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Fig. 7. Importin ß overexpression induces spindle abnormalities. (A) Localization of exogenous importin-ß/GFP (green) in transfected HeLa cells, showing stable association with the NE in interphase (left) and with spindle poles in mitosis (right). Merged signals with DAPI (blue) are shown. Scale bar, 10 µm. (B) A typical abnormal spindle in a mitotic cell expressing importin-ß/GFP: notice the chromosome misalignment (DAPI, left) and a small `extrapole' in addition to the two main poles (-tubulin, middle). Scale bar, 10 µm. (C) Induction of mitotic abnormalities (left) and delay (right) in importin-ß-expressing human and murine cell lines. Abnormalities were counted among 500 mitoses in HeLa cultures, 250 in MRC5 and 400 in NIH-3T3 (two independent experiments) and their frequency in importin-ß- (pIB-GFP) and vector- (pGFP) transfected populations was statistically evaluated using the ² test (P<<0.001 in HeLa cells, P<0.02 in MRC5 cells and P<0.001 in NIH-3T3 cells). The mitotic index was calculated in 1000 transfected cells in HeLa cultures, 800 in MRC5 cultures and 1200 in NIH-3T3 cultures (three experiments per cell line) and analysed using the ² test (P<<0.001 in all cell lines). (D) Localization of mitotic regulators to poles of importin-ß-induced abnormal spindles (GFP emission not depicted). Markers in the leftmost column are revealed by AMCA-conjugated secondary antibody (blue) and in the central column by a redemitting secondary antibody (Cy3 or Texas Red). Merged images are shown on the right. Scale bar, 10 µm.

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