1 Department of Molecular and Cellular Biology, Baylor College of Medicine, One
Baylor Plaza, Houston, Texas 77030, USA
2 Department of Biological Sciences, The University of Alabama, Tuscaloosa,
Alabama 35487, USA
3 Department of Molecular Pathology, MD Anderson Cancer Center, Houston, Texas
77030, USA
4 Program in Cell and Molecular Biology, Baylor College of Medicine, One Baylor
Plaza, Houston, Texas 77030, USA
5 Department of Immunology, Baylor College of Medicine, One Baylor Plaza,
Houston, Texas 77030, USA
6 Department of Medicine, Baylor College of Medicine, One Baylor Plaza, Houston,
Texas 77030, USA
* Author for correspondence (e-mail: yulee{at}bcm.tmc.edu)
Accepted 5 February 2003
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Summary |
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Key words: NudC, Plk1, Cytokinesis, Midbody microtubule, Small interfering RNA, Caenorhabditis elegans
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Introduction |
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We originally cloned mammalian NudC as an early mitogen-responsive
gene in T cells and showed that, in synchronized cultures, NudC mRNA
levels increase during mid-G1 and NudC protein levels double during G2/M
(Axtell et al., 1995). NudC
expression is elevated in cells with a high growth rate
(Gocke et al., 2000a
) and in
prostate tumors derived from transgenic mouse models
(Aumais et al., 2000
). Others
have shown that NudC is highly expressed in clinical bone marrow isolates from
patients with acute lymphoblastic or acute myelogenous leukemia
(Miller et al., 1999
;
Gocke et al., 2000b
). These
studies show that NudC levels correlate with the proliferative status of
various cell types, tissues and tumors. To address a role of NudC in mitosis,
one study reduced NudC mRNA levels with an inducible-ribozyme strategy and
showed an increase in the number of cells exhibiting multipolar spindles
(Zhang et al., 2002
). These
results suggest that NudC plays a role in spindle formation during mitosis but
the mechanism involved is unknown. Dawe et al.
(Dawe et al., 2001
) used
Caenorhabditis elegans to silence nud-1, the nematode
NudC ortholog, and showed that this led to embryonic lethality with
defects in centrosome-pronuclear rotation at the time of the first embryonic
cell division. In further support of a mitotic role for NudC, we recently
demonstrated that NudC is phosphorylated at the onset of mitosis, and that
NudC is both an in vitro and in vivo substrate for the mitotic polo-like
kinase Plk1 (T. H. Zhou, J. P. Aumais, X. Liu, L.-y. Yu-Lee and R. L. Erikson,
unpublished). Plk1 phosphorylates NudC in vitro on two serine residues, S274
and S326, in the highly conserved C terminus, and phosphorylation of NudC on
these two sites in vivo appears to influence cytokinesis. How phosphorylated
NudC mediates this effect is unclear.
To investigate further the role of NudC in mitosis and cytokinesis in
mammalian cells, we altered NudC levels either by small interfering RNA
(siRNA) silencing (Elbashir et al.,
2001) to reduce endogenous levels of NudC, or adenovirus-mediated
gene transfer to overexpress NudC ectopically. Here, we report that altering
NudC levels inhibited cell proliferation and led to an increase in the
proportion of multinucleated cells. Complementary studies using time-lapse
video microscopy on C. elegans embryos extended our findings by
demonstrating that cytokinetic furrows regressed when the nud-1 gene
was silenced by RNA-mediated interference (RNAi). In both NudC altered
mammalian cells and C. elegans embryos, a remarkably similar and
consistent phenotype was the loss of midzone microtubule organization, an
event that probably contributes to the observed failure in cytokinesis.
Further, altering NudC levels resulted in the mislocalization of Plk1 from
various mitotic structures, including the midbody during cytokinesis. These
results show that a proper level of NudC is crucial for mitotic progression
and completion of cytokinesis, and further suggest that NudC might function by
regulating the stability of the cytokinetic furrow and microtubule
organization during cytokinesis.
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Materials and Methods |
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Cell culture and transfection
HeLa cells were grown in Dulbecco's modified Eagle Medium (DMEM, Gibco)
supplemented with 5% fetal bovine serum (FBS, Atlanta Biologicals), 100 U
ml1 of penicillin/streptomycin (Gibco), 50 µg
ml1 Gentamycin (Sigma) and 1% (v/v) L-glutamine (Gibco).
Cells were plated 24 hours prior to transfection in 12-well plates at a
density of 25,000-30,000 cells per well. For immunocytochemical studies, cells
were plated on poly-D-lysine-coated coverslips (Fisher). For each
transfection, 200 pmol (10 µl) of the appropriate siRNA was diluted in 80
µl Opti-MEM I (Gibco). In a separate tube, 3 µl Oligofectamine
(Invitrogen) was diluted in 7 µl Opti-MEM I and incubated at room
temperature for 5 minutes. The diluted Oligofectamine was mixed gently with
the diluted siRNA and incubated at room temperature for 20 minutes. RNA
complexes were added to cells with 400 µl serum-free Opti-MEM I and
incubated for 4 hours at 37°C, 5% CO2, after which time FBS was
added to the transfections to a final concentration of 4% (v/v) and a final
volume of 0.75 ml.
Immunoblotting
Cells were harvested by scraping in 200 µl TEN (20 mM Tris, pH 7.4, 100
mM NaCl, 5 mM EDTA and 0.5% Triton X-100) supplemented with 1 mM
phenylmethylsulfonyl fluoride (PMSF) and protease-inhibitor cocktail (both
from Sigma), and subjected to two cycles of freeze-thaw. Cell lysates were
clarified by centrifugation. Protein concentrations were determined using
Bradford reagent (Bio-Rad, Hercules, CA), and 5-10 µg total protein were
resolved by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad).
Membranes were immunoblotted with affinity-purified rabbit anti-rat NudC
C-terminus peptide (C peptide) antibodies or rabbit anti-rat MBP-NudC fusion
protein antibodies (Morris and Yu-Lee,
1998) as indicated, anti-Plk1 monoclonal antibodies (Zymed), or
mouse anti-ß-actin monoclonal antibodies (AC-40, Sigma). Immunoblots were
developed by enhanced chemiluminescence.
Immunocytochemistry
After 24-96 hours, cells were fixed for 20 minutes at room temperature in
PEM (80 mM K-PIPES, 5 mM EGTA, 2 mM MgCl2) with 4% (w/v)
polyethylene glycol-6000 (PEG), 4% (v/v) ultrapure electron-microscopy-grade
formaldehyde (Electron Microscopy Sciences, Ft Washington, PA) and 0.5% Triton
X-100. To localize NudC to microtubule structures, cells were briefly treated
with 0.5% Trion-X-100 in PEM/PEG for 1 minute at room temperature to remove
soluble NudC and then fixed as described above. Cells were then blocked in PBS
with 2% (w/v) bovine serum albumin (BSA) and 2% (v/v) normal goat serum
(Sigma) and incubated with 0.5 µg ml1 anti-NudC C
peptide, anti-ß-tubulin (tub2.1, Sigma) or anti-Plk1 antibodies followed
by incubation with Texas-red goat-anti-rabbit IgG (Molecular Probes) or
FITC-conjugated goat-anti-mouse IgG (Southern Biotechnologies, Birmingham,
AL). Slides were mounted in Vectashield mounting medium (Vector Labs,
Burlingame, CA) containing 0.2 mg ml1 4',
6-diamidino-2-phenylindole (DAPI). Images were acquired using a RT Color Spot
digital camera (Diagnostic Instruments) mounted on a Leica DME microscope with
red, green or blue emission filters. Figures were compiled using Spot Software
(version 3.0) and Photoshop (version 5.5; Adobe, Mountainview, CA).
Growth curves
Untransfected HeLa cells or HeLa cells transfected with 200 pmol
NudC or luciferase (Luc) siRNA were harvested at 24 hour
intervals in 100 µl Trypsin/EDTA (Gibco) and resuspended in 400 µl
growth medium. Cells were pipetted repeatedly to dislodge cell clumps and were
counted in quadruplicates with a HY Lite Neubauer hemacytometer (Hausser
Scientific) with Trypan blue exclusion. Values are presented as (cell number
x104) ml1. HeLa cells transduced with
recombinant adenovirus containing either the NudC or the Luc
gene were also counted in a similar manner.
Recombinant adenovirus constructions and transduction
Full-length NudC was inserted into the adenovirus (Ad) shuttle
vector pXCMV at the HindIII/NotI sites to generate
pXCMV-NudC. The recombinant adenovirus Ad-NudC was generated by
co-transfecting pXCMV-NudC and pJM17, which contains the adenoviral genome
with the E1 gene deleted, into 293 cells as previously described
(Estrera et al., 2001).
Recombinant virus (Ad-NudC or Ad-Luc) was transduced into HeLa cells at an
M.O.I. of 1. After 24 hours, the growth medium was changed and the cells were
cultured for 96 hours.
C. elegans culturing methods
Wild-type C. elegans strain N2 (Bristol variety) was cultured
under standard conditions (Brenner,
1974). To perform RNAi studies, the full-length cDNA of
nud-1 (F53A2.4), the C. elegans ortholog of nudC,
was cloned into the vector L4440 (Timmons
et al., 2001
) and transformed into HT115 (DE3). Transformed
bacteria were grown for 14 hours at 37°C in 2 ml LB supplemented with 12.5
µg ml1 tetracycline and 50 µg ml1
ampicillin. These cultures were plated onto standard worm plates containing
0.8 mM IPTG and 50 µg ml1 carbenicillin and allowed to
grow overnight at room temperature. Dauer N2 larvae were then placed onto
these plates and their offspring examined after 48 hours at 26°C
(Kamath et al., 2000
). Embryos
analysed by time-lapse video microscopy were dissected in M9 medium from N2
worms treated with nud-1 dsRNA and placed on a 2% agarose pad with a
coverslip mounted on top. Early development was typically analyzed in
wild-type and RNAi-treated embryos from shortly after fertilization through
two rounds of cell division (
40 minutes).
Immunostaining of C. elegans embryos
Immunostaining of embryos was performed as previously described
(Guo and Kemphues, 1995).
Monoclonal anti-
-tubulin antibody (T9026, Sigma) was used at 1:400 and
visualized using goat-anti-mouse AlexaFluor488 (Molecular Probes) at 1:800
dilution. DNA was visualized by staining with DAPI. Embryos were analysed
using a Nikon Eclipse E800 epifluorescence microscope equipped with DIC optics
and Endow GFP HYQ and UV-2E/C DAPI filter cubes (Chroma). Images were captured
with a Spot RT CCD camera (Diagnostic Instruments). MetaMorph Software
(Universal Imaging) was used for pseuodocoloration of images and image
overlays. Images were processed with Adobe Photoshop 7.0.
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Results |
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|
NudC downregulation by siRNA inhibits cell
proliferation
To determine the role of NudC during cellular replication in mammalian
cells, we downregulated endogenous NudC by RNAi
(Elbashir et al., 2001) and
examined the effects of NudC gene silencing on NudC protein levels
and cell growth. HeLa cells were transfected with either NudC siRNA
or a firefly luciferase Luc siRNA, which served as a control against
nonspecific gene silencing. Total cell lysates were prepared 24 hours, 48
hours, 72 hours and 96 hours post transfection and immunoblotted with
anti-NudC antibodies. Cells transfected with NudC siRNA showed a
reproducible and significant (80-90%) reduction in the amount of steady-state
42 kDa NudC protein 48 hours after transfection
(Fig. 2A, lane 3). The
reduction in NudC levels was sustained for up to 96 hours after transfection
(Fig. 2A, lanes 4-5). By
contrast, NudC levels in cells transfected with Luc siRNA were
unaffected over 96 hours of incubation
(Fig. 2A, lanes 6-9) and
remained comparable to those found in the untransfected control cells
(Fig. 2A, lane 1). Total
lysates were also blotted with an anti-ß-actin antibody to show equal
loading of proteins (Fig. 2A,
lower panel). Several doses of NudC siRNA were applied over a 3-day
period to determine whether repeated applications of the siRNA duplex could
enhance NudC gene silencing. Repeated dosing with NudC siRNA
was no more efficient in depleting NudC levels than a single initial
application (data not shown). Therefore, the regimen for subsequent
experiments was a single dose of NudC siRNA transfection followed by
a 72-hour incubation. NudC siRNA transfected cells showed reduced
proliferation after 72-96 hours post transfection when compared with
Luc-siRNA-treated or untransfected control cells
(Fig. 2B). These results
support the idea that NudC plays a role in mitosis and cell proliferation.
|
NudC depletion induces multinucleation and results in a failure in
cytokinesis
To investigate the basis of the growth inhibition of NudC-depleted cells,
HeLa cells were transfected with either Luc siRNA or NudC
siRNA for 72 hours and examined by immunofluorescence microscopy.
siRNA-mediated NudC gene silencing was observed in over 90% of cells
as determined by a lack of NudC staining (red)
(Fig. 3B), which agrees with
reduced NudC protein expression by western-blot analysis
(Fig. 2A). In control cells,
NudC staining was generally observed in all the cells, with higher levels in
mitotic cells (Fig. 3A, arrow),
in agreement with previous studies showing that NudC protein levels double
during mitosis (Morris and Yu-Lee,
1998; Aumais et al.,
2000
). Downregulation of NudC by siRNA led to several
morphological phenotypes. MTOC structures were not distinct in
NudC-siRNA-treated cells, in contrast to control cells, which showed
MTOCs located characteristically on one side of the nucleus of interphase
cells (Fig. 3A, asterisk). A
significant (sixfold) increase in the number of large, flat cells containing
multiple nuclei (Figs 3B, arrows) was observed in NudC-siRNA-treated cells (12%) compared with
Luc-siRNA-treated controls (2%)
(Fig. 3C). A 60% increase in
the number of cells connected to each other by cytoplasmic bridges was found
in NudC-siRNA-compared with Luc-siRNA-treated cells
(Fig. 3B, arrowheads),
suggesting a delay or inhibition in cleavage, cell separation or
cytokinesis.
|
To investigate further the basis of multinucleation, we examined more closely the phenotypes of NudC-downregulated cells during M-phase progression. During prometaphase, spindles appeared to be less focused in NudC-siRNA-treated cells (Fig. 4A,E). Misaligned chromosomes were seen during metaphase in NudC-siRNA-treated cells (Fig. 4F, arrow and inset). Furthermore, striking abnormalities in the midzone/midbody structure at anaphase/telophase and cytokinesis were observed. Midzone/midbody microtubules were either missing or poorly organized (Fig. 4G-I, arrowheads) compared with control cells (Fig. 4C,D, arrowheads) as revealed by ß-tubulin staining (green). Many cells had divided and re-entered interphase, as judged by the decondensed DNA, but they remained connected to each other by either thick (Fig. 4J) or very long and thin (Fig. 4K) intercellular cytoplasmic bridges. These cells failed to separate, resulting in a cell structure containing two or more nuclei of abnormal size and shape (Fig. 4J). These results suggest that multinucleation in NudC-downregulated cells might be due to incomplete cytokinesis.
|
NudC downregulation results in mislocalization of the
mitotic kinase Plk1
To determine the consequence of a lack of microtubule organization at the
midzone/midbody in NudC-depleted cells, we examined the presence of proteins
that are known to localize in the midzone/midbody matrix and to play a role in
cytokinesis (Field et al.,
1999). One such factor is the mitotic kinase polo-like kinase
(Plk1) (Glover et al., 1998
),
which we have recently shown to interact with and phosphorylate NudC during
mitosis (T. H. Zhou, J. P. Aumais, X. Liu, L.-y. Yu-Lee and R. L. Erikson,
unpublished). We determined the effect of NudC downregulation on the
level and subcellular distribution of Plk1. HeLa cells were transfected with
either Luc or NudC siRNA for 72 hours and the cell lysates
were immunoblotted with anti-NudC, anti-Plk1 or anti-ß-actin antibodies.
In NudC-siRNA-treated cells, NudC levels were downregulated
(Fig. 5A, top), as also
observed in Fig. 2A, but Plk1
levels remained essentially unchanged (Fig.
5A, middle). ß-Actin was used as a loading control
(Fig. 5A, bottom).
|
We next examined Plk1 localization during M phase in control and
NudC-downregulated cells. Plk1 staining was clearly localized to the
centrosomes (Fig. 5Ba,c) and
midbody (Fig. 5Be) in control
cells, consistent with the multiple well-described roles for Plk1 during
M-phase progression (Glover et al.,
1998; Donaldson et al.,
2001
). By contrast, Plk1 was mislocalized in 40-45% of cells
progressing through various stages of M phase in the
NudC-downregulated cells. In NudC-siRNA-transfected cells,
Plk1 staining was diffuse and not localized to the centrosomes
(Fig. 5Bg,i) or midzone
(Fig. 5Bk). Lagging chromosomes
were observed in the midzone region in NudC-downregulated cells
(Fig. 5Bl, arrow), suggesting a
defect in DNA segregation. These results show for the first time that, in
NudC-downregulated cells, the mitotic kinase Plk1 is not properly
localized to the mitotic structures in dividing mammalian cells.
NudC overexpression by adenovirus-NudC transduction inhibited cell
proliferation
We next determined the effect of overexpressing NudC on cell proliferation.
Recombinant adenovirus containing the NudC gene (Ad-NudC) was used
for high efficiency expression of NudC in mammalian cells. Recombinant
adenovirus containing the firefly luciferase gene (Ad-Luc) was used as a
control. At 24 hours, 48 hours, 72 hours and 96 hours post transduction, total
cell lysates were immunoblotted with anti-NudC antibodies. Cells transduced
with Ad-NudC showed a reproducible increase in NudC protein levels 24-96 hours
post transduction (Fig. 6A,
lanes 5-8, top). By contrast, NudC levels remained unchanged in cells
transduced with the Ad-Luc control (Fig.
6A, lanes 1-4). Reblotting with anti-ß-actin antibodies
showed equal loading of proteins (Fig.
6A, bottom). Ad-NudC-transduced HeLa cells grew at the same rate
as the Ad-Luc-transduced control cells for the first 48 hours
(Fig. 6B). However, by 72-96
hours post transduction, overexpression of NudC led to an inhibition of cell
proliferation in the Ad-NudC-transduced cells as compared with the control
cells (Fig. 6B). These results
show that overexpression of NudC also led to an inhibition of cell
proliferation in mammalian cells and is not the result of viral
transduction.
|
NudC overexpression induces multinucleation and a failure in
cytokinesis
To determine the basis of Ad-NudC mediated growth inhibition, HeLa cells
were transduced with Ad-NudC or Ad-Luc for 96 hours and examined by
immunofluorescence microscopy. Overexpression of NudC led to an increase in
multinucleated cells as early as 24 hours after Ad-NudC transduction
(Fig. 7D, arrows). At 72 hours
post Ad-NudC transduction, most of the cells showed significant increases in
both cell size and nuclear size (Fig.
7F), and appeared spread out and flat. Some of the large cells
contained micronuclei (Fig. 7F,
arrow and inset). Indeed, by 96 hours post Ad-NudC transduction, greater than
90% of the cells have become very large and multinucleated. Typically, these
large cells contained multiple nuclei (Fig.
7G) or a few enlarged nuclei
(Fig. 7H), which suggests a
failure in cytokinesis. Micronuclei were observed in these large
multinucleated cells (Fig.
7G,H, arrow). Multinucleated cells with persistent cytoplasmic
connections were also observed using transient transfection of NudC expression
vectors (data not shown), thus suggesting that the observed multinucleation
phenotype is unlikely to be a result of viral transduction. By contrast, in
the Ad-Luc transduced control cells nuclear size remained constant
(Fig. 7A-C) and the cell number
increased between 24 hours and 72 hours, in agreement with the cell
proliferation data (Fig. 6B).
These results show that overexpression of NudC also caused inhibition in cell
proliferation and led to an increase in multinucleation.
|
|
NudC overexpression results in the mislocalization of the mitotic
kinase Plk1
Because reduction of NudC levels by RNAi resulted in Plk1 mislocalization
during M phase (Fig. 5B), we
next determined whether NudC overexpression has a similar effect on the
localization of Plk1. HeLa cells were transduced with Ad-NudC or Ad-Luc for 96
hours and examined by immunofluorescence microscopy. In Ad-Luc-transduced
control cells, normal Plk1 staining was observed at the centrosomes during
metaphase (Fig. 9A) and the
midbody during cytokinesis (Fig.
9C). By contrast, Plk1 was mislocalized in 30% of
Ad-NudC-transduced cells. In NudC-overexpressing cells, Plk1 exhibited
punctate and irregular staining, as shown in cells connected by an
intercellular bridge (Fig. 9E)
and in large multinucleated cells (Fig.
9G). These results show that Plk1 exhibits aberrant localization
in NudC-overexpressing cells.
|
NUD-1 depletion in C. elegans causes cleavage-furrow
regression
We further used a different model genetic system, the nematode C.
elegans, to investigate the functional consequence of depletion of NUD-1,
the C. elegans ortholog of mammalian NudC
(Dawe et al., 2001), on
mitosis. Using injection of nud-1 dsRNA, Dawe et al.
(Dawe et al., 2001
) previously
showed that the putative zygotic function of NUD-1 involves the development of
the germ line and nervous system. Embryonic cell division defects were
observed using this method. However, oogenesis fails rapidly after injection
of nud-1 dsRNA, precluding a detailed analysis of embryonic
phenotypes by this method. We used the new technique of RNAi feeding to expand
upon these prior analyses by examining cell division defects associated with
NUD-1 knockdown during development (Kamath
et al., 2000
; Timmons et al.,
2001
). In addition to a defect in pronuclear rotation in the
one-celled embryos, as previously described
(Dawe et al., 2001
),
nud-1 RNAi feeding yields a more severe, highly reproducible defect
in late cytokinesis during the first cell cycle following fertilization
(Fig. 10) (see also movies
online:
http://jcs.biologists.org/supplemental).
In both wild-type and NUD-1-depleted embryos, a spindle developed and
elongated along the longitudinal axis
(Fig. 10A,B,E,F). The first
cleavage furrow appeared at the appropriate time and position in both types of
embryos (Fig. 10C,G). The
wild-type embryo underwent cleavage-furrow ingression, which resulted in the
generation of two differently sized cells
(Fig. 10D). By contrast, in
the nud-1 RNAi embryo, the cleavage furrow stalled
(Fig. 9H, arrow indicates the
furthest point of ingression of the furrow) and then quickly regressed
(Fig. 10I-K), resulting in a
multinucleated one-celled embryo (Fig.
10L).
|
To examine further the late cytokinesis failure in the nud-1 RNAi
embryos, we determined whether midzone microtubules were present by staining
for -tubulin (green) when NUD-1 was depleted. Midzone microtubules are
typically robust in anaphase wild-type embryos
(Fig. 11A-C). However, in
nud-1 RNAi embryos, midzone microtubules were absent in 26% (10/39)
of one-cell staged embryos (Fig.
11D-F). The remaining 74% of one-cell staged embryos (29/39)
contained midzone microtubules that were less well defined
(Fig. 11G-I). Frequently,
nud-1 RNAi embryos (15/29) containing weak midzone microtubules
exhibited chromatin bridges, which are indicative of DNA mis-segregation
(Fig. 11H,I). After the first
cell cycle, cytokinetic furrows in NUD-1-depleted embryos continued to form
and regress dynamically without complete stabilization of the furrows, as
observed in live embryos (data not shown). To confirm this, older embryos were
analysed by
-tubulin staining (Fig.
11L). Multipolar spindles and additional DNA were detected in
these embryos, suggesting that embryos depleted for NUD-1 underwent multiple
rounds of the cell cycle without completing cytokinesis. Together, the
combined mammalian and C. elegans studies show that NudC/NUD-1 is
crucial for cleavage-furrow ingression, midzone microtubule organization and
the completion of cytokinesis.
|
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Discussion |
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Consistent with midzone microtubule disruption, the mitotic kinase Plk1,
which normally localizes to centrosomes and midbody microtubule matrix of
mitotic cells, is mislocalized in cells in which NudC levels were altered
(Figs 5,
9). Plk1 is known to play
several roles during M phase progression, including mitotic entry, mitotic
exit and cytokinesis (Glover et al.,
1998; Nigg, 2001
;
Donaldson et al., 2001
) and is
suggested to affect spindle organization and microtubule dynamics
(Tong et al., 2002
). Plk1
depletion by RNAi resulted in an increase in binucleate cells and cells
connected by a long cytoplasmic bridge
(Liu and Erikson, 2002
),
whereas overexpression of Plk1 resulted in large cells containing multiple,
fragmented nuclei (Mundt et al.,
1997
). These phenotypes are reminiscent of both depletion
(Fig. 3, Fig. 4J,K) and overexpression
(Figs 7,
8,
9) of NudC, respectively.
Time-lapse microscopy studies revealed that Plk1 is involved in cleavage
furrow ingression, which supports a role of Plk1 in cytokinesis
(Seong et al., 2002
;
Chase et al., 2000
). Notably,
we recently demonstrated that NudC is phosphorylated by Plk1 in vitro and that
NudC phosphorylation increases during M phase and correlates with increased
Plk1 kinase activity in vivo (T. H. Zhou, J. P. Aumais, X. Liu, L.-y. Yu-Lee
and R. L. Erikson, unpublished). Furthermore, phosphorylation of NudC by Plk1
on the highly conserved C-terminal S274 and S326 residues seems to be
important during cytokinesis. These results suggest that, although NudC
activity might be regulated by Plk1 phosphorylation, NudC might, in turn, be
involved in the temporal and spatial localization of Plk1 during M phase.
Although it is possible that Plk1 mislocalization from the midbody in NudC
altered cells is a consequence of the effects of NudC on microtubule stability
and/or organization, we suggest that Plk1 mislocalization contributes to
cytokinesis failure and the increased incidence of multinucleation and
aneuploidy in cells with altered NudC expression.
Studies in the fungus A. nidulans have shown that NudC is
genetically and functionally linked to other nud loci that encode
components of the dynein/dynactin complex
(Morris, 2000). Biochemical
evidence in mammalian cells show that NudC is associated with the microtubule
minus-end-directed dynein/dynactin motor complex
(Aumais et al., 2001
). NudC
also forms a complex with Lis1 (A. nidulans NUDF) and the NudC/Lis1
complex is localized to the MTOC and the microtubule network in neurons and
fibroblasts (Morris and Yu-Lee,
1998
; Aumais et al.,
2001
). We previously suggested that the NudC/Lis1 complex plays a
role in mediating nuclear movement and transport in neuronal progenitors as
well as in migrating neurons (Aumais et
al., 2001
). In dividing cells, Lis1 potentiates dynein function
and is suggested to regulate dynein/dynactin interactions with the cell cortex
(Faulkner et al., 2000
;
Vallee et al., 2000
) and with
the kinetochore (Tai et al.,
2002
; Coquelle et al.,
2002
), through association with microtubule plus ends and CLIP-170
(Tai et al., 2002
;
Coquelle et al., 2002
). Lis1
also regulates microtubule dynamics (Sapir
et al., 1997
; Smith et al.,
2000
). Given the observations that NudC/Lis1 interacts with the
dynein/dynactin complex (Morris et al.,
1998
; Aumais et al.,
2001
), that dynein/dynactin subunits are localized to the midbody
(Karki et al., 1998
;
Campbell et al., 1998
;
Karki and Holzbaur, 1999
) and
that NudC is localized to the cleavage furrow
(Moreau et al., 2001
) and
midbody (this study), we speculate that the NudC/Lis1/dynein/dynactin complex
functions in regulating cytokinesis either by regulating midbody matrix
organization or by targeting regulatory and effector molecules to the midzone
and midbody during cytokinesis. Our studies also raise the interesting
possibility that Plk1 might affect the formation and/or function the
NudC/Lis1/dynein/dynactin complex. In support of this notion, we recently
observed that Plk1 associates with the dynein/dynactin complex upon entry into
mitosis (T. H. Zhou, J. P. Aumais, X. Liu, L.-y. Yu-Lee and R. L. Erikson,
unpublished), the significance of which awaits further experimentation.
Many proteins are localized to the midzone and play a role during
cytokinesis. These include chromosomal passenger proteins, mitotic kinases
such as Plk1 and aurora kinases, GTPases and kinesin-like motor proteins
CHO1/MKLP1, which are needed for microtubule stability and
microtubule-dependent protein and vesicular transport to support furrow
ingression and cytokinesis (Straight and
Field, 2000). Inappropriate expression of some of these proteins
also results in cleavage furrow regression and cytokinesis failure
(Matuliene and Kuriyama, 2002
;
Raich et al., 1998
;
Liu and Erikson, 2002
;
Seong et al., 2002
;
Chase et al., 2000
). It would
be interesting to determine whether these diverse groups of proteins, which
are needed for the successful completion of cytokinesis, are also missing or
mislocalized in NudC altered cells. Our findings that NudC is involved in Plk1
localization during M phase, cleavage-furrow ingression and midzone/midbody
microtubule organization suggest novel functions for this highly conserved
protein in mitosis and in cytokinesis.
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Acknowledgments |
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Footnotes |
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References |
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