rho-dependent transfer of citron-kinase to the cleavage...

INTRODUCTION

Cytokinesis is the final step in cell division in which a parentcell is divided into two daughter cells. After segregation ofchromosomes to the opposite poles in anaphase, a cleavagefurrow is formed around the equator of a dividing cell, whichdeepens in telophase finally to separate two daughter cells. Inclassical experiments using fertilized eggs of sea urchin ornewt eggs, a ring composed of actomyosin is observed beneaththe cleavage furrow and it is suggested that the constriction ofthis ring leads to the cleavage of the cell. Indeed, disruption ofthis ring with F-actin-depolymerizing compounds results infailure of cytokinesis. However, how cytokinesis is temporallylinked with nuclear division and how the cytokinetic apparatusis constructed spatially in a dividing cell remain largelyunknown (Satterwhite and Pollard, 1992; Fishkind and Wang,1995; Glotzer, 1997; Hales et al., 1999; Robinson and Spudich,2000). The small GTPase Rho is suggested as a crucialregulator in these processes of cytokinesis. For example,inactivation of Rho with botulinum C3 exoenzyme prevents

fertilized eggs of sea urchin or Xenopusembryos from enteringinto cytokinesis after nuclear division and producesmultinucleate cells (Kishi et al., 1993; Mabuchi et al., 1993;Drechsel et al., 1997). Furthermore, injection of C3 exoenzymeinto cells undergoing cytokinesis causes dissolution of thecontractile ring and regression of the cleavage furrow, and thecells cannot continue cytokinesis (Mabuchi et al., 1993). Theseresults strongly suggest that Rho is activated during celldivision and works as a switch to induce and maintain thecytokinetic apparatus. Recently, a putative activator of Rho inthis process has been identified. This GDP-GTP exchanger forRho, Pebble in Drosophilaand ECT-2 in mammalian cells, isfound to be activated after the nuclear division (Prokopenko etal., 1999; Tatsumoto et al., 1999). Indeed, the GTP-boundactive form of Rho accumulates during division of HeLa cellsand this accumulation was abolished by expression of adominant negative form of ECT-2, resulting in formation ofmultinucleate cells (Kimura et al., 2000). Thus, Rho appearsto induce cytokinesis by organizing the cytokinetic apparatus.However, how Rho exerts this action has not been fully

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Citron-kinase (Citron-K) is a Rho effector working incytokinesis. It is enriched in cleavage furrow, but how Rhomobilizes Citron-K remains unknown. Using anti-Citronantibody and a Citron-K Green Fluorescence Protein(GFP)-fusion, we monitored its localization in cell cycle. Wehave found: (1) Citron-K is present as aggregates ininterphase cells, disperses throughout the cytoplasm inprometaphase, translocates to cell cortex in anaphase andaccumulates in cleavage furrow in telophase; (2) Rhocolocalizes with Citron-K in the cortex of ana- to telophasecells and the two proteins are concentrated in the cleavagefurrow and to the midbody; (3) inactivation of Rho by C3exoenzyme does not affect the dispersion of Citron-K inprometaphase, but prevented its transfer to the cell cortex,and Citron-K stays in association with the midzone spindlesof C3 exoenzyme-treated cells. To clarify further themechanism of the Rho-mediated transfer andconcentration of Citron-K in cleavage furrow, we expressedactive Val14RhoA in interphase cells expressing GFP-Citron-K. Val14RhoA expression transferred Citron-K to

the ventral cortex of interphase cells, where it formed band-like structures in a complex with Rho. This structure waslocalized at the same plane as actin stress fibers, and theyexclude each other. Disruption of F-actin abolished theband and dispersed the Citron-K-Rho-containing patchesthroughout the cell cortex. Similarly, in dividing cells, astructure composed of Rho and Citron-K in cleavagefurrow excludes cortical actin cytoskeleton, and disruptionof F-actin disperses Citron-K throughout the cell cortex.These results suggest that Citron-K is a novel type of apassenger protein, which is dispersed to the cytoplasm inprometaphase and associated with midzone spindles by aRho-independent signal. Rho is then activated, binds toCitron-K and translocates it to cell cortex, where thecomplex is then concentrated in the cleavage furrow by theaction of actin cytoskeleton beneath the equator of dividingcells.

Key words: Citron, Rho, Cytokinesis, Actin cytoskeleton, Cleavagefurrow

SUMMARY

Rho-dependent transfer of Citron-kinase to thecleavage furrow of dividing cellsMasatoshi Eda 1, Shigenobu Yonemura 2, Takayuki Kato 1, Naoki Watanabe 1,*, Toshimasa Ishizaki 1,Pascal Madaule 1,‡ and Shuh Narumiya 1,§

1Department of Pharmacology, Kyoto University Faculty of Medicine, Sakyo, Kyoto 606-8501, Japan2Department of Cell Biology, Kyoto University Faculty of Medicine, Sakyo, Kyoto 606-8501, Japan*Present address: Department of Cell Biology, Harvard Medical School, 250 Longwood Ave. SGM 520, Boston, MA 02115, USA‡Present address: Récepteurs et signalisation des interleukines, INSERM U 461, Faculté de Pharmacie de l’Université d’Orsay, 5 rue Jean Baptiste Clément, 92296 Châtenay-Malabry, France§Author for correspondence (e-mail: [email protected])

Accepted 7 June 2001Journal of Cell Science 114, 3273-3284 © The Company of Biologists Ltd

RESEARCH ARTICLE

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elucidated. Because of the crucial role of the actin cytoskeletonin cytokinesis, many studies have been carried out to examinethe distribution and behavior during cell division of actin itselfand several actin-binding proteins such as myosin, profilin andcofilin (Robinson and Spudich, 2000). However, their relationto the Rho signaling has not been clarified. Rho acts ondownstream effectors to elicit its actions. They include theROCK/ROK/Rho-kinase family of protein kinases, proteinkinase PKN, Citron and Citron-kinase (Citron-K) and adapterproteins such as mDia, rhophilin and rhotekin (Narumiya,1996). Among these molecules, ROCK, mDia and Citron-K arefound to localize to the cytokinetic apparatus (Madaule et al.,1998; Kosako et al., 1999; Kato et al., 2001). ROCK is foundto accumulate in the cleavage furrow and is proposed to beinvolved in elicitation of the myosin-based contractility and indisassembly of intermediate filaments during division (Kosakoet al., 2000). Involvement of mDia in cytokinesis has beensuggested by cytokinesis defect in Drosophila diaphanousmutants as well as induction of cytokinesis failure bymicroinjecting anti-mDia antibody to cultured mammaliancells (Castrillon et al., 1994; Tominaga et al., 2000). mDiacontains the polyproline-rich FH1 region that binds profilin,and is suggested to induce actin polymerization through thisinteraction (Watanabe et al., 1997). Citron is present both asN-terminally truncated nonkinase isoforms and as an N-terminally extended kinase isoform; the former is expressed ina rather limited way in the neuronal tissues but the latter isubiquitously expressed in various tissues and cells (Madaule etal., 1995; Madaule et al., 1998). We previously found thatCitron-K accumulates in the cleavage furrow in dividing cellsand persists in the midbody between divided cells. It was alsodemonstrated that overexpression of Citron-K deletion mutantscauses cytokinesis defect in cultured mammalian cells,indicating that Citron-K plays also an important role incytokinesis (Madaule et al., 1998). These results stronglysuggest that Rho mobilizes several downstream effectors toexecute its function in cytokinesis. However, the molecularmechanism through which Rho mobilizes these effectors hasnot yet been clarified. In the present study, we have takenCitron-K as an example and analyzed how activated Rhomobilizes this effector to accumulate in the cleavage furrow.Citron-K is particularly interesting in this respect, becauseprevious studies suggest that Citron-K acts only in cytokinesis(Madaule et al., 1998; Kosako et al., 2000) and a recent studyshowed that disruption of its gene results in cytokinesis defectin vivo (Di Cunto et al., 2000).

MATERIALS AND METHODS

Cell cultureHeLa cells were grown in Dulbecco’s modified Eagle’s medium(DMEM) supplemented with 10% fetal calf serum (FCS) at 37°C withan atmosphere containing 5% CO2. We enriched mitotic cells bysynchronizing the growth of cells with the thymidine block method.Briefly, cells were incubated with 10 mM thymidine in DMEMcontaining 10% FCS for 16 hours at 37°C. The cells were then washedwith Dulbecco’s phosphate-buffered saline (PBS[−]) three times andcultured in DMEM with 10% FCS at 37°C for 11 hours. For disruptionof microtubules, cells were incubated with 500 ng/ml nocodazole(Wako Pure Chemicals, Osaka, Japan) at 37°C for 45 minutes. Fordisruption of F-actin, cells were incubated either with 2 µM

cytochalasin D (Wako Pure Chemicals) at 37°C for 30 minutes, orwith 2 µg/ml latrunculin A (Wako Pure Chemicals) at 37°C for 1 hour.For inhibition of ROCK kinase, cells were incubated with 10 µM Y-27632, a specific ROCK inhibitor (Uehata et al., 1997), for 30 minutesat 37°C. Treatment with C3 exoenzyme was performed as describedpreviously (Kato et al., 2001). Briefly, C3 exoenzyme was preparedas described previously (Morii and Narumiya, 1995) and waselectroporated into HeLa cells 6 hours after the release from thethymidine block. The cells were plated on glass coverslips andcultured in DMEM supplemented with 10% FCS. After 6-16 hoursculture, cells were subjected to fixation and stained.

Plasmid construction and expressionpCAG-myc-Citron-K has been previously described (Madaule et al.,1998). Green Fluoresecent Protein (GFP)-tagged Citron-K wasproduced by subcloning the inserts in pEGFP-C1 (Clontech) usingBsiWI and NotI restriction sites newly created in this plasmid, and theconstruct was confirmed by nucleotide sequencing. Sources of pEXV-myc-Val14RhoA, pCMV-myc-Asn19RhoA, pCMV-myc-Asn17Cdc42, pEXV-myc-Val12Rac1, pCMV5-FLAG-Asn17Rac1were described previously (Ishizaki et al., 1997; Hirose et al., 1998;Kimura et al., 2000). pCMV-myc-Val12Cdc42 was kindly providedby M. Symons (Picower Institute for Medical Research, Manhasset,NY). For construction of pEGFP-RhoA, pEGFP-Asn19RhoA, andpEGFP-Val14RhoA, the respective constructs in pBTM (Watanabe etal., 1997) were digested with BamHI and EcoRI and the resultingfragments were inserted into the BglII and EcoRI sites of pEGFP-C1.Transfection of these plasmids to HeLa cells were performed usingLipofectamine Plus (Gibco/BRL) in OPTI-MEM (Gibco/BRL) asdescribed by Fujita et al. (Fujita et al., 2000).

ImmunofluorescenceHeLa cells grown on 20×20 mm glass coverslips were fixed with 4%formaldehyde in PBS[−] for 15 minutes at 4°C except in experimentsshown in Fig. 4 and Fig. 8C, where the TCA fixation method byHayashi et al. (Hayashi et al., 1999) was used. Fixed cells werewashed with PBS-Tx (0.1% Triton X-100 in PBS[−]) several times.After blocking in PBS-Tx containing 1% bovine serum albumin (PBS-Tx-BSA) for 1 hour at room temperature, immunocytochemistry wasperformed with following antibodies and fluorescence reagents. Theprimary antibodies used were rabbit polyclonal anti-Citron antibody(Madaule et al., 1998), rabbit polyclonal anti-Nedd 5 antibody(Kinoshita et al., 1997), mouse monoclonal anti-β-tubulin antibody(clone TUB 2.1, Sigma), mouse monoclonal anti-c-Myc antibody(9E10, Santa Cruz), rabbit polyclonal anti-c-Myc antibody (A-14,Santa Cruz), rabbit polyclonal anti-FLAG antibody (D8, Santa Cruz),mouse monoclonal anti-RhoA antibody (26C4, Santa Cruz) and rabbitpolyclonal anti-RhoA antibody (119, Santa Cruz). The primaryantibodies were added at 1:200 dilution in PBS-Tx-BSA andincubation was carried out at room temperature for 45 minutes. Thecells were then washed with PBS-Tx several times, and incubated withTexas Red-X phalloidin (Molecular Probes), TOPRO3 (MolecularProbes) or 4,6-diamidino-2-phenylindole (DAPI) (Molecular Probes),and/or with following secondary antibodies; FITC-conjugated donkeyanti-mouse IgG (Jackson Immuno Research), Texas Red-conjugateddonkey anti-mouse IgG (Jackson Immuno Research), Cy5-conjugateddonkey anti-mouse IgG (Jackson Immuno Research), FITC-conjugated donkey anti-rabbit IgG (Jackson Immuno Research),Texas Red-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch) and Cy5-conjugated donkey anti-rabbit IgG (JacksonImmuno Research). For blocking anti-Citron antibody, His-taggedantigenic Citron peptide (amino acid residues 674-870 of Citron-N)was prepared as described previously (Madaule et al., 1995) andadded at 1 µg/ml to the incubation with anti-Citron antibody.Fluorescence images were acquired by an MRC 1024 laser-scanningconfocal microscope imaging system (Bio-Rad) equipped with a ZeissAxiovert 100TV microscope.

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3275Active Rho mobilizes Citron-K in cytokinesis

Phase-contrast and electron microscopyHeLa cells expressing GFP-Citron-K were identified on CELLocatecoverslips (Eppendorf) using an Olympus IX70 fluorescencemicroscope equipped with a cooled CCD camera (SenSys 0400,768X512 pixels; Photometrics) and their phase-contrast andfluorescence images were recorded together with their locationinformation. Cells were then fixed with 2.5% glutaraldehyde, 0.2%tannic acid and 0.05% saponin in 0.1M cacodylate buffer, pH 7.4, for1 hour at room temperature. After washing with 0.1 M cacodylatebuffer (pH 7.4) three times (5 minutes each), cells were postfixed withice-cold 1% OsO4 in the same buffer for 45 minutes. The sampleswere rinsed with distilled water, stained with 0.5% aqueous uranylacetate for 2 hours at room temperature, dehydrated with ethanol andembedded in Epon 812. Prior to ultra-thin sectioning, the CELLocatecoverslip was detached from the Epon block. The location informationof the coverslip was transferred onto the surface of the block, whichenabled the identification of the GFP-Citron-K-expressing cells basedon the recorded images. Ultra-thin sections of the GFP-Citron-K-expressing cells were cut, doubly stained with uranyl acetate and leadcitrate and viewed with a JEM 1010 transmission electron microscope(JEOL).

RESULTS

Change of intracellular localization of Citron-Kduring cell cycleWe previously demonstrated that Citron-K is enriched in thecleavage furrow of mitotic cells during cytokinesis (Madauleet al., 1998). Because it is an effector of the small GTPase Rho,the enrichment of Citron-K in the cleavage furrow is presumedto be carried out by the action of Rho. To understand themechanism of Citron-K mobilization, we first used anti-Citronantibody and monitored the intracellular localization ofendogenous Citron-K during the cell cycle. We also addedrecombinant Citron fragment containing the antigenic epitopeto the incubation to identify specific signals. As shown in Fig.1, although the Citron-K immunostaining is relatively weak,we could successfully identify specific signals by comparingimmunofluorescence images in the absence and presence of thecompeting peptide. First, punctate signals were detected by theanti-Citron antibody and were abolished by the addition of theepitope peptide in the cytoplasm of interphase cells. Inprometaphase, these specific Citron-K signals disintegrate anddisperse diffusely in the cytoplasm. From anaphase totelophase, Citron-K accumulates in the cleavage furrow andfinally to the midbody in the post-mitotic stage. These resultsdemonstrate that Citron-K changes its localization during celldivision from dot-like structures in interphase, to the cytoplasmin prometa- and metaphases and finally to the cortex ofcleavage furrow in telophase.

To examine whether Rho is involved in this change ofCitron-K localization, and if so, to identify a step regulated byRho in this localization change, we electroporated C3exoenzyme into HeLa cells enriched in S phase. We firstexamined whether the C3 exoenzyme treatment interfered withcytokinesis of HeLa cells, because a previous study byO’Connell et al. (O’Connell et al., 1999) showed that C3exoenzyme injection into cultured NRK epithelial cellsinduced abnormal cortical activity and resulted in ectopicdivision. The C3 exoenzyme treatment of HeLa cells resultedin almost 100% production of binucleate cells 16 hours after

the treatment (Fig. 2A), suggesting that Rho also plays acrucial role in regulation of cytokinesis of mammalian cells.Localization of Citron-K in these C3 exoenzyme-treated cellsin various mitotic phases were then examined (Fig. 2B). C3exoenzyme treatment, and consequently inactivation of Rho,did not affect dispersion of Citron-K into the cytoplasm inprometa- and metaphase. However, transfer of Citron-K tocleavage furrow in ana- to telophase was completely preventedby this treatment. Instead, Citron-K in the treated cells stayedin the spindle midzone. Co-staining with microtubulesdemonstrated that Citron-K associates with the central spindlesin these cells. All of these signals appear to reflect the behaviorof endogenous Citron-K, because they were abolished by theaddition of the antigenic peptide.

To confirm these findings, we constructed GFP-taggedCitron-K and expressed it in HeLa cells. The GFP fusionprotein showed the same pattern of phase-dependent change inthe intracellular localization as endogenous Citron-K (Fig.3A). GFP-Citron-K again shows punctate signals in interphasecells, becomes dispersed in the cytoplasm in prometaphase andconcentrates in the cleavage furrow after anaphase. To clarifythe identity of the punctate signals seen in interphase cells,GFP signals were examined with electron microscopy. Asshown in Fig. 3B, they appeared as amorphous materials notenclosed with lipid bilayer, suggesting that they are proteinaggregates and not vesicles. The GFP signals in the cleavagefurrow in dividing cells often appear punctate, suggesting thataggregates of the overexpressed protein are not completelydisassembled and migrate. When HeLa cells expressing GFP-Citron-K were treated with C3 exoenzyme, GFP signals wereagain seen in association with the midzone spindles in ana- andtelophase cells (Fig. 3C). These results corroborate the abovefindings with endogenous Citron-K and suggest that the GFP-fusion of Citron-K can be used as a probe to monitor thebehavior of the endogenous protein. Essentially the samelocalization was observed when Citron-K was expressed as aMyc-tagged protein. Expression of these recombinant Citron-K proteins did not interfere with cytokinesis.

Citron colocalizes with Rho in the cleavage furrowThe above results indicate that Rho catalyzes the transfer ofCitron-K to the cell cortex in cleavage furrow, possibly fromthe midzone spindles. Because Citron binds to the GTP-bound,active form of Rho (Madaule et al., 1995), we wonderedwhether Rho and Citron-K colocalize in cleavage furrow. Wetherefore expressed GFP-Citron-K in HeLa cells and examinedthe colocalization of GFP-Citron-K and endogenous Rho inmitotic cells (Fig. 4). In interphase cells, Rho is presentdiffusely in the cytoplasm, whereas Citron-K are in dot-likestructures as described. When cells undergo cytokinesis, Rhoaccumulates in cleavage furrow and stays to the midbody. Thisis consistent with a previous finding on the Rho localization infertilized eggs of sea urchin (Nishimura et al., 1998). Whenboth Rho and Citron-K were visualized in these cells, the Rhosignal colocalizes with the Citron-K signal from the beginningof cytokinesis in the cleavage furrow to the midbody ofpostmitotic cells (Fig. 4B-E) suggesting, although not proving,that Citron-K makes a complex with Rho in this cytokineticapparatus. Given that Citron binds only to active Rho (Madauleet al., 1995), these results suggest that Rho present in thiscomplex is the GTP-bound active form. In addition to these

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structures in the cell cortex, a portion of overexpressed GFP-Citron-K remains as aggregates in the cytoplasm, where nocolocalization with Rho was observed (Fig. 4B,C, arrowheads).

Overexpressed Citron-K transfers to the cell cortexwith Val14Rho expression in interphase cellsThe above findings that inactivation of Rho interfered with the

transfer of Citron-K to the cortex and that transferred Citron-K appeared to make a complex with Rho in cleavage furrowstrongly suggest that active Rho binds Citron-K and they movetogether to the cortex. To test this hypothesis, we examined theeffect of expression of Val14RhoA (a dominant active RhoAmutant) on the localization of overexpressed Citron-K ininterphase cells. Myc-tagged Citron-K and GFP-tagged


Fig. 1.Cell-cycle-dependent change in localization of Citron-K in HeLa cells. HeLa cells in various phases of cell cycle were fixed.Endogenous Citron-K was stained with specific anti-Citron antibody (green) in the absence (left-hand pairs of panels) and presence (right-handpairs of panels) of the antigenic peptide. Microtubules and DNA were stained with anti-β-tubulin antibody (red) and TOPRO3 (blue),respectively. The left panels of each pair represent merged images. Specific signals can be identified by comparing the two pairs of panels.Nonspecific signals in the presence of the competing peptide appear the spectral overlap from strong tubulin staining. Note that endogenousCitron-K is detected as particulate staining in the cytoplasm in interphase cells (arrowheads), disperses in the cytoplasm in prometaphase, istransferred to the cortex in the cleavage furrow in telophase and is present in the midbody in post-mitotic cells. These intracellular signalsdisappear when the blocking peptide is present. Bars, 10 µm.


Val14RhoA were co-expressed in HeLa cells. Cells expressingboth constructs were identified by GFP fluorescence and theMyc-tag staining, and the localization was examined byconfocal microscopy. As shown in Fig. 5A, Citron-K formsband-like structures at the bottom of the cells co-expressingVal14RhoA. This band-like structure consists of numeroussmall patches in which the Myc-Citron-K signal and the GFP-Val14RhoA signal overlap completely. By contrast, no overlapof the Citron-K signals and GFP-Rho was found in remainingaggregates in the middle of the cells (Fig. 5A, middle slice).This was supported by examination in a vertical view of cellsco-expressing Citron-K and Val14RhoA, which shows that theband-like structures were seen only on the cell cortex (Fig. 5B).Given that Citron binds directly GTP-Rho (Madaule et al.,1995), this colocalization of Citron-K and Val14RhoA in smallpatches in band-like structures suggests that activated Rho ispresent in these structures in a complex with Citron-K. Indeed,

co-expression of either wild-type RhoA or dominant negativeAsn19RhoA with Citron-K failed to induce the formation ofthe band-like structures, and Citron-K remains as aggregates inthe cytoplasm (Fig. 5C). We also co-expressed Citron-K withother Rho-family small GTPases, Rac1 and Cdc42. AlthoughCitron is able to bind to Rac1 in a yeast two hybrid system andin an in vitro overlay assay (Madaule et al., 1995), expressionof neither dominant active nor dominant negative Rac1(Val12Rac1 and Asn17Rac1, respectively) affected thelocalization of Citron-K in interphase cells (Fig. 5D). Thelocalization of Citron-K was not affected either by expressionof a dominant active or a dominant negative Cdc42(Val12Cdc42 and Asn17Cdc42, respectively) (Fig. 5E),suggesting that Citron-K translocation to cell cortex to formband-like structures is specific to Rho activation. These resultstaken together suggest that the binding of Citron-K andVal14RhoA induces the transfers of Citron-K from the

Fig. 2.Effect of C3 exoenzyme treatment on Citron-K localization during cell division. Recombinant C3 exoenzyme was introduced into HeLacells enriched in the S-phase by electroporation, and its effects on cell division and localization of endogenous Citron-K were examined.(A) Failure of cytokinesis in HeLa cells treated with C3 exoenzyme. Almost all the treated cells became binucleate 16 hours afterelectroporation as shown by DAPI staining (blue). Microtubules are stained in green. (B) Effect of C3 exoenzyme treatment on Citron-Klocalization during cell division. HeLa cells without (left-hand pairs of panels) or with (middle and right-hand pairs of panels) C3 exoenzymetreatment were fixed in various phases of cell division and stained with anti-Citron antibody (green). Microtubules and DNA were stained withanti-β-tubulin antibody (red) and TOPRO3 (blue), respectively. The left panels of each pair represent merged images. Note that Rhoinactivation by C3 exoenzyme treatment did not affect Citron-K localization in prometa- and metaphase, but prevented the transfer of Citron-Kto the cortex in telophase, which instead was associated with the spindle midzone (middle bottom pairs of panels). The Citron-K signal in thespindle midzone was abolished in the presence of the antigenic peptide (the right bottom pair of panels). Bars, 10 µm.

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cytoplasm to the cell cortex. In these experiments, expressionof Val14RhoA appeared to disintegrate large Citron-Kaggregates in the cytoplasm. However, detailed inspectionrevealed that Citron-K is still present in small aggregates aftertransfer to the cortex, which are seen as small patches. Thus,disintegration observed in this overexpression system isprobably not the same as dispersion of endogenous Citron-Kseen in prometaphase of mitotic cells (see Discussion).

Band-like structures of Citron-K exist on the sameplane with stress fibers but they are mutuallyexclusiveIt is well known that Rho regulates actin cytoskeleton and thatactive Rho induces stress fibers (Hall, 1998). Given that Citron-

K forms the band-like structure on Rho activation at the bottomof the cells, we wondered whether it has some connection withactin stress fibers. To this end, we cotransfected pEGFP-Citron-K and pEXV-myc-Val14RhoA into HeLa cells andsubjected the cells for phalloidin staining. Covisualization ofCitron-K and F-actin revealed that the band-like structure ofCitron-K is present at the same plane as stress fibers, but thatthese two structures exclude each other as suggested by nosignal overlap of GFP-Citron-K and F-actin (Fig. 6A-C). Wewere also interested in the relationship between Citron-K anda septin, because the latter molecule also accumulates in thecleavage furrow and is involved in cytokinesis (Field andKellogg, 1999). However, when endogenous Nedd5, one of theseptins, was stained in HeLa cells co-expressing GFP-Citron-


Fig. 3.Localization of GFP-tagged Citron-K during cell cycle. HeLa cells expressing GFP-Citron-K in various phases of cell cycle were fixed.Expressed GFP-Citron-K was localized by GFP fluorescence, and microtubules and DNA were stained with anti-β-tubulin antibody (red) andTOPRO3 (blue), respectively. Note that GFP-Citron-K is again detected as punctate cytoplasmic signals in interphase cells (arrowheads in a),disperses in the cytoplasm in prometa- and metaphase (b and c), accumulate in the cortex of the cleavage furrow in ana- to telophase (d) and ispresent in the midbody after division (e and f). In about 50% of metaphase cells overexpressing Citron-K, the GFP-Citron-K aggregatesappeared to attach to the cell cortex as shown in c. (B) Electron microscopy of GFP-Citron-K aggregates in interphase. Punctate fluorescencesignals of GFP-Citron-K expressed in HeLa cells were identified first in a live cell by fluorescence microscopy and subjected to electronmicroscopy. Note that GFP-Citron-K is seen as an amorphous structure with many holes, which is not apparently enclosed by lipid bilayers.(C) Effect of C3 exoenzyme treatment on GFP-Citron-K localization in telophase. C3 exoenzyme was introduced into HeLa cells expressingGFP-Citron-K (green) by electroporation, and Citron-K localization in telophase was examined. Note that GFP signals were associated with thespindle midzone. The left-hand panels are merged images with microtubules stained in red and DNA stained in blue. Bars, 10 µm except in theright-hand panel of B.


K and Val14RhoA, it was associated with actin stress fibers, aspreviously observed (Kinoshita et al., 1997), and its signals andthe GFP-Citron-K again excluded each other (data not shown).

Citron-K and the actin cytoskeleton exclude eachother in cleavage furrowWe next examined the spatial relation between the Citron-containing structures and F-actin in mitotic cells. We studiedthis issue on both endogenous Citron-K (Fig. 7A,B) in HeLacells and cells expressing GFP-Citron-K (Fig. 7C,D). Bothstudies demonstrated a clear separation of the structurescontaining Citron-K and the F-actin in the cleavage furrow. TheCitron-containing structure appears to be concentrated beneathF-actin structure in the cleavage furrow by being encircled inall directions by F-actin during cytokinesis except in theearliest stage, where it was difficult to separate the two signals(Fig. 7A).

Accumulation of the Citron-K-Rho complex in band-like structure in interphase cells and in cleavagefurrow of mitotic cells disappears with actindepolymerizationWe next addressed the interaction between the Citron-enriched cortical structures and the cytoskeletons bydisrupting either microtubules or F-actin. Nocodazole wasused to depolymerize microtubules in cells co-expressing

Citron-K and Val14RhoA. Depolymerization ofmicrotubules did not affect the colocalization of Citron-Kand active Rho, and the band-like structures of the Citron-K-Val14RhoA complex was maintained as those found innontreated cells (data not shown), suggesting that theformation of the band-like structures does not depend on theintegrity of microtubules. By contrast, when F-actin wasdisrupted either with cytochalasin D or latrunculin Atreatment, the band-like structures were disintegrated, andCitron-K-containing small patches spread all over the ventralsurface of the cell cortex (Fig. 8A). Because stress fibers areformed by virtue of actomyosin-based contractility that isexerted by the action of a Rho effector ROCK, we examinedthe effect of the ROCK inhibitor Y-27632 on thisaccumulation. Disruption of stress fibers by inactivation ofROCK resulted again in dispersion of the accumulation ofCitron-containing patches (Fig. 8B). In some cells whichhave remaining stress fibers, some of the Citron-containingband-like structures were also conserved (Fig. 8B, right-hand cell). These results suggest that band-like arrangementof Citron-K-active Rho patches depends on orderlyorganization of F-actin by the action of the actomyosinsystem. We then examined the effect of F-actindepolymerization on the concentration of the Citron-containing structures in cleavage furrow (Fig. 8C). Mitoticcells were enriched by the use of the thymidine block and

Fig. 4.Colocalization of RhoA and Citron-K in cleavage furrowof HeLa cells. Colocalization of Rho and Citron-K was examinedby staining endogenous Rho with anti-Rho antibody (red, right-hand panels) in HeLa cells expressing GFP-Citron-K (green,middle panels) in various stages of cytokinesis. Left-hand panelsrepresent merged images. In interphase, Rho shows homogenousstaining in the cytoplasm, whereas Citron-K shows particulatesignals (A). Punctate signals for Rho in the nucleus appearnonspecific, because such signals are only found by thispolyclonal antibody 119 used in this experiment, and not by theother monoclonal antibody 26C4. GFP-Citron-K and endogenousRhoA colocalize around the cell equator of cells in the early stage(B), in the ingressing cleavage furrow in the middle stage (C),and are concentrated together at the cleavage site in the end stage(D) of cytokinesis. In addition, a portion of GFP-Citron-Kremains as aggregates in the cytoplasm, where no colocalizationwith Rho was observed (for example, see arrowheads in B andC). Confocal sections of each cell are shown. Note that Citron-Kand a part of Rho also colocalize in the midbody of post mitoticcells (E). Blue in E is β-tubulin. Bars, 10 µm.

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treated with either cytochalasin D or latrunculin A. In thedividing cells treated with either reagent, Citron-K was notseen as a ring-like structure in the cleavage furrow butdispersed as patches all around the cell cortex. However, thecolocalization of Citron-K and endogenous Rho was spared

in the dispersed patches. However, unlike Citron-K, a portionof Rho still remained at the original site of the cleavagefurrow. Y-27632 was without effect on mitotic cells, whichis consistent with the dispensable action of ROCK incytokinesis (Madaule et al., 1998; Ishizaki et al., 2000).


Fig. 5.Translocation of GFP-Citron-K to thecortex of interphase cells by expression ofdominant active Rho. (A) Effects of expressionof dominant active Rho on localization ofCitron-K. Myc-Citron-K and GFP-Val14RhoAwere co-expressed in HeLa cells. Citron-K (red)and Val14RhoA (green) are colocalized in band-like structures at the bottom of cells (bottomslice), whereas in the middle of the cell, Citron-K is present as an aggregate withoutcolocalization with Val14Rho (the middle slice,arrowheads). Band-like structure of Citron-Kand Val14RhoA at the bottom consists of smallpatches. (B) Vertical views of GFP-Citron-Klocalization in HeLa cells co-expressing Myc-Val14RhoA and GFP-Citron-K. Note thatexpression of Val14RhoA (red) disintegratescytoplasmic aggregates of GFP-Citron-K(green) and translocates it to the cell cortex toform band-like structures. Signals of myc-Val14RhoA and GFP-Citron-K appear not tocolocalize completely because the fluorescenceof Texas Red-Myc-Val14RhoA is not so strongas GFP-Citron-K, and only a small portion ofexpressed Rho colocalize with Citron-K.(C) Effects of expression of GFP-tagged wild-type RhoA (the left pair of panels) and GFP-tagged Asn19RhoA (the right-hand pair ofpanels) on localization of Myc-tagged Citron-K.Myc-Citron-K (red) remains as aggregates whenwild-type or dominant negative RhoA (green) isco-expressed. (D) Effects of Rac expression onCitron-K localization. GFP-Citron-K (green)and either Myc-tagged Val12Rac1 (red) (the leftpair of panels) or FLAG-tagged Asn17Rac1(red) (the right pair of panels) were co-expressed in HeLa cells. Note that eitherexpression did not affect the cytoplasmicaggregates of GFP-Citron-K. (E) Effects ofCdc42 expression. GFP-Citron-K (green) andeither Myc-tagged Val12Cdc42 (red) (the leftpair of panels) or Myc-tagged Asn17Cdc42(red) (the right pair of panels) were co-expressed in HeLa cells. Note that eitherexpression did not affect the cytoplasmicaggregates of GFP-Citron-K. C, D and E allshow the bottom slices of interphase cells. Bars,10 µm.


DISCUSSION

Citron-K undergoes multi-step change in itslocalization during mitosisIn this study we monitored the localization of Citron-K duringmitosis both by immunofluorescence study of endogenousprotein with anti-Citron antibody and by expression of GFP-Citron-K fusion in HeLa cells. Both studies have revealed themulti-step change of Citron-K localization during mitosis.Citron-K is present as aggregates in interphase cells, dispersesinto the cytoplasm in prometaphase, translocates to the cellcortex in anaphase and accumulates in cleavage furrow intelophase. Although not all interphase cells contain visibleaggregates in immunofluorescence, we think that Citron-K ispresent as aggregates also in these cells in a form not detectedby this method. Oligomer formation has been reported forMRCK, a kinase homologous to Citron-K (Tan et al., 2001).Using C3 exoenzyme to inactivate Rho, we have found that theabove sequential change is catalyzed by consecutive activationof a Rho-independent and a Rho-dependent mechanism. Thus,dispersion of Citron-K occurs normally in C3 exoenzyme-treated cells. However, it does not move to the cortex but staysin association with the midzone spindles in anaphase cells.These results indicate that Citron-K moves to the cortex via themidzone spindles in a Rho-dependent manner. This is anintriguing finding because, in mammalian cells, the cleavagesignal is suggested to come from the central interdigitatingspindle microtubules (Cao and Wang, 1996). Previously,several proteins have been reported to associate with themidzone spindles. They include TD-60, INCENP, aurorakinase and survivin and are collectively termed chromosomalpassenger proteins (Andreassen et al., 1991; Adams et al.,2001; Skoufias et al., 2000). However, these proteins firstlocate at centromeres of chromosomes in metaphase, thenassociate with the midzone spindle extending to the cortex inanaphase, stay there in telophase and concentrate in theintracellular bridge after mitosis. However, we did not see anyattachment of Citron-K to centromeres or chromosomes. Itappears to bind to the midzone spindles from the cytoplasmand transfers to the cortex upon Rho activation. Thus, Citron-K is likely to be a novel type of passenger protein.

The above observation also indicates the presence of a Rho-independent, yet cell-cycle-dependent signal for the initialmobilization of Citron-K. At present, we do not know the

Fig. 6.Mutual exclusion of Citron-K-containing band-like structuresand actin stress fibers. GFP-Citron-K and Myc-Val14RhoA were co-transfected into HeLa cells. Actin stress fibers (red) and band-likestructures of Citron-K (green) exist on the same plane at the bottomof interphase cells, but the signals do not overlap at all (a mergedimage, left). Bar, 10 µm.

Fig. 7.Mutual exclusion of Citron-K-containing structures and actincytoskeleton in the equatorial cell cortex during cytokinesis. (A andB) Localization of endogenous Citron-K and F-actin. Two HeLa cellsat different stages of cytokinesis were chosen, and stained forendogenous Citron-K (green), F-actin (red), and DNA (blue). Citron-K and F-actin appear to overlap in the very early stage of cytokinesis(A), but are clearly separated in the late stage of cytokinesis (B).(C,D) Localization of GFP-Citron-K (green) and F-actin (red). GFP-Citron-K-expressing HeLa cells undergoing ingression of thecleavage furrow (C) and that at the end stage of cytokinesis (D) werechosen. In C, several confocal sections encompassing the cleavagefurrow of a dividing cell are piled up and shown. β-Tubulin wasstained blue in D. Note that GFP-Citron-K and F-actin do notcolocalize. Scale bars, 10 µm.

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identity of this signal. Cell-cycle-dependent mobilization wasalso reported for other proteins working in mitosis. Forexample, expression study showed that survivin is present asdots in the cytoplasm in interphase cells but concentrates indistinct spots on chromosomes in prophase (Skoufias et al.,2000). ECT-2, the Rho exchange protein involved incytokinesis, is present in the interphase nucleus, disperses inthe cytoplasm in prometaphase, concentrates in the spindle inmetaphase and transfers to the cortex in anaphase (Tatsumotoet al., 1999). In the latter case, phosphorylation-dependentactivation was suggested to occur.

Active Rho takes Citron-K to cell cortex andcleavage furrowAs discussed, active Rho appears to transfer Citron-K to cellcortex and to concentrate it in cleavage furrow in telophase.

We mimicked the transfer of Citron-K to the cortex by co-expression of Val14RhoA and Citron-K in interphase cells.Although the experiments in interphase cells naturally do notexactly simulate the process in dividing cells, the resultsobtained have provided many implications. In the latterexperiment, the transferred proteins form band-like structuresin the ventral cortex of cells. It should be mentioned thatCitron-K is present still as small aggregates in these band-likestructures, partly because this procedure skipped the naturaldispersion process seen during mitosis and partly because ofthe high amount of overexpressed Citron-K. Because Citron-K binds to the active form of Rho selectively, this resultsuggests that the binding of Citron-K to active Rho takes Citronto cell cortex and cleavage furrow. A number of other Rhoeffector proteins including ROCK, mDia, Rhophillin, Rhotekinand PKN have been identified (Ishizaki et al., 1997; Watanabeet al., 1997; Watanabe et al., 1996; Reid et al., 1996). Althoughmembrane translocation associated with activation of Rho hasbeen reported on some of these effectors, such a markedtranslocation as that seen in Citron-K has never been observed.This is probably because other effectors are transientlytranslocated and used only in a small amount in response tolocal activation of Rho. However, transfer of Citron-K requiresextensive and widely spread activation of Rho as induced byVal14RhoA expression in interphase cells. High accumulationof GTP-Rho during mitosis was already reported (Kimura etal., 2000). It should also be mentioned that almost all ofendogenous Citron-K is transferred by Rho activation andaccumulates in the cell cortex. At its accumulation site, Citron-K appears to make a complex with active Rho becausecolocalization of Citron-K and Rho is persistently observed inthe band-like structures formed by co-expression withVal14Rho and in cleavage furrow. These results indicate thatRho in the active GTP-bound form serves as a structuralcomponent in the Citron-K-containing cytokinetic apparatus.We previously found that accumulation of GTP-Rho continuesto be present during cytokinesis after the decline of the Rhoexchange activity, and suggested the presence of a stabilizationmechanism for GTP-Rho (Kimura et al., 2000). Our presentfinding is consistent with this suggestion. However, this is in


Fig. 8.Dispersion of Citron-K-containing patches by disruption of F-actin. (A and B) Effects of latrunculin A (A) or Y-27632 (B) treatmenton band-like structures in interphase cells. HeLa cells co-expressingGFP-Citron-K (green) and Myc-Val14RhoA were subjected to eachtreatment. F-actin is stained in red. (A) Latrunculin A treatmentdisintegrated the band-like structures of Citron-K and dispersedCitron-K-containing patches throughout the cell cortex. (B) The Y-27632 treatment disrupted stress fibers and dispersed Citron-Kcontaining patches (the left cell). In cells retaining stress fibers, someof the Citron-containing band-like structures remained (the right-handcell). (C) Effects of F-actin disruption on Citron-K accumulation inmitotic cells. HeLa cells expressing GFP-Citron-K were enriched inM-phase, subjected to latrunculin A treatment, and stained forendogenous RhoA (red) and for DNA (blue). In the dividing cellstreated with latrunculin A, GFP-Citron-K (green) was not seen as aring-like structure in the cleavage furrow but dispersed as patches allaround the cell cortex. In the dispersed patches, colocalization withendogenous RhoA is also observed. Endogenous RhoA also remainsin the putative cleavage furrow (arrowheads). Confocal sections ofthree different cells are shown. The same pattern was also observedwith cytochalasin D treatment. Bars, 10 µm.


contrast to the presumed activation mechanisms for other Rhoeffectors, in which active Rho transiently interacts witheffectors. As shown by the previous study (Nishimura et al.,1998) and also shown here, most of Rho present in the cellaccumulates in the cleavage furrow during cytokinesis. Onemechanism of this Rho accumulation is the accumulation ofCitron-K in this region. The translocation and accumulation ofCitron-K appears to be linked to its function in cytokinesis. DiCunto et al. (Di Cunto et al., 2000) disrupted selectively thegene for Citron-K in the kinase domain and showed that thekinase domain of Citron-K is crucial in cytokinesis. Thus, thisstudy presents a new mode of stimulus-activated constructionof a functional cytokinetic apparatus.

Citron-K and actin cytoskeleton in cytokinesisDuring cleavage of eggs of the echinoderms and Xenopusembryos, F-actin forms a distinct structure known as thecontractile ring, which cooperates with myosin and constrictsto divide the cell. The actin cytoskeleton also plays a crucialrole in cytokinesis of mammalian cells, although the actincontractile ring is not so discernible in these cells. Because Rhois involved in reorganization of several types of the actincytoskeleton such as stress fibers, we were interested in therelationship between the Citron-containing structures and theactin cytoskeleton. Unexpectedly, we have found that theCitron-K-containing structures and the actin cytoskeleton arepresent by excluding each other. In interphase cells co-expressing Citron-K and Val14Rho, the band structure thatcontains Citron-K is present at the same plane of the ventralcell cortex as stress fibers and the two exclude each other. Inthe cleavage furrow of mitotic cells, the Citron-K-containingstructure is encircled by the F-actin. At the very bottom of thecleavage furrow, strong accumulation of Citron-K wasobserved but not of F-actin. Oegema et al. (Oegema et al.,2000) have reported the similar absence of F-actin in the centerof the cleavage furrow in BHK-21 cells. Furthermore, we havefound that disruption of F-actin with either cytochalasin D orlatrunculin A abolished the Citron-K-containing structures anddispersed small patches containing Citron-K throughout thecell cortex. These results suggest that Citron-K molecules areput together in the band-like structures in interphase cells andin the cleavage furrow of mitotic cells by the force of the actincytoskeleton. Because Citron-K plays an essential role incytokinesis at least in some populations of neuronal cells (DiCunto et al., 2000), one function of the actin cytoskeleton incytokinesis of mammalian cells is to make Citron-Kaccumulation in the cleavage furrow. It should be emphasizedthat our present data do not exclude the role of the actomyosincytoskeleton in elicitation of the contractile force incytokinesis. There has been no report showing the contractileforce generation by Citron-K. However, a number of reportshave shown the involvement of actin binding proteins includingmyosin in generation and processing of the contractile ring(Robinson and Spudich, 2000).

The role of Citron-K and Rho in cytokinesis:remaining issuesThe data reported above have clarified how activated Rhomobilizes Citron-K during cell division. Citron-K exists in thecytoplasm in interphase, moves to midzone spindles, bindsthen to activated GTP-Rho, transfers to cell cortex and

accumulates in the cleavage furrow. How do Citron moleculesaccumulated in the cleavage furrow exert a crucial function incytokinesis? One plausible possibility is that Citron-Kaccumulated there by the force of F-actin in turn regulatesfunctions of actin and other cytoskeletons, although there hasbeen no direct evidence to support this hypothesis. Citron-Kmost probably exerts this action by phosphorylating somesubstrate(s) in this process. Identification of these substrateswill clarify this issue. The above mobilization pathway has alsoraised several other important questions such as the identity ofthe initial Rho-independent, cell-cycle-dependent signal, howCitron-K moves to the midzone spindles, how Rho mobilizesCitron-K there to cell cortex and the function of the midzonespindles in this process. These questions may be solved byidentification of domains of Citron-K responsible for eachmobilization step and their binding partner there. Thisapproach may also give us a new insight into the interactionamong Rho, Citron-K, microtubules and actin cytoskeletonduring mitosis.

We thank S. Tsukita, H. Bito, T. Tsuji, M. Okamoto, Y. Takada, F.Oceguera, K. Kimura, M. Maekawa and T. Furuyashiki for helpfuladvice and discussion, M. Kinoshita and M. Noda for generous supplyof anti-Nedd 5 antibody, K. Nonomura for technical assistance, andT. Arai and H. Nose for secretarial help.

REFERENCES

Adams, R. R., Carmena, M. and Earnshaw, W. C.(2001). Chromosomalpassengers and the (aurora) ABCs of mitosis. Trends Cell Biol. 11, 49-54.

Andreassen, P. R., Palmer, D. K., Wener, M. H. and Margolis, R. L.(1991).Telophase disc: a new mammalian mitotic organelle that bisects telophasecells with a possible function in cytokinesis. J. Cell Sci.99, 523-534.

Cao, L, G. and Wang, Y. L. (1996). Signals from the spindle midzone arerequired for the stimulation of cytokinesis in cultured epithelial cells. Mol.Biol. Cell 7, 225-232.

Castrillon, D. H. and Wasserman, S. A.(1994). Diaphanous is required forcytokinesis in Drosophila and shares domains of similarity with the productsof the limb deformity gene. Development120, 3367-3377.

Di Cunto, F., Imarisio, S., Hirsch, E., Broccoli, B., Bulfone, A., Migheli,A., Atzori, C., Turco, E., Triolo, R., Dotto, G. P. et al.(2000). Defectiveneurogenesis in citron kinase knockout mice by altered cytokinesis andmassive apoptosis. Neuron28, 115-127.

Drechsel, D. N., Hyman, A. A., Hall, A. and Glotzer, M.(1997). Arequirement for Rho and Cdc42 during cytokinesis in Xenopus embryos.Curr. Biol. 7, 12-23.

Field, C. M. and Kellogg, D. (1999). Septins: cytoskeletal polymers orsignaling GTPase? Trends Cell Biol.9, 387-394.

Fishkind, D. J. and Wang, Y. L.(1995). New horizons for cytokinesis. Curr.Opin. Cell Biol.7, 23-31.

Fujita, A., Nakamura, K., Kato, T., Watanabe, N., Ishizaki, T., Kimura,K., Mizoguchi, A. and Narumiya, S.(2000). Ropporin, a sperm-specificbinding protein of rhophilin, that is localized in the fibrous sheath of spermflagella. J. Cell Sci.113, 103-112.

Glotzer, M. (1997). The mechanism and control of cytokinesis. Curr. Opin.Cell Biol. 9, 815-823.

Hales, K. G., Bi, E., Wu, J. Q., Adam, J. C., Yu, I. C. and Pringle, J. R.(1999). Cytokinesis: an emerging unified theory for eukaryotes? Curr. Opin.Cell Biol. 11, 717-725.

Hall, A. (1998). Rho GTPases and the actin cytoskeleton. Science279, 509-514.

Hayashi, K., Yonemura, S., Matsui, T. and Tsukita, S. (1999).Immunofluorescence detection of ezrin/radixin/moesin (ERM) proteins withtheir carboxyl-terminal threonine phosphorylated in cultured cells andtissues. J. Cell Sci.112, 1149-1158.

Hirose, M., Ishizaki, T., Watanabe, N., Uehata, M., Kranenburg, O.,Moolenaar, W. H., Matsumura, F., Maekawa, M., Bito, H. andNarumiya, S. (1998). Molecular dissection of the Rho-associated protein

3284

kinase (p160ROCK)-regulated neurite remodeling in neuroblastoma N1E-115 cells. J. Cell Biol.141, 1625-1636.

Ishizaki, T., Naito, M., Fujisawa, K., Maekawa, M., Watanabe, N., Saito,Y. and Narumiya, S. (1997). P160ROCK, a Rho-associated coiled-coilforming protein kinase, works downstream of Rho and induces focaladhesions. FEBS Lett.404, 118-124.

Ishizaki, T., Uehata, M., Tamechika, I., Keel, J., Nonomura, K., Maekawa,M. and Narumiya, S. (2000). Pharmacological properties of Y-27632, aspecific inhibitor of Rho-associated kinase.Mol. Pharmacol. 57, 976-983

Kato, T., Watanabe, N., Morishima, Y., Fujita, A., Ishizaki, T. andNarumiya, S.(2001). Localization of a mammalian homolog of diaphanous,mDia1, to the mitotic spindle in HeLa cells. (2001). J. Cell Sci. 114, 775-784.

Kimura, K., Tsuji, T., Takada, Y., Miki, T. and Narumiya, S. (2000).Accumulation of GTP-bound RhoA during cytokinesis and a critical role ofECT2 in this accumulation. J. Biol. Chem.275, 17233-17236.

Kinoshita, M., Kumar, S., Mizoguchi, A., Ide, C., Kinoshita, A.,Haraguchi, T., Hiraoka, Y. and Noda, M. (1997). Nedd5, a mammalianseptin, is a novel cytoskeletal component interacting with actin-basedstructures. Genes Dev.11, 1535-1547.

Kishi, K., Sasaki, T., Kuroda, S., Itoh, T. and Takai, Y.(1993). Regulationof cytoplasmic division of Xenopus embryo by rho p21 and its inhibitoryGDP/GTP exchange protein (rho GDI). J. Cell Biol.120, 1187-1195.

Kosako, H., Goto, H., Yanagida, M., Matsuzawa, K., Fujita, M., Tomono,Y., Okigaki, T., Odai, H., Kaibuchi, K. and Inagaki, M. (1999). Specificaccumulation of Rho-associated kinase at the cleavage furrow duringcytokinesis: cleavage furrow-specific phosphorylation of intermediatefilaments. Oncogene18, 2783-2788.

Kosako, H., Yoshida, T., Matsumura, F., Ishizaki, T., Narumiya, S. andInagaki, M. (2000). Rho-kinase/ROCK is involved in cytokinesis throughthe phosphorylation of myosin light chain and not ezrin/radixin/moesinproteins at the cleavage furrow. Oncogene19, 6059-6064.

Mabuchi, I., Hamaguchi, Y., Fujimoto, H., Morii, N., Mishima, M. andNarumiya, S. (1993). A rho-like protein is involved in the organisation ofthe contractile ring in dividing sand dollar eggs. Zygote1, 325-331.

Madaule, P., Furuyashiki, T., Reid, T., Ishizaki, T., Watanabe, G., Morii,N. and Narumiya, S.(1995). A novel partner for the GTP-bound forms ofrho and rac. FEBS Lett.377, 243-248.

Madaule, P., Eda, M., Watanabe, N., Fujisawa, K., Matsuoka, T., Bito, H.,Ishizaki, T. and Narumiya, S.(1998). Role of citron kinase as a target ofthe small GTPase Rho in cytokinesis. Nature394, 491-494.

Morii, N. and Narumiya, S. (1995). Preparation of native and recombinantClostridium botulinum C3 ADP-ribosyltransferase and identification of Rhoproteins by ADP-ribosylation. Methods Enzymol. 256,196-206.

Narumiya, S. (1996). The small GTPase Rho: cellular functions and signaltransduction. J. Biochem. (Tokyo)120, 215-228.

Nishimura, Y., Nakano, K. and Mabuchi, I. (1998). Localization of RhoGTPase in sea urchin eggs. FEBS Lett. 441, 121-126.

O’Connell, C. B., Wheatley, S. P., Ahmed, S. and Wang, Y. L. (1999). Thesmall GTP-binding protein rho regulates cortical activities in cultured cellsduring division. J. Cell Biol. 144, 305-313.

Oegema, K., Savoian, M. S., Mitchison, T. J. and Field, C. M.(2000).Functional analysis of a human homologue of the Drosophila actin bindingprotein anillin suggests a role in cytokinesis. J. Cell Biol.150, 539-552.

Prokopenko, S. N., Brumby, A., O’Keefe, L., Prior, L., He, Y., Saint, R.and Bellen, H. J.(1999). A putative exchange factor for Rho1 GTPase isrequired for initiation of cytokinesis in Drosophila. Genes Dev.13, 2301-2314.

Reid, T., Furuyashiki, T., Ishizaki, T., Watanabe, G., Watanabe, N.,Fujisawa, K., Morii, N., Madaule, P. and Narumiya, S.(1996). Rhotekin,a new putative target for Rho bearing homology to a serine/threonine kinase,PKN, and rhophilin in the rho-binding domain. J. Biol. Chem.271, 13556-13560.

Robinson, D. N. and Spudich, J. A. (2000). Towards a molecularunderstanding of cytokinesis. Trends Cell Biol.10, 228-237.

Satterwhite, L. L. and Pollard, T. D. (1992). Cytokinesis. Curr. Opin. CellBiol. 4, 43-52.

Skoufias, D. A., Mollinari, C., Lacroix, F. B. and Margolis, R. L. (2000).Human survivin is a kinetochore-associated passenger protein. J. Cell Biol.151, 1575-1582.

Tan, I., Seow, K. T., Lim, L. and Leung, T. (2001) Intermolecular andintramolecular interactions regulate catalytic activity of myotonic dystrophykinase-related Cdc42-binding kinase alpha. Mol. Cell. Biol.21, 2767-78.

Tatsumoto, T., Xie, X., Blummenthal, R., Okamoto, I. and Miki, T.(1999).Human ECT2 is an exchange factor for Rho GTPases, phosphorylated inG2/M phases, and involved in cytokinesis. J. Cell Biol.147, 921-928.

Tominaga, T., Sahai, E., Chardin, P., McCormick, F., Courtneidge, S. A.and Alberts, A. S.(2000). Diaphanous-related formins bridge Rho GTPaseand Src tyrosine kinase signaling. Mol. Cell 5, 13-25.

Uehata, M., Ishizaki, T., Satoh, H., Ono, T., Kawahara, T., Morishita, T.,Tamakawa, H., Yamagami, K., Inui, J., Maekawa, M. et al.(1997).Calcium sensitization of smooth muscle mediated by a Rho-associatedprotein kinase in hypertension. Nature389, 990-994.

Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N.,Mukai, H., Ono, Y., Kakizuka, A. and Narumiya, S. (1996). Proteinkinase N (PKN) and PKN-related protein rhophilin as targets of smallGTPase Rho. Science271, 645-648.

Watanabe, N., Madaule, P., Reid, T., Ishizaki, T., Watanabe, G., Kakizuka,A., Saito, Y., Nakao, K., Jockusch, B. M. and Narumiya, S.(1997).P140mDia, a mammalian homolog of Drosophila diaphanous, is a targetprotein for Rho small GTPase and is a ligand for profilin. EMBO J.16, 3044-3056.


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