Fig. 1.

TACC3 is required for de novo assembly of acentrosomal microtubules in mitosis. (A) Detection of TACC3 RNAi depletion efficiency by Western blotting. HeLa cells were transfected with control and TACC3 siRNAs, respectively. The blots were probed with anti-TACC3 (Upper) and anti–α-tubulin (Lower). (B and C) Representative images of 1 μg/mL nocodazole-arrested normal control or TACC3-knockdown HeLa cells by siRNA followed by release into medium without nocodazole (B) or with 15 ng/mL nocodazole (C) at different time points (0.5, 1.5, 3.5, 7.5, and 15 min). The data are shown as maximum intensity projections of different z sections. TACC3 is in red, tubulin in green, and DNA in blue. (D) Statistics of numbers of acentrosomal microtubule seeds in control and TACC3 knockdown cells after nocodazole treatment and release for 1.5 min. More than 50 cells for each treatment were counted. (E) Control, nocodazole-treated (50 ng/mL for 8 min), and cold-treated (10 min on ice) HeLa cells were stained with anti-TACC3 (green) and anti-Hec1 (red) antibodies. (F) Staining of HeLa cells with TACC3 (green) and indicated proteins (Hec1, Aurora B, and clathrin) (red) after nocodazole treatment for 16 h. (G) HeLa cells were transfected with different siRNAs (control, siTACC3, siAurora A) as indicated. Cells were treated with 50 ng/mL nocodazole for 16 h before fixation. (H and I) Live imaging of HeLa cells expressing GFP–tubulin under indicated different treatments (siRNA control, siTACC3, and Aurora A inhibitor MLN8237). The mitotic cells were arrested with 500 ng/mL nocodazole and released into medium without nocodazole (H) or with 15 ng/mL nocodazole (I). (J) Illustration of TACC3-dependent acentrosomal microtubule assembly and clustering. During mitosis, TACC3 containing small microtubule seeds/asters were assembled around the chromosomes and kinetochores, and these small microtubule asters were further assembled and clustered into a complete bipolar spindle structure. (Scale bar, 10 μm.)