Equatorial localization of NuMA-based complexes impacts proper chromosomes separation during anaphase. (A and B) Schematic representation for the calculation of the distance (d [μm]) between inter chromatids in cells undergoing metaphase to anaphase transition at various time intervals (A). Quantification of inter chromatids distance in HeLa Kyoto cells that are stably coexpressing AcGFP-NuMA and mCherry-H2B and are transfected with either control siRNA, siRNA-against Ect2, or siRNA-against Cyk4 (B). Note that Ect2 or Cyk4 siRNA transfected cells that show no cleavage furrow, and thus 100% penetrance of the siRNA were analyzed for quantification of inter chromatids distance. ns, P > 0.05; **, P < 0.01; ***, P < 0.001 as determined by two-tailed unpaired Student’s t test. n = 10 cells from two independent experiments as depicted in the figure panel; error bars: SD shown by shaded region-pink: control; green: Ect2 depletion; red: Cyk4 depletion. (C–E) Confocal live-imaging analysis of HeLa Kyoto cells expressing AcGFP-Ect2^r (C), or AcGFP-Ect2^r_Δmem (D), with mCherry-H2B, and the quantification of inter chromatids distance in these cells (E). In these cells, endogenous Ect2 was depleted by siRNA targeting endogenous copy of the mRNA, but not ectopically expressed transgene. The recording was initiated 26 h post-transfection of Ect2 siRNA. “0 min” time-point represents metaphase to anaphase transition. ns, P > 0.05; *, P < 0.05; **, P < 0.01 as determined by two-tailed unpaired Student’s t test. n = 10 cells as depicted in the figure panel; error bars: SD represented by the shaded region-pink: cells coexpressing AcGFP-Ect2^r and mCherry-H2B; green: cells coexpressing AcGFP-Ect2^r_Δmem and mCherry-H2B.