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INTRODUCTION |
The small GTPase Rho is required for cytokinesis as a regulator of
the actomyosin contractile ring. Immunofluorescence showed that RhoA
accumulates at the cleavage furrow during cytokinesis in Swiss 3T3
cells and HeLa cells (1, 2). Inactivation of Rho blocked cytokinesis as
demonstrated in several experimental systems including
Drosophila embryos, Xenopus embryos, mammalian cells, etc. (3-9). RhoA is also critical for cytokinesis in
hepatocytes. Overexpression of the dominant negative mouse Ect2, a
guanine nucleotide exchange factor (GEF) that activates RhoA,
caused cytokinesis failure and the formation of binucleated cells in
cultured mouse hepatocytes (10).
Rho regulates cytokinesis through its downstream targets. The
Rho-associated kinases ROCK1
I and II and Citron kinase (Citron-K) bind to Rho-GTP and have been
shown to be involved in the regulation of cytokinesis (2, 11-15).
ROCKs and Citron-K are serine/threonine kinases, and they probably
regulate cytokinesis by phosphorylating downstream targets. ROCKs
activate the regulatory myosin light chain (RMLC) directly by
phosphorylating its Ser-19 and indirectly by inhibiting myosin phosphatases (12). Citron-K also phosphorylates RMLC at Ser-19 and
Thr-18, but it does not phosphorylate myosin phosphatases (16).
The gene encoding Citron kinase has two major transcripts, the
full-length Citron-K and a form of Citron that does not contain the
kinase domain (2, 11). Citron is highly expressed in differentiated
neuronal cells (17, 18). In contrast, the expression of Citron-K
mRNA is limited to the proliferating neuroblasts, as revealed by
in situ hybridization (15). Citron-K mRNA is detected by
Northern blots in adult murine tissues including brain, kidney, spleen,
thymus, skin, lung, and at very high levels in testis, but it is not
detected in liver, heart, and skeletal muscle (11). Considering its
strong expression in highly proliferative tissues such as testis or
embryonic neuronal cells, it is possible that the expression of
Citron-K is cell cycle-dependent rather than
tissue-specific. Therefore, the absence of Citron-K in adult mouse
liver could be a consequence of the quiescent state of the tissue.
The major function of Citron-K is the control of cytokinesis. Citron-K
localizes to the cleavage furrow and midbody in anaphase and telophase
in HeLa cells (2, 9). Overexpression of truncated or kinase-dead
constructs of Citron-K induces formation of binucleated HeLa cells (2).
The requirement of Citron-K for cell division is cell type-specific as
revealed by examining the knockout animals. Loss of Citron-K causes
failure of cytokinesis and therefore triggers apoptosis in the male
germ cells and a specific population of neuroblasts (15, 19, 20).
Whether Citron-K is involved in the control of hepatocyte cell cycle
and cytokinesis is not clear.
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MATERIALS AND METHODS |
Reagents--
All chemicals were purchased from Sigma
except where otherwise indicated.
Animals--
Male adult Fisher 344 rats (2 months old) and
pregnant Fisher 344 rats were purchased from the Charles River Breeding
Laboratories (Raleigh, NC). One pair of heterozygous Wistar Kyoto rats
with a mutation at the Citron-K gene was a gift from Dr. Lo Turco, University of Connecticut, Storrs, CT. They were bred to generate the
Flathead (FH) rats, the heterozygous rats, and the homozygous wild type
(WT) rats. The autosomal recessive mutant FH rat is a spontaneous
Citron-K knockout. It was named because of an easily recognized
phenotype, a flattened skull of the neonate (21). The mutation was
confirmed to be a single base deletion in the exon 1 of the
Citron gene, resulting in an early stop codon, and no
Citron-K protein is synthesized (20, 21). Genotypes of the littermates
were determined by PCR with genomic DNA followed by restriction enzyme
cutting. A fragment of exon 1 containing the mutation site was
amplified by PCR followed by BanII (Promega, Madison, WI)
cutting of the PCR products. The wild type gene has a BanII
cutting site lost in the mutant. The primers to exon 1 of the Citron-K
gene in rat were based on the sequence of mouse exon 1 and were
5'GAGATGTTGAAGTTCAAGTA-3' and 5'CCTGGAAGAAGAGATTTAGC-3' (20).
Phenotypes of FH rats are very similar to those observed in the
Citron-K
/
mice with minor differences. Failure of cytokinesis causes the formation of binucleated neuronal cells. Massive apoptosis is detected in the developing central nervous system, which leads to
the small brain size at birth (40% of normal) (22). FH rats begin to
have seizures around postnatal day 7 and die in the first month (23).
All animal studies were approved by the Institutional Animal Care and
Use Committee of the University of North Carolina at Chapel Hill.
Experiments have been conducted in accordance with the Guide for the
Care and Use of Laboratory Animals published by the National Institutes
of Health.
Liver Perfusion--
Single cell suspensions of hepatocytes were
prepared with a modified two-step collagenase perfusion method (24).
Cell viability was >90% as measured by trypan blue exclusion.
Primary Cultures of Rat Hepatocytes--
Freshly isolated
hepatocytes were seeded onto type I collagen (5-10
µg/cm2)-coated tissue culture plates or coverslips at a
density of 15,000 cells/cm2 to 20,000 cells/cm2
for maximal cell proliferation (25). Cells were seeded in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) and 5 µg/ml insulin with 100 µg/ml
streptomycin and 100 units/ml penicillin for 4 h. The culture was
then changed to a serum-free medium; RPMI 1640 was supplemented with 10 µg/ml iron-saturated transferrin, 100 nM dexamethasone, 5 µg/ml insulin, 10 ng/ml epidermal growth factor (EGF), 1 × 10
7 M copper (CuSO4), 5 × 10
11 M zinc
(ZnSO4·7H2O), 3 × 10
10
M selenium (NaSeO3), 100 µg/ml streptomycin,
and 100 unit/ml penicillin. Both epidermal growth factor and insulin
are hepatic mitogens and provide an additive stimulus for cell
proliferation in culture.
Binucleation Ratio of Hepatocytes--
Single cell suspensions
of rat hepatocytes were fixed with 3.7% formaldehyde in PBS for 10 min
and then stained with Hoechst 33342 (5 µg/ml in PBS and 0.1% Triton
X-100, Molecular Probes, Eugene, OR). Cells were then checked under a
fluorescence microscope. At least 2000 cells were counted for each
sample, and the binucleation ratio of hepatocytes was calculated as the
percentage of binucleated cells per total number of hepatocytes.
Nuclei Isolation and Flow Cytometric Analysis--
Nuclei of rat
hepatocytes were prepared by a modified trypsin-detergent method (26).
A total of 20,000 events was collected for each sample with a FACScan
(BD Biosciences). Quantitative analysis of cell cycle parameters was
performed using the WinMDI program.
RT-PCR--
A human fetal cDNA panel was purchased from
Clontech and used as the template for PCR. Total
RNA from liver samples were isolated using the RNeasy Mini kit (Qiagen,
Valencia, CA). The cDNA was synthesized from 5 µg of total RNA
using the SuperScriptTM first strand synthesis system for
RT-PCR (Invitrogen). To detect the specific signal for Citron-K, the
forward primer was designed to locate within the kinase domain, and the
reverse primer would be in the non-kinase domain of the molecule. The
forward primer was designed according to the sequence of the rat
Citron kinase domain nucleotides 883-906,
i.e. 5'-TGGACTGTGACTGGTGGTCTGTCG-3', whereas the reverse
primer corresponds to rat Citron 1154-1177, i.e.
5'-TGGCCTCTGTGCTGGCTTTTACAG-3'. The primers amplify a 1.1-kb PCR
fragment according to the sequence information of mouse
Citron-K. These two primers are separated by at least one
intron. Thus, a positive PCR signal with the right length should
reflect the existence of the Citron-K mRNA. The PCR product was
purified and sequenced to confirm that it is an amplified fragment of
rat Citron-K cDNA. Semi-quantitation of PCR products is performed
according to the method of Relative RT-PCR (Ambion, Austin, TX) by
using
-actin as an internal standard with Ambion's
QuantumRNATM
-actin internal standards kit.
In Situ Hybridization--
In situ hybridization was
performed as described (15), with antisense RNA probes transcribed from
plasmids containing fragments of Citron-K (nucleotides
911-2056) (11).
Hep3B Cell Culture and Transfection--
Hep3B cells were
cultured on 22-mm2 coverslips in RPMI 1640 supplemented
with 10% fetal bovine serum, 100 µg/ml streptomycin, and 100 units/ml penicillin. Transfection was performed using FuGENE 6 transfection reagent (Roche). The cDNA of mouse Citron-K was cloned in-frame with an N-terminal FLAG tag in the pcDNA3 expression vector (Invitrogen) (11). Cells were cultured for 24-48 h
after transfection to allow gene expression.
Immunofluorescence--
Rat hepatocytes and Hep3B cells cultured
on coverslips were fixed in methanol/acetone (1:1) for 5 min, and
incubated with blocking buffer (phosphate-buffered saline, 2% goat
serum, 0.1% Triton X-100) for 10 min. Primary antibodies were diluted
with blocking buffer and used as follows: mAb mouse anti-
-tubulin 1:100 (Oncogene, Cambridge, MA); mouse anti-FLAG 1:200 (M2, Sigma); mouse anti-phospho-histone H3 (Ser-10) 1:100 (Cell Signaling, Beverly,
MA); and a polyclonal rabbit anti-Citron antibody at 1:4000 (prepared
by immunization of rabbits with a purified fragment of mouse Citron-K
amino acids 454-637) (15). The secondary antibodies Alexa 488 conjugated goat anti-mouse IgG and Alexa 594 conjugated goat-anti-rabbit IgG (Molecular Probes) were diluted 1:500 with blocking buffer and incubated with the fixed cells for 1 h. Cells were then stained with Hoechst 33342 (Molecular Probes) to visualize the DNA. The stained cells were examined with an Olympus IX70 microscope (Olympus, Melville, NY) or a Leica confocal microscope.
Immunohistochemistry--
Embryos were fixed by immersion in 4%
paraformaldehyde in PBS for over 12 h, embedded in paraffin, and
sectioned at 5 µm. Sections containing liver were stained with
hematoxilin-eosin. Activated Caspase-3 was detected with an
affinity-purified rabbit polyclonal antiserum (dilution 1:1000), which
recognizes the p17 subunit of cleaved caspase-3 (kindly provided by Dr.
A. Nelsbach, New England Biolabs, Beverly, MA).
Immunoprecipitation and/or Western
Blotting--
Immunoprecipitation and Western blotting were performed
as described previously (11). Liver tissue samples were homogenized in
a high stringency buffer (120 mM NaCl, 50 mM
Tris-HCl, pH 8.0, 0.5% Nonidet P-40) with supplemented protease
inhibitors (100 µg/ml phenylmethylsulfonyl fluoride, 45 µg/ml
aprotinin, 1 mM sodium orthovanadate, 10 µg/ml leupeptin,
1 mM EDTA, 2 µg/ml antipain, and 1 µg/ml pepstatin A).
Immunoprecipitation was performed with the rabbit polyclonal
anti-Citron antibody (15) at a dilution of 1:2000. Cultured Hep3B cells
and primary cultures of hepatocytes were harvested in radioimmune
precipitation (RIPA) buffer (150 mM NaCl, 50 mM
Tris-HCl, pH8.0, with 1% Nonidet P-40, 0.5% sodium deoxycholate,
0.1% SDS) supplemented with the same set of protease inhibitors.
Protein lysates or precipitates from immunoprecipitation were run on
SDS-PAGE gels and transferred to an Immobilon polyvinylidene difluoride
membrane (Millipore, Bedford, MA). Citron and Citron-K were detected
with the same anti-Citron antibody at 1:4000. Other antibodies used
were monoclonal antibody against cyclin B1, 1:200 (NeoMarker, Fremont,
CA), and proliferating cell nuclear antigen (PCNA), 1:10,000 (Sigma).
The secondary antibodies were horseradish peroxidase-conjugated goat
anti-mouse (Amersham Biosciences) and goat anti-rabbit (Jackson
Laboratories, West Grove, PA) used at a dilution of 1:10,000 and
1:40,000, respectively. The signal was detected with an ECL plus kit
(Amersham Biosciences) according to the manufacturer's instructions.
The results shown are representative of at least three independent
experiments. Densitometric analysis of Citron-K protein levels during
development was quantified with the software Scion Image (Scion
Corporation, Frederick, MD). Relative expression levels were determined
by dividing the mean densitometric value of each stage by that found in
the livers of first postnatal week animals.
Partial Hepatectomy--
Two-thirds partial hepatectomy (PH) was
performed on 2-month-old male F344 rats as described previously (27).
Liver samples removed during surgery were saved as time 0 quiescence
controls. Animals were euthanized by CO2
asphyxiation at the following hourly intervals after PH: 20, 22, 24, 26, 28, and 30 h. Portions of livers were snap frozen in liquid
nitrogen and stored at
80 °C for protein extraction. A sham
operation, including laparotomy and mobilization of liver without
tissue removal, was performed as a control.
Statistical Analysis--
Significance of differences between FH
rats and WT control was assessed by the Student's t test.
p < 0.05 means significant difference.
 |
RESULTS |
Expression of Citron Kinase Is Not Tissue-specific but Cell
Cycle-dependent--
Expression of Citron-K has been
considered tissue-specific, because Citron-K mRNA is not detected
in adult mouse liver, heart, and skeletal muscle by Northern blots
(11). In situ hybridization studies have shown that Citron-K
mRNA was detected only in proliferating but not postmitotic
neuronal cells (15), a phenomenon that could occur in other tissues. If
so, the negative signal in adult mouse liver, heart, and skeletal
muscles could be a result of their quiescent states. A positive signal
of Citron-K should be detected in these tissues if undergoing
proliferation. To address this question, Citron-K expression in
proliferating cells was evaluated. RT-PCR detected Citron-K mRNA in
human fetal heart, liver, spleen, kidney, thymus, skeletal muscle, and
lung (Fig. 1A). In
situ hybridization detected a positive signal of Citron-K mRNA
in mouse fetal liver (Fig. 1B). Citron-K is detected by
Western blots in cultured Hep3B cells, embryonic day 15 (E15) rat
liver, and cultured proliferating rat hepatocytes but not in quiescent
adult rat hepatocytes (Fig. 1, C and D). These
results strongly argue that Citron-K expression is cell
cycle-dependent rather than tissue-specific.

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Fig. 1.
Expression of Citron kinase is not
tissue-specific but cell cycle-dependent.
A, RT-PCR showed expression of Citron-K mRNA in human
fetal tissues. Lane 1, thymus; lane 2,
kidney; lane 3, skeletal muscle; lane
4, lung; lane 5, spleen;
lane 6, heart; and lane 7,
liver. B, in situ hybridization (ISH)
detected a positive signal of Citron-K mRNA in embryonic mouse
liver. H, heart; L, liver; H&E,
hematoxilin-eosin. C, Western blots detected Citron-K
protein expression in embryonic day 15 rat liver. Lane
1, adult rat cerebellum; lane 2,
embryonic day 15 rat liver. D, Western blots detected
Citron-K protein expression in the cultured Hep3B cell line and in
cultured proliferating rat hepatocytes. Lane 1,
adult rat cerebellum; lane 2, cultured Hep3B cell line;
lane 3, freshly isolated hepatocytes from
2-month-old rat; lane 4, hepatocytes in culture
for 72 h under the stimulation of insulin and epidermal growth
factor.
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The expression pattern of Citron-K was further characterized in
regenerating liver after two-thirds PH because the first round of DNA
synthesis by hepatocytes is naturally synchronized, providing a
convenient model for studying the cell cycle expression pattern of
proteins. Rat hepatocytes reach first peak of DNA synthesis at 24 h after PH as revealed by BrdUrd labeling and PCNA expression (27-30).
Thus, we followed the protein expression ~24 h after surgery. Previous studies have demonstrated that cell proliferation induced by
PH is predominantly non-binucleating (31, 32). Thus, the expression of
Citron-K in the regenerative liver after PH will provide important
information as to the relationship between Citron-K and liver cell cytokinesis.
The expression of two well studied cell cycle markers was examined to
define stages of the cell cycle. PCNA starts to appear at the end of
G1, peaks in S, and declines at the G2/M phases (33, 34). It has been shown that the protein level of PCNA parallels
DNA synthesis as revealed by BrdUrd or [3H]thymidine
incorporation in the regenerating liver after PH (27, 30). Cyclin B1 is
a critical regulator of mitosis. Expression of cyclin B1 starts at late
S phase and accumulates until metaphase (35). After that, it is
degraded quickly by a ubiquitin-dependent mechanism.
The protein level of Citron-K was detected using immunoprecipitation
followed by Western blotting. The protein level of PCNA and cyclin B1
were analyzed using Western blots directly. PCNA protein level reached
its first peak at 24 h after PH, a finding that is consistent with
the literature. Citron-K was detected as early as 26 h after PH
when cyclin B1 reached its first peak (Fig.
2B). These protein expression
changes were not observed in the sham-control animals (data not
shown).

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Fig. 2.
Expression of PCNA, cyclin B1, and Citron-K
proteins in regenerating liver after two-thirds partial
hepatectomy. Expression of cyclin B1 and PCNA protein was analyzed
by Western blotting. Total protein (50 µg) was loaded from each
samples. Immunoprecipitation followed by Western blots was used to
detect Citron-K. Total protein (3 mg) of each sample was used to pull
down Citron-K. Liver tissue removed during the surgery was saved as the
time zero (T0) control. T20-30
represented 20-30 h after PH. Shown is a representative result of
three independent experiments.
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The data presented here demonstrate that expression of Citron-K is cell
cycle-dependent, not tissue specific. Its expression starts
after that of cyclin B1 in the mitotic cell cycle, probably at late S
and/or early G2 phase.
Cellular Localization of Citron Kinase--
Comparing the
immunofluorescence signals from WT and FH hepatocytes permitted the
identification of the specific signal derived from Citron-K. An evenly
distributed positive nuclear signal was detected in a small subset of
interphase nuclei in WT hepatocytes but not FH cells (Fig.
3A). A positive nuclear signal
was also detected in cultured Hep3B cells, and this was further
confirmed by XZ scanning with a confocal microscope (Fig.
4A). An examination of
transfected FLAG-tagged Citron-K in Hep3B cells showed that overexpressed exogeneous Citron-K presented itself as puncta in interphase cells, and some puncta were localized in the nuclei as
confirmed by confocal microscopy (Fig. 4B). Double
staining with an antibody against the phosphorylated histone H3 at
serine 10 allowed the recognition of mitotic cells. Citron-K began to disperse into cytoplasm at prophase (Fig.
5, A and B) and was distributed to the whole cytosol during prometaphase (Fig.
5C), metaphase (Fig. 3B), and early anaphase
(Fig. 3A). It moved to the cleavage furrow during anaphase
(Figs. 3B and 6A) and concentrated at midbody
during telophase and cytokinesis (Fig.
6B), which is consistent with
previous reports on HeLa cells (2, 9). A punctate signal was detected
in some interphase and mitotic cells (Figs. 3B and 6,
C and D). Double staining with anti-
-tubulin antibody showed that the intracellular puncta resembled the signal from
the midbody residue after cytokinesis (Fig. 6, C and
D). The intracellular puncta could be detected in any part
of the cytosol, including cleavage furrow. In most cases, only 1-3
puncta existed in a single hepatocyte.

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Fig. 3.
Cellular localization of Citron-K. The
merged image is a double staining of Citron-K (green) and
DNA (red). A, cultured rat hepatocytes.
B, cultured Hep3B cells.
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Fig. 4.
Confocal microscopy to confirm the nuclear
localization of Citron-K. A, endogenous Citron-K
localizes in a small subset of interphase Hep3B cells.
Green, Citron-K; red, DNA. B,
exogenous Citron-K exists in the nuclei of Hep3B cells transfected with
FLAG-Citron-K plasmid. Green, FLAG-Citron-K; red,
DNA.
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Fig. 5.
Cellular localization of Citron-K in cultured
rat hepatocytes during prophase and prometaphase. A,
Citron-K began to move to cytoplasm at early prophase. B,
Citron-K localized to both nucleus and cytosol during prophase,
C, Citron-K dispersed to the whole cytosol during
prometaphase. The merged image is a double staining of Citron-K
(green) and phospho-histone H3
(Phospho-H3) at Ser-10 (red).
Bars, 5 µm.
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Fig. 6.
Cellular localization of Citron-K in cultured
rat hepatocytes. The merged image is a triple staining of
-tubulin (green), Citron-K (red), and DNA
(blue). A, Citron-K localizes to cleavage furrow
during anaphase. B, Citron-K localizes to midbody during
telophase and cytokinesis. C, Citron-K presents itself as
puncta in some interphase and mitotic cells. D, Citron-K
puncta exist at midbody at the end of cytokinesis and in some
interphase cells.
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Expression of Citron Kinase Declines Gradually during
Development--
The mRNA level of Citron-K was detected with
RT-PCR and further quantified with
-actin serving as an internal
standard. The mRNA level of Citron-K was detected in embryonic day
14 (E14) rat livers and increased to its highest level in E15 and E16
rat livers. Its expression gradually declined after birth and was beneath the detection limit of RT-PCR after the third postnatal week
(Fig. 7A).

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Fig. 7.
Citron kinase expression decreases gradually
during development in rat liver. A, relative RT-PCR
confirms the quantitative changes of Citron-K mRNA during rat liver
development. Semi-quantitative analysis of the RT-PCR products was
achieved by using -actin as an internal standard.
E14-16, embryonic days 14-16. P1-60,
postnatal days 1-60. B, Citron-K protein level gradually
decreased during development as revealed by Western blots.
E15, embryonic day 15; P1-22, postnatal days
1-22. Total protein loaded in E15 samples (50 µg) was only half of
that after birth (100 µg). C, immunoprecipitation followed
by Western blotting to show the low levels of both Citron-K and
Citron-N in the liver from P15-P35 day-old rats. Total protein used for
immunoprecipitation was 6 mg. P15-35, postnatal days 1-35.
Shown is a representative result of three independent experiments.
D, densitometric analysis to show the relative level of
Citron-K protein expression during development in rat liver. Each stage
consisted of 4-10 samples.
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Protein expression of Citron-K showed a similar pattern to that of its
mRNA. The level of Citron-K protein was highest in E15 rat liver;
it decreased after birth and stayed stable for the first week, then it
decreased again and stayed at that level until weaning, when a third
decrease happened. After that, Citron-K was no longer detected by
Western blots (Fig. 7B). In addition, expression of Citron
was also detected in rat liver and mirrored that of Citron-K with a
gradually decreasing pattern during the first 4 weeks of postnatal
life. A very low and constant level of both Citron and Citron-K protein
was detected in rat livers at weeks 4 and 5 with immunoprecipitation
followed by Western blots (Fig. 7C). Densitometric analyses
of the Western blots indicated that Citron-K protein levels in E15 rat
livers were as much as 8.25-fold higher than those observed in the
livers in the first postnatal week (Fig. 7D). In addition,
by the time of weaning Citron-K had declined to a level that was only
5% of that in the first postnatal week. Finally, the ratio of Citron-K
versus Citron decreased from four at E15 to one after
weaning. In summary, both mRNA and protein expression of Citron-K
decrease gradually after birth, reaching the lowest level after weaning.
Citron-K Knockout Mice and Rats (Flathead
Rats)--
The relevance of Citron-K to the cell cycle and cytokinesis
control in hepatocytes was examined by comparing the nuclear pattern and ploidy level of hepatocytes in FH rats with that in WT littermates. Based on the developmental expression pattern of Citron-K, neonates were examined in their first postnatal week when Citron-K is still expressed at a relatively high level. This stage also provides a better
match of the body weight of the animals because FH rats grow slower
than their littermates, and the differences in body weight and body
size become more apparent after 1 week. Finally, interference from the
natural binucleating process of hepatocytes is reduced to a minimal
level at this age of the animals. Western blotting confirmed that no
Citron-K was detected in either the freshly isolated or the cultured
proliferating hepatocytes from FH rats (Fig.
8A). Isolated hepatocytes were
cultured and checked under phase contrast microscope after 24 h.
Cells from WT and FH rats presented a similar morphology in culture
(data not shown). A low percentage of binucleated hepatocytes do exist
in both WT and FH animals but with no significant difference (2.8 ± 0.35% for FH and 2.6 ± 0.53% for WT, n = 4, p > 0.05) (Fig. 8B). These results suggest
that Citron-K is not essential to the cytokinesis of rat hepatocytes
and that some functionally redundant molecules exist in these cells.
Flow cytometric analysis of hepatocyte nuclei from one-week-old rats
revealed a significant 50% increase of the G2 tetraploid
nuclei in FH rat livers over controls (5.0 ± 0.22% for FH
versus 3.3 ± 0.28% for WT, n = 5, p < 0.05) (Fig. 8C). There were no
significant differences among the diploid G0/G1 nuclei percentages between FH and WT animals (WT 93.3 ± 0.44% versus FH 91.6 ± 0.53%, n = 5, p > 0.05) and those in S phase (WT 3.4 ± 0.20%
versus FH 3.4 ± 0.34%, n = 5, p > 0.05). Identical results have been obtained with
Citron-K
/
mice (data not shown). These data indicate that Citron-K
influences the G2/M phase transition of the cell cycle in
hepatocytes. Moreover, increased apoptosis was detected in embryonic
livers of the Citron-K
/
mice. Activated caspase 3 immunostaining,
a marker for apoptotic cells, was obvious in the liver of E14.5
Citron-K
/
mice but not WT animals (Fig. 8D), suggesting
that Citron-K is essential to some embryonic liver cells.

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Fig. 8.
Analysis of the liver from
Citron-K knockouts. A, Citron-K protein is not detected
in both freshly isolated and cultured hepatocytes of FH rats by Western
blotting. Total protein loaded for each sample was 50 µg.
B, binucleation ratio of hepatocytes isolated from Citron-K
/ and +/+ rats. Binucleated cells were counted, and binucleation
percentage was calculated as 2.8 ± 0.35% for FH and 2.6 ± 0.53% for WT, n = 4, p > 0.05. C, flow cytometrical analysis of hepatocyte nuclear ploidy
from WT and FH rats during the first week after birth. There was no
significant difference between the WT and FH group of diploid
G0/G1 (WT 93.3 ± 0.44% versus
FH 91.6 ± 0.53%) and S phase (WT 3.4 ± 0.20%
versus FH 3.4 ± 0.34%) nuclei, but there is a 50%
increase of G2 population (5.0 ± 0.22% for FH
versus 3.3 ± 0.28% for WT) (n = 5, *p < 0.05 for G2). D, increased
apoptosis in embryonic liver of Citron-K / mice. Sections of the
E14.5 liver of Citron-K +/+ and / mice were analyzed by
anti-activated caspase-3 immunohistochemistry to detect apoptotic
cells. KO, knockout. Bar, 200 µm.
Arrow points to activated caspase-3 staining. E,
Protein levels of ROCK I and II in both freshly isolated and cultured
hepatocytes from 1-week old WT and FH rats examined by Western blots.
An equal amount of 50 µg of total protein was loaded for each
sample.
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The structural and functional similarity between ROCKs and Citron-K
make ROCKs the possible candidates to compensate the lost function of
Citron-K. Protein levels of ROCKs in FH rats and the inhibition of ROCK
function in Citron-K knockout cells might reveal whether the
dispensability of Citron-K is due to the up-regulation and/or existence
of ROCKs. Western blotting detected similar protein levels of ROCK I
and ROCK II in both freshly isolated and cultured hepatocytes from WT
and FH rats (Fig. 8E). Thus, the expression of ROCKs was not
up-regulated in the Citron-K deficient situation. The requirement of
Citron-K and ROCKs for hepatocyte cytokinesis was further investigated
with the application of the specific inhibitor of ROCKs, Y-27632, to
the primary culture of isolated hepatocytes from both WT and FH rats.
The efficacy of Y-27632 was confirmed by blocking the formation of
stress fibers in NIH 3T3 cells at 10 µM after 1 h
and after 24 h of treatment (data not shown). Adding Y-27632 at
four different concentrations (10, 25, 50 and 100 µM) to
the cultured cells did not show easily detectable changes in
cytokinesis in both WT and FH cells (data not shown).
 |
DISCUSSION |
This study demonstrates that Citron-K is the first known Rho-GTP
downstream target with a cell cycle-dependent expression pattern and has a nuclear localization at interphase. In addition to a
possible involvement in cytokinesis control, it is required for the
G2/M transition of hepatocytes.
Citron-K Has a Distinct Cell Cycle-dependent Expression
Pattern and Cellular Localization--
The expression of Citron-K is
not tissue-specific but cell cycle-dependent. This result
extends the previous observations on the central nervous system,
i.e. that mRNA of Citron-K is detected only in
proliferating but not postmitotic neuronal cells (15). It also explains
that the absence of Citron-K mRNA in adult liver, and presumably
also skeletal muscle and heart, is due to the quiescent state in these
organs. The developmental expression data further confirms the cell
cycle-dependent expression pattern of Citron-K, as it was
shown previously that the percentage of cycling cells is quickly
reduced in rat liver after birth (36). On the other hand, the decline
of both Citron-K mRNA and protein after weaning is inversely
correlated with the postweaning binucleation of hepatocytes. Considering the function of Citron-K in the control of cytokinesis, this observation might provide some indirect evidence to support the
long believed hypothesis that acytokinesis is the basis of hepatocyte binucleation.
The small GTPases of the Rho family members are critical regulators in
many important cellular functions. At present, Citron-K is the only
known downstream target of Rho that presents a cell cycle-dependent expression pattern, which makes it
extremely possible that Citron-K might act to switch Rho from its other
cellular functions to its role in cytokinesis. In support of
this idea, a reduced RhoA signal at the cleavage furrow was detected in
mitotic neuronal cells isolated from Flathead rats, suggesting that
localization of RhoA to the cleavage furrow is partly
Citron-K-dependent (20).
The finding of increased G2 nuclei in the liver of the
Citron-K knockouts implies that Citron-K functions in the
G2/M transition, which is consistent with observations on
the central nervous system in Citron-K knockout mice (15). The small
difference in the numbers of G2 nuclei would be more
meaningful considering that there is only a small percentage of cycling
cells, whereas the majority of the diploid nuclei are actually in the
quiescent G0 state.
The immunofluorescence antibody staining shows that Citron-K is a
nuclear protein in a small percentage of interphase cells. Confocal
microscopy confirms that both endogenous Citron-K and some exogenous
FLAG-tagged Citron-K proteins localize in the nuclei. The fact that
overexpressed FLAG-tagged Citron-K exists in the cytosol and the
nucleus suggested that the nuclear import of Citron-K requires a
specific condition. By searching the Prosite data base, a potential
bipartite nuclear targeting sequence has been found in the Citron-K
protein (39). Double staining with the anti-phospho-histone H3 Ser-10
antibody shows that cells at later G2 and prophase have a
nuclear positive signal for Citron-K, which is consistent with our
Western blot data on the expression pattern of Citron-K. The nuclear
localization of Citron-K may provide useful information for
understanding the mechanism of Citron-K in the control of the
G2/M transition. Thus, Citron-K is a downstream target of Rho with a nuclear localization. Because Rho is a cytoplasmic protein,
the function of Citron-K in the nucleus may not be
Rho-dependent. An interesting note to mention is that mouse
Ect2/Drosophila pebble, an upstream stimulator of Rho, also has a cell
cycle-dependent expression pattern, a nuclear localization
at interphase, and a function in cytokinesis regulation (37).
An additional observation in our study is that Citron-K still diffuses
to the whole cytosol at the beginning of anaphase, right after the
sister chromatids dissociate from each other. Citron-K then quickly
moves to the cortex of the cleavage furrow, a time point we speculate
to be the initiation of cytokinesis. Whether this transition is a
precondition or a consequence of the formation of the cleavage furrow,
it suggests that Citron-K is probably involved in the earliest step of
cytokinesis, even though a truncated Citron-K construct only interfered
with the final stages of cytokinesis in transfected HeLa cells (2).
In a previous paper it was argued that Citron-K existed as puncta in
interphase cells (9). These punctas dispersed into the whole cytosol
during prometaphase in order to conduct their function during
cytokinesis. We question this interpretation for several reasons. These
punctate signals resemble the midbody residue after cytokinesis. In
most situations, only 1-3 punctas were observed in a single
hepatocyte, whereas a similar number of puncta were observed in both
interphase and mitotic cells. According to our results, Citron-K is a
cell cycle-dependent protein expressed after cyclin B1.
However, no obvious pattern was found between the number and size of
the puncta with the cell cycle progress. In addition, it is hard to
believe that the Citron-K protein that exists in this state could be
functionally active in controlling the G2/M transition. As
reported previously, abscission occurred at the intercellular bridge on
one side of the midbody, and the midbody residue was then left in the
other side's daughter cell after cytokinesis (38). We would propose
that some if not all punctas are more likely the residue of the midbody
left in one daughter cell.
Citron-K Regulates Cytokinesis in Hepatocytes--
The importance
of Citron-K in the control of cytokinesis in hepatocytes is implicated
by several observations. Citron-K localizes to the cleavage furrow and
midbody during cytokinesis. The expression of Citron-K is highly
up-regulated in proliferating dividing hepatocytes both in
vivo and in vitro. In contrast, its expression is
down-regulated during the postweaning binucleation of hepatocytes, a
process believed to be caused by acytokinesis. The knockout studies
indicate that Citron-K is not essential for cytokinesis in postnatal
hepatocytes. Several possibilities should be considered. There might be
unidentified functionally redundant molecules in these cells,
considering the importance of cytokinesis. The control mechanism of
cytokinesis could be different in varied cell types, and Citron-K might
regulate a dispensable step of cytokinesis in hepatocytes. There could be distinct isoforms of Citron-K that are functional in embryonic versus adult developmental stages. Thus, the susceptibility
to apoptotic death could be different in embryonic versus
postnatal cells as observed in the central nervous system and in the
testis (15, 19, 22). The knockout shows only a low penetrance
cytokinesis phenotype due to the elimination of the binucleated
hepatocytes by apoptosis during embryonic stages as suggested by
increased activated caspase-3 staining in the fetal liver of Citron-K
/
mice.
ROCKs, the homologues of Citron-K and also the downstream targets of
Rho-GTP involved in the regulation of cytokinesis, were the most
promising functionally redundant alternatives. Both Citron-K and ROCKs
phosphorylate the RMLC during cytokinesis (12, 16). However, the
absence of Citron-K did not result in up-regulation of ROCKs in the
neural cells (15) or the liver cells. In the Citron-K-sensitive
neuronal cells, ROCKs do not cover the lost function caused by
deficiency of Citron-K (15). Furthermore, our results show that
inhibition of the kinase activities of ROCKs by its specific inhibitor
Y-27632 does not block cytokinesis in cultured Citron-K
/
hepatocytes. This confirms that the lost function of Citron-K is not
compensated by the kinase activity of ROCKs. In addition, even if RMLC
is a substrate of both Citron-K and ROCKs during cytokinesis, it must
be activated by at least one other kinase such as the myosin light
chain kinase in these cells.