Title: A centrosomal function for the human Nek2 protein kinase, a member of the NIMA family of cell cycle regulators
Abstract: Article15 January 1998free access A centrosomal function for the human Nek2 protein kinase, a member of the NIMA family of cell cycle regulators Andrew M. Fry Andrew M. Fry Department of Molecular Biology, Sciences II, University of Geneva, 30, Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland Search for more papers by this author Patrick Meraldi Patrick Meraldi Department of Molecular Biology, Sciences II, University of Geneva, 30, Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland Search for more papers by this author Erich A. Nigg Corresponding Author Erich A. Nigg Department of Molecular Biology, Sciences II, University of Geneva, 30, Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland Search for more papers by this author Andrew M. Fry Andrew M. Fry Department of Molecular Biology, Sciences II, University of Geneva, 30, Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland Search for more papers by this author Patrick Meraldi Patrick Meraldi Department of Molecular Biology, Sciences II, University of Geneva, 30, Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland Search for more papers by this author Erich A. Nigg Corresponding Author Erich A. Nigg Department of Molecular Biology, Sciences II, University of Geneva, 30, Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland Search for more papers by this author Author Information Andrew M. Fry1, Patrick Meraldi1 and Erich A. Nigg 1 1Department of Molecular Biology, Sciences II, University of Geneva, 30, Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:470-481https://doi.org/10.1093/emboj/17.2.470 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Nek2, a mammalian protein kinase of unknown function, is closely related to the mitotic regulator NIMA of Aspergillus nidulans. Here we show by both immunofluorescence microscopy and biochemical fractionation that human Nek2 localizes to the centrosome. Centrosome association occurs throughout the cell cycle, including all stages of mitosis, and is independent of microtubules. Overexpression of active Nek2 induces a striking splitting of centrosomes, whereas prolonged expression of either active or inactive Nek2 leads to dispersal of centrosomal material and loss of a focused microtubule-nucleating activity. Surprisingly, this does not prevent entry into mitosis, as judged by the accumulation of mitotically arrested cells induced by co-expression of a non-destructible B-type cyclin. These results bear on the dynamic function of centrosomes at the onset of mitosis. Moreover, they indicate that one function of mammalian Nek2 relates to the centrosome cycle and thus provide a new perspective on the role of NIMA-related kinases. Introduction In the filamentous fungus Aspergillus nidulans, entry into mitosis requires the activation not only of the cyclin-dependent protein kinase Cdc2 (Cdk1), but also of a second, structurally distinct serine/threonine kinase, known as NIMA (Osmani et al., 1991a). Mutation of the nimA gene causes cells to arrest in G2—hence ‘nim’ for ‘never in mitosis’ (Morris, 1976; Osmani et al., 1991a), whereas massive overexpression of the wild-type gene induces certain aspects of a premature mitosis, including condensation of chromosomes and, in some cases, formation of a mitotic spindle (Osmani et al., 1988). The precise relationship between the NIMA and Cdc2 pathways at mitosis remains to be determined, but recent data suggest that they are interlinked, with NIMA possibly being a substrate of the Cdc2 kinase (Ye et al., 1995; for reviews see Fry and Nigg, 1995; Osmani and Ye, 1996). In analogy to the evolutionary conservation of the Cdk cell cycle regulators, it seems plausible that NIMA-related kinases may also be required for regulating the cell cycle of eukaryotic organisms other than Aspergillus. Indeed, overexpression of wild-type NIMA or expression of dominant-negative mutants of NIMA disrupts the cell cycle of yeast, Xenopus and human cells, providing some support to the view that NIMA-related pathways might exist in these species (O'Connell et al., 1994; Lu and Hunter, 1995a; reviewed in Lu and Hunter, 1995b). The only organism for which a genuine functional homologue of Aspergillus NIMA has so far been cloned is another filamentous fungus, Neurospora crassa (NIM-1; Pu et al., 1995). However, genes encoding protein kinases structurally related to NIMA have been isolated from several organisms, including Saccharomyces cerevisiae (KIN3/NPK1; Jones and Rosamond, 1990; Barton et al., 1992; Schweitzer and Philippsen, 1992), trypanosomes (Nrk; Gale and Parsons, 1993) and mammals (Letwin et al., 1992; Schultz and Nigg, 1993; Levedakou et al., 1994; Schultz et al., 1994). The mammalian kinases have been designated ‘Nek's for NIMA-related kinases. Interestingly, both Nek1 and Nek2 are highly expressed in male and female germ cells, suggesting that they might be important during meiosis (Letwin et al., 1992; Rhee and Wolgemuth, 1997; Tanaka et al., 1997). Among the mammalian kinases known to date, human Nek2 is most closely related to NIMA, falling into the same subfamily as fungal NIMA, NIM-1 and KIN3 (Fry and Nigg, 1997). In addition, the in vitro substrate specificity and some biochemical properties of Nek2 are reminiscent of NIMA, and both Nek2 protein levels and activity are regulated through the cell cycle (Fry et al., 1995). However, significant differences between NIMA and Nek2 have also been observed. In particular, NIMA activity peaks in mitosis and remains high during mitotic arrest (Osmani et al., 1991b), whereas Nek2 activity is high during S and G2 phases but low during M phase arrest (Fry et al., 1995). Although the Nek2 protein kinase is suspected to play some role in cell cycle progression towards mitosis, direct evidence implicating Nek2 in any process related to cell division is lacking. Here, we have investigated the subcellular localization of the human Nek2 kinase by both immunofluorescence microscopy and subcellular fractionation. We found that endogenous Nek2 is highly enriched at the centrosome, the microtubule organizing centre (MTOC) of animal cells. This organelle plays a crucial role in cell division, as it gives rise to the poles of the mitotic spindle apparatus. In turn, correct assembly of a bipolar spindle is important for the faithful segregation of sister chromatids during mitosis and homologous chromosomes during meiosis (Fuller et al., 1992; Kellogg et al., 1994). The centrosome consists of a pair of centrioles surrounded by an amorphous structure known as the pericentriolar material (PCM) from within which microtubules are nucleated. A key role in microtubule nucleation has been attributed to γ-tubulin (Oakley et al., 1990; Horio et al., 1991; Joshi et al., 1992; Felix et al., 1994; Stearns and Kirschner, 1994), which forms part of a γ-tubulin-containing ring complex, or γ-TuRC (Moritz et al., 1995; Zheng et al., 1995). During the cell cycle, the centrosome undergoes a series of morphological and functional changes. These include the semi-conservative replication of centrioles in late G1/S phase, the enlargement or ‘maturation’ of the centrosome through the recruitment of additional PCM proteins in S and G2, and the physical separation of the duplicated centrosomes and their migration to opposite sides of the nucleus at the G2 to M transition (Kochanski and Borisy, 1990; Kimble and Kuriyama, 1992; Kellogg et al., 1994). In addition, the onset of mitosis is accompanied by an abrupt increase in the microtubule-nucleating capacity of centrosomes (Karsenti, 1991). Many of these events are thought to be brought about, at least in part, through changes in the phosphorylation state of critical centrosomal components. In particular, protein kinases have been implicated in centrosome duplication, maturation and separation (Blangy et al., 1995; Glover et al., 1995; Lauze et al., 1995; Lane and Nigg, 1996), as well as in the regulation of the centrosomal microtubule nucleation capacity (Ohta et al., 1990; Verde et al., 1990, 1992; Buendia et al., 1992). The identification of Nek2 as a novel component of the centrosome has prompted us to investigate in which aspects of centrosome function this protein kinase might be involved. Our results argue that Nek2 protein is important for centrosome integrity and, moreover, may play a role in the regulation of centrosome separation. Results Nek2 localizes to the centrosome For the purposes of the present study, two new polyclonal anti-Nek2 antibodies, termed R40 and R50, were generated in rabbits. As shown by Western blotting, both antibodies recognized a major protein of 48 kDa in whole HeLa cell extracts, and this protein co-migrated exactly with Nek2 overexpressed from a recombinant baculovirus in Sf9 insect cells (Figure 1A). The two antibodies also precipitated a strong β-casein kinase activity from both HeLa and Nek2 baculovirus-infected insect cells (Figure 1B). β-Casein had previously been shown to be a good in vitro substrate for human Nek2 (Fry et al., 1995). Consistent with earlier data (Fry et al., 1995), autophosphorylation of recombinant Nek2 could also be observed (Figure 1B). Figure 1.Characterization of R40 and R50 anti-Nek2 antibodies. (A) Western blots on total cell lysates. (B) Immunoprecipitations, followed by Nek2 kinase assays using casein as a substrate, and analysis of phosphoproteins by SDS–PAGE and autoradiography. Anti–Nek2 antibody reactivities were tested on total HeLa cell extracts (lanes 2 and 4) and lysates prepared from Sf9 insect cells infected with recombinant Nek2 baculovirus (lanes 6 and 8). For control, the corresponding pre-immune sera were used under identical conditions (lanes 1 and 3), and lysates prepared from wild-type baculovirus-infected insect cells were analysed in parallel (lanes 5 and 7). P, pre-immune serum; I, immune serum; C, control lysate. The migrations of Nek2 and β-casein are marked on the right. Molecular weight markers are indicated on the left. Equivalent Western blots performed on lower percentage SDS–polyacrylamide gels confirmed that there were no high-molecular weight (>80 kDa) immunoreactive bands with either anti-Nek2 antibody (data not shown). Download figure Download PowerPoint By immunofluorescence microscopy on cultured cells, both anti-Nek2 antibodies consistently stained one or two closely spaced dots which were usually located close to the nuclear envelope (shown for R40 in Figure 2c, not shown for R50). No such staining was produced by the pre-immune serum (Figure 2a). Co-staining with an anti-α-tubulin antibody revealed that the stained dots lay at the centre of the interphase microtubule array, suggesting that they might represent centrosomes (Figure 2d). This was confirmed by double-labelling of cells with anti-Nek2 antibody and CTR453, a monoclonal antibody (MAb) specific for a pericentriolar antigen (Bailly et al., 1989; Moudjou et al., 1991; data not shown). Staining of centrosomes was seen in all cell types analysed, and was independent of fixation method, as it was observed following procedures based on either aldehyde or organic solvent fixation. Finally, pre-incubation of the anti-Nek2 antibody with baculovirus-expressed recombinant Nek2 completely abolished centrosome staining (Figure 2f), whereas mock competition had no effect (Figure 2e). Figure 2.Localization of endogenous Nek2 to the centrosome in U2OS osteosarcoma cells. Asynchronous U2OS cells, growing on coverslips, were fixed with methanol and double stained for Nek2 (a, pre-immune; c, immune serum) and α-tubulin (b and d). Note that the immune R40 antibody stains two closely opposed points which co-localize with the centre of the interphase microtubule array, suggesting an association of Nek2 with the centrosomes. No such staining is produced by the corresponding pre-immune serum. Competition with recombinant Nek2 abolished centrosome staining (f), whereas mock-competition did not (e). Scale bar, 10 μm. Download figure Download PowerPoint Enrichment of Nek2 in purified human centrosomes To corroborate and extend the above findings, the association of Nek2 with centrosomes was explored using a biochemical approach. Centrosomes were isolated from either CCRF-CEM or KE37 human leukaemic cell lines (Bornens et al., 1987; Moudjou and Bornens, 1994) and then examined by both immunofluorescence microscopy and Western blotting. Immunofluorescent staining of these preparations with MAb CTR453, a marker for the PCM, revealed a sharp, punctate staining with the frequent occurrence of two closely spaced dots, characteristic of intact centrosomes (Figure 3A, panels b and d). Similar results were obtained with antibodies against α-tubulin, a reporter of centrioles (data not shown). Double staining with anti-Nek2 antibodies revealed positive staining of all centrosomes detected by CTR453 (Figure 3A, panel c), suggesting that Nek2 was associated with centrosomes in all cells, regardless of their position in the cell cycle (see below). No centrosome staining was produced by the corresponding pre-immune antibodies (Figure 3A, panel a). Figure 3.Enrichment of Nek2 protein in isolated centrosome preparations. (A) Isolated centrosomes from KE37 cells were spun onto coverslips, fixed with methanol and then double stained with anti-Nek2 serum (panel a, pre-immune; panel c, immune serum) and MAb CTR453, marker for centrosomes (panels b and d). Co-localization of Nek2 and the CTR453 antigen on isolated centrosomes can be seen with the immune R40 antibody (arrows) but not with the pre-immune serum. Scale bar, 10 μm. (B) Centrosomes were isolated from exponentially growing KE37 human leukaemic cells as described in Materials and methods. The fractions eluting from the bottom of the final sucrose gradient were analysed for number of centrosomes by immunofluorescence observation, following staining with the anti-centrosomal marker CTR453, and for the abundance of Nek2 protein by Western blotting with anti-Nek2 antibody. The doublet seen in the Nek2 blot most likely represents full-length Nek2 and a proteolytically truncated form of Nek2 produced during isolation of centrosomes; a similarly truncated product can also be seen when overexpressed Nek2 is isolated from insect cells (Figure 1A, lanes 6 and 8). (C) Western blot analysis of total KE37 cell extracts (T) and isolated KE37 centrosome preparations (C), with antibodies against human Nek2 (lanes 1 and 2), γ-tubulin (lanes 3 and 4), lamin B2 (lanes 5 and 6) and Cdk7 (lanes 7 and 8). Antibodies against Nek2 and γ-tubulin are those described in this study; anti-lamin and anti-Cdk7 antibodies are E6 (Lehner et al., 1986) and MO-1.1 (Tassan et al., 1994), respectively. Approximately 20 times more protein was loaded in the total extract than in the centrosome fraction, as determined by a comparison of the two samples on a silver-stained gel. The positions of molecular weight markers (kDa) are indicated on the left and arrowheads mark the positions of the respective proteins on the right of each panel. Download figure Download PowerPoint The final step of the centrosome preparation procedure involves fractionation on a sucrose gradient. Analysis of several neighbouring fractions by SDS–PAGE and silver staining revealed similarly complex protein profiles (data not shown, but see Bornens et al., 1987). Yet, as judged by immunofluorescent staining with MAb CTR453, only one or two of these fractions contained large numbers of centrosomes. To determine whether the bulk of Nek2 protein co-fractionated with centrosomes, aliquots of the final sucrose gradient fractions were stained with MAb CTR453, and centrosomes were counted using a fluorescence microscope. In parallel, each fraction was subjected to Western blotting with anti-Nek2 antibodies. As shown in Figure 3B, the highest levels of Nek2 protein were indeed seen in the very same fractions that contained the vast majority of centrosomes. To provide an approximate measure for the enrichment of Nek2 in centrosome preparations, Nek2 protein levels in total KE37 cell extracts and centrosome preparations were compared by semi-quantitative Western blotting (Figure 3C). Although total cellular protein was loaded in a 20-fold excess over centrosomal protein, ∼5-fold more Nek2 was found in the centrosomal fraction (Figure 3C, lanes 1 and 2), suggesting a 100-fold enrichment in the centrosomal preparation. In comparison, γ-tubulin was found to be 2- to 3-fold more abundant in centrosome preparations than in total cell extracts (Figure 3C, lanes 3 and 4), indicating a 50-fold enrichment. As negative controls, the fractionation of two nuclear proteins was examined under identical conditions. Nuclear lamins as well as a nucleoplasmic cyclin-dependent kinase, Cdk7, were virtually absent from the centrosomal fraction (Figure 3C, lanes 5–8), arguing that the centrosomes analysed here were not significantly contaminated with either structural or soluble nuclear material. Next, we asked what proportion of the total cellular Nek2 is associated with the centrosome. By comparing the amount of Nek2 protein present in a known number of KE37 cells with that in a known number of centrosomes isolated from the same cells, we estimate that only ∼10% of endogenous Nek2 protein is associated with the centrosome (data based on Western blotting; not shown). Although this number may appear low, we emphasize that similarly low numbers have recently been determined for other bona fide centrosomal components, notably γ-tubulin and centrin (Moudjou et al., 1996; Paoletti et al., 1996). Furthermore, we have used semi-quantitative Western blotting to determine the approximate number of Nek2 molecules present in cultured human cells. Using recombinant Nek2 expressed in Escherichia coli for standardization, we estimate that Nek2 is present at ∼104 copies per (HeLa) cell (data not shown). Assuming that the non-centrosomal pool of Nek2 is diffusely distributed throughout the cytoplasm and/or the nucleus, it is not surprising that immunofluorescence microscopy preferentially reveals the Nek2 protein that is concentrated at the centrosome. Centrosome association of Nek2 is independent of microtubules To determine whether Nek2 association with the centrosome was dependent upon microtubules, U2OS cells were treated with either nocodazole or taxol, and the distributions of Nek2 and α-tubulin were examined by immunofluorescence microscopy (Figure 4). Nocodazole leads to a complete depolymerization of the cytoplasmic microtubule network, whereas taxol treatment has a stabilizing effect, resulting in long microtubule bundles and the loss of centrosome-nucleated microtubules (De Brabander et al., 1986). In cells treated with nocodazole for 4 h, interphase microtubules were completely absent (Figure 4f). Yet, in comparison with untreated cells, there was no observable decrease in the concentration of Nek2 at the centrosome (Figure 4, compare b and a). In cells incubated with taxol, on the other hand, the microtubules appeared as dense bundles, which in many interphase cells were visibly detached from the centrosome (Figure 4g). In such cells, Nek2 was clearly still associated with the centrosome and not with microtubule bundles (Figure 4c). Furthermore, when taxol-treated cells entered mitosis, the long microtubule bundles were replaced by numerous small microtubule asters (Figure 4h). In these cells, Nek2 was invariably restricted to only two asters (Figure 4d), in line with previous data showing that only two of the many taxol-induced asters contain the duplicated centrosomes (De Brabander et al., 1986). These data indicate that the centrosomal localization of Nek2 results from interactions with bona fide components of the centrosome rather than with microtubules, although a weak interaction of this kinase with microtubules cannot be rigorously excluded. Figure 4.Nek2 remains associated with the centrosome in cells treated with nocodazole or taxol. Asynchronous U2OS cells, growing on coverslips, were either untreated (a and e) or treated for 4 h with either nocodazole (6 μg/ml; b and f), or taxol (5 μM; c, d, g and h). Cells were then fixed with methanol and double stained for Nek2 (a–d) and α-tubulin (e–h). Nek2 can still be seen associated with the centrosome despite complete depolymerization of microtubules by nocodazole (b) or taxol-induced detachment of microtubule bundles from the centrosome (c). Taxol-treated mitotic cells form numerous MTOCs (h), but Nek2 remains associated with only two of these centres (d). Scale bars: (g), 10 μm, also for a–c and e–g; scale bar in (h), 10 μm, also for d and h. Download figure Download PowerPoint Nek2 localizes to the centrosome throughout the cell cycle Considering the cell cycle-dependent expression of Nek2 and its suspected role in cell cycle control (Fry et al., 1995), it was important to determine at which stage(s) of the cell cycle Nek2 associates with the centrosome. Following immunostaining of exponentially growing U2OS cells, Nek2 could unequivocally be identified at the centrosome in >90% of all cells. Moreover, Nek2 could be seen at centrosomes in paired cells that were still connected by post-mitotic bridges (early G1 cells), in cells arrested at the G1–S phase transition with aphidicolin, and in cells that were positive for bromodeoxyuridine after a 2 h pulse labelling (S or G2 cells) (data not shown). As summarized in Figure 5, Nek2 was also visible at the spindle poles throughout mitosis, from early prophase (Figure 5b), through metaphase (Figure 5c) and anaphase (Figure 5d), to telophase (Figure 5e). These data clearly show that Nek2 is associated with centrosomes at all stages of the cell cycle. However, although a rigorous quantitation of such data is difficult, we note that Nek2 antibody staining of mitotic spindle poles was at most as intense as that of interphase centrosomes, and frequently appeared to be reduced, particularly in telophase. This is in striking contrast to several other centrosomal proteins, including γ-tubulin, which accumulate at the centrosome during G2, and, as a consequence, are stained much more intensely at spindle poles than at interphase centrosomes (e.g. Zheng et al., 1991; Lane and Nigg, 1996). Figure 5.Nek2 is localized to the centrosome throughout the cell cycle. Asynchronous U2OS cells, growing on coverslips, were fixed with paraformaldehyde and stained for Nek2 (top panels; a–e) and DNA (bottom panels; f–j). Representative cells are shown from different stages of the cell cycle: interphase (a and f); prophase (b and g); metaphase (c and h); late anaphase (d and i); telophase (e and j). Arrowheads indicate the association of Nek2 with the centrosome at all stages of interphase and mitosis. Scale bar, 10 μm. Download figure Download PowerPoint Overexpression of active Nek2 leads to centrosome splitting and dispersal To gain insights into the possible function of Nek2 at the centrosome, myc epitope-tagged wild-type and catalytically inactive Nek2 were introduced into cells by transient transfection. After 24 or 48 h, transfected cells were identified by staining with anti-myc antibodies and the fate of centrosomes monitored by staining with anti-γ-tubulin antibodies. In some transfected cells, both anti-myc and anti-γ-tubulin antibodies could readily be seen to stain the same dots, indicating that ectopically expressed Nek2 kinase can localize to the centrosome (Figure 6a and b). This was particularly clear at early times after transfection and in cells expressing low levels of exogenous Nek2. In cells expressing higher levels, exogenous Nek2 was also seen in the nucleus (Figure 6a) or, in some cells, in the cytoplasm (see Figure 8A). Most interestingly, Nek2 overexpression caused two dramatic effects on centrosome structure. As visualized by γ-tubulin staining, the centrosome of many transfected cells had split into two foci, which were usually several microns apart (Figure 6c and d), and sometimes separated by more than half a cell diameter (data not shown). In other transfected cells, centrosomes were undetectable altogether (Figure 6e and f). Very similar effects could also be observed using antibodies against other centrosomal components, including the PCM marker CTR453 (Figure 6g and h), indicating that Nek2 overexpression profoundly affected centrosome structure and not merely the localization of γ-tubulin. Figure 6.Nek2 overexpression causes centrosome splitting and disappearance. U2OS cells were transfected with myc-tagged, wild-type Nek2 and double stained for the myc-epitope to identify transfectants (myc-Nek2; a, c and e) and γ-tubulin to reveal the centrosomes (γ-Tubulin; b, d and f). Some cells overexpressing Nek2 (a and b; left cell) display a normal pattern of centrosomes, as seen in untransfected cells (a and b; right cell). However, in other transfected cells, the two centrosomes show a clear splitting (c and d; right cell), and in yet others, the centrosomes are undetectable, regardless of the plane of focus (e and f; right cell). As long as centrosomes are present, the exogenous myc-Nek2 protein can be seen to co-localize with γ-tubulin (a–d, arrows). The same phenotypes could be seen using the anti-PCM monoclonal antibody, CTR453 to stain the centrosomes (g and h); in these two panels the transfected cells are indicated with a star. Scale bar, 10 μm. Download figure Download PowerPoint Figure 7.Quantitation of centrosomal phenotypes caused by overexpression of wild-type and catalytically inactive Nek2. U2OS cells were transfected with cDNAs as indicated and analysed by indirect immunofluorescence microscopy at 24 h (A) or 48 h (B) after transfection. Cells were double stained with anti-myc antibodies to identify transfected cells and anti-γ-tubulin antibodies to observe the appearance of the centrosomes, except in the case of γ-tubulin transfections where transfected cells were identified with anti-γ-tubulin antibodies and centrosomes with the CTR453 monoclonal antibody. Centrosomes were classified as either normal (two closely opposed sharp points; open bars), split (a gap of at least 3–4 μm between the two centrosomes; filled bars) or dispersed (severely diminished, fragmented or undetectable; hatched bars). Results are the mean of at least five independent experiments for Nek2 plasmids and two independent experiments for Cdk7 and γ-tubulin plasmids; at least 100 transfected cells were counted for each coverslip. Error bars represent standard deviations. Download figure Download PowerPoint Figure 8.Loss of focused microtubules in cells overexpressing Nek2. In cells transfected for 48 h with wild-type Nek2, microtubules were either untreated (a and b) or depolymerized by cold treatment and then allowed to regrow by the addition of warm medium (c and d). Cells were fixed with methanol and double stained with anti-Nek2 antibodies to identify transfected cells (a and c), and anti-α-tubulin antibodies to visualize the microtubules (b and d). In (b), the transfected cell can be seen to have a microtubule array emanating primarily from the perinuclear region, while in the regrowth assay (d) the transfected cell has failed to organize a focused microtubule regrowth. Arrowheads indicate transfected cells. Scale bar, 10 μm. Download figure Download PowerPoint To analyse these phenotypes in quantitative terms, cells were transfected with either Nek2 or control plasmids, and then classified depending on whether they had apparently normal centrosomes, distinctly split centrosomes or dispersed centrosomes (Figure 7). Cells were counted as having dispersed centrosomes when γ-tubulin staining was virtually undetectable, or, in rare cases, confined to clusters of several tiny dots (not shown). By 24 h after transfection (Figure 7A), only 10% of the Nek2-transfected cells showed a normal centrosome pattern (i.e. one or two closely opposed sharp dots). Instead, in 45% of cells centrosomes had split over several microns, and in the remaining 45% they were almost or completely invisible. By 48 h (Figure 7B), split centrosomes could still be detected in 23% of cells, but >70% of cells lacked detectable centrosomes. Interestingly, transfection of a catalytically inactive Nek2 mutant (K37R; Fry et al., 1995), also caused dispersal of centrosomal material, but did not trigger any centrosome splitting (Figure 7). This demonstrates that centrosome splitting depends on kinase activity, and suggests that centrosome splitting and dispersal are brought about by different mechanisms. Also, no centrosome splitting or dispersal could be observed in response to overexpression of either the protein kinase Cdk7 or the centrosomal component γ-tubulin (Figure 7), indicating that the observed effects were brought about specifically by Nek2 protein. Loss of functional MTOCs in cells overexpressing Nek2 The above data clearly show that overexpression of Nek2 produces very drastic effects on centrosome structure. To extend our studies beyond a structural description, we asked to what extent overexpression of Nek2 interfered with the microtubule organizing function of centrosomes. When Nek2-transfected cells were stained for α-tubulin, it was frequently difficult to find a focal point of microtubule organization; instead, a dense concentration of microtubules could often be seen at the nuclear periphery (Figure 8a and b). This suggested that microtubules might be nuclea