Fig. 7

Self-association of CCDC74A/B is required for stabilizing microtubules and K-fibers. a Immunofluorescence of Flag (red) and α-tubulin (green) in RPE1 cells transfecting with Flag-CCDC74B wild-type or 7N mutant for 48 h (7N: MLAL67-71NNNN and LLL230-234 NNN). Scale bar, 20 μm. b Percentages of cells with bundled microtubules in a and in identically treated HeLa cells (three independent experiments). c Overexpressed empty vector (EV), Flag-CCDC74B wild-type (WT), or 7N mutant into HeLa cells introduced with negative control siRNA (siNC) or CCDC74A/B siRNAs (siCCDC74A/B) for 48 h. The cells were immunostained with α-tubulin (red) and the DNA stained with DAPI. Scale bar, 5 μm. d Statistical analysis of bipolar spindle length in cells from c (three independent experiments). e Graphic of CCDC74A/B functions in bundling microtubules and stabilizing spindle microtubules especially K-fibers. CCDC74A/B bind to and bundle microtubules through both their dual microtubule-binding regions and self-association. CCDC74A/B depletion causes spindle and K-fiber organization defects, thus inducing chromosomal misalignment and cell cycle delay. In b, data are mean ± SEM (unpaired two-tailed Student’s t test, ***P < 0.001). In d, data are mean ± SEM (one-way ANOVA test, ***P < 0.001; n.s., not significant)