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Vol. 20, Issue 20, 4348-4361, October 15, 2009
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*Center for Biochemistry, Medical Faculty, Center for Molecular Medicine Cologne and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, 50931 Köln, Germany; and Institute of Anatomy and Cell Biology and Center for Integrated Protein Science, Ludwig-Maximilians-University, 80336 München, Germany
Submitted March 3, 2009;
Revised August 10, 2009;
Accepted August 11, 2009
Monitoring Editor: Stephen Doxsey
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tubulin ring complexes and regulatory molecules. They allow the centrosome to function as MTOC and to carry out its regulatory functions during cell cycle transitions, cellular responses to stress, and organization of signal transduction pathways (Keryer et al., 2003
; Doxsey et al., 2005). Accordingly, the protein composition of the centrosomes is complex and highly dynamic.
In protozoa, algae, and fungi, the centrosome morphology varies greatly. In Dictyostelium discoideum it is a nucleus associated body consisting of a box-shaped core surrounded by the corona, an amorphous matrix functionally equivalent to the PCM (Ueda et al., 1999). Only a few centrosomal proteins have been characterized in D. discoideum so far. Most of them were identified based on the homology to their mammalian counterparts such as -tubulin (Euteneuer et al., 1998); DdCP224, an XMAP215-family protein (Gräf et al., 2000); the centrin-related DdCrp (Daunderer et al., 2001); EB1 (Rehberg and Gräf, 2002); Lis1 (Rehberg et al., 2005); Spc97 and Scp98 (Daunderer and Gräf, 2002); and the kinase DdNek2 (Gräf, 2002). A 350-kDa protein, that has not been further characterized, was isolated using monoclonal antibodies generated against a nucleus-centrosome complex (Kalt and Schliwa, 1996). A further 70 candidates of the Dictyostelium centrosome were identified in a proteomic approach (Reinders et al., 2006). These proteins are components of the corona; however, during mitosis they relocate to the centrosomal core structure and stay associated with the centrosome throughout mitosis. Exceptions are DdNek2, a permanent component of the core that is also present in the cytoplasm, and DdCrp, which disappears during prometaphase and reassociates with the centrosome after telophase. DdCrp resembles with its behavior the corona that dissociates from the centrosomal core at the transition from prophase to metaphase and reforms at the end of telophase (Ueda et al., 1999).
Here we describe CP250, a novel component of the D. discoideum centrosome, and localize it to the corona. We follow its dynamic association with the centrosome during interphase and mitosis. Loss of the protein leads to defects in growth, cell polarity, and development. The results point toward a role for CP250 in centrosome–microtubule cytoskeleton interactions during interphase. Furthermore, we observed in the mutant alterations in the nuclear envelope (NE) using antibodies specific for the UNC-84 homolog Sun-1 and the KASH-domain protein interaptin pointing out the close interaction between centrosome and NE (Rivero et al., 1998; Xiong et al., 2008; Starr, 2009).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). For expression of green fluorescent protein (GFP)-tagged 
-tubulin a plasmid was used described by Koonce et al. (1999). Growth and development were done as described (Khurana et al., 2002). For generation of CP250-deficient cells a knockout vector was generated by amplifying gDNA sequences (DDB 0233900) from positions 1648–2229 and 2781–3348 and cloning them into the Cre-loxP vector pLPBLP carrying a blasticidin resistance conferring gene (Faix et al., 2004). For expression of CP250 GFP fusion proteins, sequences encompassing amino acids 756-1148 corresponding to nucleotides 2236–3444 (GFP-CP250 D1) and amino acids 447-1257 corresponding to nucleotides 1338–3770 (GFP-CP250 D2) were cloned into pBsr GFP N2 and pBsr GFP N1, respectively. Expression was under the control of the constitutively active Dictyostelium actin15 promoter. The plasmids were introduced into AX2 cells by electroporation following standard procedures (Faix et al., 2004). Selection of the transformants was with blasticidin (3.5 µg/ml). Mutant cells in which the CP250 gene was successfully targeted were identified by PCR followed by Southern blotting. Mutant analysis was done as described (Khurana et al., 2002; Noegel et al., 1999; 2004). Cell size was determined using cells that had been treated with 20 mM EDTA in Soerensen phosphate buffer (17 mM Na+/K+-phosphate buffer, pH 6.0) in order to obtain perfectly round cells. For expression as glutathione S-transferase (GST)-fusion proteins, cDNA sequences encoding amino acids 756-1148 (GST-CP250 D1) were cloned into pGEX4T (GE Healthcare, Waukesha, WI).
Generation of Antibodies
Mouse monoclonal antibodies were generated against CP250 D1 (amino acids 756-1148) as described (Xiong et al., 2008). For immunization of mice the GST-part was removed by thrombin cleavage. The identity of the CP250 polypeptide was confirmed by mass spectrometry. mAbs K68-332-3 and K68-439-8 were used in this study. They recognized the bacterially produced recombinant protein, the GFP-tagged fusion proteins and the endogenous protein in Western blots of whole cell lysates and the GFP-tagged fusion proteins and the endogenous protein in immunofluorescence analysis and immunoelectron microscopy. Rabbit polyclonal antibodies specific for Sun-1 were generated against a GST fusion protein containing the N-terminal 266 amino acids of Sun-1 (N800, Xiong et al., 2008; Pineda, Berlin, Germany) and affinity purified.
Immunofluorescence Analysis
Approximately 1 x 106 cells were transferred onto a coverslip (10-mm diameter) and allowed to adhere for 20 min at room temperature. If not mentioned otherwise, standard immunofluorescence staining was carried out using ice-cold methanol as fixative (45 min, –20°C). Cells were treated twice for 15 min (room temperature) with blocking solution (1x PBS containing 0.5% (wt/vol) BSA and 0.1% (vol/vol) fish gelatin). The appropriate antibodies were diluted in the blocking solution and applied for 1 h at room temperature; the excess of antibodies was removed by washing with the blocking solution before the 1-h incubation with the according secondary antibodies.
For cell cycle synchronization, cells were kept for 3 h in a Petri dish in Soerensen phosphate buffer, at room temperature. The buffer was then replaced by growth medium, and incubation was continued for 4 h. Fixation was with cold methanol. For the staging of the mitotic phases we used the disappearance or reappearance of microtubules as well as the size of the centrosomes.
Primary antibodies used in this study were mouse monoclonal anti-GFP antibody K3-184-2 (Noegel et al., 2004), mAb 260-60-10 against interaptin (Rivero et al., 1998), Sun-1 specific mAb K55-432-2 (Western blot analysis) and K55-460-1 (immunofluorescence; Xiong et al., 2008) as well as polyclonal antibodies (this study), rat mAb YL1/2 against -tubulin (Kilmartin et al., 1982), mouse monoclonal antibodies K29-359-31 and K29-359-10, which are subclones of a single clone, that were generated against a tubulin-fraction from the green alga Spermatozopsis similis and that recognize specifically the D. discoideum centrosome (gift from Dr. K. Herkner and Dr. M. Melkonian, (University of Cologne); Xiong et al., 2008). Polyclonal antibodies against DdCP224 were kindly provided by Dr. R. Gräf (University of Potsdam; Gräf et al., 1999). The appropriate secondary antibodies were Cy3-conjugated sheep anti-mouse IgG (Sigma, St. Louis, MO) and Alexa 488–, 568– or 647–conjugated goat anti-mouse or donkey anti-mouse and Alexa 488 and 647 donkey anti-rabbit IgG or Alexa 568–coupled goat anti-rabbit IgG (Molecular Probes, Eugene, OR). DNA was stained with 4'-6-diamidino-2-phenylindole (DAPI). Analysis was done by confocal laser scanning microscopy (Leica TCS SP5). Actin was recognized by mAb act1 (Simpson et al., 1984), CAP by mAb 230-18-8 (Gottwald et al., 1996).
Electron Microscopy
Nuclei of AX2 cells were isolated using Nucleopore membranes (5 µm, Corning Glass, Corning NY). The cells were washed twice in cold phosphate buffer and then resuspended in Dicty-PHEM (Cox et al., 1995) containing a protease inhibitor cocktail and PMSF. They were pressed through the filter assembly three to five times. The resulting mixture was layered onto a 30% sucrose cushion and centrifuged at 3500 rpm for 10 min at 4°C. The pellet containing mainly nuclei was resuspended in Dicty-PHEM and centrifuged onto 12-mm coverslips at 3500 rpm for 5 min. The nuclei were fixed with 2% formaldehyde in Dicty-PHEM for 15 min. They were washed and treated with mouse monoclonal antibodies K68-332-3. The antibodies were detected using 6 nm goat anti-mouse antibody (Aurion, Biotrend, Wageningen, the Netherlands). Dehydration and embedment were carried out according to standard procedures. Alternatively, methanol fixed nuclei were embedded in Lowicryl (Euteneuer et al., 1998). mAb K29-359-31 was applied to thin sections followed by a bridge (rabbit anti-mouse) Ab (rabbit anti-mouse) and 10 nm protein A gold. The samples were sectioned using a Reichert Ultracut E and viewed in a JEOL 1200C (Peabody, MA) equipped with a TEM camera Keenview-10/12 and iTEM imaging system (Soft Imaging System, Münster, Germany).
Analysis of Cell Shape and Cell Migration
Aggregation competent AX2 and CP250– cells were plated onto glass coverslips and allowed to settle for 15 min, and chemotaxis experiments performed with micropipettes filled with 10–4 M cAMP attached to a micromanipulator system. Images were recorded at intervals of 6 s using a Leica DM-IL inverse microscope (Deerfield, IL; 40x objective) and a conventional CCD video camera and analyzed using Dynamic Image Analysis Software (DIAS, Soll Technologies, Iowa City, IA; Wessels et al., 1998).
Miscellaneous Methods
For the yeast-two-hybrid screen residues 1–700 of the CAP cDNA sequence were cloned into pAS2 (Durfee et al., 1993; Noegel et al., 2004). A Dictyostelium cDNA library kindly provided by Dr. A. Kuspa (Baylor College of Medicine) was used for the screen (Knuth et al., 2004). The interactions were further confirmed by testing the recombinant proteins in vitro (Knuth et al., 2004) using GST-CP250 D1 and N-CAP (residues1–226) expressed as his-tagged fusion protein. The binding assay was done in 10 mM MES, pH 6.0, 138 mM KCl, 2 mM EDTA, 3 mM MgCl2, 1% Triton X-100, and 5 mM DTT. To enrich for centrosomal proteins, the procedure published by (Gräf 2001) was essentially followed. The sucrose density gradient centrifugation step was omitted. Proteins were separated on 8% SDS-polyacrylamide gels or gradient gels (3–15% acrylamide) and transferred to nitrocellulose membrane using wet blotting methods. For detection of CP250 transfer was done for 48 h at 140 mA.
For immunoprecipitation of GFP-CP250 D2* protein cells were lysed in lysis buffer (10 mM Tris/HCl, pH 8.0, 50 mM NaCl, 0.1% Triton X-100, 1 mM EDTA, complete protease inhibitor cocktail [Roche, Indianapolis, IN]), 10 mM benzamidine, 10 µg/ml aprotinin, and 10 µg/ml leupeptin). GFP specific polyclonal rabbit antibodies were coupled to protein A Sepharose and incubated with the lysate. Bound proteins were separated on 3–15% gradient gels and analyzed by Western blotting. Alternatively gels were stained with Coomassie Brilliant Blue R, bands were cut out, and the proteins were identified by LC-MS/MS.
For analysis of the effect of microtubule-specific drugs cells were treated with nocodazole (5 and 10 µM), vinblastine (1 µM), taxol (1 µM), thiabendazole (5 and 10 µM), benomyl (50 µM), and colchicine (0.5 µM) for 2 up to 45 h. The distribution of proteins after drug treatment was assayed in immunofluorescence studies, and the impact on growth was followed by determining the cell number.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Among others we identified four clones ranging from 1504 to 2106 base pairs in length and overlapping in a 1176-base pair stretch. These clones were derived from a single gene, DDB0233900, located on chromosome 6 and their sequences corresponded to the central part of this gene (Figure 1A). Gene DDB0233900 codes for a protein of 2110 aa with a molecular mass of 248,355. The protein was named CP250 based on its location and molecular mass. It has a pI of 4.4 and contains stretches for which a coiled coil structure is proposed by the Smart prediction program (Schultz et al., 1998). Several involucrin repeats and an Nt-DnaJ domain signature at position 965–984 are predicted (Figure 1A). Blast searches showed weak homologies to myosins, hook-related proteins, the Schizosaccharomyces pombe protein SPAC1F3.06c that colocalizes with the Sun-domain protein Sad1, a component of the spindle pole body that is the yeast equivalent of the centrosome, and CENPE, reflecting the coiled coil structure (Earnshaw and Cleveland, 1991; Saito et al., 2005).
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).
CP250 Is a Centrosomal Protein and Localizes to the Corona
To determine the subcellular localization of CP250, we generated monoclonal antibodies against recombinant CP250 D1 (Figure 1A). The antibodies recognized the recombinant CP250 polypeptide, the GFP-tagged CP250 fusion proteins, and the endogenous protein in Western blots of whole cell homogenates. In AX2 lysates we detected CP250 as a large protein migrating at 
280–300 kDa. In cells expressing GFP-CP250 D1 we detected with mAb K68-439-8 a strong signal at 95 kDa that was also recognized by GFP-specific mAb K3-184-2, whereas in lysates from cells that express GFP-CP250 D2 we observed a signal above 250 kDa both with mAb K3-184-2 and mAb K68-439-8 (Figure 2B and data not shown). We designate this protein GFP-CP250 D2* to indicate that it is the result of a knock in. Endogenous CP250 was absent. Further analysis revealed that in these cells a knockin event had occurred and that the vector had integrated into the CP250 gene leading to the production of a GFP fusion protein of a calculated molecular weight of 220 kDa (Figure 2, A and C). The identity of the protein was confirmed in immunoprecipitation experiments with polyclonal GFP antibodies and subsequent identification using LC-MS/MS.
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To corroborate these results, we carried out immunoelectron microscopy using mAb K68-332-3 (Figure 3A). This approach confirmed the localization of CP250 at the centrosome. K68-332-3 decorated the more open matrix of the corona of the centrosome but did not stain the core. Thus, CP250 is most likely a component of the corona. CP250 was also recently described as a centrosomal component in the course of a proteomic characterization of the Dictyostelium centrosome, and our data confirm and extend the proteomic data (Reinders et al., 2006).
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-tubulin (Figure 3B and data not shown).
mAb K29-359-31 recognizes specifically tubulin at the centrosome (Xiong et al., 2008) and localizes in immunoelectron microscopy to the corona (Figure 3A, bottom panels). Gold particles formed clusters within the corona, but were not present in the electron-dense, layered core structure of the centrosome. The staining was similar to the one of -tubulin that is detected in regularly spaced clusters within the corona (Euteneuer et al., 1998).
The data show that amino acids 756-1148 are sufficient for correct localization of CP250. Expression of the GFP-fusion protein did not affect centrosome position, centrosome number, and the nucleus number in the cells (Figure 3B). GFP-CP250 D2* was also seen at the centrosome (Figure 3B).
CP250 Is a Permanent Resident of the Corona
Next we followed the fate of CP250 during the cell cycle. The fate of the corona during mitosis was previously revealed in electron microscopy studies (Ueda et al., 1999). It disappears at the end of prophase and reassembles during late telophase. Most of the known components of the corona do not follow this pattern as they relocate to the core of the centrosome during mitosis and stay associated throughout the cell cycle. CP250 behaved differently. It was present throughout interphase, still seen in prophase but was no longer detectable during meta- and anaphase and reappeared at the centrosome at the end of late telophase (Figure 4A). The GFP-tagged polypeptide CP250 D1 behaved in the same manner and was not present at the centrosome during metaphase until late telophase (Figure 4B), whereas K29-359-31 staining was always associated with the centrosome (Figure 4C).
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12 µm, whereas AX2 cells were between 12 and 16 µm (Figure 6B). The smaller diameter was not due to an increased thickness of the cells for two reasons. First, we determine the diameter using perfectly round cells. This is achieved by EDTA treatment before the measurement. Second, we analyzed the cells using confocal microscopy and did not observe an increase in thickness. The mutant cells were mostly mononucleated.
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It is generally accepted that the centrosome and microtubules regulate cell polarity and persistence of pseudopods. In mammalian cells, for example, the orientation of the MTOC is a marker for polarization in a wound-healing assay (Lüke et al., 2008). Furthermore, disruption of the microtubule network or mutations in microtubule-associated proteins affect directed cell migration and motility (Williams et al., 2006; Li and Gundersen, 2008; Tang et al., 2008). We therefore analyzed chemotaxis and motility of the mutant cells in a cAMP gradient. We used synchronously developed cells, plated them on a glass surface, and analyzed their chemotactic movement when migrating toward a micropipette filled with 0.1 mM cAMP. Under these conditions wild-type cells elongate, are highly polarized and migrate in a directed manner toward the cAMP source extending and retracting pseudopods in the direction of the cAMP gradient. CP250– cells are also chemotactically active and migrate toward cAMP. The cells are polarized, but they are slightly slower and change their direction more frequently than wild-type cells leading to slightly less directed migration. Notably, the leading pseudopod is less stable, is frequently retracted, and a new pseudopod is formed (Table 1 and Figure 7, A and B; arrow indicates change of direction).
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). When we tested the effect of several drugs on growth of wild-type and mutant cells, we found that at the concentrations used, nocodazole, vinblastine, and colchicine mildly inhibited growth to a similar extend in wild-type and mutant cells, whereas taxol had no effect on AX2 and a stimulatory effect on the growth of the CP250– strain. Thiabendazol (10 µM) inhibited growth quite effectively in both strains. When we stained the drug-treated AX2 cells for CP250 the protein was still present at the centrosome, although the microtubule network had been destroyed by the drug treatment (see Figure 10 for nocodazole-treated AX2 cells).
The Centrosome and the Nuclear Envelope in CP250-deficient Cells
Immunofluorescence analysis of the centrosome of the mutant cells by labeling with K29-359-31 showed that each nucleus was associated with one centrosome. Additional centrosomes were not observed. Also, the distance between the nucleus and the centrosome was not altered, and -tubulin and K29-359-10 behaved as in wild type during mitosis (Figure 8A). Similarly, the centrosomal marker DdCP224 was present at the centrosome in interphase and in mitotic cells, and we observed that the DdCP224 antibodies labeled the centrosome throughout the entire cell cycle as in AX2. During mitosis, spindle microtubules were also decorated, especially in the midbody region in anaphase and telophase as has been reported for AX2 cells previously (Figure 8B; Gräf et al., 2000).
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). In S. pombe the corresponding KASH- and SUN-domain–containing proteins are Kms2 and Sad1, and in Drosophila it is Klarsicht and Klaroid (Fridkin et al., 2009; Starr, 2009). In D. discoideum we have shown that the SUN-domain protein Sun-1 links the centrosome to the NE, whereas a role for the KASH-domain protein interaptin in this process could not be demonstrated. Also, Sun-1 and interaptin do not directly interact (Xiong et al., 2008).
We analyzed Sun-1 as well as interaptin and followed their localization during progression of the cell cycle in AX2 and mutant cells. We used GFP--tubulin expressing AX2 and CP250– cells and stained for interaptin using mAb 260-60-10 and for Sun-1 using mAb K55-432-2 and polyclonal antibodies. D. discoideum undergoes a closed mitosis. Interaptin stays at the NE during mitosis in AX2 cells. During anaphase, interaptin decorates the microtubules linking both asters. In the mutant cells, the interaptin distribution during mitosis followed essentially the pattern seen in wild-type cells, but overall interaptin staining at the NE was reduced, whereas the cytoplasmic staining increased indicative of a relocation to endoplasmic reticulum (ER) membranes. Also, staining of the microtubules linking the asters was less obvious (Figure 9A). Sun-1 antibodies strongly label the NE in a continuous manner in interphase in AX2 cells (Figures 9B and 10A; Xiong et al., 2008). During mitosis, Sun-1 stays at the NE, and the intensity of staining increases near the centrosome. In mutant interphase cells, Sun-1 exhibited a strongly reduced NE staining in comparison to AX2, and during mitosis a single spot near the centrosome was detected and nearly no NE stain was observed (Figures 9B and 10A). Western blot analysis revealed a reduction of Sun-1 protein levels in the mutant to 50% of wild-type levels, confirming the observations of the immunofluorescence analysis (Figure 9C).
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), we treated AX2 and CP250– cells with the microtubule-depolymerizing drug nocodazole (10 µM, 2 h). This led to a redistribution of Sun-1 at the NE and an enrichment of the protein near the centrosome in AX2. In CP250– cells, Sun-1 staining was unaffected by nocodazole treatment, and the protein stayed in its centrosome near position (Figure 10, A and B). The mechanism is not known; however, one proposal is that an intact and dynamic microtubule system maintains the distribution of Sun-1 at the NE and, further, that changes in the microtubule system or its dynamics as they occur during mitosis, drug treatment, or after changes in the corona lead to alterations in Sun-1 distribution.
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). Mutant cells are heterogeneous in cell size and have a cytokinesis defect exhibiting multiple nuclei. Here we stained for the centrosome using mAb K68-439-8. We found that CP250 staining was unaltered; however, CAPbsr showed significant abnormalities in centrosome number. Cells with one nucleus can have two or more centrosomes, and all multinucleated cells had extra centrosomes. In the latter case they were not associated with a nucleus (Figure 11C). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Using immunofluorescence analysis and immunoelectron microscopy, we localized the protein to the corona, which, as the equivalent of the PCM, is responsible for nucleation of microtubules. CP250 not only is a component of the corona, it also behaved like a component of the corona during the cell cycle. It stayed associated with the centrosome during interphase, disappeared at the transition from prophase to metaphase, a stage where the corona has dissociated from the central core, and reappeared at the end of late telophase (Ueda et al., 1999). In general, the known Dictyostelium centrosomal proteins relocate from the corona to the core during mitosis. Only for Dictyostelium centrin (DdCrp) a loss from the centrosome was reported at the prophase to metaphase transition, similar to CP250 (Daunderer et al., 2001).
Based on these findings, a role for CP250 in interphase is more likely as compared with a role in centrosome duplication and in mitosis. In human cells and in C. elegans an interference with specific PCM proteins affected the PCM and interfered with centriole formation (Keryer et al., 2003; Dammermann et al., 2004). In CP250-deficient cells we did not note defects in centrosome number and positioning when CP250 was absent. Also, the nuclear number is unaltered, indicating that the protein is most likely not involved in centrosome duplication.
We therefore focused on growth, chemotaxis, cell motility, and development that rely on an intact centrosome and microtubule system. CP250-deficient cells were smaller in size and had a disadvantage when grown on a lawn of bacteria. The cells developed and formed mature fruiting bodies that were however smaller in size than wild-type fruiting bodies. The reduced size of the fruiting bodies is probably the result of the smaller aggregation zones formed by the mutant cells. The basis of the growth and developmental defect may result from an altered chemotactic behavior. When we analyzed chemotactic motility toward cAMP, we found that the cells were slightly slower than wild type and migrated in a less directed manner toward the cAMP source. Also, the leading pseudopod seemed less stable and was retracted more frequently than in wild-type cells.
Recently a Dictyostelium mutant was described that lacks the Dictyostelium homolog of the Fused kinase. The mutant has defects in chemotaxis and development. The cells do not polarize perfectly and are impaired in chemotaxis. Furthermore, development is delayed and fruiting bodies are smaller than in wild type. In this work the kinase was located to microtubules and release of the kinase to the cytosol during microtubule depolymerization caused a loss in cell polarity underlining the importance of the microtubule system for cell polarity and chemotaxis (Tang et al., 2008).
Alterations were also noted in CP250– cells when we stained for the NE components Sun-1 and interaptin. Early on a physical association between centrosome and the NE has been suggested from electron microscopic studies (Bornens, 1977; Nadezhdina et al., 1979). However only recently have proteins been described that might mediate this physical association. In S. pombe the Sun domain–containing protein Sad-1 together with Kms1 (Miki et al., 2004) and in S. cerevisiae the Sun-domain protein Mps3 together with Mps2 provide a physical link to the spindle pole body (Jaspersen et al., 2006). In C. elegans Sun-1/matefin functions together with Zyg-12 to attach the centrosome to the NE (Malone et al., 2003). Zyg-12 and Kms1 are KASH-domain proteins and can interact with the SUN-domain proteins. In mammalian cells, SUN2 was detected at the centrosome and at the NE (Wang et al., 2006). We showed that Sun-1 mediates centrosome-nucleus connection in D. discoideum and that deletion of the Sun-1 N-terminus (GFP-
N-Sun-1) abrogated this close connection. Also, in cells expressing GFP-N-Sun-1 the centrosomes were often connected to the nucleus by a membrane bridge that was positive for the GFP fusion protein (Xiong et al., 2008).
CP250 might also be involved in the cross-talk between NE and centrosome as its loss led to strongly reduced amounts of Sun-1 and interaptin at the NE, although a direct interaction between these proteins is not likely. The residual amounts of Sun-1 concentrated near the centrosome where the protein might cluster and collect the telomeres as has been described in fission yeast and in mammals. It has been suggested that the prime function of Sun-1 during mitosis and meiosis is near the centrosome where it anchors telomeres, allowing correct distribution of the chromosomes (Schmitt et al., 2007; Ding et al., 2007). In accordance with this, we frequently observed aneuploidy in cells expressing GFP-N-Sun-1 (Xiong et al., 2008). A possible microtubule involvement in this process is indicated by the behavior of Sun-1 in nocodazole-treated wild-type cells where Sun-1 collects near the centrosome and does not show an overall staining of the NE as in control cells. We previously reported reduced amounts of Sun-1 using siRNAi that resulted in 40% reduction of Sun-1 (Xiong et al., 2008). In these cells we observed nuclear deformations, but the centrosomes were not affected as in cells that expressed a Sun-1 protein lacking the N-terminus. Normally the N-terminus retains Sun-1 in the inner nuclear membrane. When this location is abandoned, the cells show nuclear deformation, hyperamplification of centrosomes, and an altered centrosome–nucleus distance. These results suggested to us that the residual amounts of Sun-1 are sufficient to tether the chromatin to the centrosome and to maintain the centrosome nucleus connection and mitotic spindle stability, which is in agreement with the results presented here. The absence of nuclear abnormalities in CP250-deficient cells might be explained by the additional loss of one more protein, namely CP250.
In nocodazole-treated wild-type cells Sun-1 also accumulates in NE regions near the centrosome. Nocodazole treatment depolymerizes interphase microtubules very efficiently in Dictyostelium. One explanation for the observed changes could be that upon loss of the forces that originate in the cytoskeleton and act on the nucleus, the mechanical strength might be lost and the organization of the NE affected (Starr, 2007, 2009).
Interaptin staining of the NE was also reduced in CP250-deficient cells. In contrast to mammalian cells for which we reported a direct interaction between the C-terminus of SUN-1 with the KASH-domain of Nesprin-1/Enaptin and Nesprin-2/NUANCE (Padmakumar et al., 2005) such a direct interaction does not exist for D. discoideum interaptin and Sun-1. Instead, both proteins competed with each other for NE localization (Xiong et al., 2008). The findings here are consistent with the absence of an interaptin Sun-1 interaction.
Identification of CP250 as partner of CAP was rather unexpected as CAP has been described primarily as a cytoplasmic protein present throughout the cell and in regions of high actin dynamics. A clear location at the centrosome could not be demonstrated by immunofluorescence analysis of fixed cells so far. This does not, however, rule out a transient association. In fact, centrosomes can bind many regulatory components that involve the centrosome in a multitude of cellular functions (Doxsey et al., 2005). Among these were proteins that are involved in the regulation of cytoskeleton-related functions like beta-catenin (Bahmanyar et al., 2008) or integrin-linked kinase (Fielding et al., 2008). For the Dictyostelium Lis1 homologue an interaction with Rac1A in vitro was described through which its observed effects on actin dynamics could be explained (Rehberg et al., 2005). Another link between centrosome and the actin cytoskeleton is provided by the mammalian CENPJ, a protein located at the centrosome throughout mitosis, which associates with a splice variant of protein 4.1 (Hung et al., 2000). Such a connection might explain the phenotypes we observed in CP250-deficient cells that are similar to those frequently observed in cytoskeletal mutants and is supported by the altered actin distribution with increased cytoplasmic levels.
Interestingly, mammalian CAP2 was identified as a protein present in the cytoplasm as well as in the nucleus (Peche et al., 2007). Furthermore, we have described a cytokinesis defect in D. discoideum cells deficient for CAP (CAPbsr). Forty-four percent of mutant cells in comparison to 17% of AX2 cells have multiple nuclei (Noegel et al., 1999). We now explored this phenotype and investigated the centrosome by expressing GFP--tubulin and staining for CP250. In normal-sized mononucleated CAPbsr cells CP250 distribution was unaltered, and the centrosomes were closely associated with the nuclei in mono- and binucleated cells. In contrast, multinucleated cells had additional centrosomes that were freely present in the cytoplasm. Frequently the cells displayed abnormal nuclei that were larger than normal and had different shapes. The association between CAP and CP250 could also be responsible for the defects in cell polarity and development that are observed in both cell lines (Noegel et al., 1999; 2004; this work). Both the actin and the microtubule cytoskeleton have been implicated in the generation of cell polarity, and it might well be that both systems converge at this point (Azimzadeh and Bornens, 2007). More recently, Srv2, the CAP homologue in S. cerevisiae, was identified in complex with Nur1 (Ydf089w), a component of the inner nuclear membrane (Mekhail et al., 2008).
In summary, we have identified a component of the corona that affects cell growth and chemotactic motility and is involved in the interaction with the NE. CP250 is present at the centrosome during interphase; however, it disappears during mitosis. We narrowed down the region required for centrosomal association and correct behavior during mitosis to a 46-kDa polypeptide located in the first half of the protein. Loss of CP250 does not interfere with the role of the centrosome as MTOC and for formation of an intact microtubule network nor does it affect centrosome duplication. In this respect it resembles several pericentriolar proteins in higher eukaryotes such as PCM-1 and pericentrin, which appear to be dispensable for centrosome duplication (Balczon et al., 1994; Kleylein-Sohn et al., 2007). By contrast, loss of CP250 leads to disturbance of the NE composition and affects two major proteins of the NE, Sun-1 and interaptin, and causes a reduction of their amounts at the NE. How this is achieved is presently unclear. It might well be that the MTOC as microtubule organizer is not functioning correctly in the mutant. Furthermore, we know that Sun-1 provides a link between centrosome and nucleus. This interaction appears to be altered in the mutant leading to an altered distribution of Sun-1.
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Acknowledgments |
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Footnotes |
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Present address: Department of Pathology and Laboratory Medicine, UMDNJ–Robert Wood Johnson Medical School, 675 Hoes Lane, R231, Piscataway, NJ 08854. 
Address correspondence to: Angelika A. Noegel (noegel{at}uni-koeln.de).
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