![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 14, Issue 7, 2876-2889, July 2003
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom
Submitted February 25, 2003;
Revised March 21, 2003;
Accepted March 24, 2003
Monitoring Editor: Trisha Davis
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
;
Hinchcliffe and Sluder, 2001;
Meraldi and Nigg, 2002). After
a mitotic division, cells inherit a pair of morphologically distinct
centrioles, called the mother and daughter
(Marshall, 2001). At some
point early in the cell cycle, these undergo disorientation, whereby they lose
the orthogonal arrangement and become more obviously separated. The precise
timing of centriole disorientation is a matter of debate and may be cell type
dependent. During S and G2, centriole duplication occurs with procentrioles
that can only clearly be detected by electron microscopy elongating at a
perpendicular angle from the surface of mother and daughter centrioles. As
cells enter mitosis, the original mother and daughter centriole become
detached from one another in an event termed centrosome disjunction
(Hinchcliffe and Sluder,
2001). The two pairs of duplicated centrioles then separate toward
opposite ends of the cell to facilitate mitotic spindle formation. The PCM
also undergoes structural reorganization during cell cycle progression, most
notably upon entry into mitosis when additional proteins are recruited and
there is a change in microtubule nucleating capacity
(Khodjakov and Rieder, 1999).
The remodeling of the PCM as cells enter mitosis is referred to as centrosome
maturation.
The changes in centriole and PCM organization that occur during the
centrosome duplication cycle have been well documented by electron and light
microscopy of fixed cells and isolated centrosomes. However, it is only with
the recent application of green fluorescent protein (GFP) technology, coupled
with live cell imaging, that it has become apparent that centrioles are not
static organelles within the cytoplasm. On the contrary, centrioles exhibit
dramatic and extensive movements that may be critical for initiating specific
cell cycle events as well as for determining the distribution of the
microtubule network. The mother centriole, for example, migrates toward the
midbody during late mitosis in an event that precedes, and may be necessary
for, cell abscission (Piel et
al., 2001). Meanwhile in G1, the mother centriole remains
close to the center of the cell, whereas the daughter oscillates around it
visiting different regions of the cytoplasm
(Piel et al., 2000).
The extent of these movements varies according to cell type, with centrioles
exhibiting a greater degree of movement in L929 and NIH 3T3 cells, for
instance, than HeLa cells. In HeLa cells, centrioles remain in relative
proximity throughout interphase raising the possibility that proteinaceous
material, perhaps part of the PCM, acts as a physical tether between mother
and daughter centriole after disorientation. In fact, even in those cell types
where centrioles migrate far apart, some sort of intercentriolar connection
seems to be present because the G1 movements of the two centrioles are
correlated and, in S and G2, the centrioles move back closer together and
become less dynamic (Piel et al.,
2000).
Perhaps the most persuasive evidence that a physical linkage is established
between mother and daughter centriole after disorientation is the fact that
centrioles remain paired in isolated centrosome preparations
(Bornens and Moudjou, 1999).
Electron dense material has been observed between the mother and daughter
centrioles of isolated centrosomes, as well as between the pair of basal
bodies in Chlamydomonas, but the molecular nature of these
connections remains a mystery (Bornens
et al., 1987;
Paintrand et al.,
1992; Preble et al.,
2000). Some progress, though, has been made into proteins that
regulate centriolar cohesion. Nek2A is a never in mitosis A (NIMA)-related
protein kinase that forms a complex with the catalytic subunit of protein
phosphatase 1 (PP1) and a large coiled-coil protein called C-Nap1
(Fry, 2002). Nek2A can
phosphorylate itself, PP1 and C-Nap1, whereas PP1 can dephosphorylate both
Nek2A and C-Nap1 (Fry et al.,
1998b,
1999;
Helps et al., 2000).
Transient overexpression of active Nek2A kinase or microinjection of C-Nap1
antibodies leads to premature splitting of the mother and daughter centrioles,
suggesting that these treatments either stimulate centriole dynamics or
somehow modify the intercentriolar linkage
(Fry et al., 1998a;
Mayor et al., 2000).
Coexpression of PP1 with Nek2A suppresses centriole splitting, whereas ectopic
expression of inhibitor protein 2, a physiological inhibitor of PP1,
stimulates splitting (Meraldi and Nigg,
2001; Eto et al.,
2002). Together, these studies support a model in which centriole
cohesion is carefully modulated by opposing kinase and phosphatase
activities.
Nek2 and C-Nap1 both localize to the proximal ends of centrioles, which
would be consistent with their forming an anchoring site for an
intercentriolar linkage (Fry et
al., 1998b; Mayor et
al., 2000). Moreover, C-Nap1 is displaced from spindle poles
in mitosis at a time when it is hyperphosphorylated in vivo
(Mayor et al., 2002).
These observations provide the basis for a model in which Nek2A promotes
centrosome disjunction through phosphorylation-induced displacement of C-Nap1
from centriolar ends (Mayor et
al., 1999; Fry,
2002). If this model were correct then overexpression of a
catalytically-inactive Nek2A protein might be expected to interfere with
centrosome disjunction and, subsequently, mitotic progression. However, in
transient transfection experiments such phenotypes have been difficult to
observe. We therefore decided to generate stable cell lines expressing active
and kinase-dead Nek2A from tetracycline-inducible promoters. Using these cell
lines, we have, for the first time, demonstrated a dominant-negative phenotype
for kinase-dead Nek2A with respect to centrosome disjunction, spindle
formation, and chromosome segregation. These results provide the strongest
evidence yet that Nek2 has an important function in coordinating centrosome
structure and function with mitotic progression alongside other protein
kinases such as Cdk1, Plk1, and Aurora-A
(Nigg, 2001).
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
). To induce and maintain expression from
tetracycline-responsive promoters, tetracycline or doxycycline (1 µg/ml)
was added to the culture medium every 24 or 48 h, respectively. To induce
cytokinesis failure, cytochalasin D (0.6 µg/ml) was added.
Plasmid Constructions
Generation of pEGFP-Nek2A has been described previously
(Hames and Fry, 2002).
pEGFP-Nek2A-K37R was made by excision of Nek2A-K37R from pGEM-Nek2-K37R
(Fry et al., 1995) on
a NaeI-BamHI fragment and inserting it into a pEGFP-C1
vector (BD Biosciences Clontech, Palo Alto, CA) modified to include a T7
promoter upstream of the enhanced green fluorescent protein (EGFP) coding
region. To generate vectors for inducible expression of C-terminal
myc-His–tagged proteins, full-length cDNAs encoding wild-type Nek2A or
Nek2A-K37R were excised from pEGFP-Nek2A or pEGFP-Nek2A-K37R, respectively, as
SphI-XhoI blunted fragments and subcloned into the
pcDNA4/TO/myc-HisA inducible eukaryotic expression vector (Invitrogen) cut
with XhoI and blunted. To generate vectors for inducible expression
of N-terminal EGFP–tagged proteins, EGFP-Nek2A, wild-type, or K37R, was
excised from pEGFP-Nek2A or pEGFP-Nek2A-K37R, respectively, on an
AgeI (blunted)-XbaI fragment and subcloned into the
pcDNA4/TO inducible eukaryotic expression vector (Invitrogen) cut with
EcoRV and XbaI.
Generation of Tetracycline-inducible Cell Lines
Stable cell lines were generated by introducing the relevant construct into
a 10-cm dish of T-REx-U2OS cells by using calcium phosphate-mediated
transfection. After 24 h, cells were washed and incubated in selective media
containing Zeocin (400 µg/ml; Invitrogen) and hygromycin B (200 µg/ml)
until foci containing 
50 –100 cells were detected (3–4
wk). Culture dishes were then washed with 1x phosphate-buffered saline
(PBS) before picking foci by using paper cloning discs (Sigma-Aldrich, St.
Louis, MO; Z37, 443-1). Each disk was soaked in 1x PBS containing 0.5 mM
EDTA and then placed over the foci of cells for 5–10 min at 37°C.
Discs were then transferred into individual wells of a 24-well plate and
selective medium added. After attachment of cells to plastic culture wells,
cloning discs were removed and individual clones expanded in selective media
before testing for protein expression by Western blotting and fluorescence
microscopy.
Cell Extraction, Protein Electrophoresis, and Western Blotting
To prepare cell extracts, cells were washed once with ice-cold 1x PBS
before direct lysis on the dish in hot (95°C) SDS-gel loading buffer.
Lysed cells were collected from the plate with a cell scraper and heated to
95°C for 10 min. Chromosomal DNA was sheared by passage through a 27-gauge
needle before centrifugation for 10 min. Protein gel electrophoresis and
Western blotting was performed as described previously
(Fry et al., 1998b).
Primary antibodies used were anti-Nek2 (1 µg/ml;
Fry et al., 1999) and
anti-phospho-H3 (1 µg/ml; Upstate Biotechnology, Lake Placid, NY), whereas
secondary antibodies were alkaline phosphatase-conjugated anti-rabbit or
anti-mouse IgGs (1:7500; Promega, Madison, WI).
Fixed Cell Microscopy
Immunofluorescence microscopy was performed as described previously
(Fry et al., 1998a;
Fry and Faragher, 2001),
except for GT335 staining where cells were first detergent extracted according
to MacRae et al.
(1990). Primary antibodies
used were anti-Nek2 (1 µg/ml; Zymed, South San Francisco, CA;
Fry et al., 1999),
anti-C-Nap1 (1 µg/ml; R63 anti-C-term antibody;
Fry et al., 1998b),
antimyc (9E10; undiluted supernatant), anti-Plk1 (10 µg/ml; Upstate
Biotechnology), anti-polyglutamylated tubulin (GT335; 1:200),
-cytokeratin 18 caspase cleavage product (M30 CytoDEATH; Roche
Diagnostics, Indianapolis, IN), anti-
-tubulin (0.15 µg/ml;
Sigma-Aldrich), and anti--tubulin (0.3 µg/ml; Sigma-Aldrich).
Secondary antibodies used were Alexa Fluor 488 goat anti-rabbit and rabbit
anti-mouse IgGs (1 µg/ml; Molecular Probes), donkey anti-rabbit and sheep
anti-mouse biotinylated whole antibodies (1:100; Amersham Biosciences UK,
Little Chalfont, Buckinghamshire, United Kingdom). Streptavidin Texas Red
(1:200; Amersham Biosciences UK) was used to detect biotinylated antibodies,
whereas DNA was stained with Hoechst 33258 (0.2 µg/ml; Calbiochem, San
Diego, CA). Fluorescence images were captured on a Nikon TE300 inverted
microscope using an ORCA ER charge-couple device camera (Hamamatsu, Shizuoka,
Japan) by using Openlab 3.09 software (Improvision, Coventry, United Kingdom)
and processed using Adobe Photoshop (Adobe Systems, San Jose, CA). Digital
deconvolution was performed by capturing optical z-sections at 0.1-µm steps
through the cell of interest using a high-speed Piezo focus drive device
(Orbit II). Volume deconvolution (Openlab 3.09; Improvision) by using five
nearest neighbors was used on each z-section before display as maximum
intensity projections.
Centrosome Intensity Measurements
Cells were processed for indirect immunofluorescence microscopy by using
anti-C-Nap1 or anti--tubulin antibodies, as described above. Using
constant exposure times and gain settings determined to be within the linear
range of the camera, we captured fluorescence images of centrosomes and
mitotic spindle poles. A 1.5-µm2 region of interest was
positioned to encompass each centrosome and the mean intensity (integrated
optical density) signal per pixel within this region of interest, minus
background, was measured. Measurements were made on each centrosome in 20
cells for each condition and independent experiments performed three
times.
Live Cell Imaging
For live cell microscopy, cells were grown on coverslips and induced with
doxycycline as indicated in the text. Coverslips were then washed once with
1x PBS before mounting in a steel coverslip holder (made in house) in
CO2-independent medium without glutamine (Invitrogen) but with
doxycycline. The surface of the medium was overlaid with mineral oil
(Sigma-Aldrich) to prevent evaporation. The steel coverslip holder was mounted
in a Patch Slice MicroIncubator regulated at 37°C by a TC-202A temperature
controller (Harvard Apparatus Medical Systems Research Products, supplied by
Digitimer, Hertfordshire, United Kingdom). The Patch Slice MicroIncubator was
placed on the microscope stage and images captured at 2- or 5-min intervals
with the microscope system described above. At each time point, five optical
sections (z-planes) at 0.5-µm intervals were captured using a high-speed
Piezo focus drive (Orbit II) attached to a 60x oil objective (numerical
aperture 1.4) and merged using Openlab software. Images were processed using
Adobe Photoshop or converted to QuickTime movies.
Flow Cytometry
Cell cycle distributions were determined by measuring DNA content by flow
cytometry after staining of cells with propidium iodide. Specifically, 1
x 106 cells were washed with 1x PBS and slowly
resuspended in 1 ml of 70% ethanol (–20°C) before storage at
–20°C. Fixed cells were pelleted and washed with 1x PBS before
resuspension in 1 ml of DNA stain (0.02 mg/ml propidium iodide, 0.2 mg/ml
RNase A in 1x PBS) and incubated overnight at 4°C. DNA content was
measured using a FACScan II instrument (BD Biosciences, San Jose, CA) and
analyzed using CellQuest.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
|
Fluorescence microscopy was performed on each cell line after 24-h
induction. Exogenous Nek2A proteins primarily localized to the interphase
centrosome as indicated by colocalization of the myc or GFP signal with
-tubulin (Figure 2A).
Isolation of centrosomes from these cells by sucrose gradient centrifugation
also showed enrichment of recombinant proteins by Western blot (our
unpublished data). Induction of wild-type Nek2A, either GFP or myc-His tagged,
led to premature centriole splitting as judged by the loss of close
juxtaposition of the two -tubulin–stained structures. This was
not observed with the kinase-dead versions of Nek2A. Reassuringly, these
results fall in line with those obtained in transient transfection experiments
in which it was shown that active, but not inactive, Nek2A can induce
centriole splitting (Fry et al.,
1998a).
|
On entry into mitosis, centrosome staining was lost
(Figure 2B), supporting the
finding that Nek2A is actively destroyed by the proteasome, rather than being
transcriptionally down regulated, in early mitosis
(Hames et al., 2001).
Importantly, destruction led to loss of Nek2A from spindle poles and not
simply from the noncentrosomal pool. In contrast to a recent report
(Kim et al., 2002),
no localization to mitotic chromosomes
(Figure 2B, b) or the midbody
(our unpublished data) was observed. By Western blotting for Nek2 protein in
asynchronous cells incubated with cycloheximide
(Figure 2C), the half-life of
recombinant Nek2A-K37R-myc-His was determined to be very similar to endogenous
Nek2A at 1 h (Hames et al.,
2001). Thus, based upon localization, activity and stability
measurements, these cell lines represent excellent new tools to study Nek2
function.
Analysis of Nek2A-induced Centriole Splitting in Live Cells
In certain cell types (e.g., L929) centrioles exhibit significant mobility
during interphase (Piel et al.,
2000), but in others the mother and daughter centriole remain in
proximity. We calculated that in the parental U2OS cell line, although two
dots can almost invariably be distinguished, <10% cells (n >200) have
centrioles that are >2 µm apart
(Figure 3A). However,
doxycycline-induced expression of GFP-Nek2A or Nek2A-myc-His for 24 h
stimulated a premature splitting of centrioles in 67 and 19% cells (n >
200), respectively (Figure 3A).
This correlates with the higher level of expression in the GFP-Nek2A cell line
and supports the notion that splitting depends upon achieving a certain
threshold of Nek2A activity that is presumably in excess of competing PP1
activity. Proteasomal destruction in G1 is likely to reduce the level of
recombinant Nek2A below this threshold explaining why splitting is not
observed in 100% of cells (Fry et
al., 1998a; Meraldi and
Nigg, 2001).
|
To study Nek2A localization and centriole splitting in live cells, time-lapse microscopy was performed on cells that had been induced to express wild-type GFP-Nek2A for 24 h. GFP-Nek2A protein was clearly detected at the two centrioles in live cells, providing conclusive evidence that previous staining patterns on fixed cells were not localization artifacts (Figure 3B). In most cells, the residual GFP fluorescence not associated with centrioles was detected in the cytoplasm and was faintly punctate in nature (our unpublished data). By selecting cells that had widely spaced centrioles (>5 µm) at the start of filming, it became apparent that Nek2A-induced splitting is not a transient event. In each cell, centrioles were observed for at least 3 h and, in the majority of cases (82%, n = 11), centrioles remained far apart and sometimes moved further apart (Figure 3, B and C). Moreover, fine movements of the individual centrioles seemed independent, implying that, if they were still linked, the connection was not under obvious tension (our unpublished data). Only in two cells (18%) did split centrioles return to a separation distance of <5 µm. However, as the initial intercentriolar distance was <10 µm in these cells, it is possible that any intercentriolar connections had not yet been fully dismantled. In summary, live cell imaging demonstrates that Nek2A overexpression stimulates either loss or relaxation of the attachment between mother and daughter centrioles.
C-Nap1 Is Only Partially Displaced from Split Centrioles
The hypothesis has been put forward that phosphorylation of C-Nap1 by Nek2
at the onset of mitosis triggers its displacement from the proximal ends of
centrioles which, in turn, leads to centrosome disjunction
(Mayor et al., 2002).
We therefore examined whether premature splitting of interphase centrioles by
active Nek2A kinase was accompanied by a similar loss of the C-Nap1 protein to
that seen in normal mitosis. U2OS cells were fixed in cold methanol and
processed by indirect immunofluorescence microscopy with anti-C-Nap1
antibodies. Quantitative imaging of parental U2OS cells
(Figure 4A, a) or the GFP-Nek2A
cell line without induction (Figure 4A,
b) indicated a 10-fold reduction in C-Nap1 protein on mitotic
spindle poles compared with unsplit centrosomes in neighboring interphase
cells (Figure 4B). Imaging of
C-Nap1 in the GFP-Nek2A cell line after doxycycline induction revealed that
interphase cells with split centrioles had reduced C-Nap1 abundance, although
not to the same extent as at mitotic spindle poles
(Figure 4A, c and d).
Quantitation revealed only a twofold decrease in C-Nap1 abundance on split,
compared with unsplit, centrioles (Figure
4B). To determine the specificity of this response, similar
quantitative imaging was performed on -tubulin
(Figure 4B). In contrast to
C-Nap1, the abundance of -tubulin at mitotic spindle poles was
approximately threefold higher than at interphase centrosomes in agreement
with previous studies showing recruitment of -tubulin during centrosome
maturation (Khodjakov and Rieder,
1999). -Tubulin levels though were relatively unchanged
between split and unsplit centrioles, suggesting that the moderate
displacement of C-Nap1 protein is a specific effect and not due to general
disintegration of the centrosome structure. However, whether the twofold
reduction in C-Nap1 protein is sufficient to trigger the centriole splitting
remains to be proven.
|
Kinase-Dead Nek2A Induces Accumulation of Multiple Centrosomes
Transient transfection of kinase-dead Nek2A had not revealed an obvious
mitotic defect (Fry et al.,
1998a). However, in those experiments it was difficult to detect
transfected mitotic cells. The reason for this is clear now that we know that
Nek2A is destroyed soon after mitotic entry
(Hames et al., 2001).
In contrast, kinase-dead Nek2A was capable of suppressing nocodazole-induced
centriole splitting, implying that it could exert a dominant-negative activity
(Meraldi and Nigg, 2001). We
therefore decided to reexamine the consequences of overexpressing kinase-dead
Nek2A by using the tetracycline-inducible cell lines. Careful examination of
centrosomes in the Nek2A-K37R-myc-His cell line first revealed that >50% of
cells (n = 500) had centrosomes that were abnormal in appearance, i.e., they
no longer consisted of just two discrete dots of 1 µm in diameter
(Figure 5A). This was evident
not only after immunostaining with anti-Nek2 antibodies
(Figure 5B) but also with
anti--tubulin (Figure
5C) and anti-C-Nap1 (our unpublished data) antibodies. In some
cases, only one or two structures could be distinguished, although these were
often much larger than a normal centrosome and irregular in shape
(Figure 5, B and C, d). In
other cases, however, there were clearly more than two centrosomes that again
were not necessarily of normal appearance
(Figure 5, B and C, b and c). These phenotypes were strictly dependent upon expression of the recombinant
protein because they were not observed in the parental cell line or in the
mutant cell line in the absence of tetracycline. Moreover, the abnormal
phenotypes were evident in more than one clone of the Nek2A-K37R-myc-His cell
line. Microtubule regrowth assays after cold-induced microtubule
depolymerization indicated that all of these centrosomes were still capable of
efficient microtubule nucleation (our unpublished data).
|
Where more than two dots were observed, it was important to determine
whether each dot contained a centriole rather than simply being an aggregate
of centrosomal proteins. Induced cells were therefore stained with the GT335
monoclonal antibody that recognizes polyglutamylated tubulin and hence acts as
a well-characterized marker of centrioles
(Bobinnec et al.,
1998). Whereas only two GT335 dots were clearly distinguished in
uninduced cells (Figure 5D,
a–c), more than two GT335-stained dots were evident in many
cells expressing kinase-dead Nek2A (Figure
5D, d–f). Hence, cells expressing kinase-dead Nek2A
accumulate abnormal centrosomes that fall into two categories: centrosomes
with extracentrosomal material and centrosomes with supernumerary centrioles
that remain in proximity. These two categories are not mutually exclusive
making it difficult to score each phenotype separately.
Spindle and Segregation Defects Induced by Kinase-Dead Nek2A
Expression
The presence of some cells with more than two centrioles raised the
possibility that kinase-dead Nek2A was acting in a dominant-negative manner to
prevent separation of mother and daughter centrioles at mitosis. To determine
whether such abnormalities may result from a failure of centrosome
disjunction, mitotic cells were examined. In the parental cell line, only 3.5%
(n = 300) of mitotic cells exhibited any unusual spindle structures with these
being a mix of monopolar and multipolar arrays. However, induction of
kinase-dead Nek2A for 24 h led to a 4.5-fold increase in abnormal spindles
(15.7%; n = 450) (Figure 6A).
DNA and microtubule staining revealed that the overwhelming majority of these
abnormal mitoses contained monoastral arrays of microtubules surrounded by a
chromosome rosette (Figure 6B,
b–d). By immunostaining cells with -tubulin
antibodies and capturing optical z-sections through the monopolar spindles, it
was clear that centrosomes had not substantially separated in either the x, y,
or z-axis (Figure 6C,
b). Measurements showed the average pole-to-pole distance in the
monopolar spindles to be 2.1 µm, compared with 9.2 µm in a bipolar
metaphase spindle. The frequency of spindle abnormalities did not increase
with longer times of induction, suggesting that the maximal level of
interference had been achieved for this cell line.
|
Similar monopolar spindle defects have been described under conditions
where centrosome maturation, rather than disjunction, is defective
(Lane and Nigg, 1996). We
therefore immunostained cells for Plk1, which is first recruited to
centrosomes during centrosome maturation
(Golsteyn et al.,
1995). In kinase-dead Nek2A-expressing cells, the recruitment of
Plk1 to the poles of monopolar spindles was similar to that in normal bipolar
spindles (Figure 6C, c and d).
This, together with the normal increase in -tubulin abundance
(Figure 6C, a and b), indicates
that the monopolar spindle phenotype induced by kinase-dead Nek2A is not a
result of defective centrosome maturation, at least with respect to Plk1 and
-tubulin recruitment. Importantly, the abundance of C-Nap1 at poles of
monopolar spindles was indistinguishable from that at poles of bipolar
spindles, indicating that apparently complete C-Nap1 displacement had occurred
(compare Figure 6D with
Figure 4A). Thus, the block to
centrosome separation could not be attributed to a failure to displace
C-Nap1.
Clearly, a significant proportion of cells expressing kinase-dead Nek2A was capable of forming bipolar spindles, possibly because the recombinant protein was targeted for proteasomal degradation upon mitotic entry. However, chromosome segregation defects were frequently observed in many of these cells as indicated by misaligned chromosomes in metaphase cells, lagging chromosomes in anaphase cells and thin chromatin bridges in telophase cells (Figure 6E). Further evidence for mitotic progression defects came from the appearance of multi- and micronucleated interphase cells (Figure 7A). Immunofluorescence staining of these cells with anti-C-Nap1 antibodies indicated that micronucleated cells generally contained a normal complement of two centrosomes (Figure 7B, a and b), whereas multinucleated cells usually contained clusters of more than two centrosomes suggestive of cytokinesis failure (Figure 7B, c and d). Induction for 72 h of kinase-dead Nek2A caused an approximate sixfold increase in multinucleation compared with the parental U2OS cells (Figure 7C). As a positive control, multinucleation was demonstrated in cells treated with cytochalasin D, which blocks cytokinesis through inhibiting formation of the contractile actin ring. Together, these data provide the first evidence that alteration of Nek2 activity can interfere with mitotic progression in human cells.
|
Expression of Kinase-Dead Nek2A Alters the DNA Content of the Cell
Population
To determine whether these centrosome and mitotic defects led to a major
loss of cell viability, cell cycle arrest, or apoptosis, a number of further
experiments were performed with the Nek2A-K37R-myc-His cells. Growth curves
revealed no significant change in proliferation or survival rate after 12 days
of tetracycline induction (Figure
8A). Equally, there was no sudden appearance of apoptotic cells in
the population as measured by antibody staining for cleaved cytokeratin 18
(our unpublished data). Similarly, mitotic index counts and Western blotting
for the mitosis-specific phospho-H3 epitope revealed no substantial mitotic
arrest (our unpublished data). Together, these observations indicate that the
centrosome and mitotic abnormalities do not lead to a gross loss of viability
within the cell population. Flow cytometric analysis, however, revealed a
clear reduction in the proportion of cells with a 2N DNA content after 8-days
induction, leading to the majority of cells having >2N DNA
(Figure 8, B and C). One
interpretation of this result is that there is a delay to cell cycle
progression, an intriguing possibility in light of the mutant phenotypes of
fungal NIMA-related kinases. However, our data is equally consistent with the
increasing percentage of multinucleated cells that were present in the
population as a result of either failed mitosis or abortive cytokinesis.
Indeed, because the kinase-dead Nek2A cells were derived from an osteosarcoma
cancer cell line, it is entirely plausible that they can tolerate a
chromosomal instability phenotype that overtime leads to a gradual increase in
ploidy without obvious cell death.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
;
Pihan et al.,
1998).
Inducible Cell Lines: Novel Tools to Study Nek2 Function In Vivo
Research on Nek2, and its most closely related counterparts in other
species, is providing accumulating evidence that this is an important family
of cell cycle regulators (reviewed in Fry,
2002). However, the precise functions of Nek2 in mitotic
progression remain far from understood. The generation of inducible cell lines
provides us with powerful new tools to study the regulation and function of
the Nek2A splice variant. Interestingly, transient overexpression of both
active and inactive Nek2A kinase led to dispersal of the centrosome and loss
of a functional microtubule organizing center
(Fry et al., 1998a).
This was not a major phenotype of the inducible cell lines, most likely
because the level of overexpression per cell is considerably lower than in the
transient transfection experiments. Nevertheless, during our live cell
recordings of both wild-type and kinase-dead GFP-Nek2A, we did observe
centrosomes that seemed to shed fragments or particles into the cytoplasm (our
unpublished data). This not only supports the hypothesis that Nek2 contributes
to the assembly and maintenance of an intact centrosome structure
(Fry et al., 2000b;
Uto and Sagata, 2000) but also
implies that tumor cells with significant amplification of Nek2 might lose a
focused microtubule organizing center.
As a means to unravel Nek2 function, these cell lines rely on protein
overexpression. The identification of a specific Nek2 inhibitor would
therefore be of great benefit in confirming the role of endogenous Nek2 kinase
activity in the control of chromosome segregation and mitotic progression,
particularly because it has not yet proven possible to identify effective
siRNA molecules despite much effort on our part. Because more than half the
cells expressing active GFP-Nek2A have split centrosomes and the GFP-Nek2A
protein acts as a vital marker of the centrosome, these cell lines make
excellent reagents for high-throughput microscope-based assays to screen for
in vivo inhibitors of Nek2 kinase. A small molecule inhibitor may also prove
useful as a therapeutic agent in tumors that show up-regulation of Nek2
expression (Wai et al.,
2002).
Nek2A Activation Stimulates Centrosome Disjunction
There is growing evidence that centrosome disjunction is triggered by
altering the protein phosphorylation status of core centrosomal components.
Kinases implicated in regulating centrosome cohesion include Nek2A
(Fry et al., 1998a;
Meraldi and Nigg, 2001), Cdk2
(Lacey et al., 1999;
Meraldi and Nigg, 2001), and
p160ROCK (Chevrier et al.,
2002), whereas phosphatase involvement has been proposed for PP1
(Helps et al., 2000;
Eto et al., 2002) and
Cdc14A (Mailand et al.,
2002). Nek2A directly binds PP1 via a KVHF motif in its C-terminal
noncatalytic domain. Because Nek2A is activated by autophosphorylation
(Fry et al., 1999),
PP1 can negatively regulate Nek2A by dephosphorylation. Conversely, Nek2A can
phosphorylate PP1 reducing its phosphatase activity
(Helps et al., 2000).
This double-negative feedback produces an exquisitely sensitive bistable
switch that dictates the phosphorylation status of Nek2A substrates. In these
experiments, we show that increasing the level of Nek2A expression above
fivefold is sufficient to flip the switch in favor of centrosome splitting.
Moreover, our time-lapse observations of centrosome dynamics suggest that the
intercentriolar linkage has been permanently lost. Centrosomes that were >5
µm apart at the start of filming rarely returned to within this distance
over the observation period. Clearly, in these in vivo experiments, it is
difficult to state with absolute certainty that connections have been
irrevocably broken. It remains possible that Nek2A activity alters the
conformation of the linkage such that it becomes sufficiently long and
flexible to accommodate greater intercentriolar separations. Because our
experiments were not performed on synchronized cells, the duplication state of
the split centrioles was not known. However, in all our fixed and live cell
recordings, four separated structures were never observed strongly implying
that Nek2A expression does not trigger the release of procentrioles from their
sites of synthesis adjacent to mother and daughter centrioles. We believe that
it acts specifically on the connections between mother and daughter
centriole.
Kinase-Dead Nek2A Interferes with Centrosome Disjunction and Mitotic
Progression
The most striking results of this study are the consequences of
overexpressing kinase-dead Nek2A. First, many cells accumulated unusually
large and irregularly shaped centrosomes, and centrosomes that clearly had
supernumerary (>2) centrioles. Both of these phenotypes are common in
cancer cells (Nigg, 2002). The
unusually large centrosomes may result from accumulation of excess kinase-dead
Nek2A protein, which in turn causes recruitment of other centrosomal proteins,
including at least -tubulin and C-Nap1. This is consistent with data
showing that Xenopus Nek2 is required for recruitment of
-tubulin during zygotic centrosome assembly
(Fry et al., 2000b).
Surprisingly, in transient transfection experiments, the opposite phenotype
was observed in that expression of kinase-dead Nek2A led to centrosome
dispersal. We interpret this difference in terms of the relative expression
levels of the ectopic protein: in the induced cell line, the level of
overexpression is sufficiently low that most protein can still localize to the
centrosome where it acts as a site of recruitment for other centrosomal
proteins; in the transient transfections, however, the massive overexpression
means that the bulk of the protein can no longer reside at the centrosome and
therefore titrates other centrosomal proteins out into the cytoplasm. From
either perspective, the experiments emphasize the importance of Nek2 as a
critical scaffold element of the centrosome.
Perhaps more informative with regard to Nek2 kinase function is the
presence of cells with multiple centrioles. Interpretation of this phenotype
is aided by the observation of mitotic cells with monopolar spindles and
unseparated centrosomes. Because these centrosomes had recruited both
-tubulin and Plk1, it seems reasonable to propose that they had
undergone some degree of G2/M maturation but then failed to separate. It is
interesting to note that centrosomes at the heart of the monoastral
microtubule arrays were separated on average by 2.1 µm. For comparison,
inhibition of the motor protein Eg5, either by antibody microinjection or by
the small molecule inhibitor monastrol, led to monopolar spindles with
centrosomes that were <2 µm apart
(Blangy et al., 1995;
Kapoor et al., 2000).
Although it is difficult to know whether this difference is significant based
upon the fact that these experiments have been performed in different cell
types, it is tempting to speculate that, in the kinase-dead Nek2A-expressing
cells, motor proteins have attempted to separate centrosomes but failed
because they are still physically attached.
Because we have not been able to follow live cells through mitosis, it is
not clear whether the monopolar spindles are eventually resolved into
pseudobipolar spindles that can complete mitosis. This may happen in some
cells after destruction of the recombinant Nek2A protein. However, the
frequent observation of chromosome segregation errors on bipolar spindles as
well as multinucleated and micronucleated interphase cells suggests that
aneuploidy frequently results from the expression of kinase-dead Nek2A.
Unseparated centrosomes may also directly interfere with cytokinesis, leading
to tetraploidization. This would provide an explanation for the multinucleated
cells with multiple centrosomes. Tetraploidization as a result of mitotic
failure has been proposed to account for the supernumerary centrosomes
generated by aberrant expression of another centrosomal kinase, Aurora-A
(Meraldi et al.,
2002). Indeed, this may represent a common pathway for cancer
cells to acquire multiple centrosomes and a chromosome instability phenotype,
particularly in cells lacking p53 (Nigg,
2002).
The gradual change in ploidy of the whole cell population also supports the
idea that kinase-dead Nek2A promotes chromosome instability. Besides this,
though, there was no significant decrease in cell proliferation, no striking
mitotic arrest and no induction of programmed cell death. Although this was
disappointing, similar results have been reported after spindle disruption and
aneuploidy induced by interference with another centrosomal protein,
pericentrin (Purohit et al.,
1999), and it may reflect loss of or adaptation to mitotic
checkpoints in transformed cells. Furthermore, proteasomal destruction of
Nek2A, as previously discussed, may be an important explanation for the
long-term survival, as well as why a larger fraction of mitotic cells do not
have abnormal spindles. We are currently generating cell lines expressing
nondestructible, kinase-dead Nek2A in an attempt to resolve this issue.
C-Nap1 and the Molecular Nature of the Intercentriolar Linkage
A key substrate for Nek2 at the centrosome is C-Nap1, a large coiled-coil
protein with no obvious enzymatic activity
(Fry et al., 1998b;
Mack et al., 1998).
C-Nap1 localizes to proximal ends of centrioles in interphase but is displaced
from mitotic spindle poles. This coincides with the time when it is maximally
phosphorylated in vivo (Mayor et
al., 2002). Using quantitative immunofluorescence microscopy,
however, we found that although there is a 10-fold reduction in C-Nap1
abundance at mitotic spindle poles, there is only a twofold reduction in
C-Nap1 abundance at split interphase centrioles. Furthermore, C-Nap1 was
virtually absent from the poles of monoastral spindles in cells expressing
kinase-inactive Nek2A. Our results therefore raise the possibility that Nek2A
activation alone is not sufficient to completely trigger C-Nap1 displacement
and nor is C-Nap1 displacement necessarily sufficient for centrosome
disjunction. Cohen and coworkers estimated that in vitro Nek2A was capable of
introducing 13 moles of phosphate into 1 mole of the C-terminal domain of
C-Nap1 alone (Helps et al.,
2000). Hence, it is possible that during interphase, due to the
antagonistic action of PP1, there is only partial phosphorylation of C-Nap1 by
the recombinant Nek2A and hence partial displacement. At the G2/M transition
full activation of Nek2A would lead to hyperphosphorylation and complete
displacement of C-Nap1. Alternatively, other mitotic kinases such as Plk1,
Cdk1 or Aurora-A might contribute to the full displacement of C-Nap1 from
spindle poles at the G2/M transition
(Figure 9). Whether the twofold
reduction in C-Nap1 at the centrosome is sufficient to cause disassembly of
the intercentriolar linkage is a difficult question to answer. Phosphorylation
of C-Nap1 may also cause loss of interaction with other components of the
intercentriolar linkage leading to centriole splitting. However, C-Nap1 may
not be the only target of Nek2 within the linkage and it could be the
cumulative phosphorylation of several proteins by Nek2A that triggers
centrosome disjunction. Further insight into the mechanism of centrosome
disjunction will clearly require a more detailed understanding of the
molecular nature of the intercentriolar linkage.
|
|
ACKNOWLEDGMENTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
|
Footnotes |
|---|
Abbreviations used: Nek2, NIMA-related kinase 2; NIMA, never in mitosis A; C-Nap1, centrosomal Nek2-associated protein 1; PP1, protein phosphatase 1; PCM, pericentriolar material.
* Corresponding author. E-mail address: amf5{at}le.ac.uk.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
|---|
Bobinnec, Y., Moudjou, M., Fouquet, J.P., Desbruyeres, E., Edde, B., and Bornens, M. (1998). Glutamylation of centriole and cytoplasmic tubulin in proliferating non-neuronal cells. Cell Motil. Cytoskeleton 39, 223–232.[CrossRef]
Bornens, M., and Moudjou, M. (1999). Studying the composition and function of centrosomes in vertebrates. Methods Cell Biol. 61, 13–34.[Medline]
Bornens, M., Paintrand, M., Berges, J., Marty, M.C., and Karsenti, E. (1987). Structural and chemical characterization of isolated centrosomes. Cell Motil. Cytoskeleton 8, 238–249.
Chevrier, V., Piel, M., Collomb, N., Saoudi, Y., Frank, R.,
Paintrand, M., Narumiya, S., Bornens, M., and Job, D. (2002). The
Rho-associated protein kinase p160ROCK is required for centrosome positioning.
J. Cell Biol. 157,
807–817.
Doxsey, S. (2001). Re-evaluating centrosome function. Nat. Rev Mol. Cell. Biol. 2, 688–698.[CrossRef][Medline]
Eto, M., Elliot, E., Prickett, T.D., and Brautigan, D.L.
(2002). Inhibitor-2 regulates protein phosphatase-1 complexed
with NimA-related kinase to induce centrosome separation. J. Biol.
Chem. 277,
44013–44020.
Fry, A.M. (2002). The Nek2 protein kinase: a novel regulator of centrosome structure. Oncogene 21, 6184–6194.[CrossRef][Medline]
Fry, A.M., Arnaud, L., and Nigg, E.A. (1999). Activity
of the human centrosomal kinase, Nek2, depends upon an unusual leucine zipper
dimerization motif. J. Biol. Chem.
274,
16304–16310.
Fry, A.M., Descombes, P., Twomey, C., Bacchieri, R., and Nigg, E.A. (2000b). The NIMA-related kinase X-Nek2B is required for efficient assembly of the zygotic centrosome in Xenopus laevis. J. Cell Sci. 113, 1973–1984.[Abstract]
Fry, A.M., and Faragher, A.J. (2001). Identification of centrosome kinases. Methods Cell Biol. 67, 305–323.[Medline]
Fry, A.M., Mayor, T., Meraldi, P., Stierhof, Y.-D., Tanaka, K., and
Nigg, E.A. (1998b). C-Nap1, a novel centrosomal coiled-coil
protein and candidate substrate of the cell cycle-regulated protein kinase
Nek2. J. Cell Biol. 141,
1563–1574.
Fry, A.M., Meraldi, P., and Nigg, E.A. (1998a). A centrosomal function for the human Nek2 protein kinase, a member of the NIMA-family of cell cycle regulators. EMBO J. 17, 470–481.[CrossRef][Medline]
Fry, A.M., Schultz, S.J., Bartek, J., and Nigg, E.A.
(1995). Substrate specificity and cell cycle regulation of the
Nek2 protein kinase, a potential human homolog of the mitotic regulator NIMA
of Aspergillus nidulans. J. Biol. Chem.
270,
12899–12905.
Golsteyn, R.M., Mundt, K.E., Fry, A.M., and Nigg, E.A.
(1995). Cell cycle regulation of the activity and subcellular
localization of PLK1, a human protein kinase implicated in mitotic spindle
function. J. Cell Biol. 129,
1617–1628.
Hames, R.S., and Fry, A.M. (2002). Alternative splice variants of the human centrosomal kinase Nek2 exhibit distinct patterns of expression in mitosis. Biochem. J. 361, 77–85.[CrossRef][Medline]
Hames, R.S., Wattam, S.L., Yamano, H., Bacchieri, R., and Fry, A.M. (2001). APC/C-mediated destruction of the centrosomal kinase Nek2A occurs in early mitosis and depends upon a cyclin A-type D-box. EMBO J. 20, 7117–7127.[CrossRef][Medline]
Helps, N.R., Luo, X., Barker, H.M., and Cohen, P.T.W. (2000). NIMA-related kinase 2 (Nek2), a cell cycle-regulated protein kinase localized to centrosomes, is complexed to protein phosphatase 1. Biochem. J. 349, 509–518.[CrossRef][Medline]
Hinchcliffe, E.H., and Sluder, G. (2001). "It
takes two to tango": understanding how centrosome duplication is
regulated throughout the cell cycle. Genes Dev.
15,
1167–1181.
Kapoor, T.M., Mayer, T.U., Coughlin, M.L., and Mitchison, T.J.
(2000). Probing spindle assembly mechanisms with monastrol, a
small molecule inhibitor of the mitotic kinesin, Eg5. J. Cell
Biol. 150,
975–988.
Khodjakov, A., and Rieder, C.L. (1999). The sudden
recruitment of -tubulin to the centrosome at the onset of mitosis and
its dynamic exchange throughout the cell cycle, do not require microtubules.
J. Cell Biol. 146,
585–596.
Kim, Y.H., Choi, J.Y., Jeong, Y., Wolgemuth, D.J., and Rhee, K. (2002). Nek2 localizes to multiple sites in mitotic cells, suggesting its involvement in multiple cellular functions during the cell cycle. Biochem. Biophys. Res. Commun. 290, 730–736.[CrossRef][Medline]
Lacey, K.R., Jackson, P.K., and Stearns, T. (1999).
Cyclin-dependent kinase control of centrosome duplication. Proc. Natl.
Acad. Sci. USA 96,
2817–2822.
Lane, H.A., and Nigg, E.A. (1996). Antibody
microinjection reveals an essential role for human polo-like kinase 1 (Plk1)
in the functional maturation of mitotic centrosomes. J. Cell
Biol. 135,
1701–1713.
Lingle, W.L., Lutz, W.H., Ingle, J.N., Maihle, N.J., and Salisbury,
J.L. (1998). Centrosome hypertrophy in human breast tumors:
implications for genomic stability and cell polarity. Proc. Natl. Acad.
Sci. USA 95,
2950–2955.
Mack, G.J., Rees, J., Sandblom, O., Balczon, R., Fritzler, M.J., and Rattner, J.B. (1998). Autoantibodies to a group of centrosomal proteins in human autoimmune sera reactive with the centrosome. Arthritis Rheum. 41, 551–558.[CrossRef][Medline]
MacRae, T.H., Lange, B.M., and Gull, K. (1990). Production and characterization of monoclonal antibodies to the mammalian sperm cytoskeleton. Mol. Reprod. Dev. 25, 384–392.[CrossRef][Medline]
Mailand, N., Lukas, C., Kaiser, B.K., Jackson, P.K., Bartek, J., and Lukas, J. (2002). Deregulated human Cdc14A phosphatase disrupts centrosome separation and chromosome segregation. Nat. Cell Biol. 4, 317–322.[Medline]
Marshall, W.F. (2001). Centrioles take center stage. Curr. Biol. 11, R487–R496.[CrossRef][Medline]
Mayor, T., Hacker, U., Stierhof, Y.-D., and Nigg, E.A.
(2002). The mechanism regulating dissociation of the centrosomal
protein C-Nap1 from mitotic spindle poles. J. Cell Sci.
115,
3275–3284.
Mayor, T., Meraldi, P., Stierhof, Y.-D., Nigg, E.A., and Fry, A.M. (1999). Protein kinases in control of the centrosome cycle. FEBS Lett. 452, 92–95.[CrossRef][Medline]
Mayor, T., Tanaka, K., Stierhof, Y.-D., Fry, A.M., and Nigg, E.A.
(2000). The centrosomal protein C-Nap1 displays properties
supporting a role in cell cycle-regulated centrosome cohesion. J. Cell
Biol. 151,
837–846.
Meraldi, P., Honda, R., and Nigg, E.A. (2002). Aurora-A overexpression reveals tetraploidization as a major route to centrosome amplification in p53–/– cells. EMBO J. 21, 483–492.[CrossRef][Medline]
Meraldi, P., and Nigg, E.A. (2001). Centrosome cohesion is regulated by a balance of kinase and phosphatase activities. J. Cell Sci. 114, 3749–3757.
Meraldi, P., and Nigg, E.A. (2002). The centrosome cycle. FEBS Lett. 521, 9–13.[CrossRef][Medline]
Nigg, E.A. (2001). Mitotic kinases as regulators of cell division and its checkpoints. Nat. Rev. Mol. Cell. Biol. 2, 21–32.[CrossRef][Medline]
Nigg, E.A. (2002). Centrosome aberrations: cause or consequence of cancer progression? Nat. Rev. Cancer 2, 815–825.[CrossRef][Medline]
Paintrand, M., Moudjou, M., Delacroix, H., and Bornens, M. (1992). Centrosome organization and centriole architecture: their sensitivity to divalent cations. J. Struct. Biol. 108, 107–128.[CrossRef]
) or presence (grey line, 
) of doxycycline. In the
left-hand graph, doxycycline was added at T = 0, whereas in the right-hand
graph, cells had been grown in the presence of doxycycline for 1 wk before T =
0. (B) Flow cytometry profiles for Nek2A-K37R-myc-His cells induced with
tetracycline for 0, 72, and 192 h. The positions of 2N and 4N DNA are
indicated. (C) Percentage of cells in each phase of the cell cycle was
calculated from the flow cytometry profiles shown in B.