(A,B) Kymograph analysis of mitotic progression monitored by live-cell imaging (A) and corresponding quantification of Mad1-EGFP kinetochore levels (B) of control, PP1-87B depleted or Mps1 and PP1-87B co-depleted Drosophila S2 cells expressing mCherry-Tubulin and Mad1-EGFP under control of Mad1 promoter. The mean time from nuclear envelope breakdown to anaphase onset for each condition is displayed in the corresponding kymograph. Mad1-EGFP fluorescence intensities at kinetochores were corrected for cytosolic signal (N ≥ 9 cells for each condition). Vertical dashed line indicates the frame corresponding to nuclear envelope breakdown. Horizontal scale bar: 5 min. Vertical scale bar: 5 μm. (C,D) Representative immunofluorescence images (C) and corresponding quantifications (D) of inter-kinetochore distances and relative levels of Aurora B T232 phosphorylation (AurBT232Ph) at unaligned and aligned chromosomes in control and PP1-87B depleted S2 cells. AurBT232Ph relative levels were plotted over the inter-kinetochore distance measured as the distance between centroids of CID pairs. AurBT232Ph fluorescence intensities were determined relative to CID signal (N ≥ 58 kinetochore pairs for each condition). Scale bar: 5 μm. (E) Quantification of inter-kinetochore distances measured in chromosomes aligned at the metaphase plate of control and PP1-87B depleted S2 cells. Inter-kinetochore distances were measured as the distance between centroids of identified CID pairs (N ≥ 86 kinetochores for each condition). (F,G) Representative immunofluorescence images (F) and corresponding quantification (G) of cold-stable kinetochore fibers in control, PP1-87B and Ndc80 depleted S2 cells. CID immunolocalization was used as kinetochore reference. The insets display magnifications of the outlined regions. The graph represents the % of kinetochores attached to cold-stable microtubules per cell (N ≥ 10 cells for each condition). (H,I) Analysis of microtubules turnover rates by speckle contrast fadeout of GFP-α-Tubulin. (H) Contrast fadeout–time curves of GFP-α-Tubulin fluorescent speckles (lines) and their time point means (dots) measured in rectangular areas enclosing the spindle of control and PP1-87B-depleted S2 cells in prometaphase and metaphase. The rate of speckle contrast fadeout was calculated to obtain microtubule turnover rates. (I) Table showing microtubules half-lives of non-kinetochore- (non-KT-MTs) and kinetochore-microtubules (KT-MTs) of control and PP1-87B-depleted S2 cells determined by inducible speckle imaging in prometaphase and metaphase. The average speckle intensity squared-contrast at each time point was fit to a double-exponential curve A1*exp(-k1*t)+A2*exp(-k2*t), in which t is time, A1 represent the less stable population (non-KT-MTs) and A2 the more stable population (KT-MTs) with decay rates of k1 and k2, respectively. The turnover half-life for each population was calculated as ln2/k (N ≥ 7 metaphase cells for each condition). Data information: in (A), (B), (E), (G) and (H) data are presented as mean ± SD. Numerical source data for this figure are provided in Figure 1—source data 1.