EUKARYOTIC CELL, May 2008, p. 894–905 Vol. 7, No. 51535-9778/08/$08.00�0 doi:10.1128/EC.00422-07Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Dictyostelium Aurora Kinase Has Properties of both Aurora A andAurora B Kinases�†

Hui Li,1 Qian Chen,1‡ Markus Kaller,2 Wolfgang Nellen,2 Ralph Graf,3 and Arturo De Lozanne1*Section of Molecular Cell & Developmental Biology and Institute for Cellular and Molecular Biology, University of Texas at Austin,Austin, Texas 787121; Abt. Genetik, Universitat Kassel, Heinrich-Plett-Str. 40, 34132 Kassel, Germany2; and Universitat Potsdam,

Institut fur Biochemie und Biologie, Zellbiologie, Karl-Liebknecht-Str. 24-25, Haus 26, 14476 Potsdam-Golm, Germany3

Received 19 November 2007/Accepted 27 February 2008

Aurora kinases are highly conserved proteins with important roles in mitosis. Metazoans contain twokinases, Aurora A and B, which contribute distinct functions at the spindle poles and the equatorial regionrespectively. It is not currently known whether the specialized functions of the two kinases arose after theirduplication in animal cells or were already present in their ancestral kinase. We show that Dictyosteliumdiscoideum contains a single Aurora kinase, DdAurora, that displays characteristics of both Aurora A and B.Like Aurora A, DdAurora has an extended N-terminal domain with an A-box sequence and localizes at thespindle poles during early mitosis. Like Aurora B, DdAurora binds to its partner DdINCENP and localizes oncentromeres at metaphase, the central spindle during anaphase, and the cleavage furrow at the end ofcytokinesis. DdAurora also has several unusual properties. DdAurora remains associated with centromeres inanaphase, and this association does not require an interaction with DdINCENP. DdAurora then localizes atthe cleavage furrow, but only at the end of cytokinesis. This localization is dependent on DdINCENP and themotor proteins Kif12 and myosin II. Thus, DdAurora may represent the ancestral kinase that gave rise to thedifferent Aurora kinases in animals and also those in other organisms.

Universal to all eukaryotes, Aurora kinases are known tofunction during multiple stages of cell division. The activity ofAurora kinases regulates almost every crucial stage of Mphase, from chromosome condensation and separation to thevery end of cytokinesis (10). Most animal cells contain tworelated kinases, Aurora A and B, which have distinct localiza-tions and specialized roles during mitosis (1). Aurora A islocalized primarily at the spindle poles and is implicated in theregulation of entry into mitosis, centrosome maturation, andspindle assembly (3, 28, 52). Aurora B forms the well-knownchromosome passenger complex together with INCENP, sur-vivin, and borealin (60). This complex associates with centro-meres early in mitosis and then redistributes to the centralspindle at the metaphase/anaphase transition (2, 53, 57). Ac-cordingly, Aurora B is required for correct chromosome seg-regation and cytokinesis (31). Vertebrates contain an addi-tional Aurora C kinase that is expressed exclusively in thetestis, where it may play a role similar to that of Aurora B (35,40). In contrast to animals, yeast cells contain only one Aurorakinase (Ipl1 in budding yeast and Ark1 in fission yeast) (12,48). The yeast kinases have been shown to be mainly involvedin chromosome segregation and cytokinesis (22, 39, 45, 47),processes that are usually regulated by animal Aurora B ki-nase. Similarly to animal Aurora B, yeast Auroras also associ-

ate with homologues of INCENP and survivin and localize tothe centromeres and spindle (9, 34, 51). Unlike animal AuroraA, Ark1 mutant cells exhibited only a minor defect in spindleformation and Ark1 kinase does not seem to be localized onthe spindle pole body (48).

To determine the separate roles of Aurora A and B kinases,it is important to consider their evolutionary origin. The highsequence similarity between these proteins suggests that theyarose by gene duplication. Phylogenetic analysis suggests thatthe duplication of Aurora genes has occurred independently invertebrates, invertebrates, and plants (8). However, it is notclear whether the original ancestral kinase had the propertiesand functions of Aurora A, Aurora B, or both. Given that, bymost criteria, the yeast Aurora kinases resemble Aurora B, itwould appear that the specialized features of Aurora A mayhave been a subsequent invention in animal cells after theduplication of the ancestral Aurora gene. Since fungi have aclosed mitosis and a reduced set of spindle and cytoplasmicmicrotubules, it is possible that yeasts may have dispensed withthe specialized activity of an Aurora A kinase. On the otherhand, only the kinase domain of human Aurora A, but not ofAurora B, can partially complement the function of Saccharo-myces cerevisiae Ipl1 when fused to the N terminus of Ipl1 (5).To better understand the similarities and differences amongdifferent Aurora kinases and to better appreciate what paral-lels can be drawn among them, it is important to analyze thefunction of Aurora kinases in other eukaryotes.

Other eukaryotes in which Aurora kinases have been iden-tified include plants and the parasite trypanosomes, but thefunction of these kinases has not yet been characterized ingreat detail. In higher plants, Aurora kinases have diversifiedinto two major groups, alpha and beta, that are not related tothe animal Aurora A and B kinase groups (18). Both types of

* Corresponding author. Mailing address: Molecular Cell and De-velopmental Biology, University of Texas at Austin, 1 University Sta-tion C-1000, Austin TX 78712. Phone: (512) 232-6100. Fax: (512)471-3878. E-mail: a.delozanne@mail.utexas.edu.

‡ Present address: Department of Molecular, Cellular and Develop-mental Biology, Yale University, New Haven, CT 06511.

† Supplemental material for this article may be found at http://ec.asm.org/.

� Published ahead of print on 7 March 2008.

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plant Aurora proteins localize on centromeres and spindlefibers and do not appear to be required for spindle formation(18). Given that plant mitotic spindles do not have centro-somes, it would seem reasonable to postulate that plants maynot need an Aurora A-like kinase. In trypanosomes, the Au-rora kinases have diversified into three closely related proteinsthat seem most similar in sequence and function to animalAurora B (58). However, no homologues of INCENP, survivin,or borealin seem to be present in the genomes of any plants ortrypanosomes. Therefore, it is not clear whether the mecha-nism of regulation of Aurora kinases in these organisms sharesany similarity to that in animal cells.

Dictyostelium is an excellent model system to study mitosisand cytokinesis in eukaryotic cells. In addition to the manymolecular tools available to study these cells, the morphologyof their mitotic apparatus is amenable to detailed microscopyanalysis (43, 44, 50). Dictyostelium cells contain relatively fewinterphase microtubules that are rearranged into a well-de-fined intranuclear spindle during mitosis. A large effort toidentify and characterize the function of centrosomal proteinsis well under way (25, 49), and a large collection of mutantsthat affect mitosis or cytokinesis have been gathered over theyears (16, 20, 36, 38, 46). Most importantly, Dictyostelium dis-coideum contains a protein homologous to animal INCENP,the activating partner of Aurora B (15). This protein, DdIN-CENP, displays the dynamic behavior of chromosomal passen-ger proteins and is important for mitosis and cytokinesis. Sinceanalysis of the Dictyostelium genome suggests that this organ-ism has retained many of the ancestral properties of the lastcommon ancestor of plants, animals, and fungi (19), it is anideal system to dissect the original properties of Aurora ki-nases. Our findings presented here demonstrate that the singleDictyostelium Aurora kinase displays properties found in bothAurora A and B kinases and thus suggest that the ancestralAurora kinase played both roles in primitive cells.

MATERIALS AND METHODS

Cloning of DdAurora and construction of GFP-DdAurora. A 1.6-kb sequencecorresponding to the full length of DdAurora gene was cloned from Dictyoste-lium genomic DNA by using the 5� and 3� primers AO-499 (5�-CGAGCTCATGAGTTATCCAAATAATAAAGAAAATAGTAACAATATTGGTG-3�) andAO-500 (5�-CGGATCCTTAATAAGTCATTTGAGATGGTAATGGAAGACCC-3�), respectively. The full-length DdAurora was further cloned into pTX-GFP (green fluorescent protein) vector.

Cell culture and transformation. Dictyostelium discoideum AX2 (wild-type)cells were grown in axenic HL-5 medium supplemented with penicillin andstreptomycin at 19°C. DdINCENP-null cells were grown with additional 5 �g/mlblasticidin S hydrochloride. Cells carrying pTX-based plasmids were grown inmedium with additional 10 �g/ml G418. For stationary culture, cells were grownin plastic petri dishes. Cells in suspension culture were grown in conical flasks ona rotating shaker at �200 rpm. The constructs were introduced into cells withelectroporation, and the transformants were selected in the HL-5 medium with10 �g/ml G418.

Fluorescence microscopy of live cells. For live microscopy of mitotic cells, cellsin active log-phase growth were first resuspended to a concentration of 2 � 106

cells/ml. Cell suspension (0.5 ml) was plated on a small petri dish with a coverslipmounted at the bottom (MatTek, Ashland, MA). After cells attached to thecoverslip, HL-5 medium was removed and replaced with low-fluorescence me-dium for at least 20 min before observation (7). The live imaging of the cells wasconducted by using a Nikon Eclipse TE200 microscope (Nikon Instruments,Dallas, TX) equipped with a �100 1.4 NA PlanFluor objective, shuttered illu-mination, and a Quantix 57 camera (Roper Scientific, Tucson, AZ) controlled byMetamorph (Universal Imaging Corp., West Chester, PA). The exposure time

for the GFP fluorescence was 100 ms or less, with the interval time being at least10 s.

Immunostaining and microscopy of mitotic cells. For immunostaining of mi-totic cells, cells in active log-phase growth were harvested from stationary cultureand resuspended to a concentration of 2 � 106 cells/ml. Cell suspension (200 �l)was put on each coverslip and allowed to sit still for at least 20 min. For fixation,cells were first fixed in 1� PDF buffer (20 mM KCl, 11 mM K2HPO4, 13.2 mMKH2PO4, 1 mM CaCl2, 2.5 mM MgSO4 [pH 6.4]) with 2% formaldehyde and0.01% Triton X-100 for 15 min at room temperature and then in dehydratedmethanol with 1% formaldehyde at �20°C for 5 min. The primary antibody wasaffinity-purified anti-DdAurora or monoclonal anti-DdCP224. The second anti-body was Texas red-conjugated goat anti-rabbit or fluorescein isothiocyanate-conjugated goat anti-mouse antibody (Molecular Probes, Eugene, OR). ForDAPI (4�,6�-diamidino-2-phenylindole) staining of DNA, coverslips were im-mersed in 1� PDF buffer with 0.1 �g/ml DAPI for 10 min before the final washstep.

Antibody generation and affinity purification. A full-length cDNA of DdAu-rora was cloned from Dictyostelium cDNA library (Stratagene, La Jolla, CA) byusing the 5� and 3� primers AO-554 (5�-CGAATTCATGAGTTATCCAAATAATAAAGAAAATAGTAACAATATTGGTG-3�) and AO-500 (5�-CGGATCCTTAATAAGTCATTTGAGATGGTAATGGAAGACCC-3�), respectively.The PCR product was further cloned into the pMal-c2X vector (New EnglandBiolabs, Ipswich, MA). A maltose-binding protein (MBP)–DdAurora fusionprotein was expressed in Escherichia coli and purified according to the providedprotocol. Five milliliters of amylose resin (New England Biolabs, Ipswich, MA)was used for 1 liter of Escherichia coli culture. The purified fusion protein wasinjected into rabbits to raise polyclonal anti-DdAurora antibodies (CocalicoBiologicals, Reamstown, PA). To purify polyclonal anti-DdAurora antibodies byaffinity binding, purified MBP-DdAurora was further cross-linked to AminoLinkcoupling gel (Pierce, Rockford, IL) according to the provided protocol. Tandem-affinity purification (TAP)-tagged GFP was purified from Dictyostelium cell cul-ture and used to generate polyclonal anti-GFP antibodies by the same procedure.

Affinity purification of polyclonal anti-DdAurora antibodies. One milliliter ofAminoLink coupling gel (Pierce, Rockford, IL) cross-linked with more than 10mg of MBP-DdAurora was used to purify polyclonal anti-DdAurora antibodiesfrom 2 ml of crude serum. The crude serum was first precleaned by centrifuga-tion at 13,000 rpm in a desktop centrifuge for 10 min. The supernatant wasdiluted to 10 ml with 0.1 M sodium phosphate buffer (pH 7.4) and incubated withprewashed MBP-DdAurora gel for 2 h at 4°C in a 15-ml Poly-Prep chromatog-raphy column (Bio-Rad, Hercules, CA). The antibody-gel mixture was thenpacked by gravity in the column and washed extensively with 0.1 M sodiumphosphate buffer. Bound anti-DdAurora antibodies were finally eluted by 100mM glycine (pH 2.5) and equilibrated to pH 7.4 immediately with 1 M Tris-HCl(pH 8.0).

Coimmunoprecipitation with DdAurora antibodies. Affinity-purified poly-clonal anti-DdAurora antibodies were used in all immunoprecipitation experi-ments. Cells expressing target GFP-fusion protein were cultured in suspension ina 250-ml flask. When the concentration reached �2 � 106 cells/ml, activelydividing cells are harvested by centrifugation at 1,500 rpm for 5 min. They werewashed in 1� PDF buffer once and then resuspended in 3 ml of 0.1 M sodiumphosphate buffer (pH 7.4) with fresh addition of 1:100 protease inhibitor cocktail(Sigma-Aldrich, St. Louis, MO) and 1 mM dithiothreitol. Cells were lysed bysonication six times for 15 s each with a 15-s rest in between. The lysate was thencentrifuged at 13,000 rpm for 15 min at 4°C twice to clean up cell debris. Fortymicroliters of purified anti-DdAurora antibodies was added to 1 ml of superna-tant after centrifugation. Either unspecific rabbit immunoglobulin G or no ad-dition of antibody was used as a negative control. The mixture was rotated at 4°Cfor 1 h, and then 20 �l of prewashed protein A beads (Amersham Pharmacia,Piscataway, NJ) was added to the suspension. After another 30-min incubation,the antibody-protein A complex was spun down at 3,000 rpm for 1 min andwashed by 6� 1 ml 0.1 M sodium phosphate buffer. Finally, bound proteins wereeluted by boiling the protein-bead complex in 100 �l SDS-sample buffer at 95°Cfor 10 min. The elutions were resolved on a 10% polyacrylamide gel and exam-ined by Western blot analysis with anti-GFP antibodies.

In vitro pull-down with MBP-DdAurora. Because different DdINCENP trun-cations have very different expression levels in DdINCENP-null cells, an in vitroassay was used instead of coimmunoprecipitation to keep the amount of DdAu-rora input at the same level. Cells were cultured and harvested in the same wayas that of the coimmunoprecipitation experiment. After sonication, GFP-DdIN-CENP-�C lysate was diluted 10 times before centrifugation. By this dilution, theinput levels of GFP-fusion proteins were adjusted to the same level too. Aftercentrifugation, 200 �l of MBP-DdAurora-saturated amylose beads was added tothe supernatant and incubated at 4°C for 2 h. After extensive wash, the bound

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proteins were boiled off from the beads by 100 �l SDS-sample buffer. Theelutions were also resolved on a 10% polyacrylamide gel and examined byWestern blot analysis with anti-GFP antibodies.

RESULTS

Dictyostelium has a single protein with similarities to bothAurora A and Aurora B kinases. A search of the Dictyosteliumgenome database with Aurora kinase sequences from otherorganisms yielded only one related gene, which has beennamed aurK (entry DDB0216254 in http://dictybase.org;GenBank accession no. XP_641803). The sequence of the aurKgene encodes a serine-threonine protein kinase of 43 kDa thathas a high degree of sequence similarity to Aurora kinasesfrom other organisms, including both Aurora A and Aurora Bkinases (Fig. 1). Therefore, we will refer to this protein simplyas Dictyostelium Aurora, or DdAurora.

DdAurora contains the signature motifs found in all Aurorakinases, including a KEN box at its N terminus, the activationloop in the catalytic domain (DFGWSXXXXXXXRXTXCGTXDYLPPE), and a D2-type destruction box (LLXXXPXXRXXLXXXXXHPW), near its C terminus (11, 13, 54). In ad-dition, DdAurora has a potential A box (PSXXXQRVXXQ)near its N terminus, which is a specific feature found in verte-brate Aurora A kinases. The A box has been shown to beimportant to regulate the Cdh1-dependent destruction of Au-rora A. Phosphorylation of the consensus serine site (in bold-face) in this motif is able to block destruction of Aurora A (41).

To explore the relationship between DdAurora and Aurorakinases from other organisms, we constructed a phylogenetictree with protein sequences from many organisms (Fig. 2).

Vertebrate proteins group into three distinct clades: the knownAurora A, B, and C kinases (8). Plant proteins form two clades,the Aurora alpha and beta kinase groups. The Aurora proteinsfrom different fungi also form a clearly defined group. TheDictyostelium kinase branches off near the base of the animaland plant groups as it is about equally similar to the animal,plant, and fungus kinases (57.5% to human Aurora A, 56% tohuman Aurora B, and 61.5% to Arabidopsis ATAur-1 and 47.3to budding yeast Ipl1). The protein most similar to DdAurorais an uncharacterized Aurora protein from the cycad Cycasrumphii (71.2%). Since it is generally thought that Dictyoste-lium diverged early in the evolution of eukaryotes, our analysissuggests that the archetype Aurora kinase in the commonancestor of plants, fungi, and animals already had characteris-tics of both Aurora A and Aurora B kinases. Importantly, thiskinase had the A-box signature of Aurora A kinases.

Dictyostelium Aurora has the dynamic distribution of bothAurora A and Aurora B kinases. To determine whether DdAu-rora behaves like Aurora A or Aurora B, we examined thedynamic localization of GFP-tagged DdAurora in live Dictyo-stelium cells. GFP-DdAurora accumulated to levels similar tothat of the endogenous protein and did not affect the growth ofDictyostelium cells (see Fig. S1 in the supplemental material).In interphase, GFP-DdAurora was diffuse throughout the en-tire cytoplasm, with no discernible accumulation in the nucleus(Fig. 3a). When cells entered mitosis, GFP-DdAurora wasreadily detectable at spindle poles during prometaphase (Fig.3b). GFP-DdAurora formed bright foci at both spindle poles,making it easy to identify mitotic cells among many interphasecells. Subsequently, GFP-DdAurora was visible at the meta-

FIG. 1. Alignment of DdAurora with Aurora kinases from other organisms. DdAurora has high sequence similarity to both Aurora A andAurora B kinases. DdAurora has the KEN sequence commonly found at the N terminus of Aurora kinases. DdAurora also has an N-terminal Abox (PSXXXQRVXXQ; framed in dashed box), which is important for Aurora A destruction. Within the A box, the conserved phosphorylationsite, serine 22 (boldface in the A-box sequence [asterisk on the figure]) is known to be phosphorylated in vertebrates to block Cdh1-dependentdestruction of Aurora A (41). DdAurora also contains the Aurora signature motif (DFGWSXXXXXXXRXTXCGTXDYLPPE) within theactivation loop (framed) and a D box (LLXXXPXXRXXLXXXXXHPW) near its C terminus (underlined in the figure). All Aurora kinases havethese two motifs to regulate their activation and degradation. H.s., Homo sapiens; X.l. Xenopus laevis; S.c. Saccharomyces cerevisiae.

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phase plate, probably in association with centromeres (Fig. 3c).With the onset of anaphase, GFP-DdAurora localized at thecentral spindle and remained there during telophase and earlycytokinesis (Fig. 3d to f). The Dictyostelium spindle is known todismantle during late telophase or early cytokinesis (50). Ac-cordingly, we observed that GFP-DdAurora disappeared fromthe region of the central spindle during early cytokinesis (Fig.3f). At the end of cytokinesis, GFP-DdAurora localized at thecytoplasmic bridge that connects the two daughter cells (Fig.3g). When this bridge finally severed, GFP-Aurora persisted atthe breaking point for a short time (Fig. 3i and see Movies S1and S2 in the supplemental material). From anaphase to theend of cytokinesis, GFP-DdAurora could also be constantlyobserved near the spindle pole regions (Fig. 3d to f; not shownin panels g to i because of the focus plane).

Our observations indicate that DdAurora displays the dis-tribution of both Aurora A (polar localization) and Aurora B(central spindle localization). DdAurora also has novel prop-erties not previously reported for any other Aurora kinase(localization at the cytoplasmic bridge in the absence of mid-body microtubules). As expected for a protein participating inmultiple steps during mitosis, we were not able to generate aknockout of the DdAurora gene (data not shown).

Polar localization of DdAurora. To investigate the localiza-tion of DdAurora in greater detail, we raised polyclonal anti-bodies against DdAurora. The anti-DdAurora antibodies spe-cifically recognized DdAurora as a single band in Western blotanalysis of whole-cell lysates (see Fig. S1 in the supplementalmaterial). Immunofluorescence microscopy with affinity-puri-fied anti-DdAurora antibodies revealed a distribution of en-dogenous DdAurora similar to that observed with GFP-DdAu-rora (Fig. 4). The only difference between GFP-DdAurora andendogenous DdAurora distribution was their relative intensityon centromeres during prometaphase and metaphase plate.Endogenous DdAurora appeared brighter on centromeresthan at the poles (Fig. 3a to b), whereas GFP-DdAurora wasbrighter at the poles than on centromeres (Fig. 4b and c). Thismight indicate a weaker association of GFP-DdAurora withthe centromeres at these stages or a stronger association withthe poles.

We then used double-immunofluorescence microscopy todetermine the localization of DdAurora in relation to that ofthe centrosomal protein DdCP224 (24). In Dictyostelium, thenuclear envelope is not dismantled during mitosis (44). In-stead, the centrosome duplicates and becomes embedded inthe nuclear envelope early in mitosis (59). The embedded

FIG. 2. Phylogenetic tree of Aurora kinases from different species. The core kinase domains of Aurora sequences from the indicated specieswere aligned by ClustalW, and the corresponding phylogenic tree was generated using MegAlign. Aurora kinases in vertebrates are divided intothree groups of Aurora A, B, and C subfamilies. Plants have two groups of Aurora kinases: the alpha and beta subfamilies. Most fungi have a singleAurora kinase, similar to other members within this clade. Caenorhabditis elegans and Drosophila contain divergent Aurora A and B kinases thatprobably arose through an independent duplication from that giving rise to vertebrate kinases (8). The Dictyostelium DdAurora kinase (asterisk)is about equally similar to the kinases of animals, plants, and fungi.

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centrosomes subsequently nucleate an intranuclear spindle.DdCP224, an ortholog of XMAP215, regulates microtubuledynamics (26) and is colocalized with -tubulin exactly at themitotic spindle poles (24).

Close observation of the double-stained wild-type cells re-vealed that, rather than displaying an overlapping colocaliza-tion with DdCP224, DdAurora localized in close proximity buteither distal to DdCP224 during prometaphase and metaphase(Fig. 4a to c) or proximal to DdCP224 during anaphase andtelophase (Fig. 4d and e). Therefore, our images suggestedthat DdAurora was localized on the outer (cytosolic) side ofthe spindle poles during prometaphase and metaphase (Fig. 4ato c). Upon entry into anaphase, the outer polar DdAuroralocalization disappeared, to be replaced by DdAurora on theinner (intranuclear) side of the spindle poles (Fig. 4d and e).

DdAurora remains bound to centromeres after anaphase.Dictyostelium centromeres migrate in close proximity to spin-dle poles in anaphase and remain associated with spindle polesthrough the rest of cell division (44). Thus, the presence ofDdAurora on the inner side of the spindle poles, in closeapposition with the segregated chromosomes (Fig. 4d), sug-gested the possibility that DdAurora remains associated with

centromeres after metaphase. To test this possibility, we com-pared the localization of endogenous DdAurora with that ofHcpA, a centromeric protein homologous to mammalian HP1(32). Examination of cells expressing HcpA-GFP and immu-nostained with anti-DdAurora antibodies revealed that DdAu-rora colocalized with HcpA-GFP at centromeres in metaphase(Fig. 5a). During metaphase, Dictyostelium cells contain allcentromeres congregated as a single dot at the center of themetaphase spindle and the chromosome arms spread outwardperpendicularly to the spindle (50). At this stage, DdAurorawas also present on the outer (cytosolic) side of the spindlepoles (Fig. 4b).

We confirmed the observation that the localization of HcpA-GFP changes early in anaphase (33). HcpA-GFP was bound tocentromeres at metaphase but became diffuse inside the nu-cleus of cells in early anaphase (Fig. 5b). This suggests that

FIG. 3. GFP-DdAurora localization in different stages of M phase.Shown are fluorescence images of live wild-type cells expressing GFP-DdAurora. In interphase, GFP-DdAurora was diffuse in the cytoplasm(a). During prometaphase, GFP-DdAurora concentrated at spindlepoles (arrows) (b). At metaphase, GFP-DdAurora localized to themetaphase plate (arrowhead) in addition to spindle poles (c). GFP-DdAurora localized to the central spindle (arrowhead) from earlyanaphase and faded from this location as the cell progressed throughcytokinesis (d to f). Notice that GFP-DdAurora did not accumulate atthe cleavage furrow early in cytokinesis (e) or even when the furrowwas very advanced (f). In contrast, DdINCENP has been shown tolocalize at the cleavage furrow early in cytokinesis (15). Near the endof cytokinesis, GFP-DdAurora accumulated at the cytoplasmic bridgeformed between the two daughter cells (g and h). GFP-DdAurorapersisted at the breaking point of the cytoplasm bridge for a time aftercytokinesis (i). Bar, 5 �m. (See Movies S1 and S2 in the supplementalmaterial.)

FIG. 4. DdAurora localizes at the spindle poles, centromeres, andcentral spindle. Shown is colocalization of endogenous DdAurora andDdCP224 (a centrosomal and spindle marker) (24). Wild-type cellswere fixed and stained with antibodies against DdAurora andDdCP224. In the merged images, DNA is shown in blue, DdAurora isshown in red, and DdCP224 is shown in green. (a and b) In prometa-phase and metaphase, DdAurora localized to spindle poles (smallarrows) and the metaphase plate (large arrows). DdAurora localized inclose proximity but distal to DdCP224 at the poles. The metaphaseplate localization corresponds to centromeric localization (Fig. 5). (c)In early anaphase, DdAurora localized simultaneously to spindle poles(small arrows), centromeres (large arrows), and the central spindle(arrowhead). (d and e) In late anaphase and telophase, DdAurora waslost from the distal portion of the spindle poles and was instead foundon the inner side of the poles. Compare the locations of the polarDdAurora (red) in panels a and d in relation to the DdCP224-labeledcentrosome (green). The DdAurora on the inner side of the polesrepresents protein associated with centromeres (Fig. 5). The stainingof DdAurora at the central spindle was very bright in anaphase andfaded during telophase (arrowheads). Bars, 5 �m.

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HcpA-GFP dissociates from the centromeres at this time. HP1,the mammalian homologue of HcpA, is known to be displacedfrom mitotic chromosomes by Aurora B kinase during mitosis(21, 29). Later in telophase, HcpA-GFP was again localized atcentromeres (Fig. 5c), which are closely apposed to the innerside of the spindle poles (32). At this stage, DdAurora clearlycolocalized with HcpA-GFP at the centromeres of the telo-phase cells (Fig. 5c). The spindle poles of these cells, asmarked by DdCP224 or -tubulin, are located farther awayfrom the center of the spindle (Fig. 4e). These observationsindicate that DdAurora remained bound to the centromeres ofthe separated chromatids during anaphase and telophase.HcpA is known to remain associated with the centromeresthrough interphase (33). The retention of Aurora kinase oncentromeres after chromosomes have separated is unusual andhas not been observed in any other species.

The Aurora B-like properties of DdAurora are dependent onits interaction with the IN-box domain of DdINCENP. Wehave shown that DdAurora displays the distribution character-istic of both Aurora A and Aurora B kinases. Interestingly, thechromosomal passenger protein DdINCENP was found atsome, but not all, of these locations in a mitotic cell (15).DdINCENP localized on centromeres during prometaphaseand metaphase and on the central spindle during anaphase andtelophase. These observations suggest the possibility that theDdAurora protein that colocalizes with DdINCENP may bepart of a chromosomal passenger complex that has the prop-erties of the similar complex formed by animal Aurora B ki-

nase. On the other hand, the DdAurora found at the spindlepoles would not be part of the chromosomal passenger com-plex and would behave as an Aurora A kinase. If this scenariois correct, then DdINCENP should be important only forthe Aurora B-like localization of DdAurora. To test thishypothesis, we examined the localization of DdAurora inDdINCENP-null cells expressing the centromeric markerHcpA-GFP (32).

The localization of DdAurora on the spindle poles of meta-phase cells was not influenced by the absence of DdINCENP(Fig. 6a and see Movie S3 in the supplemental material). Thisobservation supports the idea that the polar localization ofDdAurora is analogous to the polar localization of Aurora Aand therefore does not require DdINCENP. In contrast,DdAurora failed to localize to the central spindle of DdIN-CENP-null cells during anaphase and telophase (Fig. 6b andc). The requirement of DdINCENP for the central spindlelocalization of DdAurora strengthens the model that these twoproteins form a complex at the central spindle. However,DdINCENP was not required for the centromeric localizationof DdAurora. DdAurora colocalized with HcpA-GFP on thecentromeres of DdINCENP-null cells from metaphase to te-lophase (Fig. 6a, b, and c). Remarkably, HcpA-GFP was notdisplaced from the centromeres of DdINCENP-null cells dur-ing early anaphase (Fig. 6b) as it was displaced in wild-typecells (Fig. 5b). We interpret this observation as indicative thatthe loss of DdINCENP renders the centromeric DdAurorainactive and unable to displace HcpA from the centromere at

FIG. 5. DdAurora remains bound to centromeres even after ana-phase. Wild-type cells expressing the centromeric marker HcpA-GFP(32) were stained with antibodies against DdAurora. In the mergedimages, DNA is shown in blue, DdAurora in red, and HcpA-GFP ingreen. (a) In metaphase, DdAurora localized to spindle poles (smallarrows) and the metaphase plate. DdAurora colocalized with HcpA-GFP at the metaphase plate (large arrows), suggesting that DdAuroralocalizes to the centromeres at this stage. (b) In anaphase, DdAurorawas found at the central spindle (arrowhead) in addition to the spindlepoles (small arrows) and centromeres (large arrows). HcpA-GFP wasdiffusely distributed in the nucleus in anaphase, possibly by beingdisplaced from centromeres by activated DdAurora (Fig. 6). (c) Intelophase, HcpA-GFP could be observed again at the centromeres andcolocalized extensively with centromeric DdAurora (large arrows).DdAurora also localized to the central spindle (shown by arrowhead)at this stage. Bars, 5 �m.

FIG. 6. DdINCENP is essential for DdAurora localization at thecentral spindle but not on centromeres. DdINCENP-null cells express-ing the centromeric marker HcpA-GFP were stained with DdAuroraantibodies. In the merged images, DNA is shown in blue, endogenousDdAurora is shown in red, and HcpA-GFP is shown in green. (a) Inmetaphase, DdAurora localized normally to the centromeres (largearrows) and spindle poles (small arrows). DdAurora colocalized withHcpA-GFP on the centromeres at the metaphase plate. (b) In ana-phase, DdAurora localized to the spindle poles (small arrows) andcentromeres (large arrows) but not at the central spindle. In contrastto wild-type cells (Fig. 5), HcpA was not displaced from the centro-meres of DdINCENP-null cells. In these cells, HcpA-GFP remainedassociated with the centromeres and colocalized with DdAurora (largearrows) during anaphase. (c) In telophase, DdAurora continued itsassociation with centromeres and colocalized with HcpA-GFP (largearrows). Bars, 5 �m.

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the onset of anaphase. This is also consistent with the obser-vation that DdINCENP-null cells have defects in chromosomesegregation (15), which may be caused by failure to activateDdAurora at the centromeres.

To verify that the loss of DdAurora localization at the cen-tral spindle was caused by the absence of DdINCENP, weexpressed GFP-DdINCENP in DdINCENP-null cells. Wehave shown previously that GFP-DdINCENP can fully rescuethe mitosis and cytokinesis defects observed in DdINCENP-null cells (15). Similarly, we found that the localization ofDdAurora to the central spindle was rescued by GFP-DdIN-CENP and both proteins colocalized extensively at the centralspindle (Fig. 7). This result strongly suggests that DdINCENPand DdAurora formed a chromosomal passenger complex thatlocalized at the central spindle at the metaphase/anaphasetransition. To confirm the interaction between these two pro-teins we precipitated endogenous DdAurora or TAP-taggedDdAurora and found that endogenous DdINCENP or GFP-DdINCENP coprecipitated with DdAurora (see Fig. S2 in thesupplemental material).

The conserved C-terminal IN-box domain of INCENP isknown to be essential for its interaction with Aurora B andactivation of the kinase (6, 30). In addition, the N-terminaldomain of DdINCENP is necessary and sufficient to localizeDdINCENP to the cleavage furrow (14). To investigate the

contribution of these domains to the localization of DdAurora,we determined the distribution of endogenous DdAurora inDdINCENP-null cells expressing two different DdINCENPtruncation mutants (Fig. 8A). The localization of DdAurora atthe central spindle of DdINCENP-null cells was rescued byexpression of DdINCENP-�N but not of DdINCENP-�C (Fig.8B). Even though DdAurora was absent from the central spin-dle, DdINCENP-�C was clearly visible at the central spindle,indicating that DdINCENP can localize at the central spindleindependently of DdAurora. In all cases, DdAurora localizednormally to the spindle poles in metaphase (data not shown)and to the centromeres of the separated chromosomes in telo-phase (Fig. 8B).

To test whether the localization results described above aremediated by the interaction between various DdINCENP trun-cations and DdAurora, we performed pull-down experimentson all different cell lines. We found that the proteins thatcontained the IN-box domain (full-length DdINCENP andDdINCENP-�N) coprecipitated with DdAurora (see Fig. S3 in

FIG. 7. GFP-DdINCENP rescues the localization of DdAurora tothe central spindle in DdINCENP-null cells. DdINCENP-null cellsexpressing GFP-DdINCENP were stained with anti-DdAurora anti-bodies. In the merged images, DNA is shown in blue, GFP-DdIN-CENP is shown in green, and endogenous DdAurora is shown in red.(a) In prophase, DdAurora and DdINCENP colocalized at the cen-tromeres (arrow). (b) In metaphase, DdAurora colocalized with GFP-DdINCENP at the centromeres (large arrow). GFP-DdINCENP alsolabels the spindle poles (small arrows). (c) In anaphase, a portion ofDdAurora redistributed to the central spindle. GFP-DdINCENP la-beled most of the anaphase spindle and colocalized with DdAurora atthe central spindle (arrowhead). (d and e) During telophase, DdAu-rora colocalized with GFP-DdINCENP at the central spindle (ar-rowhead). Bars, 5 �m.

FIG. 8. The IN-box domain of DdINCENP is required for thelocalization of DdAurora to the central spindle. (A) Schematic dia-gram of different truncation mutants of DdINCENP. The conservedIN-box domain, which is essential for Aurora B binding and activation,is shown as a gray box. (B) The truncated GFP-DdINCENP proteinswere expressed in DdINCENP-null cells, and the central spindle lo-calization of endogenous DdAurora was examined by immunofluores-cence microscopy. During anaphase, DdAurora localized at the centralspindle of cells expressing GFP-DdINCENP and GFP-DdIN-CENP-�N (arrowheads), but not in those expressing GFP-DdIN-CENP-�C. As shown in Fig. 6, the centromeric localization of DdAu-rora near the spindle poles (arrows) was independent of DdINCENP.Bar, 5 �m.

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the supplemental material). In contrast, the truncated form ofDdINCENP lacking the IN-box domain (DdINCENP-�C)could not be pulled down with DdAurora. These results indi-cate that the C-terminal IN-box domain of DdINCENP iscritical for its interaction with DdAurora and is required forthe recruitment of DdAurora to the central spindle.

The motor protein Kif12 is important for the localization ofDdAurora at the central spindle. In mammalian cells, thekinesin 6-like protein MKLP2 is essential for the localizationof the chromosomal passenger complex to the central spindle(27). In Dictyostelium, Kif12 was identified as a kinesin 6-likeprotein required for myosin localization to the furrow (37). InKif12-null cells, GFP-DdINCENP can still localize to the cen-tral spindle but fails to localize at the cleavage furrow (14). Totest whether Kif12 influences the distribution of DdAurora, wedetermined the localization of endogenous DdAurora inKif12-null cells expressing HcpA-GFP. We found that DdAu-rora localized normally at the spindle poles and colocalizedwith HcpA-GFP at the centromeres of Kif12 mutant cells (Fig.9a). However, the localization of DdAurora at the centralspindle was abrogated in the Kif12 mutant cells (Fig. 9b and cand see Movie S4 in the supplemental material). Similar to ourobservations in wild-type cells (Fig. 5b), HcpA-GFP was dif-fusely distributed in the nucleus of Kif12-null cells during earlyanaphase (Fig. 9b). This finding suggests that Kif12 is notrequired for the activation of DdAurora at the centromeres inmetaphase/anaphase.

Intriguingly, while we found that DdAurora failed to localizeat the central spindle of Kif12-null cells, GFP-DdINCENP did

localize at the central spindle in these mutant cells (14). Thisdiscrepancy could reflect different mechanisms of localizationof these two proteins at the central spindle or may be due tothe overexpression of GFP-DdINCENP. To distinguish be-tween these possibilities, we stained Kif12-null cells expressingGFP-DdINCENP for endogenous DdAurora localization (Fig.10). We found that DdAurora was restored at the centralspindle of these cells indicating that overexpression of GFP-DdINCENP overcomes the defect caused by loss of Kif12.Immunoprecipitation assays also showed that GFP-DdIN-CENP interacted with DdAurora in Kif12-null cells (see Fig.S4 in the supplemental material).

Localization of DdAurora at the cleavage furrow. Aurora Band INCENP are known to localize at the cleavage furrow andmidbody of dividing animal cells but not at the mother-budneck of yeast cells (9, 42, 57) Accordingly, Aurora B is requiredfor cytokinesis in animal cells but the requirement is not sostringent in yeast cells (4, 48, 57). In Dictyostelium, DdINCENPlocalizes at the cleavage furrow early in cytokinesis and con-centrates at the cytoplasmic bridge connecting the two daugh-ter cells (15). DdINCENP is also required for the scission ofthe cytoplasmic bridge at the end of cytokinesis (15). Liveimaging of GFP-DdAurora and immunofluorescence micros-copy of endogenous DdAurora demonstrated that DdAuroralocalized at the region of the cleavage furrow, but only late incytokinesis (Fig. 4f to h and see Movie S2 in the supplementalmaterial). DdAurora was visible at the cleavage furrow onlynear the end of cytokinesis when the furrow becomes a thincytoplasmic bridge between the two daughter cells (Fig. 4 h).When this bridge finally severed, GFP-Aurora persisted at thebreaking point for a short time (Fig. 4i).

Since little is known about how Aurora B localizes at thecleavage furrow, we determined the ability of GFP-DdAurorato localize at the furrow of different mutant cell lines. Wefound that GFP-DdAurora failed to localize at the cleavagefurrow of DdINCENP-null cells (see Movie S3 in the supple-mental material). Given that DdINCENP is required for thelocalization of DdAurora at the central spindle, it is reasonableto expect a similar requirement for DdAurora localization at

FIG. 9. Kif12 is essential for the localization of DdAurora at thecentral spindle. The localization of endogenous DdAurora in Kif12-null cells expressing HcpA-GFP was determined by immunofluores-cence microscopy. In the merged images, DNA is shown in blue,DdAurora is shown in red, and the centromeric marker HcpA-GFP isshown in green. (a) In metaphase, DdAurora colocalized with HcpA-GFP at the centromeres (large arrows). DdAurora also localized at thespindle poles (small arrows). (b) In early anaphase, DdAurora local-ized to the spindle poles (small arrows) and centromeres (large ar-rows), but was absent from the central spindle. Again, HcpA wasdiffusely distributed in the nucleus of Kif12-null cells during anaphase,similar to its distribution in wild-type cells at this stage (Fig. 5b). (c) Intelophase, DdAurora continued to be associated with centromeres andcolocalized with HcpA-GFP at the congregated centromeres near thespindle poles (arrows). Bars, 5 �m.

FIG. 10. Overexpression of GFP-DdINCENP in Kif12-null cellsrescues translocation of DdAurora to the central spindle. The local-ization of endogenous DdAurora in Kif12-null cells expressing GFP-DdINCENP was determined by immunofluorescence microscopy. Inanaphase and telophase, the central spindle localization (arrowheads)of DdAurora was rescued by the overexpression of GFP-DdINCENP.Bars, 5 �m.

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the cleavage furrow. It seems likely that the absence of DdAu-rora at the cleavage furrow is the basis of the cytokinesis defectobserved in DdINCENP-null cells (15).

Similarly, GFP-DdAurora did not localize at the cleavagefurrow of Kif12-null cells (see Movie S4 in the supplementalmaterial). Since DdINCENP does not localize at the cleav-age furrow in these mutant cells (14), it is likely that this isthe cause for the failure to recruit DdAurora to the cleavagefurrow.

We have shown previously that the distribution of DdIN-CENP at the cleavage furrow was disturbed by the absence ofmyosin II (15). In myosin II mutant cells, GFP-DdINCENPlocalized as a narrow band at the furrow instead of the broadcleavage furrow cortex localization observed in wild-type cells(15). To determine whether myosin II also influences the dis-tribution of DdAurora, we determined the localization of en-dogenous DdAurora and of GFP-DdAurora in myosin IIheavy-chain-null cells. The localization of endogenous DdAu-rora to the spindle poles, centromeres, and central spindle wasnormal in these mutant cells (Fig. 11A). In contrast, the local-ization of GFP-DdAurora at the cytoplasmic bridge during latecytokinesis was completely abrogated (Fig. 11B). Thus, it ap-pears that an intact contractile ring is essential for the properorganization of the chromosomal passenger protein complex atthe cleavage furrow.

DISCUSSION

We have presented here the characterization of the singleAurora kinase from Dictyostelium. Among the several Aurorakinases that have been studied to date, DdAurora is the firstkinase with demonstrable characteristics of both Aurora A andAurora B kinases. The similarity of DdAurora to both Aurora

kinase subfamilies is shown not only by sequence homology,but also by subcellular localization at different stages of mitosisand by interactions with DdINCENP. These studies offer anevolutionary perspective on the properties of Aurora kinases inthe common ancestor of animals, fungi, and plants. We suggestthat the ancestral Aurora kinase already had the dual role ofAurora A and B kinases and that these roles were subsequentlyseparated in the animal Aurora kinases. Our studies also re-vealed novel properties of the Dictyostelium kinase that havenot been recognized in Aurora kinases from other organisms.Given the evolutionary conservation of these kinases, it seemslikely that similar properties will be found in other Aurorakinases.

DdAurora has properties of Aurora A kinase. Aurora Akinases are distinct from Aurora B kinases in having a longerN-terminal domain that contains an A-box sequence importantfor the proteolytic regulation of these proteins. The Dictyoste-lium DdAurora kinase contains an A-box sequence within itsN-terminal domain similar to that of Aurora A kinases. Asecond property characteristic of Aurora A kinases is theirlocalization at the spindle poles, where they play an importantrole in the maturation of centrosomes and spindle assembly.We have shown that DdAurora is also localized at the spindlepoles from the initiation of mitosis until the beginning of ana-phase. Given its sequence similarity to Aurora A kinases andsimilar localization during mitosis, we conclude that DdAuroraplays the role of Aurora A during Dictyostelium mitosis. It willbe interesting to identify proteins that may regulate the local-ization and function of DdAurora at the spindle poles. Sincethe vertebrate Aurora A regulators Aip1 and TPX2 do notappear to have orthologs in invertebrates, fungi or protists, it islikely that other proteins yet to be discovered regulate theactivity of this kinase at the spindle poles.

Colocalization of DdAurora with the centrosomal markerDdCP224 revealed that DdAurora is localized on the outerside of the spindle poles. In Dictyostelium, the centrosome isembedded in the nuclear envelope during mitosis (44). Thus,one centrosomal surface remains in the cytosol while the otherresides inside the nucleus. Our observations suggest that theremay be functional differences between these two surfaces andthat DdAurora may play a role on the cytosolic side of thecentrosome during prometaphase and metaphase. In mamma-lian cells, Aurora A kinase was found to localize to centro-somes and the adjacent spindle microtubules (55). It is possiblethat this pericentrosomal localization of Aurora A is equiva-lent to the localization of DdAurora on the outer portion ofthe spindle poles.

DdAurora has properties of Aurora B kinase. Aurora Bkinases are characterized by their association with the proteinsof the chromosomal passenger complex and dynamic sequen-tial localization at centromeres, central spindle, and midbody.We have shown that DdAurora displays all these properties ofAurora B kinases. DdAurora is found on centromeres as earlyas prometaphase; it localizes at the central spindle duringanaphase and at the cytoplasmic bridge at the end of cytoki-nesis. DdAurora also interacts with the chromosomal passen-ger protein DdINCENP and this interaction requires the con-served IN-box domain at the C terminus of DdINCENP. By allof these criteria, it is clear that DdAurora plays the role ofAurora B in Dictyostelium mitosis.

FIG. 11. Localization of GFP-DdAurora in myosin II-null cells.(A) Localization of endogenous DdAurora in myosin II-null cells wasdetermined by immunostaining. In the merged images, DNA is shownin blue and endogenous DdAurora is shown in red. The polar andcentromeric (arrow) localization during metaphase was normal inthese cells. In addition, the central spindle localization of DdAurora(arrowheads) in anaphase was normal. Bar, 5 �m. (B) Live images ofmyosin II (myosin heavy chain)-null cells expressing GFP-Aurora dur-ing cytokinesis. The central spindle localization of GFP-DdAuroracould still be observed at 01:19 (arrowhead). GFP-DdAurora failed tolocalize at the cytoplasmic bridge (arrows) formed between the twodaughter cells. Bar, 5 �m.

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We demonstrated that DdAurora is found on centromeresby colocalization with the centromeric protein HcpA. In con-trast with Aurora B from other organisms, we found thatDdAurora remains associated with the centromeres even dur-ing anaphase and telophase. In Dictyostelium, the centromeresof the daughter chromatids congregate closely to the spindlepoles during anaphase (44), and therefore, DdAurora andHcpA are visible as a single dot on the inner surface of thespindle poles. In contrast, animal Aurora B leaves the centro-meres at the onset of anaphase and relocates to the centralspindle (2, 53, 57). Interestingly, one of the Aurora isoforms(AtAur-1) found in Arabidopsis appears to move with centro-meres during anaphase (18). While this localization has notbeen proven to be centromeric, it opens the possibility thatAurora kinases play an additional centromeric function duringanaphase, at least in plants and Dictyostelium.

In contrast to DdAurora, its binding partner, DdINCENP,does not remain associated with anaphase centromeres. Likeits animal counterpart, DdINCENP redistributes to the centralspindle during anaphase (15). Therefore, the association ofDdAurora with the centromere appears to be independent ofDdINCENP. We confirmed this possibility by showing thatDdAurora is still found on centromeres in DdINCENP-nullcells.

The localization of Aurora B at the centromeres has beenshown to be important for the regulation of proper microtu-bule attachment to kinetochores (17, 56). This activity is de-pendent on the proteins of the chromosomal passenger com-plex (51). Aurora B is also known to be required for thedissociation of human HP1 proteins from chromosomes duringmitosis (21, 29). We found that Dictyostelium HcpA, the or-tholog of human HP1, also dissociates from centromeres, butonly during early anaphase. Importantly, we observed thatHcpA failed to dissociate from anaphase centromeres in theDdINCENP-null mutants. We interpret these observations asindicative that the activity of the DdAurora/DdINCENP com-plex at the centromeres dissociates HcpA at the onset of ana-phase. Concomitantly, as in other organisms, DdINCENP dis-sociates from the centromeres and the DdAurora that remainsassociated with the anaphase centromeres would not have Au-rora B-like activity. DdINCENP and a portion of DdAuroraredistribute to the central spindle and eventually to the cleav-age furrow.

DdAurora forms a chromosomal passenger complex withDdINCENP. We have shown that DdAurora interacts withDdINCENP and that this interaction requires the IN-box do-main at the carboxyl terminus of DdINCENP. Therefore, itappears that Dictyostelium has a chromosomal passenger com-plex similar to that described in other organisms. However, theDictyostelium genome does not encode any protein with simi-larity to the other chromosomal passenger complex proteinssurvivin/Bir1 and borealin. Elucidation of whether other pro-teins participate in the Dictyostelium chromosomal passengercomplex will require the purification of this complex.

While it is clear that DdAurora and DdINCENP form acomplex, we also provide evidence that their interaction mustbe transient and spatially regulated. The central spindle is alocation where these two proteins clearly interact. DdAuroradepends on DdINCENP to localize to the central spindle,although the reverse is not true. Similarly, both proteins are

enriched at the cytoplasmic bridge connecting the two daugh-ter cells at the end of cytokinesis. The localization of DdAu-rora at this location also requires the presence of DdINCENP.However, DdINCENP localizes to the invaginating cleavagefurrow from the beginning of cytokinesis, whereas DdAuroradoes not. This would suggest that DdINCENP associates withthe cleavage furrow and later recruits DdAurora. It seemslikely that the late cytokinesis defect observed in DdINCENP-null cells is caused by a lack of DdAurora recruitment to thecytoplasmic bridge.

The differences in DdAurora and DdINCENP localizationalso suggest that, instead of being solely a DdAurora cofactor,DdINCENP may play additional roles in mitosis and cytoki-nesis by interacting with other proteins. For example, it hasbeen shown recently that phosphorylated INCENP can bind toPolo-like kinase (23), another important mitotic kinase essen-tial for mitotic entry, spindle formation, and cytokinesis (61).DdINCENP may also regulate mitosis by interacting with aPolo-like kinase protein in Dictyostelium.

The cytoskeletal proteins Kif12 and myosin II modulate asubset of DdAurora localizations. Similarly to the role of theanimal motor protein MKLP2, Dictyostelium Kif12 is essentialfor the localization of DdAurora to the central spindle at themetaphase/anaphase transition. However, when GFP-DdIN-CENP is overexpressed in Kif12-null cells, it rescues the local-ization of DdAurora to the central spindle. It seems likely thatKif12 is also important for the localization of DdINCENP tothe central spindle but that overexpression of GFP-DdIN-CENP overcomes the loss of Kif12. Unfortunately, the anti-bodies raised against DdINCENP have not been useful forimmunofluorescence studies to determine the distribution ofendogenous DdINCENP. Nonetheless, while the overexpres-sion of GFP-DdINCENP rescues the localization of DdAurorato the central spindle, it is not sufficient to recover the local-ization of either protein to the cleavage furrow of Kif12-nullcells. This indicates that this motor protein is responsible,directly or indirectly, for the localization of the chromosomalpassenger complex to the cleavage furrow.

The localization of proteins at the cleavage furrow is apoorly understood process. We showed previously that thedistribution of DdINCENP at the cleavage furrow is abnormalin myosin II-null cells (15). We showed here that the localiza-tion of DdAurora was also affected by the absence of myosinII. Although GFP-DdAurora was still localized at the centralspindle, it was absent from the cleavage furrows of myosinmutant cells. This suggests that the absence of myosin II notonly affects localization of DdINCENP at the contractile fur-row, but also disrupts the furrow localization of DdAurora.Since Aurora kinase activity is important for the completion ofcytokinesis, the cytokinesis defect of myosin II-null cells couldbe explained by both failure of contraction and the absence ofAurora kinase activity at the furrow during late cytokinesis.

ACKNOWLEDGMENTS

We would like to thank the members of the De Lozanne andO’Halloran labs for their comments and help throughout the develop-ment of this project. We also thank J. A. Spudich and G. S. Lakshmi-kanth for providing us with the Kif12-null cell line and D. A. Laro-chelle for an unpublished small interfering RNA construct.

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This research was supported by grant GM48745 from the NationalInstitutes of Health to A.D. and a grant from the Deutsche For-schungsgemeinschaft (SPP 1129) to W.N.

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