Fig. 2. RanBP1 overexpression induces centriole splitting in mitosis. (A) L929 cell cultures stably expressing a centrin 1-GFP chimera (Piel et al., 2000) were transfected with RanBP1-RFP, synchronized as described in the text, and mitotic cells recovered by `shake-off' were analysed. In the upper row, the non-transfected cell (upper left corner, negative for RFP emission) shows correctly aligned chromosomes (DNA panel) and centriole pairs in each centrosome, as shown in the magnified insert (a). In RanBP1-transfected cells (positive for RFP emission), single split centrioles are visible: two examples are shown, magnified in inserts b and c. Scale bar, 10 µm. (B) Quantification of centrosome defects induced by RanBP1 overexpression. Possible distributions of centrioles in mitosis are: I, normal arrangement; II, overduplicated centrosomes; III, split centrioles; IV, overduplicated and split centrosomes. Only tetrapolar spindles are represented, for simplicity. Histograms in the left panel show the frequency of centrosome overduplication (gray), calculated by grouping patterns II and IV (i.e. all cells with more than four centrioles) as abnormal. The same samples were re-analysed for the frequency of centriole splitting (histograms in the right panel), calculated by grouping patterns III and IV as abnormal (i.e. all cells showing single centrioles, regardless of total centriole number). 200 mitotic cells from vector- and RanBP1-RFP-transfected cultures were scored. The asterisks mark a highly significant difference (P<0.001).