Figure 6(A) Representative immunofluorescence images of Spindly localization to unaligned KTs in Drosophila S2 cells expressing either PoloWT-EGFP or PoloT182D-EGFP. Insets display magnifications of the outlined regions. Cells were treated with MG132 prior to fixation to increase the number of late prometaphase figures and allow better identification of late congressing KTs. CID was used as a KT reference. (B) Graph represents the percentage of Spindly levels at late congressing and non-oriented (not oriented parallel to the spindle axis) KTs for cells shown in (A). Spindly levels were determined relative to CID and all values were normalized to the mean fluorescence intensity quantified in cells expressing PoloWT-EGFP, which was set to 100% (n≥121 KTs from at least 39 cells for each condition, n=3 independent experiments). Data information: Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm. (C) Selected frames from live imaging analysis of SpindlyWT-EGFP behavior during KT alignment in Drosophila S2 cells expressing either PoloWT-mRFP or PoloT182D-mRFP. Insets show magnifications of the outlined regions that highlight KTs with delayed congression to the spindle equator (yellow dashed line). Time 0 is the first shift in KT direction from poleward to anti-poleward movement. (D) Graph represents the variation in SpindlyWT-EGFP levels at KTs highlighted in (C) and its correlation with efficiency of alignment. Spindly-EGFP mean fluorescence intensity (MFI) levels were corrected for photobleaching during acquisition and normalized to the first frame of analysis to account for differences in expression levels. KTs were tracked during the process of congression (using Polo-mRFP signal as a KT reference) and the distance from the unaligned KT pair to the spindle equator was measured. All values were normalized to the last frame of analysis (stable integration in metaphase plate). Time 0 is the first shift in KT direction from poleward to anti-poleward movement (n≥9 KTs from at least 5 cells for each condition, n≥4 independent experiments). Data information: Statistical analysis was calculated using an unpaired t-test (Mann-Whitney). p values: ****, <0.0001. Data are shown as mean ± SD. Scale bar: 5 μm. (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. (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.