DictyosteliumDdCP224 is a microtubule-associated protein ...INTRODUCTION From an evolutionary...

INTRODUCTION

From an evolutionary perspective, the Dictyosteliumcentrosome is an intriguing organelle. Like its counterpart inyeast, the spindle pole body, it exhibits a compact, layeredstructure that lacks the centrioles typical of metazoan cells, butas in the latter, the core is surrounded by an electron-dense,amorphous matrix. With vertebrate centrosomes it shares acytoplasmic localization in interphase, but it inserts itself intoan opening of the nuclear envelope during mitosis like the yeastspindle pole body, which is a permanent resident of the nuclearenvelope. This centrosome with its hybrid features resides ina cell type that resembles mammalian cells in terms ofbehavioral repertoire and motile properties, makingDictyostelium amoebae an important model system for theanalysis of centrosome structure, function, and evolution.

The Dictyostelium centrosome is located in close proximityto the nucleus (Roos, 1975) to which it is tightly connected viaa fibrous linkage (Omura and Fukui, 1985). It consists of a box-shaped core structure with three major layers surrounded by acorona, which is composed of regularly spaced, dense nodulesembedded in an amorphous matrix (Moens, 1976; Roos, 1975).The nodules seem to be the sites of microtubule nucleationsince they contain γ-tubulin, and since all interphasemicrotubules emanate from these nodules (Euteneuer et al.,1998). Thus the corona seems to be the functional equivalent

of the pericentriolar matrix of animal cells. Recently, the modeof centrosome duplication in Dictyostelium was elucidated indetail by a combination of electron microscopic analysis offixed cells and observation of living cells tranformed with γ-tubulin-GFP, which possess green fluorescing centrosomes(Ueda et al., 1999). In contrast to animal cells and yeast, theentire duplication process occurs during mitosis. Centrosomeduplication starts in prophase with an enlargement of the three-layered core. In late prophase, the corona dissociates and theinterphase microtubules are lost. The central layer disappearsat the transition to prometaphase, and the two outer layers peelapart and become the mitotic centrosomes, or spindle poleplaques. Microtubules are nucleated at the formerly innersurfaces of the two layers to form a nascent spindle that startsto separate the spindle pole plaques. During the separationprocess the edges of the plaques bend away from the nucleusuntil, in telophase, each plaque folds back onto itself. As aresult of this folding process the microtubule-nucleatingsurface turns into the outside of the new daughter centrosomewhile the former outer surface becomes buried inside (Ueda etal., 1999). These morphological changes during centrosomeduplication are unique among eukarytic cells.

At present, relatively little is known about the centrosomalcomponents that orchestrate this intriguing duplication processand carry out all other centrosomal activities. So far, molecularanalyses were based on the expected similarity of

1747Journal of Cell Science 113, 1747-1758 (2000)Printed in Great Britain © The Company of Biologists Limited 2000JCS1194

A cDNA encoding a 224-kDa Dictyostelium discoideumcentrosomal protein (DdCP224) was isolated byimmunoscreening. DdCP224 was detected at thecentrosome and, more weakly, along microtubulesthroughout the entire cell cycle. Centrosomal localizationdoes not require microtubules, suggesting that DdCP224is a genuine centrosomal component. DdCP224 exhibitssequence identity to a weakly conserved class ofmicrotubule-associated proteins including human TOGpand yeast Stu2p. Stu2p has a size of only ~100 kDa andcorresponds to the N-terminal half of DdCP224. Thefunctions of the N- and C-terminal halves of DdCP224 wereinvestigated in the corresponding GFP-fusion mutants.Surprisingly, the N-terminal construct showed onlycytosolic localization, whereas the C-terminal construct

localized exclusively to the centrosome. This is unexpectedbecause Stu2p is localized at the spindle pole body. Full-length DdCP224-GFP was present both at centrosomes andalong microtubules. Furthermore, it bound to microtubulesin vitro, unlike the two truncated mutants. Thuscentrosome binding is determined by the C-terminal halfand microtubule binding may require the interaction of theN- and C-terminal halves. Interestingly, cells expressingfull-length DdCP224-GFP exhibit supernumerarycentrosomes and show a cytokinesis defect, suggesting thatDdCP224 plays an important role in centrosomeduplication. These features are unique among the knowncentrosomal proteins.

Key words: Dictyostelium, Centrosome, MTOC, DdCP224

SUMMARY

Dictyostelium DdCP224 is a microtubule-associated protein and a permanent

centrosomal resident involved in centrosome duplication

Ralph Gräf*, Christine Daunderer and Manfred Schliwa

Adolf-Butenandt-Institut, Zellbiologie, Schillerstr. 42, D-80336 München, Germany*Author for correspondence (e-mail: [email protected])

Accepted 3 March; published on WWW 18 April 2000

1748

Dictyostelium centrosomal proteins to their homologues inother species. Thus, Dictyostelium γ-tubulin was cloned by aPCR approach using degenerated primers (Euteneuer et al.,1998). Recently, four more centrosomal proteins wereidentified by database analysis (Gräf et al., 2000) of theDictyostelium cDNA project (Morio et al., 1998) and genomeproject (http://genome.imb-jena.de/Dictyostelium). Theyinclude the Dictyostelium homologues of centrin (Schiebel andBornens, 1995), Spc97p, Spc98p (Knop and Schiebel, 1997)and human Nek2, a centrosomal NIMA-related kinase involvedin centrosome duplication (Fry et al., 1998a,b).

However, novel or weakly conserved centrosomal proteinscannot be uncovered by database searches. An alternativeaproach is immunoscreening of DNA libraries with specificmonoclonal antibodies (mAbs), as successfully performed inyeast (Donaldson and Kilmartin, 1996; Kilmartin et al., 1993;Wigge et al., 1998). Following the development of a protocolfor the isolation for Dictyostelium centrosomes in highquantity and purity (Gräf et al., 1998), 14 new monoclonalantibodies against Dictyostelium centrosomes were generated(Gräf et al., 1999). One of these antibodies was used here toclone a 224-kDa centrosomal component which we havenamed DdCP224 (Dictyostelium discoideum centrosomalprotein). Sequence comparison revealed that this proteinbelongs to a family of weakly conserved MAPs including,among others, human TOGp (Charrasse et al., 1995), andyeast Stu2p (Wang and Huffaker, 1997). The analysis ofDdCP224 in wild-type cells and GFP mutants providesinsights into the centrosome and microtubule-bindingproperties of this protein. Moreover, unexpected andpotentially significant differences to other members of thisprotein family are revealed, and a novel function incentrosome duplication is suggested.

MATERIALS AND METHODS

Cell cultureVegetative Dictyostelium discoideum amebae and hybridoma cellswere cultivated as described previously (Gräf et al., 1999). DdCP224-GFP mutants were grown at 21°C in HL-5c medium containing4 µg/ml blasticidin S (ICN Biomedicals, Eschwege, Germany) or5 µg/ml G418 (Sigma, Deisenhofen, Germany), respectively.

Isolation of cytosolic DdCP224 and binding tomicrotubules in vitroApproximately 2×109 Dictyostelium cells yielding ~100 mg of totalcytosolic protein were used for enrichment of DdCP224 and its GFPversions. All steps were performed at 4°C or on ice exceptchromatography which was done at room temperature and allsolutions contained a protease inhibitor cocktail (1 mM Pefabloc SC,25 µg/ml leupeptin, 10 µg/ml tosyl-arginine-methyl ester, 10 µg/mlsoybean trypsin inhibitor, 1 µg/ml aprotinin, 1 µg/ml pepstatin, 1 mMbenzamidine, all from Biomol, Hamburg, or Sigma, Deisenhofen,Germany). Cells (prepared as described by Gräf et al., 1998) weresuspended in 10 ml SPLB (20 mM Na-Pipes (pH 6.9), 30 mM NaCl,2 mM MgCl2, 10% sucrose, 1 mM ATP, 1 mM DTT) and lysis wasachieved by filtration through 5 µm polycarbonate filters (Nuclepore,Corning Costar, Bodenheim, Germany). The lysate was cleared bycentrifugation with 20000 rpm for 15 minutes and the supernatant wasloaded onto a SP-Sepharose cation exchange column (Amersham-Pharmacia, Freiburg, Germany). After washing with WB (20 mM Na-Pipes, pH 6.9, 30 mM NaCl, 2 mM EGTA 4 mM MgCl2, 1 mM DTT),

DdCP224 was eluted with 0.5 ml 100 mM Na-Pipes (pH 6.9), 150mM NaCl, 2 mM EGTA, 4 mM MgCl2, 1 mM DTT.

For microtubule binding, the eluate was diluted with an equalvolume of 4 mM MgCl2/2 mM EGTA. The diluted protein samplewas cleared by centrifugation at 260000 g for 10 minutes (BeckmanTLA100.3 rotor). Porcine brain tubulin (Mandelkow et al., 1985) waspolymerised for 15 minutes at 37°C by addition of 1 mM GTP and,after 5 minutes, of 24 µM taxol. 25 µl (3 mg) of microtubules weremixed with 500 µl of the cleared protein sample (~0.3 mg of protein),supplemented with 24 µM taxol and incubated for 5 minutes at 25°C.Microtubules and associated proteins were sedimented through asucrose cushion (100 µl of 40% sucrose, 100 mM Na-Pipes, 2 mMMgCl2, 24 µM taxol) at 260000 g for 10 minutes at 25°C (BeckmanTLA100.3 rotor). Microtubule pellets were resuspended in 50 µl of0.5× urea sample buffer (4.5 M urea, 5% SDS, 2.5% β-mercaptoethanol, 125 mM Tris/HCl, pH 6.8). Proteins in thesupernatants were precipitated with TCA (Bollag et al., 1996) anddissolved in 50 µl of 0.5× urea sample buffer.

Sucrose density gradient centrifugation1 ml of cytosolic extract prepared as described above was diluted withWB to reduce the sucrose concentration to 7% and loaded onto asucrose step gradient of 0.5 ml each of 10%, 15%, 20%, 25%, 30%,35% and 40% sucrose in WB including protease inhibitors (seeabove). The gradient was centrifuged for 4 hours at 234000 g(Beckman SW50.1 rotor) and was fractionated from the bottom in 0.3ml steps. The proteins in each fraction were precipitated with TCAand dissolved in 30 µl 0.5× urea sample buffer.

Cloning of DdCP2242 µg of double-stranded cDNA with EcoRI/NotI adaptors wereprepared from mRNA of vegetative myosin null mutants (Manstein etal., 1989; QuickPrep mRNA micro and TimeSaver cDNA SynthesisKit from Amersham-Pharmacia, Freiburg, Germany) and size-fractionated on a 1.5% agarose gel. An agarose block including allcDNAs longer than 4.5 kb was excised. The cDNA was recovered bya glass milk procedure (JetSorb, Genomed, Bad Oeynhausen,Germany), ligated into λZAPII-EcoRI arms and packaged in vitro(GigapackIII, Stratagene, Amsterdam, Netherlands). The titer of theprimary library was 1×105 pfu. Immunoscreeing of ~3×105 pfu of theamplified cDNA library with the mAb 4/148 (Gräf et al., 1999) leadto the isolation of 6 positive clones. 4 clones contained a ~6.3 kb insertand the two other clones were false positives since they encodedEF1α. Thus, only one of the 6.3 kb clones was sequenced completelyon both strands (MWG Biotech, Ebersberg, Germany).

Construction of GFP-expression vectorsGFP constructs with DdCP224 were created in vectors based on theC-terminal GFP fusion vector pB15-GFP (Ueda et al., 1997), aderviative of pDXA-3C (Manstein et al., 1995). The SacI-sitedownstream of the S65T-GFP stop codon was destroyed by PCR-based point mutation resulting in pB15GFPXSac. Fusion proteinexpression is driven by the constitutive actin 15 promoter. A furthercloning vector, p1ABsr8, was constructed from pB15GFPXSac byreplacing the G418 resistance cassette by the blasticidin resistancecassette from pUCBsr∆Bam (XbaI-XhoI fragment; Adachi et al.,1994). All three constructs described below were cloned intopB15GFPXSac and p1ABsr8 after BamHI/SacI double digestion. Thepolypeptide chain of all DdCP224 mutants is preceded by the vector-derived peptide MDGTEL and the DdCP224 and GFP sequences areseparated by a glycine and a serine encoded by the BamHI restrictionsite.

For expression of the N-terminal part of DdCP224 (∆C-GFP), thecDNA encoding the N-terminal half of DdCP224 (amino acids 1-813)was amplified by PCR with a sense SacI-primer/linker and anantisense BamHI-primer/linker. For the construction of expressionvectors for full-length DdCP224 and its C-terminal part with GFP

R. Gräf, C. Daunderer and M. Schliwa

http://genome.imb-jena.de/Dictyostelium

1749DdCP224 at the Dictyostelium centrosome

(DdCP224-GFP and ∆N-GFP) the last 78 bp of coding sequence andthe 3′-untranslated region of the original DdCP224 clone inpBluescript were excised by cleavage with SpeI and SacI and replacedby a 86mer custom-synthesized linker substituting the excised codingsequence and introducing a BamHI recognition site at the 3′-end. Inan independent step, the first 543 bp of coding sequence wereamplified by PCR using a sense SacI-primer/linker and an antisenseprimer downstream from the NcoI restriction site. Then the SacI/NcoI5′-fragment of the original DdCP224 clone (745 bp of cDNAincluding the 5′-untranslated sequence) was replaced by the PCRproduct. The insert of this plasmid contained only the complete codingsequence of DdCP224. Similarly, in the case of the ∆N-GFPexpression vector, 304 bp of coding sequence starting at base position2660 were amplified by PCR using a sense SacI-primer/linker and asuitable antisense primer downstream from the SphI restriction site.The first 2938 bp including the 5′-unstranslated region were deletedby SacI/SphI double digestion and replaced by the PCR product.

All PCR-generated sequences of DdCP224 were verified by DNAsequencing. Plasmids were transformed into AX2 cells by usingelectroporation or the calcium phosphate method (Mann et al., 1998;Nellen et al., 1987). All data shown in the figures and tables of thispaper were obtained with the blasticidin-resistant mutants; the G418-resistant strains showed the same phenotypes and were only used forcomparison.

Other methodsSDS polyacrylamide electrophoresis, immunoblotting, silver staining,indirect immunofluorescence microscopy and confocal lightmicroscopy were performed as described previously (Gräf et al., 1998,1999; Ueda et al., 1997).

RESULTS

Cloning and sequence analysis of DdCP224Three of the 14 mAbs against purified Dictyosteliumcentrosomes obtained recently (2/165, 4/95 and 4/148) reactedwith the same ~200 kDa protein band in western blots ofisolated centrosome preparations (Gräf et al., 1999). Furtherimmunoblot analysis revealed the antigen to be ~5-fold moreabundant in cytosolic than in nucleus/centrosome extracts (Fig.1). Since gel electrophoresis and immunoblotting showedthat the ~200 kDa protein was a minor protein component,we expected the corresponding cDNA to be highlyunderrepresented in conventional cDNA libraries. Indeed,initial attempts to clone the corresponding cDNA byimmunoscreening of a random-primed Dictyostelium cDNAlibrary with the three mAbs failed. Thus we generated a size-fractionated cDNA library containing mainly cDNAs longerthan 4.5 kb. These cDNAs comprise only a minor amount ofthe total cDNA, hence the cDNA encoding the ~200 kDaprotein should be highly enriched in this library. Indeed, sixpositive clones could be isolated by immunoscreening withmAb 4/148. All positives were also recognized by mAbs 2/165and 4/95 confirming that all three mAbs are directed to thesame antigen. Complete sequencing of one of these clonesyielded 6325 bp of cDNA sequence (EMBL accession no.AJ012088) with one complete open reading frame encoding abasic protein (pI = 8.06) of 2015 amino acids with a calculatedmolecular mass of 224,126 Da, which was designatedDdCP224. No functional sequence motifs known from otherproteins could be detected, and unlike many other centrosomalproteins it apparently does not contain long coiled-coil regions.

However, employing the coilscan program at a window size of21 amino acids, two short coiled-coil regions (amino acidposition 1211-1234 and 1989-2015) were predicted (Lupas etal., 1991). These stretches are probably too short to promotestable interactions based on a coiled-coil.

Amino acid sequence comparison with EMBL data libraryentries revealed a number of weakly homologous proteins. Theclosest homologues are the human TOG protein, theDrosophila Msps protein and Xenopus XMAP215, whosecomplete sequence was published during the revision processof this work (Charrasse et al., 1995; Cullen et al., 1999;Tournebize et al., 2000). All three proteins share ~40%similarity and ~30% identity with the DdCP224 sequence (Fig.2A). The homology group also includes Caenorhabditiselegans ZYG-9 (Matthews et al., 1998), Saccharomycescerevisiae Stu2p (Wang and Huffaker, 1997), andSchizosaccharomyces pombe p93dis1 (Nabeshima et al., 1995).All these proteins bind to microtubules and appear to havecentrosomal functions, at least during mitosis, but they exhibitonly weak amino acid similarity to each other and differmarkedly in length (Fig. 2A). Thus the two yeast proteins havea size of only ~100 kDa and are homologous to the N-terminalhalf only of the Dictyostelium and human proteins. ZYG-9 hasa size of 150 kDa and, compared to TOGp, the Msps proteinand DdCP224, it possesses only two conserved ~250 aminoacid sequences (Fig. 2B,C). They were called region 1 and 2(Matthews et al., 1998). Region 2, which is missing in theyeasts, is located more N-terminal in ZYG-9 than in DdCP224and TOGp. Region 1 (designated region 1a and 1b in Fig. 2B)is clearly duplicated in ZYG-9, and at a lower level ofconservation, both regions may occur in more than one copyin the other species as well (Nakaseko et al., 1996; Cullen etal., 1999). Recently, Cullen et al. (1999) suggested that evenfour tandemly arranged copies of region 1 exist in the N-terminal half of the Msps and TOG protein sequence, whereasregion 2 is duplicated in both animal proteins. However, in thecase of DdCP224 both sequence motifs are not clearlyrepeated. With respect to region 1, only the short sequenceKKILADVNVM (amino acid position 318-327) is clearlyrepeated more N-terminally where it reads KKILADINPM(amino acid position 53-62). Yet, there is no experimentalevidence for the biological function of the repeated motifs inany of these proteins.

Localization of DdCP224 throughout the cell cycleThe dynamics of DdCP224 localization through the cell cyclewere studied by confocal microscopy. Cells were double-stainedwith anti-DdCP224 mAbs and anti-γ-tubulin as a centrosomalmarker (Fig. 3). During interphase, DdCP224 was localized to

Fig. 1. Relative amounts of cytosolicand centrosomal DdCP224. Westernblot of a 6% SDS-polyacrylamidegel containing proteins of thecentrosomal/nuclear (lane 1) and thecytosolic fraction (lane 2) of approx.5×106 cells. DdCP224 was labeledby mAb 4/148.

1750

the centrosomal corona which appears as a doughnut-shapedstructure since it surrounds the unstained core of theDictyostelium centrosome (Fig. 3A′). This staining pattern wassimilar to that of γ-tubulin antibodies (Fig. 3A). However,DdCP224 seems to reside more in the periphery of the coronacompared to γ-tubulin because the diameter of the structurestained with anti-DdCP224 was always bigger than the anti-γ-tubulin labeling at the same centrosome (Fig. 3A′′ ). WeakDdCP224 staining could sometimes be detected along interphasemicrotubules as well (see below).During mitosis, strong centrosomalstaining persisted without anydetectable change in intensity (Fig. 3B′-G′). In prophase, the doughnut-shapedstructure stained with anti-DdCP224and anti-γ-tubulin became larger andmore oval, but DdCP224 was stilllocated farther outside than γ-tubulin(Fig. 3B-B′′ ). Later on, both antigenscolocalized almost exactly at thespindle poles (Fig. 3D′′ ). In addition tocentrosomal labeling, anti-DdCP224mAbs stained the region wherekinetochores reside in metaphase cells(Fig. 3D′) in a manner similar to theMPM2 antibody (Fig. 3E′; Engle et al.,1988), a good centrosomal andkinetochore marker in mitoticDictyostelium cells (Ueda et al., 1999).Spindle microtubules are also stained,especially in the midbody region inanaphase and telophase (Fig. 3D′-G′).

DdCP224 localization at thecentrosome, but not atkinetochores, is unlikely torequire microtubulesDdCP224 was found at the centrosomethroughout the entire cell cycle,irrespective of the dramatic changes incentrosomal morphology duringmitosis, which includes breakdown of

the interphase microtubule cytoskeleton together withdissociation of the corona in prophase (Kitanishi-Yumura andFukui, 1987; Ueda et al., 1999). This implies that DdCP224 isredistributed from the corona to the two outer layers of the corestructure which start to form the spindle poles in prometaphase.DdCP224 was also present at the corona of isolatedcentrosomes (Fig. 4A) which lack microtubules (Gräf et al.,1998), strongly suggesting that its localization at thecentrosome is not dependent on the presence of an intact


DdCP224 269 LPKLTSEFYEGLQAKKWQERSEQMDKL-VTILTNTPKIETA--DFSELCKALKKILDm-msps 276 LSKMPKDFYDKLEEKKWTLRKESLEVL-EKLLTDHPKLENG--EYGALVSALKKVIHs-TOGp 277 LSKLPKDFYDKIEAKKWQERKEALES --VEVLIKNPKLEAG--DYADLVKALKKVVCeZyg9-1a 13 LPKLPPNFDELRESKKWQERKEALEAL-LKVLTDNERLSTKA-SYAELIGHLQMVLCeZyg9-1b 299 LSKMPDGFDTNIESKKWQERKEALEGL-LQLITANPKLDPKA-NYGALVERLQKVLSp-p93dis1 314 LSKLTPEFHTALSSPKWKDRKEALESM-VPV-CSNPVYQEG--DYSELLRVIAKSLSc-Stu2p 326 LDKLPKDFQERITSSKWKDRVEALEEFWDSVLSQTKKLKSTSQNYSNLLGIYGHII

DdCP224 A-DVNVMIVQKAVVSIGLLADSLR-GGFTS-YVKPFITPIL-EKFKEKKTSVLQSVHTTMDDm-msps TKDSNVVLVAMAGKCLALLAKGLA-KRFSN-YASACVPSLL-EKFKEKKPNVVTALREAIDHs-TOGp GKDTNVMLVALAAKCLTGLAVGLR-KKFGQ-YAGHVVPTIL-EKFKEKKPQVVQALQEAIDCeZyg9-1a AKDANINCQALAAKCIGKFATGLR-AKFSS-FAGP-LLPVIFEKMKEKKPMLREPLVDCSNCeZyg9-1b EKDANINVAALAANCITGIANGLR-TKFQP-FAVS-VTPIIFEKFKEKKPTLRDPLVACIDSp-p93dis1 -KDANVVVVGVAALLLTHIAKALR-KGFLP-YTGIVL-PSLFDRFKERKSSLVHSLLDAANSc-Stu2p QKDANIQAVALAAQSVELICDKLKTPGFSKDYVSLVFTPLL-DRTKEKKPSVIEAIRKALL

DdCP224 SL------VGKSISLSDIIDELTATMQSK-VPQIKQEVLVFICNSITNTKKPADITKVT-KDm-msps AI------YA-STSLEAQQESIVESLANK-NPSVKSETALFIARALTRT-QPTALNKKLLKHs-TOGp AI-------FLTTTLQNISEDVLAVMDNK-NPTIKQQTSLFIARSFRHCTAS-TLPKSLLKCeZyg9-1a EV------GRTMQSLETGQEDILAALA-KPNPQIKQQTALFVARQLDLVV-PAKQPKGFIKCeZyg9-1b AV------V-ATTNLEAVGEIVLAALG-KPNPSIKTQTDLFLQRCFMKLN-SQTMPKKTLKSp-p93dis1 AI-------FESCGLNDIMDETLEFLKHK-NPQVKTETLRWLNRCL-QLTD-VCPPRASLESc-Stu2p TICKYYDPLASSGRNEDMLKDILEHMKHK-TPQIRMECTQLFNASMKEEKDGYSTLQRYLK

DdCP224 -QLTKIFMEALNDTDSNIRDNASKAFAALGGIIGERAMTPYLNQI--DPIKAKKIKD 485Dm-msps -LLTTSLVKTLNEPDPTVRDSSAEALGTLIKLMGDKAVTPLLADV--DPLKMAKIKE 492Hs-TOGp -PFCAALLKHINDSAPEVRDAAFEALGTALKVVGEKAVKPFLADV--DKLKLDKIKE 492CeZyg9-1a -AVVPVFGKLTGDADQDVREASLQGLGAVQRIIGDKNVKNLLGDASSDEGKMKKIGE 233CeZyg9-1b -TLIPSLIKHSGDSDSEVREASYAAMGAMMRAIGEKPSLQLLADIASDNLKMSKIKE 518Sp-p93dis1 -TLCSLCVTLINDTFEPVRMATTNVLATLVQIFSQPVLSKYIVGL--DPKKLPKILE 527Sc-Stu2p DEVVPIVIQIVNDTQPAIRTIGFESFAILIKIFGMNTFVKTLEHL--DNLKRKKIEE 556

Alignment of homologous region 1B

DdCP224 1118 EFIIFDINGKMNRQKTNQIPS---WHFIEPTEEVVEILQDQVLQCFTEEFANLMFSSLDm-msps 1150 LCANSAK NQRLLEQKM-KVLK---WTFVTPREEFTELLRDQMM---T---ANVN-KALHs-chTOG 1160 IFIVVP-NGKEQRMKDEKGLKVLKWNFTTPRDEYIEQLKTQMSSCVAKWLQDEMFHSDCeZyg9-2 603 ELLLSDNEDKKQRIKEEKQLKLVKWNFQAPTDEHISQLQTLLGNQAKVSLMSQLFHKD

DdCP224 PSNSQHMSDLMIGM--IEQNPEAIIS-------V--LDILFRWITFKLFDTGLASQKRVLKILDm-msps IANMFH-DDFRYHLKVIEQLSEDLAGNSKAL--VCNLDLILKWLTLRFYDTNPSVLIKGLEYL Hs-TOGp FQHHNKALAVMVDH--LESEKEGVIG--------C-LDLILKWLTLRFFDTNTSVLMKALEYLCeZyg9-2 FKQHLAALDSLVRL--ADTSPRSLLS---------NSDLLLKWCTLRFFETNPAALIKVLELC

DdCP224 EILLNKLIDSEYSIGEYEASCLVPILLEKSGSATNEQIKQIFKQSIQQ-LEELCL---PNVLFDm-msps VQVFQVLIDEEYILAENEGSSFVPHLLLKA--NPKDAVRNGVRRVLRQVI----LVFPFVKVFHs-TOGp KLLFTLLSEEEYHLTENEASSFIPYLVVKVG-----EPKDVIRKDVRAILNRMCLVYPASKMFCeZyg9-2 KVIVELIRDTETPMSQEEVSAFVPYLLLKTGEA-----KDNMRTSVRDIVNVLSDVVGPLKMT

DdCP224 GASVCRFAIEMVTSQNWRTRVEVLNVMASIIDKN 1317Dm-msps GMNICGYVMEGLKSKNARQRTECLDELTFLIESY 1345Hs-TOGp GMNVCPFIMEGTKSKNSKQRAECLEELGCLVESY 1360CeZyg9-2 GISPLPMLLDALKSKNARQRSECLLVIEYYITNA 804

Alignment of homologous region 2C

DdCP224

Hs-TOGp

Ce-ZYG-9

Sp-p93dis1

Sc-Stu2p 42/28

49/36

41/2941/30

44/30

42/28

42/25

Overall similarity/identity of DdCP224:

40%/28%

not possible

32%/24%

34%/23%

A

100 amino acids

Dm-msps 50/35 44/31 40%/30%

Fig. 2. (A) Comparison of the domainorganization of DdCP224, Msps protein,TOGp, ZYG-9, p93dis1 and Stu2p.Sequence region 1 (black boxes) isconserved in all five sequences whereasregion 2 (dark shaded boxes) is missing inthe two yeast sequences. Similar regionsnot shared by ZYG-9 are shown in grey andthe ZYG-9 sequences with no similarity tothe other proteins is shown in white.Numbers in the boxes refer to the aminoacid similarity/identity to the DdCP224sequence (EMBL accession no. AJ012088)and the overall similarity/identity is statedon the right. (B,C) Amino acid sequencealignments of region 1 and 2. Amino acidsidentical in at least two sequences areunderlined and highlighted in boldface;gaps are indicated by dashes.


microtubule system. When cells were treated with themicrotubule depolymerizing drug thiabendazole (Fig. 4C-E;Kitanishi et al., 1984) for 3 hours, long microtubules emanatingfrom the centrosome were absent (Fig. 4C′,D′), but thecentrosomal presence of DdCP224 was unchanged (Fig. 4C-E′). In contrast, kinetochores in metaphase-like mitotic figureswere unlabeled with DdCP224 antibodies but still showedlabeling by the MPM2 antibody (Fig. 4D,E). Since mitoticcells are unable to form proper spindles in the presence ofthiabendazole (Kitanishi et al., 1984; Kitanishi-Yumura et al.,1985) DdCP224 localization in the kinetochore region requireseither its transport along microtubulestowards the spindle midzone or thepresence of microtubules at thekinetochore.

GFP mutants reveal molecularfunctions of DdCP224The localization of DdCP224throughout the cell cycle is similar tothat of yeast Stu2p, the only otherprotein of this family that is alsopermanently located at themicrotubule-organizing center (Wangand Huffaker, 1997). As DdCP224,Stu2p binds weakly alongmicrotubules in interphase and mitosisas well. Since Stu2p is only half aslong as DdCP224 and corresponds tothe N-terminal half only, we wonderedabout the function of the C-terminalhalf of DdCP224. We generated threetypes of Dictyostelium mutantsexpressing the full-length protein(DdCP224-GFP), the N-terminal half(∆C-GFP, amino acids 1-813) and theC-terminal half (∆N-GFP, amino acids809 to 2016) of DdCP224 as a C-terminal fusion to GFP. The siteseparating the two halves of DdCP224was chosen based on sequencealignments with Stu2p and p93dis1

using the GAP and PILEUP programsof the GCG package, which placed theC-termini of the Stu2p and p93dis1 atapproximately the same position. Thetransformation plasmids insertrandomly into the genome andexpression is driven by the actin 15promoter with wild-type DdCP224 asbackground. All three GFP-mutantswere viable in axenic shaking cultureand were able to form fruiting bodiesand spores. No phenotypic differenceswere observed between severalindependent transformants. Therefore,all the data reported here are based onone representative mutant of eachtype.

The expression of the expectedfusion protein was confirmed by

immunoblots stained with anti-GFP and anti-DdCP224 mAbs(Fig. 5). The electrophoretic mobility of DdCP224-GFP, ∆C-GFP and ∆N-GFP in SDS-gels was as predicted for GFP-fusion proteins with calculated molecular masses of 251, 127and 162 kDa, respectively. The epitope for the anti-DdCP224mAbs is located in the C-terminal half of DdCP224, since ∆C-GFP could not be labeled by these mAbs (data shown only for4/95 in Fig. 5). Thus the expression levels of the fusion proteinscan be estimated from protein band intensities only in the caseof DdCP224-GFP and ∆N-GFP. Cytosolic extracts ofDdCP224-GFP cells contain equal amounts of fusion protein

Fig. 3. Confocal light microscopy of the localization of DdCP224 through the cell cycle. Singleoptical sections of the centrosome or spindles are shown. Cells were fixed with methanol orformaldehyde and double-labeled with antibodies against DdCP224 (2/165 or 4/148) (A-D,F′,G′)or MPM2 (E′), and γ-tubulin (A-G). The graphs in (A′′ ), (B′′ ) and (D′′ ) are tracings offluorescence intensity (in arbitrary units) along a line through the center of the centrosomes in(A,A′) and (B,B′), or the long axis of the spindle in (D,D′), respectively. They demonstrate thatthe distribution of DdCP224 (black curve) is slightly broader than that of γ-tubulin (grey curve) ininterphase (A′′ ) and prophase (B′′ ), whereas both proteins colocalize exactly in metaphase (D′′ ).Bar, 1 µm.

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and endogenous DdCP224, whereas ∆N-GFP cells showapproximately 10-fold overexpression of the fusion protein.

As predicted, DdCP224-GFP cells exhibited strong GFPfluorescence at the centrosome in interphase and mitosis (Figs6A′-B′, 9). Fluorescence along interphase microtubules wasalso clearly visible (Figs 6A′, 9A′,B′). Hence, the labelingpattern was indistinguishable from that of endogenousDdCP224 visualized by indirect immunofluorescence inuntransformed cells (Fig. 3A′). In contrast to full-lengthDdCP224, the GFP localization pattern of the two truncatedmutants was quite unexpected. ∆C-GFP was not localized ateither microtubules or centrosomes; rather, it was distributeduniformly over the cytosol (Fig. 6C-C′′ ), and there was nodifference between interphase and mitosis (not shown). ∆N-GFP mutants, on the other hand, displayed strong GFPfluorescence at the centrosome and no detectable labeling ofmicrotubules in interphase (Fig. 6D′). During mitosis, spindlemicrotubules were labeled in addition to the centrosomes andthe kinetochore region (Fig. 6E′,F′). These results show thatthe centrosome targeting domain of DdCP224 resides in the C-terminal half of the molecule, which is missing in the spindlepole body (SPB) component Stu2p, and they confirm thatcentrosomal localization and microtubule binding areseparable functions. However, microtubule binding evidentlyrequires the interplay of both parts of the protein.

DdCP224 binds to microtubules in vitro and behavesas a monomeric proteinThe microtubule-binding properties of DdCP224 were studiedin more detail in vitro. DdCP224 and all three GFP-mutantswere enriched from cytosolic Dictyostelium extracts by cationexchange chromatography. Further purification attempts werehampered by problems with proteolysis and low protein yields,but this partially purified preparation was found sufficient totest for binding to microtubules in a spin-down assay. Inuntransformed AX2 cells, most of the cytosolic DdCP224 co-sedimented with taxol-stabilized pig brain microtubules (Fig.7), whereas almost no DdCP224 was detected in the pellet in

the absence of microtubules. Co-sedimentation experimentswith the three GFP fusion proteins were fully consistent withthe microscopic observations. Most of the DdCP224-GFPfusion protein could be sedimented with microtubules, but ∆C-GFP and ∆N-GFP exhibited no detectable microtubulebinding. These findings show that DdCP224 binds tomicrotubules, but we cannot judge from this result whetherDdCP224 interacts directly with microtubules or via anotherassociated protein.

To determine whether DdCP224 can self-associate or forma complex with other proteins, Dictyostelium cytosolic extracts


Fig. 4. Centrosomal localizationof DdCP224 does not requiremicrotubules. (A) Confocal imageof isolated centrosomes (Gräf etal., 1998) stained with the anti-DdCP224 mAb 2/165.(B-E) Thiabendazole treatment ofvegetative Dictyostelium cells.(B) Untreated cells in interphase,(C,C′) a dinucleate,thiabendazole-treated interphasecell and (D,D′, E,E′)thiabendazole-treated mitoticcells. Cells were stained with therat-anti-α-tubulin antibody YL1/2(B,C′,D′), the anti-DdCP224 mAb2/165 (C,C), rabbit-anti-Dictyostelium-γ-tubulin (E′),MPM2 (E) and 4,6-diamidino-2-phenylindole (DAPI) (blue). Note that mitotic figures in thiabendazole-treated cells have an unusual appearance since Dictyostelium cells areunable to form a mitotic spindle in the presence of the drug. Therefore, the duplicated centrosomes are often found at one side of the condensedchromosome mass and the kinetochores (arrow) are not centered between the two centrosomes (E) as in Fig. 3D′,E′. Double-labeling of thesame cell with an anti-DdCP224 mAb and MPM2 was not possible since both antibodies were generated in mice. Bars: 1 µm (A); 10 µm (B-E).

Fig. 5. Size determination and expression level of DdCP224/GFPfusion proteins. Western blot of a cytosolic extract of full-lengthDdCP224-GFP (FL), ∆C-GFP (∆C) and ∆N-GFP (∆N), separated ona 6% SDS-polyacrylamide gel. Protein of ~5×106 cells was loaded oneach lane. The blot was stained with either the anti-DdCP224 mAb4/95 or an anti-GFP mAb (Chemicon, Temecula, California) asindicated on the bottom. Bands were visualized by color detectionusing nitrobluetetrazolium chloride and bromo-chloro-indolyl-phosphate in case of 4/95-labeling and by enhancedchemiluminescence in case of anti-GFP labeling. The band at ~205kDa (arrow) corresponds to endogenous DdCP224. The length ofstandard proteins is indicated on the right.


were loaded onto sucrose density gradients. DdCP224fractionated almost exactly as catalase (232 kDa) and thusbehaved like a monomeric protein in these gradients (Fig. 8).Fractionation was unaffected by the presence of 0.1% TritonX-100 (data not shown).

The DdCP224-GFP mutant shows that DdCP224 isinvolved in centrosome duplicationDdCP224-GFP mutants displayed informative defects incentrosome duplication and cytokinesis (Table 1). In axenicshaking culture, only ~25% of the transformants contained onenucleus and one centrosome (Fig. 6A), whereas more than 50%had more than one centrosome per nucleus (Fig. 9) Thesesupernumerary centrosomes were usually not linked to thenucleus but seemed to be functional in microtubule nucleationsince they were the center of amicrotubule aster (Fig. 9A′,B′).Furthermore, labeling ofsupernumerary centrosomes withantibodies against γ-tubulin and the350-kDa centrosomal antigen (Kaltand Schliwa, 1996) wasindistinguishable from that of nucleus-associated centrosomes (data notshown), and the doughnut-likeappearance of anti-DdCP224-labeledsupernumerary centrosomes inconfocal microscopy suggests thatthey possess a normal corona as well(Fig. 9F-F′′′ ). Unusual centrosomalshapes such as large elongated,dumbbell-, or kidney-shapedcentrosomes at the nucleus were alsoobserved (Fig. 9C,D,E). Theseaberrant shapes may be the result ofincomplete centrosome duplication.Moreover, 50% of all cells possessedmore than one nucleus, and giant cellswith 6 and more nuclei were quitefrequent. Moreover, unequalcytokinesis is suggested by anabundance of tri- and hexanucleatecells. Taken together, theseobservations indicate that DdCP224-GFP cells have a cytokinesis defect aswell (Fig. 9A). Since multinuclearcells exhibited supernumerarycentrosomes with about the samefrequency as mononuclear cells (Table1), the centrosome duplication defectand the cytokinesis defect seem to beindependent of each other.

As DdCP224-GFP cells,approximately 50% of all ∆C-GFPcells had two or more nuclei (Fig. 6C),but immunofluorescence analysis withthe mAb 4/148 revealed that only asmall fraction of these mutant cellscontained supernumerary centrosomes(4%; Table 1). The occurrence of morethan one nucleus did not correlate with

GFP fluorescence intensity (not shown). In contrast, ∆N-GFPmutants showed no centrosome duplication or cytokinesisdefect at all (Table 1). Supernumerary centrosomes were notobserved and less than 30% of all cells contained more thanone nucleus, and hardly any cell more than two. These cellstherefore were essentially indistinguishable from theuntransformed AX2 strain.

Surprisingly, the strikingly frequent centrosomal defects ofDdCP224-GFP mutants and the cytokinesis defects of bothDdCP224-GFP and ∆C-GFP cells did not affect the growth ratesof these cell lines. As the normal-appearing ∆N-GFP cells, thesetwo mutants had maximum doubling times between 10 and 11hours in axenic shaking culture at 21°C. Untransformed cellsdivide only slightly faster under these growth conditions(doubling time 9-10 hours; data not shown). Thus the extra

Fig. 6. Localization of DdCP224-GFP, ∆C-GFP and ∆N-GFP. Phase contrast imagesare shown in (A,B,C,D,E,F) and GFPfluorescence images (green) in(A′,B′,C′,D′,E′,F′). Since there is no GFPfluorescence at the centrosome in ∆C-GFPmutants, centrosomes in C′′ were labeledby mAb 4/148 (red). DAPI staining is

shown in blue. Cells in A, C and D are in interphase, whereas the cells in B, E and F are intelophase, anaphase and metaphase, respectively. All cells were fixed with methanol. Bar, 10 µm.

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centrosomes apparently did not interfere significantly withmitosis. We frequently observed mitotic cells in meta- oranaphase with normal spindles and supernumerary centrosomesin the cytosol not associated with the chromosomes or spindles(Fig. 9G′,H′). Dictyostelium undergoes closed mitosis, so thenuclear envelope might protect the mitotic spindle frominterference by these supernumerary, cytosolic centrosomes. Thecytokinesis defect does not seem to be severe, and it apparentlycan be overcome in a subsequent cell cycle. Fig. 9J shows a oncetrinucleate cell in telophase undergoing cytokinesis that appearsto lead to 6 daughter cells. Multiple fission events such as thesemight explain the good viability of DdCP224-GFP cells despitetheir centrosomal and cytokinesis defects.

DISCUSSION

Using mAbs raised against isolated Dictyostelium centrosomeswe have obtained the complete cDNA sequence of a proteinwe have named DdCP224. It is a new member of a family ofmicrotubule-binding proteins consisting of human TOGp, theDrosophila Msps protein, Xenopus XMAP215, C. elegansZYG-9, S. cerevisiae Stu2p and S. pombe p93dis1 (Charrasse etal., 1995; Cullen et al., 1999; Matthews et al., 1998; Nabeshimaet al., 1995; Tournebize et al., 2000; Wang and Huffaker, 1997).The primary structures of these proteins are only weaklyconserved and show some striking differences in domainorganization. These likely reflect unique cell biological

properties of these proteins. Among the sequences publishedso far, DdCP224 is the first non-animal member of this familywhich displays sequence similarity over the entire sequence tothe human, Xenopus and Drosophila proteins and which hasapproximately the same size. Surprisingly, DdCP224 is moreclosely related to the Drosophila protein than ZYG-9, the otherinvertebrate member of this protein family.

The cellular distribution of DdCP224 is most similarto yeast Stu2pCuriously, the cell biological properties of DdCP224 shows theclosest similarity to Stu2p (Wang and Huffaker, 1997) whoseamino acid sequence is the most distantly related. Whereas allseven proteins localize to spindle microtubules and spindlepoles during mitosis (Charrasse et al., 1998; Cullen et al., 1999;Gard et al., 1995; Matthews et al., 1998; Nabeshima et al.,1995; Wang and Huffaker, 1997), only DdCP224 and Stu2p aregenerally localized at the centrosome (SPB in yeasts) ininterphase as well. In both cases, the presence at thecentrosome is unaffected by microtubule depolymerisingdrugs, suggesting that microtubules are not required forcentrosomal localization. The presence of DdCP224 at isolatedcentrosomes and the association of the ∆N-GFP protein withcentrosomes but not microtubules (see below) are consistent


Table 1. Ratio of centrosomes per nucleus and frequency of multinuclear cells in DdCP224 mutantsCentrosome/nucleus ratio 1C/1N ≥2C/≥2N, C=N ≥2C/1N ≥2C/≥2N, C>N

DdCP224-GFP 27% (n=52) 21% (n=40) 23% (n=45) 29% (n=55)∆C-GFP 49% (n=109) 47% (n=103) 3% (n=6) 1% (n=3)∆N-GFP 71% (n=150) 29% (n=60) Not observed Not observedAX2 (control) 64% (n=158) 36% (n=90) Not observed Not observed

The number of centrosomes and nuclei in DdCP224-GFP cells, ∆C-GFP cells, ∆N-GFP cells and the control strain AX2 was counted. The table gives thepercentage of cells with one nucleus and one centrosome (1C/1N), cells with two or more nuclei and one centrosome per nucleus (≥2C/≥2N, C=N),supernumerary centrosomes but only one nucleus (≥2C/1N) and cells with supernumerary centrosomes and two or more nuclei (≥2C/≥2N, C>N). The number ofcells (n) evaluated is given in brackets.

Fig. 7. In vitro microtubule binding properties of DdCP224 and itsGFP mutants. Partially purified wild-type DdCP224 (WT), full-length DdCP224-GFP (FL), ∆C-GFP (∆C) and ∆N-GFP (∆N) wereincubated with microtubules (MT). Control samples (C) contained nomicrotubules. After centrifugation, pellets (P) and supernatants (S)were proteins were separated on a 6% SDS-polyacrylamide gel andblotted. The western-blot was stained with either the anti-DdCP224mAb 2/165 or an anti-GFP mAb, as indicated on the bottom. Bandswere visualized by color detection (see Fig. 5) in case of 2/165-labeling and by enhanced chemiluminescence in case of anti-GFPlabeling. The length of standard proteins is indicated on the left.

Fig. 8. Sedimentation behavior of cytosolic DdCP224 in sucrosegradients. A cytosolic extract was loaded onto a sucrose gradient andthe sedimentation behavior of DdCP224 was compared to nativestandard proteins with molecular masses of 670 kDa (thyroglobulin),232 kDa (catalase), 140 kDa (lactate dehydrogenase) and 67 kDa(bovine serum albumin). After centrifugation and fractionation,proteins were separated on a 6% SDS-polyacrylamide gel. DdCP224was visualized on an immunoblot stained with the anti-DdCP224mAb 2/165, and standard proteins were stained with CoomassieR250 (shown for catalase here). The peak fractions of the standardproteins are marked by an arrow at the bottom.


with this conclusion. Thus DdCP224 is a genuine centrosomalcomponent, unlike the DdCP224-like proteins in animalswhich have other localizations in interphase. For example,TOGp colocalizes with markers of the endoplasmic reticulum(Charrasse et al., 1998), while ZYG-9 is distributed in thecytoplasm (Matthews et al., 1998). Less is known about thecellular distribution of XMAP215 (Gard et al., 1995). TheMsps protein in cellularized Drosophila embryos is also absentfrom the centrosome during interphase, but it localizes to thecentrosome also in interphase during the first 13 cell cycles ofthe syncycial embryo (Cullen et al., 1999). But these early cellcycles are unique: They are extremely fast since there are nogap phases, DNA replication checkpoints or cell divisions.Thus the permanent centrosomal presence of the Msps proteinduring these early cell cycles might be due to the very shortinterphase which lasts only ~10 minutes. The two yeastproteins Stu2p and p93dis1 were localized along microtubulesin interphase by fluorescence microscopy just as DdCP224,suggesting that they bind laterally along microtubules. In thecase of Stu2p, this lateral association could be confirmed bymicrotubule binding experiments in vitro (Wang and Huffaker,1997). Taken together, the members of this protein familyexhibit considerable variety in their localization patterns.

The C-terminal half of the protein targets DdCP224to the centrosome...The strikingly similar localization patterns of DdCP224 andStu2p raises the question of the role of the C-terminal half ofDdCP224, since Stu2p corresponds to the N-terminal half ofDdCP224 only. Therefore, we generated the ∆C-GFP mutant,which is the topological equivalent of the yeast proteins, its ∆N-GFP counterpart, and the full-length mutant, DdCP224-GFP,for reference. DdCP224-GFP behaved as expected, i.e. its majorlocalization was at the centrosome during the entire cell cycleas well as along interphase-microtubules. Unexpectedly, ∆N-GFP localized to centrosomes in interphase and mitosis to thesame extent as full-length DdCP224-GFP. Thus the centrosomaltargeting domain of DdCP224 resides in its C-terminal half. Thebinding of Stu2p to the SPB must therefore be achieved by adifferent mechanism and might involve different proteinbinding partners. One possible candidate is the SPB componentSpc72p which also serves as the docking site for yeast γ-tubulincomplexes (Tub4p-complexes) at the SPB (Knop and Schiebel,1998). Chen et al. (1998) have shown that Stu2p and Spc72pbind to each other and form a cytosolic complex. At the SPB,Spc72p likely anchors both the Tub4p complex and Stu2p. Nohomologue for Spc72p has so far been found in higherorganisms or in Dictyostelium. Thus one might speculate that aSpc72p homologue does not exist in Dictyostelium becauseanchoring of DdCP224 is accomplished by its C-terminaldomain, which serves the function of a Spc72p-like protein.Furthermore, unlike Stu2p, cytosolic DdCP224 behaves as amonomer in sucrose density gradients suggesting that there isno Spc72p-like binding partner of DdCP224. The lack ofsequence similarity between the DdCP224 C-terminal half andSpc72p does not exclude the possibility that they arefunctionally equivalent, at least in part.

...but binding to interphase microtubules requiresthe N-terminal half of DdCP224The issue of microtuble binding seems to be more complicated.

In interphase, the full-length GFP fusion protein (DdCP224-GFP) is localized along microtubules, whereas ∆N-GFP ispresent at centrosomes only. During mitosis both constructswere detected at the mitotic spindle and at the spindle poles.This suggests that the N-terminal half is required for bindingalong cytoplasmic microtubules but not involved in binding tospindle microtubules. Hence binding to cytoplasmic andspindle microtubules, respectively, might be independentactivities of DdCP224. However, the N-terminal half alonedoes not seem to be sufficient for binding to cytoplasmicmicrotubules because ∆C-GFP was present neither atmicrotubules nor at the centrosome. The topologicallyequivalent yeast protein, on the other hand, has to provide thebinding sites for both localizations. Consequently, microtubulebinding appears to require interaction between the N- and C-terminal half of DdCP224. The microscopic observations wereconfirmed by in vitro microtubule binding assays where wild-type DdCP224 and DdCP224-GFP but neither of the twotruncated GFP fusion proteins cosediment with taxol-stabilizedpig brain microtubles. Since DdCP224 could not be purified tohomogeneity we cannot exclude that microtubule binding isaccomplished via a DdCP224 associated protein. However,since the vast majority of cytosolic DdCP224 seems to bemonomeric it is likely that DdCP224 binds directly tomicrotubules.

Sequence comparison does not reveal the microtubulebinding determinants of DdCP224. TOGp possesses a motifclose to its C terminus that is similar to the microtubule bindingrepeats of MAP2, MAP4 and tau (Charrasse et al., 1998), butDdCP224 and both yeast proteins do not contain such aconsensus sequence. The microtubule binding sites of p93dis1

and Stu2p could be mapped to a ~100 amino acid regionstarting approximately at amino acid position 550 (Nakasekoet al., 1996; Wang and Huffaker, 1997). In Stu2p, this regionincludes two imperfect repeats that both contribute tomicrotubule binding, but the corresponding region in p93dis1

does not contain these repeats and shows no striking similarityto Stu2p. DdCP224 does not exhibit any similarity in thisregion to either of the two yeast proteins. Thus the sequencemotifs or domains that contribute to microtubule binding haveyet to be identified.

The functions of DdCP224-like proteins at microtubules arenot known. It is conceivable that these functions vary indifferent organisms as suggested by their considerablesequence divergence. However, particularly in the case ofXMAP215, the microtubule binding properties have beenthoroughly studied in vitro (Andersen, 1998; Gard andKirschner, 1987; Tournebize et al., 2000; Vasquez et al., 1994,1999). These studies suggest that XMAP215 binds along theentire length of microtubules, and that it mainly functions as astimulator of microtubule plus-end assembly and turnoverwithout blocking catastrophes. Since XMAP215 seems to beregulated by CDK1/cyclin B (Vasquez et al., 1999; Charrasseet al., 2000) it may play a role in promoting the increasedmicrotubule dynamics at the transition from interphase tomitosis (Desai and Mitchison, 1997).

DdCP224 is involved in centrosome duplication andcytokinesisThe analysis of the DdCP224-GFP and ∆C-GFP mutantsprovides strong evidence for an important role of DdCP224 in

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centrosome duplication. Such a functionhas not been discussed for any othermember of this protein family so far.More than 50% of all DdCP224-GFP cells contained supernumerarycentrosomes not associated with thenucleus. Furthermore, these mutants hada cytokinesis defect as well, since about50% were multinuclear, often with morethan two nuclei per cell. However,the two defects do not seem to bedirectly linked since supernumerarycentrosomes did not occur at a higherfrequency in multinuclear than inmononuclear cells. It is unlikely thatthese defects are caused by integrationof the transformation vector into anothergene also involved in centrosomeduplication or cytokinesis, mainly fortwo reasons: first, the mutantphenotypes were observed in fiveindependent DdCP224-GFP and threeindependent ∆C-GFP mutants,respectively; and second, ∆N-GFPmutants based on the same GFP-transformation plasmid possess normalphenotypes. Moreover, the presence ofGFP at the centrosome can also beexcluded as a reason for these defectssince ∆N-GFP cells and γ-tubulin-GFPcells (Ueda et al., 1997) appear normal.Thus the centrosomal and cytokinesisdefects likely are caused by theexpression of supernumerary copiesof DdCP224-GFP and ∆C-GFP,respectively, in a wild-type backgroundof DdCP224. The GFP forms, which areslightly bulkier due to the GFP-tag, maycompete with the endogenous DdCP224and slightly impair its function. Thedefects observed here then imply thatDdCP224 plays a role in centrosomeduplication. Interestingly, ∆N-GFP doesnot impair the function of wild-typeDdCP224 even though it shows correctcentrosomal localization. Conceivably,due to the N-terminal deletion, it may beunable to interact with microtubulesor other potential protein partnersinvolved in centrosome duplication orcytokinesis. Since DdCP224 is both acentrosomal and a microtubule bindingprotein, defects in its crosstalk withmicrotubules might cause thecentrosome duplication deficiency.Microtubules are not required forcentrosome duplication per se but ratherfor completion of the centrosomecycle, which includes separation ofthe duplicated centrosomes. Thisconclusion is supported by earlier


Fig. 9. Defects incentrosome duplication andcytokinesis of full-lengthDdCP224-GFP cells. Phasecontrast images are shownin A,B,G,H,J and GFPfluorescence images inA′,B′,C,D,F,G′,H′,J′. DAPIstainings are shown in blue.DdCP224-GFP cells areshown in interphase(A,B,C,D,E,F) and mitosis(G,H,J). (A) shows a

hexanuclear cell with 12 centrosomes, (B) a mononucleate cell with 2 centrosomes. Cells inC,D,E are examples of aberrant interphase centrosomes. The dinucleated cell in D has onenormal and one dumbbell-shaped centrosome. (F-F′′′ ) Confocal microscopy of a mononucleatedcell with supernumerary centrosomes labeled with mAb 2/165 (green); DNA (red) was stainedwith 5 µg/ml propidium iodide containing 100 µg/ml RNAse A; merged image (F). All threecentrosomes (F′-F′′′ ) are doughnut-shaped indicating an intact corona. The anaphase cells in Gand H have three (G′) and two (H′) supernumerary centrosomes, respectively. The oncetrinucleate cell in (J) undergoes cytokinesis, probably leading to six daughter cells. All cellswere fixed with methanol. Bars: 10 µm (A,B,F,G,H,J); 2 µm (C,D,E); 0.5 µm (F′-F′′′ ).


experiments with thiabendazole-treated Dictyostelium cells(Kitanishi et al., 1984; Kitanishi-Yumura et al., 1985) whichhave defects similar to DdCP224-GFP cells.

Spindle microtubules interact not only with the centrosomebut also with kinetochores. DdCP224 and C. elegans ZYG-9were both detected at the kinetochore region in metaphase, inaddition to their localization at the spindle poles (Matthews etal., 1998). Moreover, in several dis1 mutants of S. pombe, lossof functional p93dis1 inhibits sister chromatid separationwithout blocking spindle elongation (Nabeshima et al., 1995,1998). Similarly, functionally compromised DdCP224-GFPcould interfere with sister chromatid separation inDictyostelium, but the spindle poles may still be separated bythe elongating spindle. After breakdown of the spindle, thechromosomes would then be associated with one pole only andthe second centrosome would be set free. However, in the caseof dis1 mutants, viable cells with supernumerary SPBs havenot been reported, and the occurence of large elongated,dumbbell- and kidney-shaped centrosomes in DdCP224-GFPcells cannot be explained solely by a defect in chromatidseparation. Furthermore, the nuclear size as a measure forploidy of DdCP224-GFP cells with supernumerarycentrosomes is usually not increased (data not shown). TheDrosophila msps mutant, which displays a strongly reducedexpression of the Msps protein, exhibits chromosomesegregation defects as well (Cullen et al., 1999). However, inthis case chromosome segregation seems to be disturbed by adisruption of the mitotic spindle. This is characterized by theappearance of one or more small additional bipolar ormonopolar spindles, so-called mini spindles. Frequently,spindles are totally disorganized and contain no more distinctspindle poles at all. Thus, Cullen et al. (1999) concluded thatone of the functions of the Msps protein might be aninvolvement in microtubule bundling which holds the mitoticspindle together. Supernumerary centrosomes as in DdCP224-GFP cells were not described and there is so far no evidencefor the presence of any centrosomal marker protein at the mini-spindle poles of mutant Drosophila embroys. By contrast, threelines of evidence indicate that supernumerary centrosomes ofthe DdCP224-GFP mutant closely resemble normalcentrosomes. First, they contain DdCP224, γ-tubulin and the350-kDa antigen (Kalt and Schliwa, 1996); second, confocalmicroscopy suggests that they possess a normal corona; andthird, they are capable of microtubule organization ininterphase. Interestingly, the phenotype of the msps mutant isquite reminiscent of the C. elegans zyg-9 mutant embyosshowing disorganized spindles and numerous cytoplasmicclusters of short microtubules during meiosis (Kemphues et al.,1986). Taken together, within this protein family, DdCP224-GFP is the first mutant exhibiting supernumerary centrosomesand no significant defects in spindle integrity and mitoticprogression which are characteristic for all functionallydefective mutants of DdCP224-related proteins in the otherspecies.

The role of DdCP224 in cytokinesis is more difficult toexplain. There is no evidence for an association of DdCP224with proteins directly involved in cytokinesis. However, in latetelophase, the spindle midbody to which DdCP224-GFP islocalized comes in close contact with the cytokineticconstriction zone. This might allow DdCP224 to fulfill a latefunction in cytokinesis. ∆C-GFP cells have a similar

cytokinesis defect but in these cells no GFP labeling at anystructure involved in mitosis is observed. Thus the role ofDdCP224 in cytokinesis may be quite indirect.

This work shows DdCP224 to be a multifacetted andmultifunctional molecule involved in serveral aspects ofmicrotubule and centrosome biology. The roles of the membersof the interesting protein family to which it belongs may havechanged in the course of evolution, resulting in a highlyadaptive and therefore relatively poorly conserved moleculewith nevertheless essential roles in centrosome function andmitosis.

We thank Nicole Brusis for expert technical assistance and TimoZimmermann for his help at the confocal microscope, UrsulaEuteneuer for helpful discussion and John V. Kilmartin for generouslyproviding us with the YL1/2 antibody. This work was supported bythe Deutsche Forschungsgemeinschaft (SFB184) and the Fonds derChemischen Industrie.

REFERENCES

Adachi, H., Hasebe, T., Yoshinaga, K., Ohta, T. and Sutoh, K. (1994).Isolation of Dictyostelium discoideum cytokinesis mutants by restrictionenzyme-mediated integration of the blasticidin S resistance marker.Biochem. Biophys. Res. Commun. 205, 1808-1814.

Andersen, S. S. (1998). Xenopus interphase and mitotic microtubule-associated proteins differentially suppress microtubule dynamics in vitro.Cell Motil. Cytoskeleton. 41, 202-213.

Bollag, D. M., Rozycki, M. D. and Edelstein, S. J. (1996). Protein Methods.New York: Wiley-Liss Inc.

Charrasse, S., Mazel, M., Taviaux, S., Berta, P., Chow, T. and Larroque,C. (1995). Characterization of the cDNA and pattern of expression of a newgene over-expressed in human hepatomas and colonic tumors. Eur. J.Biochem. 234, 406-413.

Charrasse, S., Schroeder, M., Gauthier Rouvieve, C., Ango, F., Cassimeris,L., Gard, D. L. and Larroque, C. (1998). The TOGp protein is a newhuman microtubule-associated protein homologous to the XenopusXMAP215. J. Cell Sci. 111, 1371-1383.

Charrasse, S., Lorca, T., Doree, M. and Larroque, C. (2000). The XenopusXMAP215 and its human homologue TOG proteins interact with cyclin B1to target p34cdc2 to microtubules during mitosis. Exp. Cell Res. 254, 249-256.

Chen, X. P., Yin, H. and Huffaker, T. C. (1998). The yeast spindle pole bodycomponent Spc72p interacts with Stu2p and is required for propermicrotubule assembly. J. Cell Biol. 141, 1169-1179.

Cullen, C. F., Deák, P., Glover, D. M. and Ohkura, H. (1999). Minispindles: a gene encoding a conserved microtubule-associated proteinrequired for the integrity of the mitotic spindle in Drosophila. J. Cell Biol.146, 1005-1018.

Desai, A. and Mitchison, T. J. (1997). Microtubule polymerization dynamics.Annu. Rev. Cell Dev. Biol. 13, 83-117.

Donaldson, A. D. and Kilmartin, J. V. (1996). Spc42p: a phosphorylatedcomponent of the S. cervisiae spindle pole body (SPB) with an essentialfunction during SPB duplication. J. Cell Biol. 132, 887-901.

Engle, D. B., Doonan, J. H. and Morris, N. R. (1988). Cell-cycle modulationof MPM-2-specific spindle pole body phosphorylation in Aspergillusnidulans. Cell Motil. Cytoskel. 10, 434-437.

Euteneuer, U., Gräf, R., Kube-Granderath, E. and Schliwa, M. (1998).Dictyostelium γ-tubulin: molecular characterization and ultrastructurallocalization. J. Cell Sci. 111, 405-412.

Fry, A. M., Mayor, T., Meraldi, P., Stierhof, Y. D., Tanaka, K. and Nigg,E. A. (1998a). C-Nap1, a novel centrosomal coiled-coil protein andcandidate substrate of the cell cycle-regulated protein kinase Nek2. J. CellBiol. 141, 1563-1574.

Fry, A. M., Meraldi, P. and Nigg, E. A. (1998b). A centrosomal function forthe human Nek2 protein kinase, a member of the NIMA family of cell cycleregulators. EMBO J. 17, 470-481.

Gard, D. L., Cha, B. J. and Schroeder, M. M. (1995). Confocalimmunofluorescence microscopy of microtubules, microtubule-associated

1758

proteins, and microtubule-organizing centers during amphibian oogenesisand early development. Curr. Top. Dev. Biol. 31, 383-431.

Gard, D. L. and Kirschner, M. W. (1987). A microtubule-associated proteinfrom Xenopus eggs that specifically promotes assembly at the plus-end. J.Cell Biol. 105, 2203-2215.

Gräf, R., Euteneuer, U., Ueda, M. and Schliwa, M. (1998). Isolation ofnucleation-competent centrosomes from Dictyostelium discoideum. Eur. J.Cell Biol. 76, 167-175.

Gräf, R., Daunderer, C. and Schliwa, M. (1999). Cell cycle-dependentlocalization of monoclonal antibodies raised against isolated Dictyosteliumcentrosomes. Biol. Cell. 91, 471-477.

Gräf, R., Brusis, N., Daunderer, C., Euteneuer, U., Hestermann, A.,Schliwa, M. and Ueda, M. (2000). Comparative structural, molecular andfunctional aspects of the Dictyostelium discoideum centrosome. Curr. Top.Dev. Biol. (in press).

Kalt, A. and Schliwa, M. (1996). A novel structural component of theDictyostelium centrosome. J. Cell Sci. 109, 3103-3112.

Kemphues, K. J., Wolf, N., Wood, W. B. and Hirsh, D. (1986). Two locirequired for cytoplasmic organization in early embryos of Caenorhabditiselegans. Dev. Biol. 113, 449-460.

Kilmartin, J. V., Dyos, S. L., Kershaw, D. and Finch, J. T. (1993). A spacerprotein in the Saccharomyces cerevisiae spindle poly body whose transcriptis cell cycle-regulated. J. Cell Biol. 123, 1175-1184.

Kitanishi, T., Shibaoka, H. and Fukui, Y. (1984). Disruption of microtubulesand retardation of development of Dictyostelium with ethyl N-phenylcarbamate and thiabendazole. Protoplasma. 120, 185-196.

Kitanishi-Yumura, T., Blose, S. H. and Fukui, Y. (1985). Role of the MT-MTOC complex in determination of the cellular locomotory unit inDictyostelium. Protoplasma. 127, 133-146.

Kitanishi-Yumura, T. and Fukui, Y. (1987). Reorganization of microtubulesduring mitosis in Dictyostelium: dissociation from MTOC and selectiveassembly/disassembly in situ. Cell Motil. Cytoskel. 8, 106-117.

Knop, M. and Schiebel, E. (1997). Spc98p and Spc97p of the yeast γ-tubulincomplex mediate binding to the spindle pole body via their interaction withSpc110p. EMBO J. 16, 6985-6995.

Knop, M. and Schiebel, E. (1998). Receptors determine the cellularlocalization of a γ-tubulin complex and thereby the site of microtubuleformation. EMBO J. 17, 3952-3967.

Lupas, A., VanDyke, M. and Stock, J. (1991). Predicting coiled coils fromprotein sequences. Science. 252, 1162-1164.

Mandelkow, E. M., Herrmann, M. and Rühl, U. (1985). Tubulin domainsprobed by limited proteolysis and subunit-specific antibodies. J. Mol. Biol.185, 311-327.

Mann, S. K. O., Devreotes, P. N., Eliott, S., Jermyn, K., Kuspa, A.,Fechheimer, M., Furukawa, R., Parent, C. A., Segall, J., Shaulsky, G. etal. (1998). Cell biological, molecular genetic, and biochemical methodsused to examine Dictyostelium. In Cell Biology: A Laboratory Handbook,vol. 1. (ed. J. E. Celis), pp. 431-465, San Diego: Academic Press.

Manstein, D. J., Titus, M. A., De Lozanne, A. and Spudich, J. A. (1989).Gene replacement in Dictyostelium generation of myosin null mutants.EMBO J. 8, 923-932.

Manstein, D. J., Schuster, H.-P., Morandini, P. and Hunt, D. M. (1995).Cloning vectors for the production of proteins in Dictyostelium discoideum.Gene 162, 129-134.

Matthews, L. R., Carter, P., Thierry Mieg, D. and Kemphues, K. (1998).ZYG-9, a Caenorhabditis elegans protein required for microtubule

organization and function, is a component of meiotic and mitotic spindlepoles. J. Cell Biol. 141, 1159-1168.

Moens, P. B. (1976). Spindle and kinetochore morphology of Dictyosteliumdiscoideum. J. Cell Biol. 68, 113-122.

Morio, T., Urushihara, H., Saito, T., Ugawa, Y., Mizuno, H., Yoshida, M.,Yoshino, R., Mitra, B. N., Pi, M., Sato, T. et al. (1998). The Dictyosteliumdevelopmental cDNA project: generation and analysis of expressedsequence tags from the first-finger stage of development. DNA Res. 5, 335-340.

Nabeshima, K., Kurooka, H., Takeuchi, M., Kinoshita, K., Nakaseko, Y.and Yanagida, M. (1995). p93dis1, which is required for sister chromatidseparation, is a novel microtubule and spindle pole body-associating proteinphosphorylated at the Cdc2 target sites. Genes Dev. 9, 1572-1585.

Nabeshima, K., Nakagawa, T., Straight, A. F., Murray, A., Chikashige, Y.,Yamashita, Y. M., Hiraoka, Y. and Yanagida, M. (1998). Dynamics ofcentromeres during metaphase-anaphase transition in fission yeast: Dis1 isimplicated in force balance in metaphase bipolar spindle. Mol. Biol. Cell. 9,3211-3225.

Nakaseko, Y., Nabeshima, K., Kinoshita, K. and Yanagida, M. (1996).Dissection of fission yeast microtubule associating protein p93Dis1: regionsimplicated in regulated localization and microtubule interaction. GenesCells 1, 633-644.

Nellen, W., Datta, S., Reymond, C., Sivertsen, A., Mann, S., Crowley, T.and Firtel, R. A. (1987). Molecular biology in Dictyostelium: tools andapplications. Meth. Cell Biol. 28, 67-100.

Omura, F. and Fukui, Y. (1985). Dictyostelium MTOC: Structure and linkageto the nucleus. Protoplasma. 127, 212-221.

Roos, U. P. (1975). Fine structure of an organelle associated with the nucleusand cytoplasmic microtubules in the cellular slime mould Polysphondyliumviolaceum. J. Cell Sci. 18, 315-326.

Schiebel, E. and Bornens, M. (1995). In search of a function for centrins.Trends Cell Biol. 5, 197-201.

Tournebize, R., Popov, A., Kinoshita, K., Ashford, A. J., Rybina, S.,Pozniakovsky, A., Mayer, T. U., Walczak, C. E., Karsenti, E. andHyman, A. A. (2000). Control of microtubule dynamics by the antagonisticactivities of XMAP215 and XKCM1 in Xenopus egg extracts. Nature CellBiol. 2, 13-19.

Ueda, M., Gräf, R., MacWilliams, H. K., Schliwa, M. and Euteneuer, U.(1997). Centrosome positioning and directionality of cell movements. Proc.Nat. Acad. Sci. USA 94, 9674-9678.

Ueda, M., Schliwa, M. and Euteneuer, U. (1999). Unusual centrosome cyclein Dictyostelium: correlation of dynamic behavior and structural changes.Mol. Biol. Cell 10, 151-160.

Vasquez, R. J., Gard, D. L. and Cassimeris, L. (1994). XMAP from Xenopuseggs promotes rapid plus end assembly of microtubules and rapidmicrotubule polymer turnover. J. Cell Biol. 127, 985-993.

Vasquez, R. J., Gard, D. L. and Cassimeris, L. (1999). Phosphorylation byCDK1 regulates XMAP215 function in vitro. Cell Motil. Cytoskel. 43, 310-321.

Wang, P. J. and Huffaker, T. C. (1997). Stu2p: A microtubule-binding proteinthat is an essential component of the yeast spindle pole body. J. Cell Biol.139, 1271-1280.

Wigge, P. A., Jensen, O. N., Holmes, S., Soues, S., Mann, M. andKilmartin, J. V. (1998). Analysis of the Saccharomyces spindle pole bymatrix-assisted laser desorption/ionization (MALDI) mass spectrometry. J.Cell Biol. 141, 967-977.


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