). (D) Determining cone synaptic pedicles width by TEM. Left panel: Representative images of retinal cross sections. Scale bar 500nm. Right panel: Quantification of synaptic width confirmed the significantly reduced width of cone synaptic pedicles in USH1C pigs. (1 pig, 1y, 1 retina each, number of examined synapses indicated,


( A - C ) Ciliary length measured by fluorescence microscopic analysis at 1 year of age ( 1y ) . ( A ) Representative immunostaining of the ciliary marker Gt335 ( green ) in longitudinal cryosections of PRC ciliary regions in WT and USH1C retinas . Counterstaining by DAPI , scale bar 10µm . ( B ) Gt335 staining at higher magnification and principle of quantifying ciliary length by Gt335 staining . Scale bar 500nm . ( C ) Length of connecting cilia ( CC ) appear elongated in USH1C when applying a Fiji script to immunostained histological sections ( 2 pigs , 1y , 1 retina each , number of examined connecting cilia is indicated , error bars represent SD , two - tailed Student ' s t tests , * * * * p < 0 . 0001 ) .

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(A-C) Ciliary length measured by fluorescence microscopic analysis at 1 year of age (1y). (A) Representative immunostaining of the ciliary marker Gt335 (green) in longitudinal cryosections of PRC ciliary regions in WT and USH1C retinas. Counterstaining by DAPI, scale bar 10µm. (B) Gt335 staining at higher magnification and principle of quantifying ciliary length by Gt335 staining. Scale bar 500nm. (C) Length of connecting cilia (CC) appear elongated in USH1C when applying a Fiji script to immunostained histological sections (2 pigs, 1y, 1 retina each, number of examined connecting cilia is indicated, error bars represent SD, two-tailed Student's t tests, ****p<0.0001).


( D , E ) Measuring CC length in TEM sections . ( D ) Representative images of longitudinal cross sections and principle of determining CC length . Scale bar 500nm . ( E ) Elongation of CC length is confirmed in TEM sections of USH1C PRC . ( 1 pig , 1y , 1 retina each , number of examined connecting cilia indicated , error bars represent SD , two - tailed Student ' s t tests , * * * * p < 0 . 0001 ) .

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(D, E) Measuring CC length in TEM sections. (D) Representative images of longitudinal cross sections and principle of determining CC length. Scale bar 500nm. (E) Elongation of CC length is confirmed in TEM sections of USH1C PRC. (1 pig, 1y, 1 retina each, number of examined connecting cilia indicated, error bars represent SD, two-tailed Student's t tests, ****p<0.0001).


Length of primary cilia in dermal fibroblasts . ( F ) Staining of ciliary structures in fibroblasts stained with ARL13B ( ciliary shaft , green ) and PCNT2 ( ciliary base , red ) , DAPI ( nucleus , blue ) . Scale bar upper panel 5 µm , lower panel 2 . 5 µm . ( H ) Similarly , ciliary length of fibroblasts from a human USH1CR31X / R80fs patient are elongated , compared to a healthy control ( 7 TR , number of cells examined indicated , KS - test , * * * * p < 0 . 0001 ) .

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Length of primary cilia in dermal fibroblasts. (F) Staining of ciliary structures in fibroblasts stained with ARL13B (ciliary shaft, green) and PCNT2 (ciliary base, red), DAPI (nucleus, blue). Scale bar upper panel 5 µm, lower panel 2.5 µm. (H) Similarly, ciliary length of fibroblasts from a human USH1CR31X/R80fs patient are elongated, compared to a healthy control (7 TR, number of cells examined indicated, KS-test, ****p<0.0001).


( I - M ) Outer segment ( OS ) PRC architecture by TEM of longitudinal retinal sections . ( I ) At 3w , WT show normal sub - cellular organization , including parallel stacking of photosensitive discs and calyceal processes ( black arrows , representative image from 2 pigs , 2 retinas , scale bar 600nm ) . ( J ) In 3w USH1C rods , membrane discs appear also in vertical orientation ( white arrows , representative image from 2 pigs , 2 retinas , scale bar 750nm ) . At 1y , interstitial gaps appeared in disc stacks of the OS in USH1C pigs ( K , arrow , scale bar 550nm ) and vesicles are found at the OS base ( L , asterisk , scale bar 500nm ) . ( M ) The disc architecture in 1y animals was substantially distorted ( arrows , representative image from 2 pigs , 2 retinas , scale bar 850nm ) .

O O O O O B-TISSUE I-TISSUE O B-TISSUE O B-CELL_TYPE O O B-EXP_ASSAY O O B-TISSUE O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-ORGANISM O O B-TISSUE O O O O O O O O O O O B-GENEPROD B-CELL_TYPE O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O B-ORGANISM O O B-TISSUE O O O O O O O O O O O O O B-SUBCELLULAR O O O B-TISSUE O B-GENEPROD B-ORGANISM O O O O O O O O O O B-SUBCELLULAR O O O O B-TISSUE O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-ORGANISM O O B-TISSUE O O O O O O

(I-M) Outer segment (OS) PRC architecture by TEM of longitudinal retinal sections. (I) At 3w, WT show normal sub-cellular organization, including parallel stacking of photosensitive discs and calyceal processes (black arrows, representative image from 2 pigs, 2 retinas, scale bar 600nm). (J) In 3w USH1C rods, membrane discs appear also in vertical orientation (white arrows, representative image from 2 pigs, 2 retinas, scale bar 750nm). At 1y, interstitial gaps appeared in disc stacks of the OS in USH1C pigs (K, arrow, scale bar 550nm) and vesicles are found at the OS base (L, asterisk, scale bar 500nm). (M) The disc architecture in 1y animals was substantially distorted (arrows, representative image from 2 pigs, 2 retinas, scale bar 850nm).


( A ) AAV transduction of retinal layers . AAV8 , AAV9 and Anc80 encoding eGFP under the ubiquitous CMV promoter were injected subretinally into the eyes of mature WT pigs in vivo . Upper Panel : Epifluorescence of dissected eyes demonstrates eGFP fluorescence in the bleb region of whole mount preparations of all three AAV samples . Dashed circles indicate bleb size . Lower panel : Epifluorescence images of longitudinal retina sections revealed eGFP expression in the PRC layer ( OS , IS and ONL ) for all AAV . Increased exposure ( 4x ) documented transduction of MGC ( arrows ) by AAV9 and Anc80 but not by AAV8 ( representative images from 1 pig , 1 retina each , 2 TR , scale bars 25 µm ) .

O O O B-ORGANISM O O B-TISSUE O O B-ORGANISM O B-ORGANISM O B-ORGANISM O B-GENEPROD O O O B-ORGANISM O O O O O O B-TISSUE O O O B-ORGANISM O O O O O O B-EXP_ASSAY O O B-TISSUE O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O B-ORGANISM O O O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY O O O B-TISSUE O O B-GENEPROD O O O B-CELL_TYPE O O B-TISSUE O B-TISSUE O B-TISSUE O O O B-ORGANISM O O O O O O O O O B-CELL_TYPE O O O O B-ORGANISM O B-ORGANISM O O O B-ORGANISM O O O O O B-ORGANISM O O B-TISSUE O O O O O O O O O O O

(A) AAV transduction of retinal layers. AAV8, AAV9 and Anc80 encoding eGFP under the ubiquitous CMV promoter were injected subretinally into the eyes of mature WT pigs in vivo. Upper Panel: Epifluorescence of dissected eyes demonstrates eGFP fluorescence in the bleb region of whole mount preparations of all three AAV samples. Dashed circles indicate bleb size. Lower panel: Epifluorescence images of longitudinal retina sections revealed eGFP expression in the PRC layer (OS, IS and ONL) for all AAV. Increased exposure (4x) documented transduction of MGC (arrows) by AAV9 and Anc80 but not by AAV8 (representative images from 1 pig, 1 retina each, 2 TR, scale bars 25 µm).


( D ) Efficacy of AAV gene therapy was examined in vivo by sub - retinal injection of Anc80 virus capsid expressing harmonin _ a1 under a ubiquitous promoter into a USH1C pig ( see also Figure EV5 ) . Left panel : Sub - retinal injection reconstituted harmonin expression in the AAV - treated eye but not in the sham - treated eye ( 3 TR ) . Protein extract from a WT retina served as positive control , actin staining as loading control . The 72kD band correlates with harmonin _ a isoforms while higher MW indicates putative dimer formation . Middle panel : Immunofluorescence reveals absence of harmonin in the retina of the PBS - injected control eye and harmonin abundance ( green ) in the retina of the AAV - treated eye . ( Representative image from a single animal . Blue : DAPI , scale bar 25 µm ) . Right panel : Photopic gfERG reveal increased a - and b - wave response to single flash and flicker stimulation in the AAV - treated ( red ) , compared to the sham - treated ( black ) eyes of two USH1C pigs .

O O O O O B-ORGANISM O O O O O O O O O B-TISSUE O O B-ORGANISM B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O O B-TISSUE O O B-GENEPROD O O O B-ORGANISM O O B-TISSUE O O O O O O O B-TISSUE O O O O O O O O O O B-TISSUE O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-TISSUE O O B-SMALL_MOLECULE O O O B-TISSUE O B-GENEPROD O O O O O O B-TISSUE O O B-ORGANISM O O B-TISSUE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O 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-ORGANISM O O O O O O O O O O O O O O O B-TISSUE O O B-GENEPROD B-ORGANISM O

(D) Efficacy of AAV gene therapy was examined in vivo by sub-retinal injection of Anc80 virus capsid expressing harmonin_a1 under a ubiquitous promoter into a USH1C pig (see also Figure EV5). Left panel: Sub-retinal injection reconstituted harmonin expression in the AAV-treated eye but not in the sham-treated eye (3 TR). Protein extract from a WT retina served as positive control, actin staining as loading control. The 72kD band correlates with harmonin_a isoforms while higher MW indicates putative dimer formation. Middle panel: Immunofluorescence reveals absence of harmonin in the retina of the PBS-injected control eye and harmonin abundance (green) in the retina of the AAV-treated eye. (Representative image from a single animal. Blue: DAPI, scale bar 25 µm). Right panel: Photopic gfERG reveal increased a- and b-wave response to single flash and flicker stimulation in the AAV-treated (red), compared to the sham-treated (black) eyes of two USH1C pigs.


A . Pull - down assays of yTBP ( 61 - 240 ) with Kap95p , Kap114p and Kap121p . GST - fused yTBP ( 61 - 240 ) was incubated with recombinant Kap95p , Kap114p and Kap121p . Unbound ( S ) and bound ( B ) samples were analyzed by SDS - PAGE and stained with Coomassie blue . B . Band intensities of Kap - βs from the SDS - PAGE gels in ( A ) were quantified and normalized by GST - yTBP intensity . Quantitative plots show percent Kap - βs binding on yTBP .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O

A. Pull-down assays of yTBP (61-240) with Kap95p, Kap114p and Kap121p. GST-fused yTBP (61-240) was incubated with recombinant Kap95p, Kap114p and Kap121p. Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. B. Band intensities of Kap -βs from the SDS-PAGE gels in (A) were quantified and normalized by GST-yTBP intensity. Quantitative plots show percent Kap-βs binding on yTBP.


C - E . ITC titration curves ( upper ) and binding isotherms ( lower ) of ( C ) Kap114p , ( D ) Kap95p , and ( E ) Kap121p with yTBP ( 61 - 240 ) . Salt concentration and Kd values are indicated .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O

C-E. ITC titration curves (upper) and binding isotherms (lower) of (C) Kap114p, (D) Kap95p, and (E) Kap121p with yTBP (61-240). Salt concentration and Kd values are indicated.


A , B . Purified recombinant Kap114p was mixed with RanQ69L or yTBP and analyzed by SEC . SEC ( Superdex 200 ) elution profiles of the ( A ) Kap114p  RanQ69L and ( B ) Kap114p  yTBP complexes . Peak fractions were analyzed by SDS - PAGE and stained with Coomassie blue . The void volume ( Vo ) of the peak fraction and absorbance ( a . u . ) at 280 nm for each complex is indicated .

O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O

A, B. Purified recombinant Kap114p was mixed with RanQ69L or yTBP and analyzed by SEC. SEC (Superdex 200) elution profiles of the (A) Kap114pRanQ69L and (B) Kap114pyTBP complexes. Peak fractions were analyzed by SDS-PAGE and stained with Coomassie blue. The void volume (Vo) of the peak fraction and absorbance (a.u.) at 280 nm for each complex is indicated.


SV - AUC analysis of ( C ) yTBP The experimental data were analyzed using the Sedfit program and are presented above molecular weights calculated using standards ( in brackets ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

SV-AUC analysis of (C) yTBP The experimental data were analyzed using the Sedfit program and are presented above molecular weights calculated using standards (in brackets).


SV - AUC ( D ) Kap114p and ( E ) the Kap114p  yTBP complex . The experimental data were analyzed using the Sedfit program and are presented above molecular weights calculated using standards ( in brackets ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

SV-AUC (D) Kap114p and (E) the Kap114pyTBP complex. The experimental data were analyzed using the Sedfit program and are presented above molecular weights calculated using standards (in brackets).


F . SE - AUC analysis of the Kap114p  yTBP complex . The average molecular mass determined by the Sedfit program was ~ 133 . 5 kDa . The lower panel shows the residual difference ( standard deviation ) between experimental and fitted values .

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F. SE-AUC analysis of the Kap114pyTBP complex. The average molecular mass determined by the Sedfit program was ~133.5  kDa. The lower panel shows the residual difference (standard deviation) between experimental and fitted values.


Plots of the distance distribution function P ( r ) for the ( G ) Kap114p  RanQ69L complexes derived from experimental SAXS data using the GNOM program . Rg - based dimensionless Kratky plot of the Kap114p  RanQ69L ( H ) complexes . Intersection of the two dotted gray lines indicates features of a compact protein .

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Plots of the distance distribution function P(r) for the (G) Kap114pRanQ69L complexes derived from experimental SAXS data using the GNOM program. Rg-based dimensionless Kratky plot of the Kap114pRanQ69L (H) complexes. Intersection of the two dotted gray lines indicates features of a compact protein.


The ab initio envelopes determined by GASBOR for the ( I ) Kap114p  RanQ69L complexes . Models of the ( J ) Kap114p  RanQ69L complexes derived from SAXS profiles using FoXSDock and docked into the ab initio envelopes obtained from GASBOR .

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The ab initio envelopes determined by GASBOR for the (I) Kap114pRanQ69L complexes. Models of the (J) Kap114pRanQ69L complexes derived from SAXS profiles using FoXSDock and docked into the ab initio envelopes obtained from GASBOR.


Plots of the distance distribution function P ( r ) for the ( K ) Kap114p  yTBP complexes derived from experimental SAXS data using the GNOM program . Rg - based dimensionless Kratky plot of the Kap114p  yTBP ( L ) complexes . Intersection of the two dotted gray lines indicates features of a compact protein .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

Plots of the distance distribution function P(r) for the (K) Kap114pyTBP complexes derived from experimental SAXS data using the GNOM program. Rg-based dimensionless Kratky plot of the Kap114pyTBP (L) complexes. Intersection of the two dotted gray lines indicates features of a compact protein.


B . Pull - down assays of yTBP with Kap114p , Kap114p ( Δ347 - 371 ) and Kap114p ( Δ899 - 956 ) . GST - fused yTBP ( 61 - 240 ) was incubated with recombinant Kap114p , Kap114p ( Δ347 - 371 ) and Kap114p ( Δ899 - 956 ) . Unbound ( S ) and bound ( B ) samples were analyzed by SDS - PAGE and stained with Coomassie blue . C . Band intensities of Kap - βs from the SDS - PAGE gels in ( B ) were quantified and normalized by GST - yTBP intensity . Quantitative plots show percent Kap - βs binding on yTBP .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O

B. Pull-down assays of yTBP with Kap114p, Kap114p (Δ347-371) and Kap114p (Δ899-956). GST-fused yTBP (61-240) was incubated with recombinant Kap114p, Kap114p (Δ347-371) and Kap114p (Δ899-956). Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. C. Band intensities of Kap-βs from the SDS-PAGE gels in (B) were quantified and normalized by GST-yTBP intensity. Quantitative plots show percent Kap-βs binding on yTBP.


D , E . ITC titration curves ( upper ) and binding isotherms ( lower ) of ( D ) Kap114p ( Δ347 - 371 ) and ( E ) Kap114p ( Δ899 - 956 ) with yTBP ( 61 - 240 ) . Salt concentration and Kd values are indicated .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O

D, E. ITC titration curves (upper) and binding isotherms (lower) of (D) Kap114p (Δ347-371) and (E) Kap114p (Δ899-956) with yTBP (61-240). Salt concentration and Kd values are indicated.


F . Relative fluorescence GFP intensities in the nucleus and cytoplasm of wild type , KAP114 knockout strains , and KAP114 knockout strains rescued by KAP114 deletion mutants .

O O O B-EXP_ASSAY B-GENEPROD B-EXP_ASSAY O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O

F. Relative fluorescence GFP intensities in the nucleus and cytoplasm of wild type, KAP114 knockout strains, and KAP114 knockout strains rescued by KAP114 deletion mutants.


G . His - tagged RanQ69L was incubated with recombinant Kap114p , Kap114p ( Δ347 - 371 ) and Kap114p ( Δ899 - 956 ) . Unbound ( S ) and bound ( B ) samples were analyzed by SDS - PAGE and stained with Coomassie blue . H . Band intensities of Kap - βs from the SDS - PAGE gels in ( G ) were quantified and normalized by His - Ran intensity . Quantitative plots show percent Kap - βs binding on His - Ran .

O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O

G. His-tagged RanQ69L was incubated with recombinant Kap114p, Kap114p (Δ347-371) and Kap114p (Δ899-956). Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. H. Band intensities of Kap-βs from the SDS-PAGE gels in (G) were quantified and normalized by His-Ran intensity. Quantitative plots show percent Kap-βs binding on His-Ran.


I . GST pull - down assays of yTBP ( 61 - 240 ) with Kap114p or Kap114p ( Δ347 - 371 ) in the presence or absence of RanQ69L . GST - fused yTBP ( 61 - 240 ) was incubated with recombinant Kap114p and His - tagged RanQ69L at a molar ratio of 1 : 4 : 8 Kap114p : yTBP : RanQ69L . Unbound ( S ) and bound ( B ) samples were analyzed by SDS - PAGE and stained with Coomassie blue . J . Analysis of Kap114p dissociation from yTBP in the presence of RanQ69L . Band intensities of Kap114p and Kap114p ( Δ347 - 371 ) from the SDS - PAGE gels were used to determine the average fraction of bound protein .

O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O O O O O

I. GST pull-down assays of yTBP (61-240) with Kap114p or Kap114p (Δ347-371) in the presence or absence of RanQ69L. GST-fused yTBP (61-240) was incubated with recombinant Kap114p and His-tagged RanQ69L at a molar ratio of 1:4:8 Kap114p:yTBP:RanQ69L. Unbound (S) and bound (B) samples were analyzed by SDS-PAGE and stained with Coomassie blue. J. Analysis of Kap114p dissociation from yTBP in the presence of RanQ69L. Band intensities of Kap114p and Kap114p (Δ347-371) from the SDS-PAGE gels were used to determine the average fraction of bound protein.


B . EMSA analysis of yTBP binding to DNA . We incubated 5 nM FAM - labeled oligonucleotide ( TGTATGTATATAAAAC ) with indicated concentrations of purified yTBP ( 0 . 125 to 8 μM ) and then analyzed binding by 6 % non - denaturing PAGE . Band intensities of free DNA from the gels ( Top panel ) were quantified and plotted ( Bottom panel ) .

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B. EMSA analysis of yTBP binding to DNA. We incubated 5 nM FAM-labeled oligonucleotide (TGTATGTATATAAAAC) with indicated concentrations of purified yTBP (0.125 to 8 μM) and then analyzed binding by 6% non-denaturing PAGE. Band intensities of free DNA from the gels (Top panel) were quantified and plotted (Bottom panel).