Dido3 localizes at centrosomes/spindle poles. (A) Dido3 has a distinct two-dot localization pattern in HeLa cells, reminiscent of centrosomes (interphase). (B) During mitosis, Dido3 translocates to mitotic poles in early metaphase and remains pole-associated throughout mitosis. (Upper) Dido3 localization early in mitosis. (Middle and Bottom) Mid- and late mitosis, respectively. (C) GFP-Dido1 is seen mainly in cytoplasm of HeLa cells (Upper), whereas GFP-Dido2 is nuclear (Lower). These isoforms did not associate with centrosomes or spindle poles.

Endogenous human Dido3 colocalizes with γ-tubulin. (A) HeLa cells expressing GFP-Dido3 (green), stained with anti-Dido3 (red), showed overlapping localization. (B) In untransfected HeLa cells, anti-Dido3 (red) revealed the same pattern for endogenous human Dido3 as for GFP-Dido3. (C) Staining of HeLa cells with anti-Dido3 (green) and anti-γ-tubulin (red) confirmed Dido3 centrosome localization. Confocal microscopy images of Dido3 (green) and γ-tubulin (red) localization indicated possible differences in the amount of Dido3 associated with each centrosome when centrosomes were in proximity (Middle and Bottom).

Targeted Dido gene disruption results in an N-terminal-truncated Dido3 form that loses centrosome association. (Ai) Dido gene architecture. A common N-terminal domain (blue) is followed by a specific C-terminal domain for each of the three splice variants (pink, green, yellow, and red). The domain targeted in mutant mice is indicated. (Aii) Dido3ΔNT is the truncated Dido3 form in mutant cells after disrupting the targeted region of the Dido gene, as in Ai. (Aiii) Western blot of WT and Dido-targeted (mutant) MEF lysates stained with anti-Dido3. The lower band is the truncated form of Dido3. (B) MEFs were stained with anti-Dido3 (red) and DAPI (blue). (Bi and Bii) In interphase (Bi) and mitotic (Bii) WT cells, Dido3 clearly associates with centrosomes/spindle poles. (Biii) In interphase mutant cells, the Dido3-truncated form shows a diffuse nuclear localization without the two-dot centrosome pattern. (Biv) In mitotic mutant cells, there is no spindle pole association and chromosomes appear to be misaligned (arrows).

Targeted Dido disruption leads to centrosome amplification and spindle malformation. (Ai) Anti-γ-tubulin staining revealed mutant MEFs with 4, 8, or 12 centrosomes. (Aii) Anti-centrin staining confirmed centrosome amplification in mutant cells (Lower), whereas WT MEFs showed normal numbers (Upper). (Aiii) MEFs were stained with anti-α-tubulin (red) to show spindle formation. DNA was stained with SYBR-Green. WT MEFs (Left) had normal spindle formation. Mutants formed a spindle with four poles (Center) or a completely disorganized spindle (Right). (Bi) We counted centrosomes in anti-γ-tubulin-stained WT and mutant MEFs. The x axis shows centrosomes per cell. Approximately 23% of Dido-mutant cells had three or more centrosomes, whereas only 3.9% of WT cells showed this abnormal number. (Bii) Percentage of WT and mutant MEFs showing ≤2 or >2 centrosomes per cell after hydroxyurea treatment. The percentage of mutant cells with aberrant centrosome numbers increased with time, reaching >40% of total cells at 48 h. The x axis depicts time of hydroxyurea treatment. (Biii) Freshly isolated BM samples stained with anti-γ-tubulin. A significantly larger percentage of cells from mutant mice had more than two centrosomes compared with cells from WT mice (Student's t test, P = 0.008).

Dido disruption leads to a dysfunctional SAC and enhanced chromosome instability. (A) WT and mutant cells were presynchronized with aphidicolin and released into nocodazole (Upper) or taxol (Lower) to trigger mitotic arrest. Cells were fixed, propidium iodide-stained, and analyzed by FACS. The majority of WT cells were arrested as a 4N population, whereas mutant cells failed to maintain arrest for longer than 16–18 h, giving rise to an additional 2N population. The figure is representative of at least four independent experiments of drug treatment for 16–18 h. (Bi) WT and mutant cells, taxol-treated as above for the times indicated, were tested in Western blot with equal amounts of total cell lysates by using anti-cyclin B1 (Upper). Coincident with the rapid decrease in the mitotic population of mutant cells at 16–18 h after taxol treatment (A), cyclin B1 levels dropped drastically after 16 h. cdc2 protein levels were unchanged in both cell types (Lower). (Bii) Quantification of cyclin B1 levels from WT and mutant cell lysates. Quantification was performed on the Western blots by using ImageJ software. (Ci) Cell lysates as in B were tested in Western blot with anti-BubR1 (Top). The double band probably represents hyper- and hypophosphorylated BubR1 forms. At 16 h after taxol treatment, BubR1 levels were no longer detected in mutant cells. Mad2 protein levels were unchanged and were similar for WT and mutant cells (Center). Anti-actin was used as loading control (Bottom). (Cii) Quantification of BubR1 (Left) and Mad2 levels (Right), as in Ci. (D Left and Center) Metaphase spreads from WT (Left) and mutant (Center) MEFs treated with colcemid for 4 h. (Right) Individual chromosome counts from WT (left) and mutant (right) MEFs were grouped as cells with normal karyotype (diploid, N) and cells with abnormal karyotype (aneuploid, ≠N). The percentage of aneuploid mutant cells was significantly higher than that of WT cells (Student's t test, P = 0.0088).

Dido mutants have cytokinesis defects. (A) Dido-mutant MEFs were stained with anti-γ-tubulin (red) and DAPI (blue). (Ai) Cells connected by an intercellular bridge (arrow). (Aii) A typical double-nucleus phenotype, indicating cell division failure. (Aiii) To confirm the existence of binucleated cells, we used a cytoplasmic membrane probe in confocal microscopy; DNA was stained with SYBR-Green. Two adjacent but separate WT cells are seen (Left). Binucleated mutant cells show a single membrane around the nuclei (Right). (Aiv) Representative images of bone marrow samples from Dido-targeted mice; arrows indicate binucleated cells in freshly isolated (Left) or 6-day cultured BM cells (Right). (B) Quantification of defective divisions. A significantly larger percentage of Dido-mutant than WT cells remained joined by intercellular bridges (Student's t test, P = 0.0015). A larger percentage of mutant than WT cells were binucleated (Student's t test, P = 0.0031).