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Centrosome cohesion is regulated by a balance of kinase and phosphatase activities

Department of Cell Biology, Max-Planck-Institute for Biochemistry, D-82152 Martinsried, Germany

View larger version (34K): [in a new window]   Fig. 1. Survey of protein kinases for their ability to induce centrosome splitting. U2OS cells were transfected with constructs encoding either wild-type or catalytically inactive protein kinases, as indicated. After 24 hours, centrosomes were analyzed by immunofluorescent staining with antibodies against -tubulin; transfected cells were identified using antibodies against the myc- or HA-tag, as appropriate. (A,B) Cells were classified as harboring either paired (‘un-split’) (A) or split (B) centrosomes, using the criteria described in Materials and Methods. Bar, 10 µm. As illustrated in schematic form, centrosome splitting occurs between the two parental centrioles, each with its associated PCM. Thus, splitting of an S or G2 phase centrosome produces two products that each comprises one parental centriole and one tightly associated procentriole. The histogram (C) indicates the percentages of cells showing split centrosomes in response to expression of each kinase construct.

View larger version (18K): [in a new window]   Fig. 2. Nek2 and Cdk2 induce centrosome splitting through independent pathways. U2OS cells were co-transfected with different combinations of wild-type Cdk2 and Nek2, or catalytically inactive Cdk2 (D145N) and Nek2 (K37R), together with cyclins A or E as indicated. Centrosome splitting was then analyzed as described in Fig. 1 legend.

View larger version (16K): [in a new window]   Fig. 3. Identification of PP1 as a likely Nek2 antagonist. (A) U2OS cells were treated for 1 hour with the indicated concentrations of okadaic acid or caliculin A. Then, centrosome splitting was analyzed as described. As a positive control, Nek2-transfected cells were examined in parallel. (B) U2OS cells were transfected with PP1, either alone or in combination with Nek2, Cdk2/cyclin E or Cdk2/cyclin A, and the extent of centrosome splitting was determined.

View larger version (89K): [in a new window]   Fig. 4. MT-regrowth assay on Nek2-transfected cells. U2OS cells were transfected for 24 hours with myc-Nek2. Then, MTs were depolymerized by cold treatment for 30 minutes and allowed to re-grow for 45 seconds by addition of pre-warmed medium. Cells were immediately fixed for immunofluorescence microscopy and double-stained with antibodies against Nek2, to identify transfected cells (upper panel; arrows point to split centrosomes), and -tubulin, to reveal the MTs (lower panel). Bar, 10 µm.

View larger version (16K): [in a new window]   Fig. 5. Effect of anti-cytoskeletal drugs on centrosome cohesion. (A) U2OS cells were treated for 1 hour with nocodazole (5 µg/ml), taxol (5 µM) or cytochalasin D (1.25 µg/ml) and then analyzed for centrosome splitting. (B) U2OS cells were transfected for 24 hours with myc-Nek2. One hour prior to fixation, they were treated with the above concentrations of nocodazole, taxol or cytochalasin D, and the extent of centrosome splitting was determined.

View larger version (66K): [in a new window]   Fig. 6. Nocodazole-induced centrosome splitting depends on protein kinase activity. (A,B) U2OS cells either treated for 1 hour with nocodazole, either alone or in combination with staurosporine (500 µM) or 6-DMAP (2.5 mM), before centrosome splitting was analyzed. Alternatively, MT disassembly was induced by incubating cells for 1 hour on ice. As assessed by immunofluorescent staining with antibodies against -tubulin, all treatments caused complete disassembly of MTs (not shown). (A) Histogram indicating the percentages of cells with split centrosomes; (B) -tubulin stained centrosomes, illustrating the suppression of nocodazole-induced centrosome splitting by staurosporine. Bar, 10 µm. (C,D) U2OS cells were transfected for 24 hours with catalytically inactive mutants of the kinases indicated, and 1 hour prior to fixation they were treated with nocodazole. For control, untransfected cells were treated with nocodazole alone or with nocodazole plus staurosporine. Then, all samples were scored for centrosome splitting (C). (D) -tubulin stained centrosomes, illustrating the suppression of nocodazole-induced centrosome splitting by the catalytically inactive Nek2 K37R mutant. Bar, 10 µm.

View larger version (24K): [in a new window]   Fig. 7. A model illustrating the contribution of MT dynamics to the regulation of centrosome cohesion. We propose that parental centrioles are held together by a flexible proteinaceous linker whose assembly and disassembly is controlled by phosphorylation. If some of the regulatory kinases and/or phosphatases display affinity for MTs, their relative concentrations at the centrosome will depend on the MT dynamics typical of an intact MT network (particularly a constant flux of tubulin subunits towards the centrosome-associated MT minus ends). Whereas phosphatase activity will tend to stabilize the linker, any excess of kinase activity will cause its disruption.

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