Centralspindlin (Cyk4/Mklp1) and Ect2 are required for NuMA/p150^Glued exclusion from the equatorial membrane. (A) Immunofluorescence (IF) analysis of HeLa cells in anaphase. Cells were fixed and costained using anti-RhoA (green) and anti-NuMA (red) antibodies, as indicated. In this and other IF-analysis panels, DNA is shown in blue unless specified. More than 30 cells were visually analyzed from three independent experiments, and the representative cell is shown here. Scale bar in this and for the following panels represent 10 μm. (B and C) Schematic representation of line scan analysis (B) and the outcome (C) of such analysis for RhoA and NuMA normalized intensity (in arbitrary unit [au]) for the specified membrane region (in orange). (n = 5 cells were used for this quantification as depicted; shaded region indicates SEM). (D) Schematic representation of an evolutionarily conserved pathway required for RhoA activation in metazoans. (E–I) IF analysis of HeLa cells that are either transfected with control siRNA (E) or siRNA-against Cyk4 (F), Mklp1 (G), Ect2 (H), or Anillin (I). Cells were fixed after 36 h of siRNA transfection and stained with anti-p150^Glued antibodies (green). Multinucleation percentage (%) is shown for each siRNA condition (n > 500 cells each from three independent experiments). Knockdown efficiency was also determined by immunoblot analysis (see Fig. S1, A–D). (J and K) Schematic representation of line scan analysis (J) and quantification method (K) used for analyzing equatorial membrane enrichment of NuMA fluorescence intensity. bkgd., eq. mem., and cyt. represent background, equatorial membrane, and cytoplasmic intensity, respectively. (L–P) IF analysis of HeLa cells transfected with control siRNA (L) or siRNA against- Cyk4 (M), Mklp1 (N), Ect2 (O), or Anillin (P). Cells were fixed after 36 h of siRNA transfection and thereafter costained with anti-NuMA (green) and anti-p150^Glued (red) antibodies. The percentage on the figure panels represents the fraction of anaphase cells that show equatorial NuMA enrichment for each condition as analyzed by visual quantification (n > 100 cells from three independent experiments). Yellow arrowheads depict NuMA localization at the equatorial membrane. Line scan analysis on the right represents NuMA fluorescence intensity as indicated in J. Red asterisk represents intensity at the equatorial membrane. (Q) Quantification of equatorial membrane NuMA intensity in cells transfected with control siRNA or siRNA against- Cyk4, Mklp1, Ect2, or Anillin as indicated in Figure panel K (n = 25 cells for all conditions, as listed; error bars: SD). ns, P > 0.05; ***, P < 0.001 as determined by two-tailed unpaired Student’s t test. (R and S) Confocal live-imaging analysis of HeLa Kyoto cells stably coexpressing AcGFP-NuMA (green) and mCherry-H2B (magenta) that are transfected with control siRNA (R) or siRNA against Ect2 (S). The recording was initiated 26 h post-transfection for control and Ect2 siRNA. “0 min” time-point represents metaphase to anaphase transition. Yellow arrowheads depict NuMA localization at the equatorial membrane. (T) Quantification of equatorial membrane AcGFP-NuMA intensity as described in K. ns, P > 0.05; **, P < 0.01; ***, P < 0.001 as determined by two-tailed unpaired Student’s t test. n = 10 cells for control siRNA, and n = 15 for Ect2 siRNA transfected cells; error bars: SD.