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The mechanism regulating the dissociation of the centrosomal protein C-Nap1 from mitotic spindle poles

Thibault Mayor1,*, Ulrike Hacker1, York-Dieter Stierhof2 and Erich A. Nigg1,{ddagger}

1 Department of Cell Biology, Max-Planck-Institute for Biochemistry, Am Klopferspitz 18a, D-82152 Martinsried, Germany
2 Department of Membrane Biology, Max-Planck-Institute for Biology, Corrensstrasse 38, D-72076 Tübingen, Germany
* Present address: Division of Biology, California Institute of Technology, 1200 E. California Blvd, Pasadena, CA 91125, USA



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  		Fig. 1. Loss of C-Nap1 staining during mitosis. (A) HeLa cells were synchronized at the indicated cell cycle stages. Equal amounts of protein were then separated by 10% SDS-PAGE and probed by immunoblotting with the antibodies indicated. (B) HeLa cells were collected in G2 or blocked in mitosis using either nocodazole or monastrol, as indicated. Equal amounts of protein were separated by 7.5% SDS-PAGE, and probed by immunoblotting. For the control, insoluble fractions were also resuspended and corresponding volumes (1:1) analyzed in parallel. (C) Mitotic HeLa cells (M) were collected by mechanical shake off and asynchronously growing cells (asy) by trypsinization. Levels of C-Nap1 were then determined by immunoblotting, using either C-Ab or M-Ab (Fry et al., 1998aGo; Mayor et al., 2000Go). All C-Nap1 signals were standardized relative to the {alpha}-tubulin signal. The histogram shows the relative levels of C-Nap1 in M-phase and asynchronously growing cells.

 


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  		Fig. 2. C-Nap1 is not degraded during mitosis. (A) HeLa cells were collected in G2 or blocked in mitosis in the presence or absence of the proteasome inhibitor MG132. Equal amounts of protein were separated by 7.5% SDS-PAGE and probed by immunoblotting using the antibodies indicated. (B) Destruction assay in Xenopus egg extract. [35S]-labeled cyclin B and C-Nap1 were added to a mitotic extract and samples taken at the indicated times (minutes). Proteins were separated by 10% SDS-PAGE and exposed for autoradiography.

 


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  		Fig. 3. C-Nap1 is phosphorylated during mitosis. (A) Alkaline phosphatase treatment enhances the western blot signal of mitotic C-Nap1. C-Nap1 was immunoprecipitated from asynchronously growing (asy) or nocodazole-arrested (noc) HeLa cells and treated with either active (+) or heat-inactivated (-) calf intestinal phosphatase (CIP). Samples were separated by 7.5% SDS-PAGE and probed by immunoblotting as indicated (IgG are shown to demonstrate equal loading). (B) In vivo labeling of C-Nap1 with [32P]orthophosphate. C-Nap1 was immunoprecipitated from the indicated cell cycle stages, using C-Ab. Samples were separated by 7.5% SDS-PAGE, and gels exposed for autoradiography (top panel). In parallel, recovery of C-Nap1 was controlled by western blotting with C-Ab (bottom panel).

 


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  		Fig. 4. Overexpression of full-length C-Nap1 causes patch formation. U2OS cells were transiently transfected with full-length C-Nap1 and, 24 hours later, processed for immunofluorescence or immunoelectron microscopy with C-Ab. For immunofluorescence microscopy (A), cells were fixed with methanol; for immunoelectron microscopy (B) cells were fixed with 3% paraformaldehyde/2% sucrose. C-Nap1 overexpression resulted in the formation of single (a) or multiple (b) patches. The arrowhead in Ab points to the patch containing the centrosome, as determined by counter-staining with anti-{gamma}-tubulin antibodies (not shown). Bars, 10 µm (Ab); 250 nm (Bb).

 


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  		Fig. 5. Protein recruitment to C-Nap1 patches. (A) U2OS cells were transfected with either myc-C-Nap1 or C-Nap1 and fixed with methanol 24 hours later. In the former case, they were stained with a monoclonal anti-myc antibody (9E10) for C-Nap1 (a) and counterstained with a rabbit anti-Nek2 antibody (b). In the latter case, C-Nap1 was stained with C-Ab (c), and {gamma}-tubulin was visualized with a monoclonal antibody (d). No signal was observed in cells stained with either anti-rabbit or anti-mouse secondary antibodies alone (data not shown). Bar, 10 µm (d). (B) MT re-growth assay. Following overexpression of C-Nap1, MTs were depolymerized by cold treatment, and MT re-polymerization was induced for 60 seconds by placing cells into pre-warmed medium (37°C). Cells were then fixed with methanol and stained with C-Ab (a) and a mouse anti-{alpha}-tubulin antibody (b). The arrowhead points to a transfected cell harboring a C-Nap1 patch; note that MT nucleation is severely suppressed in this cell. Bar, 10 µm (b). (C) Mutational domain analysis of C-Nap1. On the right, the C-Nap1 domain structure and the ability of different C-Nap1 deletion mutants to form patches is summarized schematically. The dark boxes designate the predicted coiled-coil domains in C-Nap1; wt (wild-type) denotes the full-length C-Nap1 protein. On the left, the phenotype observed after overexpression of the T4 C-Nap1 deletion mutant is illustrated. U2OS cells were analyzed by immunofluorescence microscopy, using anti-myc (9E10) antibodies for staining of T4. Bar, 10 µm.

 


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  		Fig. 6. C-Nap1 overexpression does not prevent cell cycle progression. (A) U2OS cells were co-transfected with two plasmids, one encoding a non-destructible cyclin B2 mutant and the second C-Nap1, GFP or Cdk1-DN, as indicated. At 36 hours post-transfection, cells were fixed and the percentages of cells in interphase (black bars) and mitosis (light gray bars) determined. Transfected cells were detected using the 9E10 mAb for C-Nap1 and anti-HA mAb for Cdk1-DN. Enhanced GFP was detected using the FITC filter. In two independent experiments, more than 200 cells were counted. (B) Integration and expression of GFP-C-Nap1 in TREx modified U2OS cells. Expression of the fusion protein was induced for 24 hours with 1 µg/ml tetracycline. Induced (i) or uninduced (u) cells were lysed directly in sample buffer, and extracts were analyzed by immunoblotting using the indicated antibodies. The positions of molecular weight markers are indicated (in kDa) and the arrowhead marks C-Nap1. (C) Growth rates were determined for stable cell lines in which the expression of GFP-C-Nap1 had not been induced (uninduced), or induced (1 µg/ml tetracycline) at either time 0 (induced) or 24 hours before the initiation of the experiment (pre-induced). Cells were collected at the times indicated and counted.

 


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  		Fig. 7. Cells overexpressing GFP-C-Nap1 establish functional mitotic spindles. (A) GFP-C-Nap1 expression was induced for 24 hours and cells were stained for MTs in red (anti-{alpha}-tubulin mAb), DNA in blue (Hoechst dye) and C-Nap1 in green (GFP fluorescence). Representative cells are shown in interphase (a), metaphase (b) and anaphase (c). Bars, 10 µm. (B) Further immunofluorescent analysis of GFP-C-Nap1-expressing cells. Staining in red shows poly-glutamylated tubulin that predominantly localizes to centrioles (GT335; a,b) and spindle poles (anti-{gamma}-tubulin; c); staining in blue shows DNA and in green shows C-Nap1. Shown are representative cells in interphase (a) and mitosis (b,c). Bar, 10 µm. (C) Prophase cells were stained in red for {alpha}-tubulin (a) and {gamma}-tubulin (b), in blue for DNA and in green for C-Nap1. Enlarged (2x) insets of the C-Nap1 patches surrounding the centrosome are shown in black and white (b); z0 and z1 designate two different focal planes. Bar, 10 µm.

 


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  		Fig. 8. Active Nek2 modulates C-Nap1 patch formation. (A) Histograms illustrate the percentage of transfected cells falling into three defined categories (as defined in B), after co-transfection of mycC-Nap1 with either active Nek2 (Nek2-wt) or a catalytically inactive Nek2 mutant (Nek2-K37R). Results are shown as averages of two independent experiments. (A total of 588 cells were counted for co-transfections with Nek2-K37R and 600 cells for co-transfections with Nek2-wt.) (B) Representative examples of three types of C-Nap1 structures, used for classification of transfected cells. Class I, C-Nap1 localized to centrosomes without inducing the formation of grossly abnormal structures (a). Class II, C-Nap1 formed one predominant patch closely associated with the centrosome, with or without accompanying smaller patches (<10 patches in total) (b). Class III, C-Nap1 formed many (>10), often small, patches (speckles) (c). Bar, 10 µm (Ac).

 

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