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Address correspondence to Ryoko Kuriyama, Dept. of Genetics, Cell Biology, and Development, 6-160 Jackson Hall, 321 Church St. SE, University of Minnesota, Minneapolis, MN 55455. Tel.: (612) 624-0471. Fax: (612) 626-6140. email: ryoko{at}lenti.med.umn.edu; or Yasuhiko Terada, Dept. of Genetics, Cell Biology, and Development, 6-160 Jackson Hall, 321 Church St. SE, University of Minnesota, Minneapolis, MN 55455. Tel.: (612) 626-4089. Fax: (612) 626-6140. email: terad002{at}umn.edu
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Abstract |
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Key Words: centrosomes; -tubulin; microtubule nucleation; mitotic spindle; MTOC
Abbreviations used in this paper: CNN, centrosomin;
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Introduction |
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Results and discussion |
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To confirm the microtubule-nucleating activity of the CNN aggregates, we polymerized microtubules in vitro by incubating isolated GFP-tagged CNN dots with X-rhodamineconjugated brain tubulin. Fig. 4 (AD) shows microtubule asters detected by phase-contrast and fluorescence microscopy. There is always a dot positive in GFP fluorescence at the center of the microtubule asters. Although variable numbers of microtubules emanated from the center, more microtubules tended to polymerize onto the GFP dots in larger sizes (Fig. 4, BD). In Fig. 4 E, the process of aster formation was monitored by time-lapse microscopy. A fluorescence image taken 10 min after mounting the sample on a microscopic stage revealed several microtubules growing from a GFP-positive site. As time progressed, more microtubules appeared to emanate from the center, indicating that microtubules were formed by direct polymerization onto the CNN-containing foci, rather than that preformed microtubules were gathered around the center.
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Multiple centrosomes/MTOCs have been detected in cells in which the mechanism of centrosome duplication coupled with the cell cycle control becomes deregulated (Hinchcliffe and Sluder, 2001). In the case of CNN-containing MTOCs, their number and size formed during relatively short periods (812 h) varied greatly according to the level of protein expression. Moreover, no centrioles were found at ectopic MTOCs by EM (Fig. 4 F) and immunostaining with centriole-specific centrin-2 antibodies (Fig. 4 G, arrows). Therefore, it is plausible that CNN expression causes the formation of protein aggregates that acquire the microtubule-nucleating capacity by recruiting -tubulin/
-TuRC. This unique property of CNN to generate microtubule-nucleating sites by interacting with
-tubulin/
-TuRC allowed us to speculate that CNN may function as an adaptor for connecting
-tubulin to the centrosome.
By expressing truncated polypeptides, it was concluded that CNN's ability to interact with -tubulin/
-TuRC and induce ectopic microtubule-nucleating sites resides in the NH2-terminal sequence of CNN from which the Aurora-Abinding domain is omitted. In contrast, cytoplasmic aggregates formed in cells expressing the COOH-terminal domain failed to initiate microtubule formation in both S2 and mammalian cells (unpublished data). These results lead us to conclude that CNN consists of two functionally distinct subdomains: the Aurora-Abinding site is at the COOH terminus capable of formation of the protein complex to be recruited to the spindle pole, and the NH2-terminal sequence is involved in assembling centrosomes/MTOCs by recruiting
-tubulin/
-TuRC. Although no CNN homologues have yet been identified outside Drosophila, Aurora-A would likely be involved in the control of microtubule nucleation through its association with the COOH terminus of a CNN-related molecule(s) in mammalian cells.
Control of mitotic spindle assembly onto the centrosome could be achieved by several mechanisms, including nucleation of individual microtubules onto -tubulincontaining protein complexes (Zheng et al., 1995), stimulation of microtubule nucleation and stabilization of polymerized microtubules by MAPs (Popov et al., 2002), and recruitment of minus ends of preexisting microtubules by the action of motor activity to the centrosome (Heald et al., 1997). Aurora-A binds not only CNN but also the D-TACC/MSPS/XMAP215 complex (Giet et al., 2002) and a spindle component of TPX2 (Kufer et al., 2002). These components appear to be required for microtubule assembly on mitotic centrosomes/poles controlled through the distinct mechanisms from that of
-tubulin recruitment. Therefore, it is reasonable that Aurora-A plays a role in regulating the overall process of centrosome maturation by orchestrating multiple pathways of microtubule assembly during mitosis. It is worth mentioning that individual mechanisms of microtubule assembly may show a distinct requirement for protein phosphorylation and the Aurora-A kinase activity; although both Aurora-A and CNN were still able to locate at the centrosome, D-TACC/MSPS complex failed to be recruited to spindle poles in the absence of enzymatic activity of Aurora-A kinase (Giet et al., 2002).
Aurora kinases are highly expressed in cells derived from many human tumor cell types (Bischoff et al., 1998; Tatsuka et al., 1998; Zhou et al., 1998), which frequently contain multiple centrosomes. Because defects in the number, structures, and function of centrosomes are closely associated with the genetic instability in transformed cells (Nigg, 2002), Aurora-A might be involved in tumorigenesis by inducing abnormal numbers of MTOCs as a result of inappropriate distribution of CNN-like molecule(s).
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Materials and methods |
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RNAi in S2 cells
cDNAs of Aurora-A and CNN were amplified by PCR using primers as follows: Aurora-A sense, 5'-TAATACGACTCACTATAGGATGTCCCATCCGTCTGACCA-3' and antisense 5'-TAATACGACTCACTATAGGAGTTGTTGAGCTCCCAGGTC-3'; CNN sense, 5'-TAATACGACTCACTATAGGGAAACAGCTGTTCCAGCGCGCTTCATTTTC-3' and antisense 5'-TAATACG-ACTCACTATAGGCACCAGTTCCTCCTCCAATCTCTGCACCTC-3'. dsRNAs were synthesized using the MEGAscriptTM T7 transcription kit (Ambion), denatured for 20 min at 94°C, and were then annealed at RT overnight. dsRNA samples were run on 1% agarose gels to ensure that they migrated as a single band.
For RNAi, S2-R cells (a gift from Dr. F. Kafatos, Harvard University, Cambridge, MA) were transferred into 6-well plates 1 d before transfection. This particular cell line appears to yield high efficiency of protein depletion by RNAi. 20 µg dsRNA was incubated with 40 µl of 4 mg/ml dimethyl dioctadecyl ammonium bromide and 80 µl M3 insect medium (Sigma-Aldrich). After transfection, cells were incubated for 3 or 4 d before fixation.
Transfection and immunofluorescence staining
The entire coding sequences of Aurora-A and CNN were cloned by PCR from a Drosophila embryo cDNA library (a gift from Dr. F. Kafatos) and ligated into expression vectors of pcDNA3 (Invitrogen) and pFBDA (a gift from Dr. A. Straight, Harvard Medical School, Boston, MA), respectively. 1.5 µg purified plasmid DNA was mixed with FuGENETM 6 (Roche Diagnostics), and added to S2 cells in a 3.5-cm dish cultured in 10% FCS containing Schneider's Drosophila medium. Cells were further cultured up to 3 d before fixing with cold methanol. CHO and 293 cells were grown as monolayers and transfected with CNN/-tubulin constructs as described before (Kofron et al., 1998; Ohta et al., 2002).
For immunostaining, fixed cells were rehydrated with 0.05% Tween 20 containing PBS, and were incubated at 37°C for 30 min with primary antibodies as follows: polyclonal anti-centrosomin (a gift from Dr. W. Theurkauf, University of Massachusetts Medical School, Worcester, MA), polyclonal anti-Drosophila Aurora-A (a gift from Dr. J. Knoblich, Research Institute of Molecular Pathology, Vienna, Austria), polyclonal antihuman Aurora-A (a gift from Dr. M. Kimura, Gifu University, Gifu, Japan), monoclonal anti--tubulin (Sigma-Aldrich), monoclonal anti-
-tubulin (Sigma-Aldrich), monoclonal rat anti-HA (Roche Diagnostics), and polyclonal anti-HA (Santa Cruz Biotechnology, Inc.). Antibodies specific to CP60, CP190 (gifts from Dr. M. Moritz, University of California, San Francisco, San Francisco, CA), pericentrin (Covalence), human centrin-2 (a gift from Dr. J. Salisbury, Mayo Clinic Foundation, Rochester, MN), and Cep135 (Ohta et al., 2002) were used to probe additional centrosomal components.
Protein-binding assays
S2 cells expressing HA-tagged CNN were lysed in TBSN (20 mM Tris-HCl at pH 8.0, 150 mM NaCl, 1.5 mM EDTA, 5 mM EGTA, and 0.5% Nonidet P-40) containing protease inhibitors. The cytosolic fraction recovered after centrifugation at 10,000 g for 20 min was incubated with polyclonal anti-HA antibody for 1 h at 4°C followed by incubation with Protein Aconjugated Sepharose beads for 3 h. Beads were subsequently washed three times with TBSN, and boiled in an SDS-containing sample buffer. Endogenous kinases were detected by immunoblot analysis using monospecific anti-Aurora-A and anti-Aurora-B antibodies (Aurora-B antibody was a gift from Dr. M. Carmena, Wellcome Center for Cell Biology, Edinburgh, UK).
For identification of the Aurora-Abinding domain of CNN, 35S-labeled full (F), NH2-terminal (N), and COOH-terminal (C) sequences of CNN were synthesized in vitro using a TNT T7/T3-coupled reticulocyte lysates system kit (Promega). The full-length Drosophila Aurora-A was subcloned into pREST B (CLONTECH Laboratories, Inc.), and T7/His6-tagged protein was expressed in the Escherichia coli strain BL21DE3 (Novagen) at 30°C for 1 h. After purification through nickel Sepharose beads according to the manufacturer's instructions, Aurora-A fusion proteins were mixed with HA-tagged CNN polypeptides in a buffer (50 mM Hepes at pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1% Tween 20, and protease inhibitors), and were further incubated overnight at 4°C in the presence of anti-T7 antibodies (Novagen).
To assay the -tubulinbinding activity of CNN, His-tagged CNN fusion proteins corresponding to F, N, and C in Fig. 1 A were first prepared in bacteria. After conjugation with nickel agarose, beads were incubated with extracts of mitotic S2 and CHO cells prepared in TBSN as above.
-Tubulin sedimented with nickel beads was detected by immunoblot analysis.
In vitro microtubule nucleation
CHO cells expressing full-length GFP-CNN were lysed in a medium containing 2 mM Pipes, pH 6.8, and 0.25% Triton X-100, and centrifuged at 1,500 rpm for 2 min. CNN-containing aggregates recovered in a post-nuclear fraction were sedimented on a sucrose cushion at 10,000 g for 20 min and resuspended in PEMG (100 mM Pipes, pH 6.8, 1 mM EGTA, 1 mM MgCl2, and 1 mM GTP). Next, the sample was mixed with purified bovine brain tubulin supplemented with X-rhodamineconjugated tubulin (Sammak and Borisy, 1988), and microtubule polymerization was monitored by fluorescence microscopy as described previously (Tournebize et al., 1997). Microtubule formation was also assayed by phase-contrast microscopy using the procedure described previously (Sellitto and Kuriyama, 1988).
EM
Transfected CHO cells were cultured for 812 h to express GFP-tagged full-length CNN. After incubation with 2 µg/ml nocodazole for 2 h, cells were briefly recovered from drug treatment, and were extracted in PEMG containing 0.1% Triton X-100 and 10% glycerol for 30 s at 37°C before fixation with 2% glutaraldehyde. Positions of GFP-positive cells were identified by fluorescence microscopy, and the sample was postfixed with 1% OsO4, embedded in Epon-Araldite according to the standard procedures.
Online supplemental material
Fig. S1 shows that mammalian Aurora-A (Aik; arrows) does not colocalize with the full-coding (CNN-F), COOH-terminal (CNN-C), and NH2-terminal (CNN-N) sequence of Drosophila CNN expressed in mammalian U-2OS cells. In Fig. S2, CNN-F was expressed in human cells from which Aik was depleted by RNAi. After monitoring immunostaining pattern of Aik and CNN, the same cells were further stained with anti--tubulin antibody. The CNN aggregates retained their capacity to nucleate microtubules in vivo in the absence of mammalian Aurora-A. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200305048/DC1.
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health (GM55735) and the Minnesota Medical Foundation to R. Kuriyama, and from the Uehara Memorial Foundation to Y. Terada.
Submitted: 12 May 2003
Accepted: 14 July 2003
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