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First published online 2 July 2003
doi: 10.1242/jcs.00624

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Mammalian RanBP1 regulates centrosome cohesion during mitosis

¹ CNR Institute of Molecular Biology and Pathology, Section of Genetics, c/o University `La Sapienza', 00185 Rome, Italy
² Institut Curie, Section Recherche, UMR144-CNRS, 75248 Paris Cedex, France

View larger version (58K): [in a new window]   Fig. 1. Supernumerary poles in RanBP1-overexpressing cells contain centrosomal components. (A) RanBP1-HA-transfected NIH/3T3 cells were double-stained with antibodies to glutamylated tubulin (GT335) and anti-HA-antibody to identify transfected cells (a); with antibodies coupled to centrosomal markers (b,c); and with anti--tubulin to label the spindle (d) and anti-centrin 2 to visualize centrioles. Experiments were carried out using all combinations of coupled antibodies to HA, -tubulin and centrosomal markers, and selected examples are shown. DNA was counterstained with DAPI (third column from the left). Merged pictures are shown on the rightmost column. Scale bar, 10 µm. (B) Quantification of RanBP1-induced abnormalities in spindle polarity (visualized by -tubulin staining) and in centrosomes (shown as either >4 or abnormally separated centrin spots). Data from three independent experiments were pooled and 100 mitoses per group were scored in each experiment. Histograms show the proportion of cells with abnormalities in vector-transfected (gray) and RanBP1-transfected (white) cultures. P values calculated using the 2 test were highly significant.

View larger version (68K): [in a new window]   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).

View larger version (34K): [in a new window]   Fig. 3. Split centrioles organize functional spindle poles. (a) A RanBP1-RFP-transfected mitosis from centrin 1-GFP stably expressing L929 cell cultures. Centrin-1/GFP allows the visualization of centrioles (b); the spindle is stained with anti--tubulin, revealed with an AMCA-conjugated secondary antibody (c). Merging of b and c produces d, which depicts single split centrioles (green) at each spindle pole (AMCA-stained MTs, in blue). Scale bar, 10 µm.

View larger version (11K): [in a new window]   Fig. 4. Centriole splitting requires microtubule integrity. Centrosomal abnormalities [i.e. overduplication (hatched columns) and splitting (black columns)] were recorded in cultures transfected with vector, wild-type RanBP1 or RanBP1L186A/V188A mutant (indicated as NES) during mitosis following thymidine release, or after treatment with nocodazole (NOC), or released after NOC arrest. For each condition, three to five experiments were carried out with wild-type RanBP1 and at least two with the RanBP1L186A/V188A mutant. Data were pooled and analysed using the 2 test. *, P<0.05; **, P<0.001.

View larger version (41K): [in a new window]   Fig. 5. Centriole splitting requires Eg5 activity. (A) Examples of MA-arrested mitoses from NIH/3T3 cultures transfected with GFP vector or with RanBP1-GFP and stained with antibody to -tubulin (right): no obvious difference in the centrosomal pattern is observed. Chromosomes are stained with DAPI and merged pictures are shown on the left. Scale bar, 10 µm. (B) Patterns of centrioles in monoastral mitoses revealed by anti-centrin-2 antibody: (a) normal arrangement with two pairs of centrioles; (b) supernumerary centriole pairs; (c) split centrioles. The rightmost column shows a magnification of the centrin 2 panels. Scale bar, 10 µm. (C) Frequency of centrosomal arrangements in NIH/3T3 MA-arrested and MA-released mitoses transfected with vector or RanBP1-GFP. Pooled data from four experiments were analysed using the 2 test. *, P<0.01.

View larger version (57K): [in a new window]   Fig. 6. A fraction of RanBP1 localizes at the centrosome. (A) Centrosomal RanBP1 in interphase (a), metaphase (b) and anaphase (c) NIH/3T3 cells. Examples of L929 (d) and HeLa (e) mitotic cells are also shown. Endogenous RanBP1 (second column) and -tubulin (third column) were revealed with FITC- and rhodamine-conjugated secondary antibodies, respectively. DNA was counterstained with DAPI (first column on the left). Signals are merged in the rightmost column. Scale bar, 10 µm. (B) Confocal signals for RanBP1 (FITC) (left) and -tubulin (rhodamine, middle) in a typical NIH/3T3 metaphase. Merged images are shown on the right. 15 focal planes of 2.28 µm thickness were scanned. (C) Anti-RanBP1 antibody (bottom) labels isolated KE37 centrosomes, stained by CTR453 (top). Scale bar, 50 µM. (D) RanBP1 is tightly associated with the centrosome fraction. Isolated centrosomes were extracted with buffers of increasing strength and analysed by western immunoblotting with the indicated antibodies. Abbreviations: p, pellet containing centrosome-associated proteins; s, supernatant containing solubilized proteins. The interaction of RanBP1 with centrosomes (bottom) is more resistant to detergents than that of -tubulin, a major PCM-recruited component (top). (E) Overexpressed RanBP1 localizes at spindle poles. An example of NIH/3T3 metaphase is shown. Anti-HA antibody, directed against exogenous RanBP1, is revealed with a rhodamine-conjugated secondary antibody. Centrosomes are stained with anti--tubulin antibody revealed with an AMCA-conjugated secondary antibody. The merged image is shown in the right panel. Scale bar, 10 µm.

View larger version (56K): [in a new window]   Fig. 7. RanBP1 retains its centrosomal localization after exposure to either nocodazole (a) or Taxol (b). Endogenous RanBP1 (second column) and -tubulin (third column) were revealed with FITC- and rhodamine-conjugated secondary antibodies, respectively. DNA was counterstained with DAPI (first column on the left). The rightmost column shows the merged signals of RanBP1, -tubulin and DAPI staining. Scale bar, 10 µm.