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First published online 27 February 2008
doi: 10.1242/dev.019075
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Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology (EPFL), Lausanne, Switzerland.
* Author for correspondence (e-mail: pierre.gonczy{at}epfl.ch)
Accepted 31 January 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: C. elegans, Embryo, Polo-like kinase, Asymmetry, Cell division timing
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
stg mutant embryos arrest at the G2/M transition of cycle 14, whereas
stg overexpression in interphase of cycle 14 leads to premature entry
into mitosis and subsequent patterning defects
(Edgar and O'Farrell, 1990).
In Xenopus, both the Wee1 kinase and the Cdc25C phosphatase are
essential for entry into mitosis and alterations in their levels result in
morphogenesis defects (Murakami et al.,
2004). Whereas these examples illustrate the importance of
coupling cell division timing and embryonic development, in general the
mechanisms underlying such coupling remain incompletely understood.
In C. elegans, a difference in cell cycle timing is apparent
already in two-cell stage embryos, when the larger anterior blastomere AB
divides 
2 minutes earlier than the smaller posterior blastomere
P1. As in early embryos of many species, the cell cycle is rapid
and alternates between S and M phases in early embryos of C. elegans.
Thus, the 2 minutes time difference correspond to 15% of the entire
duration of the AB cell cycle. The asynchrony between AB and P1 is
entirely under the control of A-P polarity cues that are established by six
PAR proteins and associated components, in concert with the actomyosin network
(Aceto et al., 2006;
Etemad-Moghadam et al., 1995;
Guo and Kemphues, 1995;
Guo and Kemphues, 1996;
Levitan et al., 1994;
Morton et al., 2002;
Tabuse et al., 1998;
Watts et al., 1996;
Watts et al., 2000). When A-P
polarity cues are defective, as for example in par-2 or
par-3 mutant embryos, the first division is equal and the two
resulting daughter cells enter mitosis in synchrony
(Kemphues et al., 1988).
How A-P polarity cues control the asynchrony between AB and P1
is only partially understood. An aspect of the answer lies in an
ATL-1/CHK-1-dependent checkpoint that is activated preferentially in
P1, thus retarding entry into mitosis more in that blastomere and
explaining 40% of the time difference
(Brauchle et al., 2003).
Preferential checkpoint activation is largely abrogated in embryos that have
normal A-P polarity, but which undergo an equal first cleavage following
defective spindle positioning (Brauchle et
al., 2003). These observations suggest that ATL-1/CHK-1 activation
normally occurs preferentially in P1 because this smaller
blastomere inherits less of a rate-limiting component for DNA replication
following unequal first cleavage (Brauchle
et al., 2003). These findings indicate also that there must be an
ATL-1/CHK-1 independent and size-independent mechanism that regulates the
remaining 60% time difference. Compatible with the existence of a
size-independent mechanism, microsurgery experiments established that AB
enters mitosis earlier even if it becomes smaller than P1 because
of the removal of cytoplasmic material
(Schierenberg and Wood,
1985).
One attractive candidate for regulating the remaining time difference is
the polo-like kinase PLK-1. First, Polo-like kinases 1 (Plk1) promote mitotic
entry in several species (Abrieu et al.,
1998; Lane and Nigg,
1996; Lenart et al.,
2007; Sumara et al.,
2004). Plk-1 is thought to act at this cell cycle transition by
phosphorylating the Myt1 kinase and the Cdc25 phosphatase. As a result, Myt1
is inactivated and Cdc25 is activated, which together promotes activation of
Cdk1 and entry into mitosis (Inoue and
Sagata, 2005; Kumagai and
Dunphy, 1996; Qian et al.,
2001; Roshak et al.,
2000). In C. elegans, PLK-1 is also essential for M phase
entry. Oocytes in plk-1(RNAi) animals fail to undergo
nuclear envelope breakdown (NEDB) and do not complete the meiotic divisions
(Chase et al., 2000). As
anticipated, this phenotype is identical to that observed after RNAi-mediated
inactivation of NCC-1, the Cdk1 homolog acting during the meiotic and the
mitotic divisions in early C. elegans development
(Boxem et al., 1999). A second
reason for considering PLK-1 as an attractive candidate for regulating the
remaining time difference between AB and P1 is that examination of
published data reporting the distribution of PLK-1 using N-terminal peptide
antibodies (Chase et al., 2000)
indicates that PLK-1 is more abundant in AB than in P1. Therefore,
we set out to investigate whether PLK-1 plays a role in regulating the
asynchrony between AB and P1 by promoting entry into mitosis
preferentially in AB.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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),
GFP-PLK-1 (Leidel and Gönczy,
2003) or GFP-HIS-11 (Histone 2B)
(Praitis et al., 2001) were
grown at 24°C. Mutants of genotypes par-3(it71)
unc-32(e189)/qC1 (Kemphues et
al., 1988), par-1(it51) rol-4(sc8)/DnT1
(Guo and Kemphues, 1995) and
pie-1(zu154) unc-25(e156)/qC1
(Tenenhaus et al., 1998) were
grown at 20°C, whereas temperature-sensitive
par-4(it57ts) mutants
(Morton et al., 1992) were
grown at 15°C and shifted to 25°C for 24 hours before analysis.
The atl-1(tm853) deletion allele was obtained from the
National Bioresource Project, Japan. We balanced the mutation using
nT1[GFP-MYO-2 unc-56] and genotyped the animals by PCR using
atl-1 specific primers. atl-1(tm853) is probably a
null allele because it harbors a 720 bp deletion that creates a frameshift and
results in a premature stop codon
(Garcia-Muse and Boulton,
2005). We found that homozyous mutant
atl-1(tm853) animals are fertile, but give rise only to dead
embryos, as reported previously
(Garcia-Muse and Boulton,
2005).
RNAi-mediated inactivation using bacterial feeding strain of plk-1
(covering nucleotides 890 to 1887 of the plk-1 cDNA) were performed
as follows: for examining the specificity of PLK-1 C-terminal antibodies
(Fig. 1B,G), L4 larvae were
selected and feeding performed for 15 hours at 20°C; for partial depletion
to measure PLK-1 ratio in AB versus P1
(Fig. 2E,F), young adult worms
were selected and feeding performed for 8 hours at 20°C; for mild
inactivation whereby entry into mitosis is delayed only in P1
(Fig. 6B,
Fig. 7G; see Fig. S1B,D in the
supplementary material), young adult worms were selected and feeding performed
for 6 hours at 20°C. Other RNAi-mediated inactivation experiments were
performed by selecting L4 larvae and feeding them as follows:
par-5(RNAi), par-2(RNAi) and
zen-4(RNAi), 24 hours at 20°C;
div-1(RNAi) (Brauchle et
al., 2003), 26 hours at 20°C; tba-2(RNAi)
(Sonneville and Gönczy,
2004) and nmy-2(RNAi)
(Guo and Kemphues, 1995), 24
hours at 24°C; and goa-1/gpa-16(RNAi)
(Colombo et al., 2003), 48
hours at 20°C. Bacterial RNAi feeding strains for par-2 and
zen-4 were from the Ahringer collection
(Kamath et al., 2003) and that
for par-5 from the Vidal collection
(Rual et al., 2004).
PLK-1 C-terminal antibodies
The last 159 bp of the plk-1-coding region were cloned into
pGEX-6P-2 (Amersham Biosciences). The resulting fusion protein was expressed,
column-purified using Glutathione Sepharose 4 Fast Flow resin (Amersham
Biosciences), eluted with 20 mM reduced glutathione pH 7.5, dialyzed and
injected into a rabbit (Eurogentec). For affinity purification, the same
fragment was cloned into pMAL-p2E (NEB). The resulting fusion protein was
expressed, column-purified using amylose resin (NEB), eluted with 20 mM
maltose, dialyzed and coupled to affigel resin (BioRad). The serum was
purified on this column, eluted with 0.2 M glycine pH 2.2 in 0.5 M NaCl,
dialyzed against PBS containing 8.7% glycerol and stored at -20°C in 50%
glycerol. The final concentration of PLK-1 antibodies is 1 µg/µl.
SDS-PAGE and western blot analyses were carried out according to standard
procedures. Rabbit anti-PLK-1 C terminal and mouse anti-
tubulin
antibodies were used at 1/1000 and the HRP-conjugated goat anti-rabbit and
anti-mouse secondary antibodies (Promega) at 1/5000; the signal was revealed
with standard chemiluminesence (Roche).
Indirect immunofluorescence
Fixation and indirect immunofluorescence were performed essentially as
described (Gönczy et al.,
1999). The following primary mouse antibodies were used:
-tubulin (DM1A, Sigma; 1/200) and GFP (MAB3580, Chemicon; 1/200, to
detect GFP-PAR-2). The following primary rabbit antibodies were used: PLK-1
C-terminal fusion protein (1/500; this study), GFP (a gift from Viesturs
Simanis; 1/300, to detect GFP-PLK-1), NCC-1 (a gift from Andy Golden; 1/1000)
and phospho-specific tyrosine 15 (pT15) antibody to detect NCC-1
phosphorylation (Calbiochem, 1/100). Secondary antibodies were goat anti-mouse
coupled to Alexa 488 (1/500) and goat anti-rabbit coupled to Alexa 568
(1/1000) (Molecular Probes). Slides were counterstained with 1 µg/ml
Hoechst 33258 (Sigma) to reveal DNA. Images were taken on a LSM510 Zeiss
confocal microscope. For quantification of the ratio of PLK-1 in AB versus
P1, images were taken in the linear intensity range with a
63x lens on a Zeiss Axioplan 2 microscope equipped with a 12-bit
Diagnostic Instrument Spot RT cooled CCD camera and analyzed using MetaMorph
software. For pT15 staining, 20 confocal sections encompassing the AB and
P1 nuclei were taken every 0.45 µm with a 63x objective
and the maximum intensity projection was constructed using ImageJ.
FRAP experiments and image analysis
FRAP was performed on an LSM510 Meta Zeiss confocal microscope, acquiring
one image every 10 seconds at each time point using a 63x lens, with
zoom 2, average 2 and unidirectional scanning of 512x512 pixels at 9%
laser power, resulting in acquisition times of 800 mseconds. For
photobleaching, the whole anterior or posterior half of a metaphase one-cell
stage GFP-PLK-1 embryo was bleached with 30 iterations at 100% laser power.
The data were analyzed using ImageJ by measuring the average intensity of the
anterior or posterior cytoplasmic area, excluding centrosomes and
kinetochores, whose frequent movements could generate measurement errors. The
intensity in the embryo anterior in the five images before photobleaching was
averaged and assigned a value of 1, whereas the intensity in the bleached area
just after photobleaching was assigned a value of 0.
Time-lapse microscopy
Embryos were analyzed by time-lapse DIC microscopy at 23°C as
described (Gönczy et al.,
1999). The timing of cytokinesis in P0 (measured at the
time of cleavage furrow initiation) as well as that of NEBD in AB and
P1 (measured at the time of nuclear membrane disappearance) were
determined. The time separating cytokinesis in P0 from NEBD in
either AB or P1 corresponds to interphase.
Embryos expressing GFP-Histone 2B were imaged using dual time-lapse
fluorescent and DIC microscopy as described
(Brauchle et al., 2003), and
the timing of the metaphase to anaphase transition determined in
P0, AB and P1.
Quantification of PLK-1 levels
PLK-1 levels were quantified in fixed wild-type,
par-2(RNAi) and par-3(it71) embryos. To
obtain reliable values, we normalized cytoplasmic PLK-1 levels with respect to
centrosomal PLK-1 levels in the same embryo. Moreover, the analysis was
conducted in metaphase and early anaphase one-cell stage embryos because the
centrosomal signal is strong and reproducible at these stages, and because
anterior and posterior levels can be quantified simultaneously in the same
cell. As PLK-1 is distributed symmetrically in par-2(RNAi)
and par-3(it71) embryos, PLK-1 levels in the anterior and
the posterior were averaged in these cases. Embryos which were partially
stained or subjected to incomplete RNAi-mediated inactivation of
par-2 were not considered.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) reflects bona fide PLK-1 distribution, and not merely that
of a particular epitope, we raised and affinity-purified polyclonal antibodies
against a C-terminal fragment of the protein
(Fig. 1A). These antibodies
recognize a single band at the expected size of 70 kDa in wild-type
embryonic extracts by western blot analysis
(Fig. 1B, lane 1). This band is
significantly reduced in plk-1(RNAi) embryonic extracts,
demonstrating specificity (Fig.
1B, lane 2). We used these antibodies to examine PLK-1
distribution in early C. elegans embryos. As shown in
Fig. 1C, we found that PLK-1 is
distributed in a uniform manner in the cytoplasm just after meiotic exit.
PLK-1 distribution becomes asymmetric shortly thereafter, with more protein
present on the anterior side of the embryo
(Fig. 1D). This asymmetry
persists throughout the one-cell stage and until the end of the two-cell stage
(Fig. 1E,F; data not shown).
Line scans quantifying pixel intensities confirm these observations
(Fig. 1C-F). These antibodies
also label centrosomes (Fig.
1E, arrowheads), the midbody
(Fig. 1E,F, arrows) and
kinetochores (data not shown), as reported previously
(Chase et al., 2000). The
signals detected by these antibodies in wild-type embryos are absent from
embryos depleted of PLK-1 by RNAi (Fig.
1G), demonstrating specificity. We found an analogous distribution
using antibodies directed against GFP in transgenic animals expressing
GFP-PLK-1, although the asymmetry is somewhat less pronounced in this case
(Fig. 1H).
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Achieving PLK-1 asymmetry
We next set out to investigate how PLK-1 asymmetry is achieved. We first
addressed whether the underlying mechanism is active solely in the one-cell
stage and results in asymmetry at the two-cell stage merely because the first
cleavage separates the anterior and posterior pools of PLK-1. Alternatively,
the mechanism at the root of PLK-1 asymmetry could be active also during the
second cell cycle. To distinguish between these possibilities, we examined
PLK-1 asymmetry at the second cell cycle in zen-4(RNAi)
embryos, which are defective in cytokinesis
(Raich et al., 1998). As shown
in Fig. 2B, PLK-1 asymmetry is
still apparent in such embryos, demonstrating that PLK-1 asymmetry is
maintained during the second cell cycle.
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-tubulin using tba-2(RNAi)
(Sonneville and Gönczy,
2004). We found that PLK-1 asymmetry is still apparent and even
slightly accentuated in tba-2(RNAi) embryos
(Fig. 2C), indicating that
microtubules are dispensable for PLK-1 asymmetry. To examine the role of the
actomyosin network, we depleted the non-muscle myosin II NMY-2
(Guo and Kemphues, 1995). As
shown in Fig. 2D, PLK-1
asymmetry is abolished in nmy-2(RNAi) embryos, indicating
that the actomyosin network is required, directly or indirectly, for PLK-1
asymmetry.
A fraction of PLK-1 is retained at the embryo anterior
Next, we considered whether PLK-1 distribution is asymmetric because a
fraction of the protein is retained in the embryo anterior. To test this
possibility, we designed a Fluorescence Recovery After Photobleaching (FRAP)
experiment to probe the mobility of the posterior and anterior pools of PLK-1.
To probe the mobility of GFP-PLK-1 located in the embryo posterior, we
photobleached the anterior half of the embryo and assayed fluorescence
intensity over time in both posterior and anterior halves
(Fig. 3A,B). We found that the
posterior pool of GFP-PLK-1 moves readily into the anterior half, such that
fluorescence intensity in the two halves equilibrates 90 seconds after
photobleaching (Fig. 3B).
Conversely, to probe the mobility of GFP-PLK-1 located into the anterior, we
photobleached the posterior half of the embryo and similarly assayed
fluorescence intensity (Fig.
3C,D). Importantly, we found in this case that even though the
bulk of the anterior pool of GFP-PLK-1 moves readily into the posterior half,
a fraction of GFP-PLK-1 is retained in the embryo anterior, such that
fluorescence intensity in the two halves remains different at 90 seconds after
photobleaching and thereafter (Fig.
3D).
As PLK-1 can be retained in the embryo anterior, we reasoned that the extent of asymmetry may increase when overall PLK-1 levels are diminished, because the remaining protein would be preferentially retained in the anterior, leaving less protein left for the posterior. To test this possibility, we compared the ratio of PLK-1 in AB versus P1 in the wild type with that observed after partial RNAi-mediated inactivation of PLK-1. We found this ratio to be 1.52±0.05 in the wild-type (Fig. 2E,G) and 1.97±0.13 in embryos partially depleted of PLK-1 (Fig. 2F,G). Although more complex mechanisms can be envisaged, this increase in ratio, together with the outcome of the FRAP experiment, is compatible with a model in which PLK-1 asymmetry is achieved through protein retention in the embryo anterior.
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), if PLK-1 were to play a role in controlling this time
difference, then PLK-1 asymmetry should be regulated by polarity cues. To
address this possibility, we first examined when PLK-1 becomes asymmetric with
respect to establishment of A-P polarity. Using GFP-PAR-2 as a polarity
marker, we found that PLK-1 distribution becomes asymmetric shortly after
polarity establishment (Fig.
4A,B), compatible with PLK-1 asymmetry being regulated by A-P
polarity cues. To test whether this is the case, we examined PLK-1
distribution in embryos with impaired PAR-1, PAR-2, PAR-3, PAR-4 or PAR-5
function. Importantly, we found that PLK-1 distribution is symmetric in such
embryos (Fig. 4D-H). Similarly,
PLK-1 distribution is symmetric in pkc-3(RNAi) and
mex-5/6(RNAi) embryos, which also have defective A-P
polarity (Cuenca et al., 2003;
Tabuse et al., 1998) (data not
shown). Therefore, we conclude that PLK-1 asymmetry is regulated by A-P
polarity cues.
We then investigated whether polarity cues regulate PLK-1 asymmetry
separately from asymmetric spindle positioning and cell fate determination,
both of which are also controlled by A-P polarity (reviewed by
Gönczy and Rose, 2005).
To this end, we examined goa-1/gpa-16(RNAi)
embryos, in which the first division is equal as a result of symmetric spindle
positioning (Colombo et al.,
2003; Gotta and Ahringer,
2001), as well as pie-1(zu154) mutant embryos,
in which the fate of the P1 blastomere is altered
(Tenenhaus et al., 1998;
Tenenhaus et al., 2001). We
found that PLK-1 distribution is asymmetric in such embryos
(Fig. 4I,J), indicating that
A-P polarity cues regulate PLK-1 asymmetry separately from spindle positioning
and cell fate determination.
PLK-1 levels correlate with the timing of mitotic entry upon PAR-2 and PAR-3 inactivation
We set out to test whether PLK-1 levels are important for dictating the
timing of mitosis onset. We reasoned that an analysis of PLK-1 levels and
mitotic timing in embryos depleted of PAR-2 and PAR-3 may shed light on this
issue. In the absence of PAR-2, both blastomeres at the two-cell stage
resemble AB in terms of spindle orientiation and cortical marker distribution
(Boyd et al., 1996;
Kemphues et al., 1988).
Conversely, in the absence of PAR-3, both blastomeres resemble P1
in these respects (Etemad-Moghadam et al.,
1995; Kemphues et al.,
1988). Accordingly, we found that the timing of mitotic entry of
both blastomeres in par-2(RNAi) embryos resembles most that
of AB in the wild type, whereas the timing of both blastomeres in
par-3(it71) mutant embryos is similar to that of
P1 in the wild-type (Fig.
5A-F; see Table S1 and Movies 1-4 in the supplementary material).
Quantification of fixed specimens established that PLK-1 levels in
par-2(RNAi) embryos are similar to those in the anterior of
wild-type embryos, whereas those in par-3(it71) embryos are
similar to those in the posterior of wild-type embryos
(Fig. 5G). We conclude that
levels of PLK-1 correlate with the timing of mitotic entry upon depletion of
PAR-2 and PAR-3.
PLK-1 asymmetry contributes to asynchronous entry into mitosis in AB and P1
We next set out to address whether the asymmetric distribution of PLK-1 is
important for asynchronous division of AB and P1. Strong
RNAi-mediated inactivation of plk-1 prevents completion of the
meiotic divisions (Chase et al.,
2000) and conditional mutant alleles are not available, precluding
an analysis in two-cell stage embryos using strong reduction of function
conditions. Thus, we performed partial plk-1(RNAi) and
analyzed cell cycle progression using time-lapse DIC microscopy. We found that
mitotic entry in partial plk-1(RNAi) embryos is delayed
somewhat in AB, but more extensively in P1, resulting in a
significant increase in the ratio of the duration of interphase in
P1 over the duration of interphase in AB (RI) (see Tables S1 and S2
in the supplementary material). Although this observation indicates that PLK-1
levels are crucial for timely entry into mitosis, chromosomes are often
mis-segregated in these partial plk-1(RNAi) embryos, which
in theory could contribute to altering cell cycle progression. Therefore, we
conducted yet milder RNAi-mediated inactivation of plk-1, such that
chromosome segregation defects are not observed. Importantly, we found that
whereas the timing of cell cycle progression in AB is not affected in these
mild plk-1(RNAi) embryos, entry into mitosis is delayed in
P1 (Fig. 6A-B; see
Table S1, Movies 5 and 6 in the supplementary material). As a result, RI is
increased compared with the wild type (Fig.
6E; see Table S2 in the supplementary material). To corroborate
these observations, we analyzed the timing of events in embryos expressing
GFP-Histone 2B to monitor precisely the metaphase to anaphase transition. We
found again that whereas cell cycle duration is not perturbed in AB in mild
plk-1(RNAi) embryos, a delay is observed in P1
(see Fig. S1A,B, and Movies 11 and 12 in the supplementary material),
resulting in an increased ratio of cell cycle duration in P1 over
that in AB (see Fig. S1E and Table S3 in the supplementary material). As
P1 has lower levels of PLK-1 than AB in the wild type, it is
presumably more sensitive to PLK-1 diminution, accounting for the observed
increased asynchrony upon mild RNAi-mediated depletion. Taken together, these
results suggest that PLK-1 levels are important for timing mitotic entry and
that PLK-1 asymmetry contributes to the asynchronous division of AB and
P1.
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), but is
activated first in AB, as evidenced by the fact that phospho-specific
antibodies that recognize the inactive form of NCC-1 disappear first from AB
(see Fig. S2D-F in the supplementary material)
(Hachet et al., 2007). As
anticipated, we found that entry into mitosis in mild
ncc-1(RNAi) embryos is delayed to a larger extent in
P1 than in AB (Fig.
6C; see Table S1 and Movie 7 in the supplementary material),
resulting in an increased RI compared with the wild type
(Fig. 6E; see Table S2 in the
supplementary material), reminiscent of the increase observed in mild
plk-1(RNAi) embryos. Even though in principle factors other
than PLK-1 could be responsible for differential activation of NCC-1, this
phenotypic similarity is compatible with the notion that PLK-1 asymmetry
results in differential NCC-1 activation.
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). Although cell cycle progression is considerably slower
overall than in the wild-type, we found that mitotic entry is delayed to the
same extent in AB and P1 in atp-2(RNAi) embryos
(Fig. 6D; see Table S1 and
Movie 8 in the supplementary material), such that RI is indistinguishable from
the wild type (Fig. 6E; see
Table S2 in the supplementary material). Taken together, these results
indicate that mild depletion of PLK-1 or of the downstream Cdk1 kinase NCC-1,
but not any delay in cell cycle progression, retards entry into mitosis
preferentially in P1.
PLK-1-mediated differential timing is independent of ATL-1
As differential timing in AB and P1 is known to rely in part on
preferential retardation of mitotic entry in P1 through activation
of an ATL-1/CHK-1 dependent checkpoint
(Brauchle et al., 2003), we
investigated whether the impact of PLK-1 depends on this checkpoint.
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), as
well as in div-1(RNAi) embryos, which engage this checkpoint
more than in the wild type owing to defective DNA replication and thus exhibit
increased RI (Brauchle et al.,
2003; Encalada et al.,
2000). If ATL-1/CHK-1 regulates PLK-1 asymmetry, then the ratio of
PLK-1 in AB versus P1 should decrease in
atl-1(tm853) embryos and increase in
div-1(RNAi) embryos. We found instead that PLK-1 asymmetry
is indistinguishable from the wild type in both cases
(Fig. 7A-D), demonstrating that
alterations in checkpoint signaling do not affect PLK-1 asymmetry. To address unambiguously whether PLK-1 functions independently of ATL-1/CHK-1, we compared atl-1(tm853) embryos with atl-1(tm853) embryos treated with mild plk-1(RNAi). If the impact of PLK-1 on cell cycle progression requires ATL-1/CHK-1 function, then mild plk-1(RNAi) should not alter the timing of events observed in atl-1(tm853) embryos. In contrast to this prediction, however, we found that mild plk-1(RNAi) treatment of atl-1(tm853) embryos retards entry into mitosis in P1 and thus increases RI compared with atl-1(tm853) mutant embryos (Fig. 7G,H; see Table S2 and Movie 10 in the supplementary material), as expected if the impact of PLK-1 is independent of ATL-1/CHK-1. To corroborate these observations, we conducted analogous experiments in embryos expressing GFP-Histone 2B. As reported in Fig. S1C-E in the supplementary material, these experiments similarly established that mild plk-1(RNAi) in atl-1(tm853) mutant embryos retards entry into mitosis in P1 and thus increases RI compared with atl-1(tm853) mutant embryos (see Table S3, and Movies 13 and 14 in the supplementary material).
Taken together, these results establish that the impact of PLK-1 asymmetry on differential timing of mitotic entry in AB and in P1 does not invoke ATL-1/CHK-1.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Mechanisms underlying PLK-1 asymmetry
How is PLK-1 asymmetry established? In principle, asymmetry could result
from plk-1 mRNA being enriched at the embryo anterior. This does not
appear to be the case, however, because mRNA localization is typically
mediated through sequences in the 3' UTR
(Evans et al., 1994;
Ogura et al., 2003), which are
absent from the gfp-plk-1 construct that nevertheless results in
anterior enrichment (Leidel and
Gönczy, 2003). Accordingly, in situ hybridization indicates
that plk-1 mRNA is distributed uniformly in two-cell stage embryos
(http://nematode.lab.nig.ac.jp/db2/ShowGeneInfo.php?celk=CELK02520).
Therefore, PLK-1 asymmetry is regulated at the protein level. Although other
mechanisms can be envisaged, including preferential degradation in the embryo
posterior, our findings taken together support a model in which PLK-1
asymmetry is established through preferential PLK-1 protein retention in the
embryo anterior.
It will be interesting to investigate the molecular underpinning of PLK-1
anterior retention. Three putative PKC-3 phosphorylation sites are present in
PLK-1 (Y.B. and P.G., unpublished) and perhaps phosphorylation of PLK-1 by
anteriorly localized PKC-3 contributes to PLK-1 asymmetry. Another possibility
is suggested by the observation that PLK-1 distribution is uniform in
nmy-2(RNAi) embryos. Although this may be an indirect
consequence from the polarity defects known to arise following NMY-2 depletion
(Cuenca et al., 2003;
Guo and Kemphues, 1996), the
requirement for NMY-2 in PLK-1 localization could be more direct. NMY-2 is
enriched on the anterior cortex of one- and two-cell stage embryos
(Munro et al., 2004), and may
help enrich PLK-1 in the vicinity of a structure or complex that promotes
anterior retention. In Drosophila, the non-muscle myosin II Zipper is
needed for asymmetric localization of Miranda to the basal side of neuroblasts
(Barros et al., 2003).
|
). Here, we
demonstrate in addition that levels of PLK-1 are important for dictating the
timing of mitotic entry. Indeed, partial depletion of PLK-1 delays mitotic
entry in AB and more so in P1, whereas milder depletion of PLK-1
delays mitotic entry merely in P1.
Where in the cell are the substrates that are phosphorylated by PLK-1 to
promote entry into mitosis? Such substrates may reside in the cytoplasm, where
PLK-1 enrichment is most apparent. Alternatively, as centrosomes are important
for dictating the timing of mitosis in C. elegans
(Hachet et al., 2007), and as
PLK-1 is present at centrosomes, perhaps it is centrosomal PLK-1 that is
relevant for mitotic timing. Because centrosomal PLK-1 readily exchanges with
PLK-1 in the cytoplasm (Leidel and
Gönczy, 2003), an excess of cytoplasmic PLK-1 in the anterior
may enhance PLK-1 loading at centrosomes in AB compared with P1.
Accordingly, levels of centrosomal PLK-1 increase during the cell cycle and
are high in AB earlier than in P1 (Y.B. and P.G., unpublished).
Coupling polarity cues and cell cycle progression
Our findings taken together support a model in which two mechanisms
together ensure coupling between A-P polarity cues and cell cycle progression
in two-cell stage C. elegans embryos
(Fig. 7I). The first mechanism
entails preferential activation of ATL-1/CHK-1 in the P1 blastomere
and contributes to asynchrony by retarding entry into mitosis in P1
more than in AB (Brauchle et al.,
2003). Such preferential checkpoint activation may result from the
unequal first cleavage, as preferential activation is largely abrogated in
embryos that divide symmetrically. Thus, A-P polarity cues regulate this first
mechanism by ensuring asymmetric spindle positioning and unequal cleavage.
These earlier findings predicted the existence of another mechanism, also
regulated by A-P polarity cues, but independent of size difference and of
ATL-1/CHK-1.
Our present work indicates that this second mechanism entails PLK-1
asymmetry, which promotes entry into mitosis in AB earlier than in
P1. The evidence supporting such a mechanism can be summarized as
follows. First, PLK-1 is distributed asymmetrically, with more protein present
in AB than in P1. Second, PLK-1 asymmetry is regulated by A-P
polarity cues, independently of cell size, as PLK-1 is still asymmetric in
goa-1/gpa-16(RNAi) embryos. Third, the time
difference between AB and P1 increases when PLK-1 is depleted, and
this occurs in an ATL-1/CHK-1 independent manner. Interestingly, Polo is also
asymmetrically distributed in the germarium of Drosophila, where it
promotes entry into meiosis and oocyte determination
(Mirouse et al., 2006).
Furthermore, in Drosophila neuroblasts, Polo phosphorylates Pon and
thus ensures efficient segregation of the fate determinant Numb during
asymmetric cell division (Wang et al.,
2007). Together with our own work, these findings indicate that
the evolutionarily conserved Polo-like kinases may broadly serve to couple
cell cycle timing and developmental decisions.
Note added in proof
While this manuscript was under review, another study reported that PLK-1
also regulates MEX-5/6 function (Nishi et
al., 2008).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/7/1303/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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