Cytokinesis failure caused by the KIF23 c.2747C>G (MKLP1 p.P916R) mutation. Efficacy of depletion of endogenous MKLP1 (A) and equal expression of wild-type (WT) and P916R GFP-MKLP1 (B) constructs was confirmed by western blotting of the whole cell lysates with anti-MKLP1 (A) or anti-GFP (B) antibody. Tubulin was also blotted as a loading control. (C) Quantitation of cytokinesis failures from live cell recordings of HeLa cells treated with MKLP1 siRNA and transfected with GFP vector or the MKLP1 variants as indicated. The graph shows the percentage of cells that failed cytokinesis averaged from 3 independent experiments in which at least 80 GFP-MKLP1 or GFP-expressing cells were analyzed (supplemental Table 4). *P < .001 after Turkey correction for multiple comparisons. Error bars indicate the standard deviation. (D-E) Stills from the live recordings (A-C) by differential interference contrast (DIC) and GFP fluorescence microscopy. Number indicates the time after midbody formation. GFP-MKLP1 P916R mutant (E) was localized to the spindle midzone (−7 min, arrow) and condensed to form the midbody (0 and 102 min, arrowheads) in a similar manner to the WT GFP-MKLP1 (D). However, the cell membrane of the intercellular bridge was detached from the midbody 217 min after its formation, creating a binucleate cell (245 min). Note that in the WT GFP-MKLP1 cell, abscission happened at 252 min and the 2 daughter cells were separated (D) at 322 min, double arrow. The remnant of the midbody (arrowhead) was incorporated to the cell on the right. Bar, 10 µm. Yellow arrowheads and dotted circles indicate segregating chromosomes and reformed nuclei, respectively.