RESEARCH ARTICLE
3273
Rho-dependent transfer of Citron-kinase to the
cleavage furrow of dividing cells
Masatoshi Eda1, Shigenobu Yonemura2, Takayuki Kato1, Naoki Watanabe1,*, Toshimasa Ishizaki1,
Pascal Madaule1,‡ and Shuh Narumiya1,§
1Department
2Department
of Pharmacology, Kyoto University Faculty of Medicine, Sakyo, Kyoto 606-8501, Japan
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 ChâtenayMalabry, France
§Author for correspondence (e-mail: [email protected])
Accepted 7 June 2001
Journal of Cell Science 114, 3273-3284 © The Company of Biologists Ltd
SUMMARY
Citron-kinase (Citron-K) is a Rho effector working in
cytokinesis. It is enriched in cleavage furrow, but how Rho
mobilizes Citron-K remains unknown. Using anti-Citron
antibody and a Citron-K Green Fluorescence Protein
(GFP)-fusion, we monitored its localization in cell cycle. We
have found: (1) Citron-K is present as aggregates in
interphase cells, disperses throughout the cytoplasm in
prometaphase, translocates to cell cortex in anaphase and
accumulates in cleavage furrow in telophase; (2) Rho
colocalizes with Citron-K in the cortex of ana- to telophase
cells and the two proteins are concentrated in the cleavage
furrow and to the midbody; (3) inactivation of Rho by C3
exoenzyme does not affect the dispersion of Citron-K in
prometaphase, but prevented its transfer to the cell cortex,
and Citron-K stays in association with the midzone spindles
of C3 exoenzyme-treated cells. To clarify further the
mechanism of the Rho-mediated transfer and
concentration of Citron-K in cleavage furrow, we expressed
active Val14RhoA in interphase cells expressing GFPCitron-K. Val14RhoA expression transferred Citron-K to
the ventral cortex of interphase cells, where it formed bandlike structures in a complex with Rho. This structure was
localized at the same plane as actin stress fibers, and they
exclude each other. Disruption of F-actin abolished the
band and dispersed the Citron-K-Rho-containing patches
throughout the cell cortex. Similarly, in dividing cells, a
structure composed of Rho and Citron-K in cleavage
furrow excludes cortical actin cytoskeleton, and disruption
of F-actin disperses Citron-K throughout the cell cortex.
These results suggest that Citron-K is a novel type of a
passenger protein, which is dispersed to the cytoplasm in
prometaphase and associated with midzone spindles by a
Rho-independent signal. Rho is then activated, binds to
Citron-K and translocates it to cell cortex, where the
complex is then concentrated in the cleavage furrow by the
action of actin cytoskeleton beneath the equator of dividing
cells.
INTRODUCTION
fertilized eggs of sea urchin or Xenopus embryos from entering
into cytokinesis after nuclear division and produces
multinucleate cells (Kishi et al., 1993; Mabuchi et al., 1993;
Drechsel et al., 1997). Furthermore, injection of C3 exoenzyme
into cells undergoing cytokinesis causes dissolution of the
contractile ring and regression of the cleavage furrow, and the
cells cannot continue cytokinesis (Mabuchi et al., 1993). These
results strongly suggest that Rho is activated during cell
division and works as a switch to induce and maintain the
cytokinetic apparatus. Recently, a putative activator of Rho in
this process has been identified. This GDP-GTP exchanger for
Rho, Pebble in Drosophila and ECT-2 in mammalian cells, is
found to be activated after the nuclear division (Prokopenko et
al., 1999; Tatsumoto et al., 1999). Indeed, the GTP-bound
active form of Rho accumulates during division of HeLa cells
and this accumulation was abolished by expression of a
dominant negative form of ECT-2, resulting in formation of
multinucleate cells (Kimura et al., 2000). Thus, Rho appears
to induce cytokinesis by organizing the cytokinetic apparatus.
However, how Rho exerts this action has not been fully
Cytokinesis is the final step in cell division in which a parent
cell is divided into two daughter cells. After segregation of
chromosomes to the opposite poles in anaphase, a cleavage
furrow is formed around the equator of a dividing cell, which
deepens in telophase finally to separate two daughter cells. In
classical experiments using fertilized eggs of sea urchin or
newt eggs, a ring composed of actomyosin is observed beneath
the cleavage furrow and it is suggested that the constriction of
this ring leads to the cleavage of the cell. Indeed, disruption of
this ring with F-actin-depolymerizing compounds results in
failure of cytokinesis. However, how cytokinesis is temporally
linked with nuclear division and how the cytokinetic apparatus
is constructed spatially in a dividing cell remain largely
unknown (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 crucial
regulator in these processes of cytokinesis. For example,
inactivation of Rho with botulinum C3 exoenzyme prevents
Key words: Citron, Rho, Cytokinesis, Actin cytoskeleton, Cleavage
furrow
3274
JOURNAL OF CELL SCIENCE 114 (18)
elucidated. Because of the crucial role of the actin cytoskeleton
in cytokinesis, many studies have been carried out to examine
the distribution and behavior during cell division of actin itself
and several actin-binding proteins such as myosin, profilin and
cofilin (Robinson and Spudich, 2000). However, their relation
to the Rho signaling has not been clarified. Rho acts on
downstream effectors to elicit its actions. They include the
ROCK/ROK/Rho-kinase family of protein kinases, protein
kinase PKN, Citron and Citron-kinase (Citron-K) and adapter
proteins such as mDia, rhophilin and rhotekin (Narumiya,
1996). Among these molecules, ROCK, mDia and Citron-K are
found to localize to the cytokinetic apparatus (Madaule et al.,
1998; Kosako et al., 1999; Kato et al., 2001). ROCK is found
to accumulate in the cleavage furrow and is proposed to be
involved in elicitation of the myosin-based contractility and in
disassembly of intermediate filaments during division (Kosako
et al., 2000). Involvement of mDia in cytokinesis has been
suggested by cytokinesis defect in Drosophila diaphanous
mutants as well as induction of cytokinesis failure by
microinjecting anti-mDia antibody to cultured mammalian
cells (Castrillon et al., 1994; Tominaga et al., 2000). mDia
contains the polyproline-rich FH1 region that binds profilin,
and is suggested to induce actin polymerization through this
interaction (Watanabe et al., 1997). Citron is present both as
N-terminally truncated nonkinase isoforms and as an Nterminally extended kinase isoform; the former is expressed in
a rather limited way in the neuronal tissues but the latter is
ubiquitously expressed in various tissues and cells (Madaule et
al., 1995; Madaule et al., 1998). We previously found that
Citron-K accumulates in the cleavage furrow in dividing cells
and persists in the midbody between divided cells. It was also
demonstrated that overexpression of Citron-K deletion mutants
causes cytokinesis defect in cultured mammalian cells,
indicating that Citron-K plays also an important role in
cytokinesis (Madaule et al., 1998). These results strongly
suggest that Rho mobilizes several downstream effectors to
execute its function in cytokinesis. However, the molecular
mechanism through which Rho mobilizes these effectors has
not yet been clarified. In the present study, we have taken
Citron-K as an example and analyzed how activated Rho
mobilizes this effector to accumulate in the cleavage furrow.
Citron-K is particularly interesting in this respect, because
previous studies suggest that Citron-K acts only in cytokinesis
(Madaule et al., 1998; Kosako et al., 2000) and a recent study
showed that disruption of its gene results in cytokinesis defect
in vivo (Di Cunto et al., 2000).
MATERIALS AND METHODS
Cell culture
HeLa cells were grown in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal calf serum (FCS) at 37°C with
an atmosphere containing 5% CO2. We enriched mitotic cells by
synchronizing the growth of cells with the thymidine block method.
Briefly, cells were incubated with 10 mM thymidine in DMEM
containing 10% FCS for 16 hours at 37°C. The cells were then washed
with Dulbecco’s phosphate-buffered saline (PBS[−]) three times and
cultured in DMEM with 10% FCS at 37°C for 11 hours. For disruption
of microtubules, cells were incubated with 500 ng/ml nocodazole
(Wako Pure Chemicals, Osaka, Japan) at 37°C for 45 minutes. For
disruption of F-actin, cells were incubated either with 2 µM
cytochalasin D (Wako Pure Chemicals) at 37°C for 30 minutes, or
with 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 Y27632, a specific ROCK inhibitor (Uehata et al., 1997), for 30 minutes
at 37°C. Treatment with C3 exoenzyme was performed as described
previously (Kato et al., 2001). Briefly, C3 exoenzyme was prepared
as described previously (Morii and Narumiya, 1995) and was
electroporated into HeLa cells 6 hours after the release from the
thymidine block. The cells were plated on glass coverslips and
cultured in DMEM supplemented with 10% FCS. After 6-16 hours
culture, cells were subjected to fixation and stained.
Plasmid construction and expression
pCAG-myc-Citron-K has been previously described (Madaule et al.,
1998). Green Fluoresecent Protein (GFP)-tagged Citron-K was
produced by subcloning the inserts in pEGFP-C1 (Clontech) using
BsiWI and NotI restriction sites newly created in this plasmid, and the
construct was confirmed by nucleotide sequencing. Sources of pEXVmyc-Val14RhoA,
pCMV-myc-Asn19RhoA,
pCMV-mycAsn17Cdc42, pEXV-myc-Val12Rac1, pCMV5-FLAG-Asn17Rac1
were described previously (Ishizaki et al., 1997; Hirose et al., 1998;
Kimura et al., 2000). pCMV-myc-Val12Cdc42 was kindly provided
by M. Symons (Picower Institute for Medical Research, Manhasset,
NY). For construction of pEGFP-RhoA, pEGFP-Asn19RhoA, and
pEGFP-Val14RhoA, the respective constructs in pBTM (Watanabe et
al., 1997) were digested with BamHI and EcoRI and the resulting
fragments were inserted into the BglII and EcoRI sites of pEGFP-C1.
Transfection of these plasmids to HeLa cells were performed using
Lipofectamine Plus (Gibco/BRL) in OPTI-MEM (Gibco/BRL) as
described by Fujita et al. (Fujita et al., 2000).
Immunofluorescence
HeLa cells grown on 20×20 mm glass coverslips were fixed with 4%
formaldehyde in PBS[−] for 15 minutes at 4°C except in experiments
shown in Fig. 4 and Fig. 8C, where the TCA fixation method by
Hayashi et al. (Hayashi et al., 1999) was used. Fixed cells were
washed with PBS-Tx (0.1% Triton X-100 in PBS[−]) several times.
After blocking in PBS-Tx containing 1% bovine serum albumin (PBSTx-BSA) for 1 hour at room temperature, immunocytochemistry was
performed with following antibodies and fluorescence reagents. The
primary 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 rabbit
polyclonal anti-RhoA antibody (119, Santa Cruz). The primary
antibodies were added at 1:200 dilution in PBS-Tx-BSA and
incubation was carried out at room temperature for 45 minutes. The
cells were then washed with PBS-Tx several times, and incubated with
Texas Red-X phalloidin (Molecular Probes), TOPRO3 (Molecular
Probes) or 4,6-diamidino-2-phenylindole (DAPI) (Molecular Probes),
and/or with following secondary antibodies; FITC-conjugated donkey
anti-mouse IgG (Jackson Immuno Research), Texas Red-conjugated
donkey anti-mouse IgG (Jackson Immuno Research), Cy5-conjugated
donkey anti-mouse IgG (Jackson Immuno Research), FITCconjugated donkey anti-rabbit IgG (Jackson Immuno Research),
Texas Red-conjugated donkey anti-rabbit IgG (Jackson Immuno
Research) and Cy5-conjugated donkey anti-rabbit IgG (Jackson
Immuno Research). For blocking anti-Citron antibody, His-tagged
antigenic Citron peptide (amino acid residues 674-870 of Citron-N)
was prepared as described previously (Madaule et al., 1995) and
added at 1 µg/ml to the incubation with anti-Citron antibody.
Fluorescence images were acquired by an MRC 1024 laser-scanning
confocal microscope imaging system (Bio-Rad) equipped with a Zeiss
Axiovert 100TV microscope.
Active Rho mobilizes Citron-K in cytokinesis
Phase-contrast and electron microscopy
HeLa cells expressing GFP-Citron-K were identified on CELLocate
coverslips (Eppendorf) using an Olympus IX70 fluorescence
microscope equipped with a cooled CCD camera (SenSys 0400,
768X512 pixels; Photometrics) and their phase-contrast and
fluorescence images were recorded together with their location
information. Cells were then fixed with 2.5% glutaraldehyde, 0.2%
tannic acid and 0.05% saponin in 0.1M cacodylate buffer, pH 7.4, for
1 hour at room temperature. After washing with 0.1 M cacodylate
buffer (pH 7.4) three times (5 minutes each), cells were postfixed with
ice-cold 1% OsO4 in the same buffer for 45 minutes. The samples
were rinsed with distilled water, stained with 0.5% aqueous uranyl
acetate for 2 hours at room temperature, dehydrated with ethanol and
embedded in Epon 812. Prior to ultra-thin sectioning, the CELLocate
coverslip was detached from the Epon block. The location information
of the coverslip was transferred onto the surface of the block, which
enabled the identification of the GFP-Citron-K-expressing cells based
on the recorded images. Ultra-thin sections of the GFP-Citron-Kexpressing cells were cut, doubly stained with uranyl acetate and lead
citrate and viewed with a JEM 1010 transmission electron microscope
(JEOL).
RESULTS
Change of intracellular localization of Citron-K
during cell cycle
We previously demonstrated that Citron-K is enriched in the
cleavage furrow of mitotic cells during cytokinesis (Madaule
et al., 1998). Because it is an effector of the small GTPase Rho,
the enrichment of Citron-K in the cleavage furrow is presumed
to be carried out by the action of Rho. To understand the
mechanism of Citron-K mobilization, we first used anti-Citron
antibody and monitored the intracellular localization of
endogenous Citron-K during the cell cycle. We also added
recombinant Citron fragment containing the antigenic epitope
to 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 comparing
immunofluorescence images in the absence and presence of the
competing peptide. First, punctate signals were detected by the
anti-Citron antibody and were abolished by the addition of the
epitope peptide in the cytoplasm of interphase cells. In
prometaphase, these specific Citron-K signals disintegrate and
disperse diffusely in the cytoplasm. From anaphase to
telophase, Citron-K accumulates in the cleavage furrow and
finally to the midbody in the post-mitotic stage. These results
demonstrate that Citron-K changes its localization during cell
division from dot-like structures in interphase, to the cytoplasm
in prometa- and metaphases and finally to the cortex of
cleavage furrow in telophase.
To examine whether Rho is involved in this change of
Citron-K localization, and if so, to identify a step regulated by
Rho in this localization change, we electroporated C3
exoenzyme into HeLa cells enriched in S phase. We first
examined whether the C3 exoenzyme treatment interfered with
cytokinesis of HeLa cells, because a previous study by
O’Connell et al. (O’Connell et al., 1999) showed that C3
exoenzyme injection into cultured NRK epithelial cells
induced abnormal cortical activity and resulted in ectopic
division. The C3 exoenzyme treatment of HeLa cells resulted
in almost 100% production of binucleate cells 16 hours after
3275
the treatment (Fig. 2A), suggesting that Rho also plays a
crucial role in regulation of cytokinesis of mammalian cells.
Localization of Citron-K in these C3 exoenzyme-treated cells
in various mitotic phases were then examined (Fig. 2B). C3
exoenzyme treatment, and consequently inactivation of Rho,
did not affect dispersion of Citron-K into the cytoplasm in
prometa- and metaphase. However, transfer of Citron-K to
cleavage furrow in ana- to telophase was completely prevented
by this treatment. Instead, Citron-K in the treated cells stayed
in the spindle midzone. Co-staining with microtubules
demonstrated that Citron-K associates with the central spindles
in these cells. All of these signals appear to reflect the behavior
of endogenous Citron-K, because they were abolished by the
addition of the antigenic peptide.
To confirm these findings, we constructed GFP-tagged
Citron-K and expressed it in HeLa cells. The GFP fusion
protein showed the same pattern of phase-dependent change in
the intracellular localization as endogenous Citron-K (Fig.
3A). GFP-Citron-K again shows punctate signals in interphase
cells, becomes dispersed in the cytoplasm in prometaphase and
concentrates in the cleavage furrow after anaphase. To clarify
the identity of the punctate signals seen in interphase cells,
GFP signals were examined with electron microscopy. As
shown in Fig. 3B, they appeared as amorphous materials not
enclosed with lipid bilayer, suggesting that they are protein
aggregates and not vesicles. The GFP signals in the cleavage
furrow in dividing cells often appear punctate, suggesting that
aggregates of the overexpressed protein are not completely
disassembled and migrate. When HeLa cells expressing GFPCitron-K were treated with C3 exoenzyme, GFP signals were
again seen in association with the midzone spindles in ana- and
telophase cells (Fig. 3C). These results corroborate the above
findings with endogenous Citron-K and suggest that the GFPfusion of Citron-K can be used as a probe to monitor the
behavior of the endogenous protein. Essentially the same
localization was observed when Citron-K was expressed as a
Myc-tagged protein. Expression of these recombinant CitronK proteins did not interfere with cytokinesis.
Citron colocalizes with Rho in the cleavage furrow
The above results indicate that Rho catalyzes the transfer of
Citron-K to the cell cortex in cleavage furrow, possibly from
the midzone spindles. Because Citron binds to the GTP-bound,
active form of Rho (Madaule et al., 1995), we wondered
whether Rho and Citron-K colocalize in cleavage furrow. We
therefore expressed GFP-Citron-K in HeLa cells and examined
the colocalization of GFP-Citron-K and endogenous Rho in
mitotic cells (Fig. 4). In interphase cells, Rho is present
diffusely in the cytoplasm, whereas Citron-K are in dot-like
structures as described. When cells undergo cytokinesis, Rho
accumulates in cleavage furrow and stays to the midbody. This
is consistent with a previous finding on the Rho localization in
fertilized eggs of sea urchin (Nishimura et al., 1998). When
both Rho and Citron-K were visualized in these cells, the Rho
signal colocalizes with the Citron-K signal from the beginning
of cytokinesis in the cleavage furrow to the midbody of
postmitotic cells (Fig. 4B-E) suggesting, although not proving,
that Citron-K makes a complex with Rho in this cytokinetic
apparatus. Given that Citron binds only to active Rho (Madaule
et al., 1995), these results suggest that Rho present in this
complex is the GTP-bound active form. In addition to these
3276
JOURNAL OF CELL SCIENCE 114 (18)
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-hand
pairs 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 endogenous
Citron-K is detected as particulate staining in the cytoplasm in interphase cells (arrowheads), disperses in the cytoplasm in prometaphase, is
transferred to the cortex in the cleavage furrow in telophase and is present in the midbody in post-mitotic cells. These intracellular signals
disappear when the blocking peptide is present. Bars, 10 µm.
structures in the cell cortex, a portion of overexpressed GFPCitron-K remains as aggregates in the cytoplasm, where no
colocalization with Rho was observed (Fig. 4B,C, arrowheads).
Overexpressed Citron-K transfers to the cell cortex
with Val14Rho expression in interphase cells
The above findings that inactivation of Rho interfered with the
transfer of Citron-K to the cortex and that transferred CitronK appeared to make a complex with Rho in cleavage furrow
strongly suggest that active Rho binds Citron-K and they move
together to the cortex. To test this hypothesis, we examined the
effect of expression of Val14RhoA (a dominant active RhoA
mutant) on the localization of overexpressed Citron-K in
interphase cells. Myc-tagged Citron-K and GFP-tagged
Active Rho mobilizes Citron-K in cytokinesis
3277
Fig. 2. Effect of C3 exoenzyme treatment on Citron-K localization during cell division. Recombinant C3 exoenzyme was introduced into HeLa
cells 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 after
electroporation as shown by DAPI staining (blue). Microtubules are stained in green. (B) Effect of C3 exoenzyme treatment on Citron-K
localization during cell division. HeLa cells without (left-hand pairs of panels) or with (middle and right-hand pairs of panels) C3 exoenzyme
treatment were fixed in various phases of cell division and stained with anti-Citron antibody (green). Microtubules and DNA were stained with
anti-β-tubulin antibody (red) and TOPRO3 (blue), respectively. The left panels of each pair represent merged images. Note that Rho
inactivation by C3 exoenzyme treatment did not affect Citron-K localization in prometa- and metaphase, but prevented the transfer of Citron-K
to the cortex in telophase, which instead was associated with the spindle midzone (middle bottom pairs of panels). The Citron-K signal in the
spindle midzone was abolished in the presence of the antigenic peptide (the right bottom pair of panels). Bars, 10 µm.
Val14RhoA were co-expressed in HeLa cells. Cells expressing
both constructs were identified by GFP fluorescence and the
Myc-tag staining, and the localization was examined by
confocal microscopy. As shown in Fig. 5A, Citron-K forms
band-like structures at the bottom of the cells co-expressing
Val14RhoA. This band-like structure consists of numerous
small patches in which the Myc-Citron-K signal and the GFPVal14RhoA signal overlap completely. By contrast, no overlap
of the Citron-K signals and GFP-Rho was found in remaining
aggregates in the middle of the cells (Fig. 5A, middle slice).
This was supported by examination in a vertical view of cells
co-expressing Citron-K and Val14RhoA, which shows that the
band-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 small
patches in band-like structures suggests that activated Rho is
present in these structures in a complex with Citron-K. Indeed,
co-expression of either wild-type RhoA or dominant negative
Asn19RhoA with Citron-K failed to induce the formation of
the band-like structures, and Citron-K remains as aggregates in
the cytoplasm (Fig. 5C). We also co-expressed Citron-K with
other Rho-family small GTPases, Rac1 and Cdc42. Although
Citron is able to bind to Rac1 in a yeast two hybrid system and
in an in vitro overlay assay (Madaule et al., 1995), expression
of neither dominant active nor dominant negative Rac1
(Val12Rac1 and Asn17Rac1, respectively) affected the
localization of Citron-K in interphase cells (Fig. 5D). The
localization of Citron-K was not affected either by expression
of a dominant active or a dominant negative Cdc42
(Val12Cdc42 and Asn17Cdc42, respectively) (Fig. 5E),
suggesting that Citron-K translocation to cell cortex to form
band-like structures is specific to Rho activation. These results
taken together suggest that the binding of Citron-K and
Val14RhoA induces the transfers of Citron-K from the
3278
JOURNAL OF CELL SCIENCE 114 (18)
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) and
TOPRO3 (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 is
present in the midbody after division (e and f). In about 50% of metaphase cells overexpressing Citron-K, the GFP-Citron-K aggregates
appeared to attach to the cell cortex as shown in c. (B) Electron microscopy of GFP-Citron-K aggregates in interphase. Punctate fluorescence
signals of GFP-Citron-K expressed in HeLa cells were identified first in a live cell by fluorescence microscopy and subjected to electron
microscopy. 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 expressing
GFP-Citron-K (green) by electroporation, and Citron-K localization in telophase was examined. Note that GFP signals were associated with the
spindle midzone. The left-hand panels are merged images with microtubules stained in red and DNA stained in blue. Bars, 10 µm except in the
right-hand panel of B.
cytoplasm to the cell cortex. In these experiments, expression
of Val14RhoA appeared to disintegrate large Citron-K
aggregates in the cytoplasm. However, detailed inspection
revealed that Citron-K is still present in small aggregates after
transfer to the cortex, which are seen as small patches. Thus,
disintegration observed in this overexpression system is
probably not the same as dispersion of endogenous Citron-K
seen in prometaphase of mitotic cells (see Discussion).
Band-like structures of Citron-K exist on the same
plane with stress fibers but they are mutually
exclusive
It is well known that Rho regulates actin cytoskeleton and that
active Rho induces stress fibers (Hall, 1998). Given that Citron-
K forms the band-like structure on Rho activation at the bottom
of the cells, we wondered whether it has some connection with
actin stress fibers. To this end, we cotransfected pEGFPCitron-K and pEXV-myc-Val14RhoA into HeLa cells and
subjected the cells for phalloidin staining. Covisualization of
Citron-K and F-actin revealed that the band-like structure of
Citron-K is present at the same plane as stress fibers, but that
these two structures exclude each other as suggested by no
signal overlap of GFP-Citron-K and F-actin (Fig. 6A-C). We
were also interested in the relationship between Citron-K and
a septin, because the latter molecule also accumulates in the
cleavage furrow and is involved in cytokinesis (Field and
Kellogg, 1999). However, when endogenous Nedd5, one of the
septins, was stained in HeLa cells co-expressing GFP-Citron-
Active Rho mobilizes Citron-K in cytokinesis
3279
Fig. 4. Colocalization of RhoA and Citron-K in cleavage furrow
of HeLa cells. Colocalization of Rho and Citron-K was examined
by staining endogenous Rho with anti-Rho antibody (red, righthand panels) in HeLa cells expressing GFP-Citron-K (green,
middle panels) in various stages of cytokinesis. Left-hand panels
represent merged images. In interphase, Rho shows homogenous
staining in the cytoplasm, whereas Citron-K shows particulate
signals (A). Punctate signals for Rho in the nucleus appear
nonspecific, because such signals are only found by this
polyclonal antibody 119 used in this experiment, and not by the
other monoclonal antibody 26C4. GFP-Citron-K and endogenous
RhoA 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-K
remains as aggregates in the cytoplasm, where no colocalization
with Rho was observed (for example, see arrowheads in B and
C). Confocal sections of each cell are shown. Note that Citron-K
and a part of Rho also colocalize in the midbody of post mitotic
cells (E). Blue in E is β-tubulin. Bars, 10 µm.
K and Val14RhoA, it was associated with actin stress fibers, as
previously observed (Kinoshita et al., 1997), and its signals and
the GFP-Citron-K again excluded each other (data not shown).
Citron-K and the actin cytoskeleton exclude each
other in cleavage furrow
We next examined the spatial relation between the Citroncontaining structures and F-actin in mitotic cells. We studied
this issue on both endogenous Citron-K (Fig. 7A,B) in HeLa
cells and cells expressing GFP-Citron-K (Fig. 7C,D). Both
studies demonstrated a clear separation of the structures
containing Citron-K and the F-actin in the cleavage furrow. The
Citron-containing structure appears to be concentrated beneath
F-actin structure in the cleavage furrow by being encircled in
all directions by F-actin during cytokinesis except in the
earliest stage, where it was difficult to separate the two signals
(Fig. 7A).
Accumulation of the Citron-K-Rho complex in bandlike structure in interphase cells and in cleavage
furrow of mitotic cells disappears with actin
depolymerization
We next addressed the interaction between the Citronenriched cortical structures and the cytoskeletons by
disrupting either microtubules or F-actin. Nocodazole was
used to depolymerize microtubules in cells co-expressing
Citron-K
and
Val14RhoA.
Depolymerization
of
microtubules did not affect the colocalization of Citron-K
and active Rho, and the band-like structures of the CitronK-Val14RhoA complex was maintained as those found in
nontreated cells (data not shown), suggesting that the
formation of the band-like structures does not depend on the
integrity of microtubules. By contrast, when F-actin was
disrupted either with cytochalasin D or latrunculin A
treatment, the band-like structures were disintegrated, and
Citron-K-containing small patches spread all over the ventral
surface of the cell cortex (Fig. 8A). Because stress fibers are
formed by virtue of actomyosin-based contractility that is
exerted by the action of a Rho effector ROCK, we examined
the effect of the ROCK inhibitor Y-27632 on this
accumulation. Disruption of stress fibers by inactivation of
ROCK resulted again in dispersion of the accumulation of
Citron-containing patches (Fig. 8B). In some cells which
have remaining stress fibers, some of the Citron-containing
band-like structures were also conserved (Fig. 8B, righthand cell). These results suggest that band-like arrangement
of Citron-K-active Rho patches depends on orderly
organization of F-actin by the action of the actomyosin
system. We then examined the effect of F-actin
depolymerization on the concentration of the Citroncontaining structures in cleavage furrow (Fig. 8C). Mitotic
cells were enriched by the use of the thymidine block and
3280
JOURNAL OF CELL SCIENCE 114 (18)
treated with either cytochalasin D or latrunculin A. In the
dividing cells treated with either reagent, Citron-K was not
seen as a ring-like structure in the cleavage furrow but
dispersed as patches all around the cell cortex. However, the
colocalization of Citron-K and endogenous Rho was spared
Fig. 5. Translocation of GFP-Citron-K to the
cortex of interphase cells by expression of
dominant active Rho. (A) Effects of expression
of dominant active Rho on localization of
Citron-K. Myc-Citron-K and GFP-Val14RhoA
were co-expressed in HeLa cells. Citron-K (red)
and Val14RhoA (green) are colocalized in bandlike structures at the bottom of cells (bottom
slice), whereas in the middle of the cell, CitronK is present as an aggregate without
colocalization with Val14Rho (the middle slice,
arrowheads). Band-like structure of Citron-K
and Val14RhoA at the bottom consists of small
patches. (B) Vertical views of GFP-Citron-K
localization in HeLa cells co-expressing MycVal14RhoA and GFP-Citron-K. Note that
expression of Val14RhoA (red) disintegrates
cytoplasmic aggregates of GFP-Citron-K
(green) and translocates it to the cell cortex to
form band-like structures. Signals of mycVal14RhoA and GFP-Citron-K appear not to
colocalize completely because the fluorescence
of Texas Red-Myc-Val14RhoA is not so strong
as GFP-Citron-K, and only a small portion of
expressed Rho colocalize with Citron-K.
(C) Effects of expression of GFP-tagged wildtype RhoA (the left pair of panels) and GFPtagged Asn19RhoA (the right-hand pair of
panels) on localization of Myc-tagged Citron-K.
Myc-Citron-K (red) remains as aggregates when
wild-type or dominant negative RhoA (green) is
co-expressed. (D) Effects of Rac expression on
Citron-K localization. GFP-Citron-K (green)
and either Myc-tagged Val12Rac1 (red) (the left
pair of panels) or FLAG-tagged Asn17Rac1
(red) (the right pair of panels) were coexpressed in HeLa cells. Note that either
expression did not affect the cytoplasmic
aggregates of GFP-Citron-K. (E) Effects of
Cdc42 expression. GFP-Citron-K (green) and
either Myc-tagged Val12Cdc42 (red) (the left
pair of panels) or Myc-tagged Asn17Cdc42
(red) (the right pair of panels) were coexpressed in HeLa cells. Note that either
expression did not affect the cytoplasmic
aggregates of GFP-Citron-K. C, D and E all
show the bottom slices of interphase cells. Bars,
10 µm.
in the dispersed patches. However, unlike Citron-K, a portion
of Rho still remained at the original site of the cleavage
furrow. Y-27632 was without effect on mitotic cells, which
is consistent with the dispensable action of ROCK in
cytokinesis (Madaule et al., 1998; Ishizaki et al., 2000).
Active Rho mobilizes Citron-K in cytokinesis
3281
DISCUSSION
Citron-K undergoes multi-step change in its
localization during mitosis
In this study we monitored the localization of Citron-K during
mitosis both by immunofluorescence study of endogenous
protein with anti-Citron antibody and by expression of GFPCitron-K fusion in HeLa cells. Both studies have revealed the
multi-step change of Citron-K localization during mitosis.
Citron-K is present as aggregates in interphase cells, disperses
into the cytoplasm in prometaphase, translocates to the cell
cortex in anaphase and accumulates in cleavage furrow in
telophase. Although not all interphase cells contain visible
aggregates in immunofluorescence, we think that Citron-K is
present as aggregates also in these cells in a form not detected
by this method. Oligomer formation has been reported for
MRCK, a kinase homologous to Citron-K (Tan et al., 2001).
Using C3 exoenzyme to inactivate Rho, we have found that the
above sequential change is catalyzed by consecutive activation
of a Rho-independent and a Rho-dependent mechanism. Thus,
dispersion of Citron-K occurs normally in C3 exoenzymetreated cells. However, it does not move to the cortex but stays
in association with the midzone spindles in anaphase cells.
These results indicate that Citron-K moves to the cortex via the
midzone spindles in a Rho-dependent manner. This is an
intriguing finding because, in mammalian cells, the cleavage
signal is suggested to come from the central interdigitating
spindle microtubules (Cao and Wang, 1996). Previously,
several proteins have been reported to associate with the
midzone spindles. They include TD-60, INCENP, aurora
kinase and survivin and are collectively termed chromosomal
passenger proteins (Andreassen et al., 1991; Adams et al.,
2001; Skoufias et al., 2000). However, these proteins first
locate at centromeres of chromosomes in metaphase, then
associate with the midzone spindle extending to the cortex in
anaphase, stay there in telophase and concentrate in the
intracellular bridge after mitosis. However, we did not see any
attachment of Citron-K to centromeres or chromosomes. It
appears to bind to the midzone spindles from the cytoplasm
and transfers to the cortex upon Rho activation. Thus, CitronK is likely to be a novel type of passenger protein.
The above observation also indicates the presence of a Rhoindependent, yet cell-cycle-dependent signal for the initial
mobilization of Citron-K. At present, we do not know the
Fig. 6. Mutual exclusion of Citron-K-containing band-like structures
and actin stress fibers. GFP-Citron-K and Myc-Val14RhoA were cotransfected into HeLa cells. Actin stress fibers (red) and band-like
structures of Citron-K (green) exist on the same plane at the bottom
of interphase cells, but the signals do not overlap at all (a merged
image, left). Bar, 10 µm.
Fig. 7. Mutual exclusion of Citron-K-containing structures and actin
cytoskeleton in the equatorial cell cortex during cytokinesis. (A and
B) Localization of endogenous Citron-K and F-actin. Two HeLa cells
at different stages of cytokinesis were chosen, and stained for
endogenous Citron-K (green), F-actin (red), and DNA (blue). CitronK 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). GFPCitron-K-expressing HeLa cells undergoing ingression of the
cleavage furrow (C) and that at the end stage of cytokinesis (D) were
chosen. In C, several confocal sections encompassing the cleavage
furrow of a dividing cell are piled up and shown. β-Tubulin was
stained blue in D. Note that GFP-Citron-K and F-actin do not
colocalize. Scale bars, 10 µm.
3282
JOURNAL OF CELL SCIENCE 114 (18)
Fig. 8. Dispersion of Citron-K-containing patches by disruption of Factin. (A and B) Effects of latrunculin A (A) or Y-27632 (B) treatment
on band-like structures in interphase cells. HeLa cells co-expressing
GFP-Citron-K (green) and Myc-Val14RhoA were subjected to each
treatment. F-actin is stained in red. (A) Latrunculin A treatment
disintegrated the band-like structures of Citron-K and dispersed
Citron-K-containing patches throughout the cell cortex. (B) The Y27632 treatment disrupted stress fibers and dispersed Citron-K
containing patches (the left cell). In cells retaining stress fibers, some
of the Citron-containing band-like structures remained (the right-hand
cell). (C) Effects of F-actin disruption on Citron-K accumulation in
mitotic cells. HeLa cells expressing GFP-Citron-K were enriched in
M-phase, subjected to latrunculin A treatment, and stained for
endogenous RhoA (red) and for DNA (blue). In the dividing cells
treated with latrunculin A, GFP-Citron-K (green) was not seen as a
ring-like structure in the cleavage furrow but dispersed as patches all
around the cell cortex. In the dispersed patches, colocalization with
endogenous RhoA is also observed. Endogenous RhoA also remains
in the putative cleavage furrow (arrowheads). Confocal sections of
three different cells are shown. The same pattern was also observed
with cytochalasin D treatment. Bars, 10 µm.
identity of this signal. Cell-cycle-dependent mobilization was
also reported for other proteins working in mitosis. For
example, expression study showed that survivin is present as
dots in the cytoplasm in interphase cells but concentrates in
distinct spots on chromosomes in prophase (Skoufias et al.,
2000). ECT-2, the Rho exchange protein involved in
cytokinesis, is present in the interphase nucleus, disperses in
the cytoplasm in prometaphase, concentrates in the spindle in
metaphase and transfers to the cortex in anaphase (Tatsumoto
et al., 1999). In the latter case, phosphorylation-dependent
activation was suggested to occur.
Active Rho takes Citron-K to cell cortex and
cleavage furrow
As discussed, active Rho appears to transfer Citron-K to cell
cortex and to concentrate it in cleavage furrow in telophase.
We mimicked the transfer of Citron-K to the cortex by coexpression of Val14RhoA and Citron-K in interphase cells.
Although the experiments in interphase cells naturally do not
exactly simulate the process in dividing cells, the results
obtained have provided many implications. In the latter
experiment, the transferred proteins form band-like structures
in the ventral cortex of cells. It should be mentioned that
Citron-K is present still as small aggregates in these band-like
structures, partly because this procedure skipped the natural
dispersion process seen during mitosis and partly because of
the high amount of overexpressed Citron-K. Because CitronK binds to the active form of Rho selectively, this result
suggests that the binding of Citron-K to active Rho takes Citron
to cell cortex and cleavage furrow. A number of other Rho
effector proteins including ROCK, mDia, Rhophillin, Rhotekin
and PKN have been identified (Ishizaki et al., 1997; Watanabe
et al., 1997; Watanabe et al., 1996; Reid et al., 1996). Although
membrane translocation associated with activation of Rho has
been reported on some of these effectors, such a marked
translocation as that seen in Citron-K has never been observed.
This is probably because other effectors are transiently
translocated and used only in a small amount in response to
local activation of Rho. However, transfer of Citron-K requires
extensive and widely spread activation of Rho as induced by
Val14RhoA expression in interphase cells. High accumulation
of GTP-Rho during mitosis was already reported (Kimura et
al., 2000). It should also be mentioned that almost all of
endogenous Citron-K is transferred by Rho activation and
accumulates in the cell cortex. At its accumulation site, CitronK appears to make a complex with active Rho because
colocalization of Citron-K and Rho is persistently observed in
the band-like structures formed by co-expression with
Val14Rho and in cleavage furrow. These results indicate that
Rho in the active GTP-bound form serves as a structural
component in the Citron-K-containing cytokinetic apparatus.
We previously found that accumulation of GTP-Rho continues
to be present during cytokinesis after the decline of the Rho
exchange activity, and suggested the presence of a stabilization
mechanism for GTP-Rho (Kimura et al., 2000). Our present
finding is consistent with this suggestion. However, this is in
Active Rho mobilizes Citron-K in cytokinesis
contrast to the presumed activation mechanisms for other Rho
effectors, in which active Rho transiently interacts with
effectors. As shown by the previous study (Nishimura et al.,
1998) and also shown here, most of Rho present in the cell
accumulates in the cleavage furrow during cytokinesis. One
mechanism of this Rho accumulation is the accumulation of
Citron-K in this region. The translocation and accumulation of
Citron-K appears to be linked to its function in cytokinesis. Di
Cunto et al. (Di Cunto et al., 2000) disrupted selectively the
gene for Citron-K in the kinase domain and showed that the
kinase domain of Citron-K is crucial in cytokinesis. Thus, this
study presents a new mode of stimulus-activated construction
of a functional cytokinetic apparatus.
Citron-K and actin cytoskeleton in cytokinesis
During cleavage of eggs of the echinoderms and Xenopus
embryos, F-actin forms a distinct structure known as the
contractile ring, which cooperates with myosin and constricts
to divide the cell. The actin cytoskeleton also plays a crucial
role in cytokinesis of mammalian cells, although the actin
contractile ring is not so discernible in these cells. Because Rho
is involved in reorganization of several types of the actin
cytoskeleton such as stress fibers, we were interested in the
relationship between the Citron-containing structures and the
actin cytoskeleton. Unexpectedly, we have found that the
Citron-K-containing structures and the actin cytoskeleton are
present by excluding each other. In interphase cells coexpressing Citron-K and Val14Rho, the band structure that
contains Citron-K is present at the same plane of the ventral
cell cortex as stress fibers and the two exclude each other. In
the cleavage furrow of mitotic cells, the Citron-K-containing
structure is encircled by the F-actin. At the very bottom of the
cleavage furrow, strong accumulation of Citron-K was
observed but not of F-actin. Oegema et al. (Oegema et al.,
2000) have reported the similar absence of F-actin in the center
of the cleavage furrow in BHK-21 cells. Furthermore, we have
found that disruption of F-actin with either cytochalasin D or
latrunculin A abolished the Citron-K-containing structures and
dispersed small patches containing Citron-K throughout the
cell cortex. These results suggest that Citron-K molecules are
put together in the band-like structures in interphase cells and
in the cleavage furrow of mitotic cells by the force of the actin
cytoskeleton. Because Citron-K plays an essential role in
cytokinesis at least in some populations of neuronal cells (Di
Cunto et al., 2000), one function of the actin cytoskeleton in
cytokinesis of mammalian cells is to make Citron-K
accumulation in the cleavage furrow. It should be emphasized
that our present data do not exclude the role of the actomyosin
cytoskeleton in elicitation of the contractile force in
cytokinesis. There has been no report showing the contractile
force generation by Citron-K. However, a number of reports
have shown the involvement of actin binding proteins including
myosin in generation and processing of the contractile ring
(Robinson and Spudich, 2000).
The role of Citron-K and Rho in cytokinesis:
remaining issues
The data reported above have clarified how activated Rho
mobilizes Citron-K during cell division. Citron-K exists in the
cytoplasm in interphase, moves to midzone spindles, binds
then to activated GTP-Rho, transfers to cell cortex and
3283
accumulates in the cleavage furrow. How do Citron molecules
accumulated in the cleavage furrow exert a crucial function in
cytokinesis? One plausible possibility is that Citron-K
accumulated there by the force of F-actin in turn regulates
functions of actin and other cytoskeletons, although there has
been no direct evidence to support this hypothesis. Citron-K
most probably exerts this action by phosphorylating some
substrate(s) in this process. Identification of these substrates
will clarify this issue. The above mobilization pathway has also
raised several other important questions such as the identity of
the initial Rho-independent, cell-cycle-dependent signal, how
Citron-K moves to the midzone spindles, how Rho mobilizes
Citron-K there to cell cortex and the function of the midzone
spindles in this process. These questions may be solved by
identification of domains of Citron-K responsible for each
mobilization step and their binding partner there. This
approach may also give us a new insight into the interaction
among Rho, Citron-K, microtubules and actin cytoskeleton
during mitosis.
We thank S. Tsukita, H. Bito, T. Tsuji, M. Okamoto, Y. Takada, F.
Oceguera, K. Kimura, M. Maekawa and T. Furuyashiki for helpful
advice and discussion, M. Kinoshita and M. Noda for generous supply
of anti-Nedd 5 antibody, K. Nonomura for technical assistance, and
T. Arai and H. Nose for secretarial help.
REFERENCES
Adams, R. R., Carmena, M. and Earnshaw, W. C. (2001). Chromosomal
passengers 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 telophase
cells 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 are
required 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 for
cytokinesis in Drosophila and shares domains of similarity with the products
of the limb deformity gene. Development 120, 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). Defective
neurogenesis in citron kinase knockout mice by altered cytokinesis and
massive apoptosis. Neuron 28, 115-127.
Drechsel, D. N., Hyman, A. A., Hall, A. and Glotzer, M. (1997). A
requirement for Rho and Cdc42 during cytokinesis in Xenopus embryos.
Curr. Biol. 7, 12-23.
Field, C. M. and Kellogg, D. (1999). Septins: cytoskeletal polymers or
signaling 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-specific
binding protein of rhophilin, that is localized in the fibrous sheath of sperm
flagella. 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. Science 279, 509514.
Hayashi, K., Yonemura, S., Matsui, T. and Tsukita, S. (1999).
Immunofluorescence detection of ezrin/radixin/moesin (ERM) proteins with
their carboxyl-terminal threonine phosphorylated in cultured cells and
tissues. 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. and
Narumiya, S. (1998). Molecular dissection of the Rho-associated protein
3284
JOURNAL OF CELL SCIENCE 114 (18)
kinase (p160ROCK)-regulated neurite remodeling in neuroblastoma N1E115 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-coil
forming protein kinase, works downstream of Rho and induces focal
adhesions. 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, a
specific inhibitor of Rho-associated kinase. Mol. Pharmacol. 57, 976-983
Kato, T., Watanabe, N., Morishima, Y., Fujita, A., Ishizaki, T. and
Narumiya, S. (2001). Localization of a mammalian homolog of diaphanous,
mDia1, to the mitotic spindle in HeLa cells. (2001). J. Cell Sci. 114, 775784.
Kimura, K., Tsuji, T., Takada, Y., Miki, T. and Narumiya, S. (2000).
Accumulation of GTP-bound RhoA during cytokinesis and a critical role of
ECT2 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 mammalian
septin, is a novel cytoskeletal component interacting with actin-based
structures. Genes Dev. 11, 1535-1547.
Kishi, K., Sasaki, T., Kuroda, S., Itoh, T. and Takai, Y. (1993). Regulation
of cytoplasmic division of Xenopus embryo by rho p21 and its inhibitory
GDP/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). Specific
accumulation of Rho-associated kinase at the cleavage furrow during
cytokinesis: cleavage furrow-specific phosphorylation of intermediate
filaments. Oncogene 18, 2783-2788.
Kosako, H., Yoshida, T., Matsumura, F., Ishizaki, T., Narumiya, S. and
Inagaki, M. (2000). Rho-kinase/ROCK is involved in cytokinesis through
the phosphorylation of myosin light chain and not ezrin/radixin/moesin
proteins at the cleavage furrow. Oncogene 19, 6059-6064.
Mabuchi, I., Hamaguchi, Y., Fujimoto, H., Morii, N., Mishima, M. and
Narumiya, S. (1993). A rho-like protein is involved in the organisation of
the contractile ring in dividing sand dollar eggs. Zygote 1, 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 of
rho 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 of
the small GTPase Rho in cytokinesis. Nature 394, 491-494.
Morii, N. and Narumiya, S. (1995). Preparation of native and recombinant
Clostridium botulinum C3 ADP-ribosyltransferase and identification of Rho
proteins by ADP-ribosylation. Methods Enzymol. 256, 196-206.
Narumiya, S. (1996). The small GTPase Rho: cellular functions and signal
transduction. J. Biochem. (Tokyo) 120, 215-228.
Nishimura, Y., Nakano, K. and Mabuchi, I. (1998). Localization of Rho
GTPase in sea urchin eggs. FEBS Lett. 441, 121-126.
O’Connell, C. B., Wheatley, S. P., Ahmed, S. and Wang, Y. L. (1999). The
small GTP-binding protein rho regulates cortical activities in cultured cells
during 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 binding
protein 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 is
required for initiation of cytokinesis in Drosophila. Genes Dev. 13, 23012314.
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, 1355613560.
Robinson, D. N. and Spudich, J. A. (2000). Towards a molecular
understanding of cytokinesis. Trends Cell Biol. 10, 228-237.
Satterwhite, L. L. and Pollard, T. D. (1992). Cytokinesis. Curr. Opin. Cell
Biol. 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 and
intramolecular interactions regulate catalytic activity of myotonic dystrophy
kinase-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 in
G2/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 GTPase
and 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-associated
protein kinase in hypertension. Nature 389, 990-994.
Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N.,
Mukai, H., Ono, Y., Kakizuka, A. and Narumiya, S. (1996). Protein
kinase N (PKN) and PKN-related protein rhophilin as targets of small
GTPase Rho. Science 271, 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 target
protein for Rho small GTPase and is a ligand for profilin. EMBO J. 16, 30443056.