1 Department of Biophysics and Biochemistry, Graduate School of Science,
University of Tokyo, Hongo, Tokyo 113-0033, Japan
2 PRESTO, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012,
Japan
3 Laboratory for Developmental Genomics, RIKEN Center for Developmental Biology,
Kobe, Hyogo 650-0047, Japan
* Author for correspondence (e-mail: myamamot{at}ims.u-tokyo.ac.jp )
Accepted 6 January 2002
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Summary |
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Key words: C. elegans, PP4, Centrosome, -Tubulin, Spindle assembly, Chiasma, Meiosis
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Introduction |
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Protein phosphatase 4 (PP4; alternatively called PPP4, or PPX) is one of
the few phosphatases that localize to centrosomes
(Brewis et al., 1993). PP4
belongs to the PPP family of protein serine/threonine phosphatases, which
includes PP1, PP2A, PP2B and PP5 (Cohen,
1997
). In mammalian cells and Drosophila embryos, PP4
predominantly localizes to PCM (Brewis et
al., 1993
; Helps et al.,
1998
). PP4 has been implicated in the regulation of microtubule
growth or its organization at centrosomes, because centrosomes with no
attached polar microtubules were often observed in Drosophila mutant
embryos that have a reduced amount of PP4 protein
(Helps et al., 1998
).
Furthermore, the amount of
-tubulin at centrosomes is significantly
reduced in these mutant embryos (Helps et
al., 1998
). However, in vivo function of PP4 has not been
clarified in other organisms, and it is not known whether the biological
function of PP4 is conserved among species.
The nematode Caenorhabditis elegans is an excellent experimental
system for centrosome research. The early blastomeres are large, and the
centrosomes and nuclei are visible by differential interference contrast
(Nomarski) microscopy. In addition, C. elegans embryos undergo
virtually invariant cell divisions, including the timing of the events of each
cell cycle, throughout development. Availability of the complete genome
sequence (The C. elegans
Sequencing Consortium, 1998) as well as development of the
RNA-mediated interference (RNAi) technique
(Fire et al., 1998
) to
knock-out gene functions enables functional analysis of evolutionarily
conserved genes. Making use of these advantages, we characterized the function
of PP4 in C. elegans in mitosis and meiosis.
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Materials and Methods |
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Identification of pph-4.1 and pph-4.2
A BLAST search of the C. elegans genome sequence identified two
open reading frames (ORFs) with homology to PP4, namely Y75B8A.30 and
Y49E10.3. We call them pph-4.1 and pph-4.2, respectively, in
this article. Two cDNA clones yk505c6 and yk373h6 corresponding to
pph-4.1, and two cDNA clones yk241a8 and yk655g3 corresponding to
pph-4.2 were supplied by Yuji Kohara (National Institute of Genetics,
Mishima, Japan). Nucleotide sequencing of the cDNA clones confirmed the
predicted coding sequences of these genes. The phylogenic tree was composed
using the CLUSTALW multiple sequence alignment.
RNA interference
RNA-mediated interference was performed as previously described
(Fire et al., 1998). The
following cDNA clones and fragments were used as templates to prepare
double-stranded RNA (dsRNA): yk505c6 and yk373h6 for pph-4.1, yk241a8
for pph-4.2, and PCR-amplified fragment from a C. elegans
cDNA library (Hayashizaki et al.,
1998
) for ncl-1 (used as a control for RNAi).
Double-stranded RNA, 3 µg/µl, was microinjected into the gonad
or the intestine of wild-type, fem-1 (hc17ts) and fem-2
(b245ts) young adult hermaphrodites. Their progenies (F1 generation),
collected 4-24 hours after the injection, were examined for phenotypes.
Because the F2 generation showed a higher penetrance of the embryonic
lethality, we used F2 embryos from fertile pph-4.1 (RNAi) F1 adults
for characterization of early embryonic phenotypes. For analysis of paternal
effects, wild-type hermaphrodites were injected with dsRNA and mated with
wild-type males to generate males in F1 generation. The rde-1(ne219)
hermaphrodites were mated with F1 males and the cross-progeny was examined for
phenotypes. For generating pph-4.1(m-/p+) embryos,
fem-1(hc17ts) or fem-2(b245ts) were grown at 15°C and
injected with pph-4.1 RNA. The F1 progeny were grown at 25°C to
adults and mated with wild-type males. Resulting cross-progeny were examined
for phenotypes.
Observation of spermatid and spermatocytes
Spermatids and spermatocytes were dissected from males in sperm medium (SM)
(5 mM HEPES (pH 7.8) with 50 mM NaCl, 25 mM KCl, 5 mM CaCl2 and 1
mM MgSO4) (Ward et al.,
1981) containing 1 µg/ml Hoechst 33342 dye (Sigma). Samples
were mounted onto a slide glass and examined with Nomarski and fluorescence
optics to visualize cells and their nuclei. To avoid abnormal spermatogenesis,
observation was carried out within 5 minutes after the dissection.
Generation of anti-PPH-4.1 antibodies and western blotting
A fragment of pph-4.1 cDNA corresponding to the first 197 amino
acids was cloned into pGEX-KG vector (Pharmacia) to create a GST-PPH-4.1
fusion construct. The fusion protein was expressed in Escherichia
coli and purified using a glutathione column (Qiagen). Antibodies against
this protein were raised in rabbits (Sawady Technologies). PPH-4.1-specific
antibodies were affinity purified against 10xHis-PPH-4.1 protein as
follows. To create a 10xHis-PPH-4.1 fusion construct, a fragment of
pph-4.1 cDNA containing coding sequence of full-length PPH-4.1
protein was cloned into pET19b vector (Novagen). The fusion protein was
expressed in E. coli and purified by using a nickel NTA
(nitro-tri-acetic acid) agarose (Qiagen). The His-PPH-4.1 fusion protein
obtained was used for blot affinity purification of the antibodies. For
western blotting, SDS-soluble total nematode extracts were run on a 10%
polyacrylamide gel, blotted and then probed with 1:500 affinity-purified
anti-PPH-4.1 antibodies. For an immunodepletion experiment, 1:500 diluted
anti-PPH-4.1 antibodies were preincubated with the membrane blotted with 1 mg
of 10xHis-PPH-4.1 fusion protein or bovine serum albumin as a
control.
Immunofluorescence and DAPI staining
Embryos were processed for staining as described previously
(Miller and Shakes, 1995).
Briefly, embryos permeabilized by the freezecrack method were fixed by placing
in methanol for 5 minutes at room temperature. Rehydrated embryos were treated
with blocking solution [1% skim milk, 5% fetal bovine serum in PBST (phosphate
buffered saline containing 0.5% Tween-20)] for 30 minutes at room temperature,
and incubated with the primary antibody overnight at 4°C and then with the
secondary antibody for 1-2 hours at room temperature. DAPI was added to a
final concentration of 2 µg/ml, and the sample was mounted for
epifluorescence microscopy. Spermatocytes were processed for immunostaining as
described previously (Varkey et al.,
1995
), except that permeabilization with PBST was carried out for
30 minutes.
Antibodies used were: anti--tubulin antibody DM1A (Sigma),
anti-
-tubulin antibody (Bobinnec et
al., 2000
) provided by Yves Bobinnec and Eisuke Nishida (Graduate
School of Biostudies, Kyoto University, Kyoto, Japan), anti-PLK-1 antibody
(Chase et al., 2000
) provided
by Andy Golden (National Cancer Institute, Frederick, MD, USA), anti-PPH-4.1
antibody, FITC-conjugated sheep anti-mouse IgG antibody (Organon Teknika), and
Cy3-labeled goat anti-rabbit IgG antibody (AP132C, Chemicon).
For DAPI staining of adult gonads, adult hermaphrodites were fixed and stained by ethanol with 2 µ/ml DAPI at room temperature for 5 minutes. Rehydrated samples were mounted for microscopy.
Microscopy
For confocal imaging, LSM510 system attached to Axioplan 2 microscope
(Zeiss) was used. Other images were taken digitally by any of the following
combinations: a CCD camera Quantix (Photometrics) attached to a Zeiss Axioplot
2 microscope with MetaMorph Imaging System Ver.4.5 (Universal Imaging
Corporation); a cooled CCD camera C4742-95-10NR (Hamamatsu Photonics) attached
to a Zeiss Axiophot microscope with Fish Imaging Software (Hamamatsu
Photonics); or a cooled CCD camera C5985 (Hamamatsu Photonics) attached to a
Zeiss Axioplan 2 microscope with NIH Image or Fish Imaging Software (Hamamatsu
Photonics).
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Results and Discussion |
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The function of PPH-4.1 and PPH-4.2 was inhibited by RNA-mediated interference (RNAi). Injection of C. elegans hermaphrodites with pph-4.1 dsRNA gave rise to dead embryos in F1 generation with low penetrance (58/292, 20%), and in F2 generation with higher penetrance (200/202, 99%). F1 larval lethality was also observed (41/234, 18%). However, pph-4.2(RNAi) resulted in F1 larval lethality with low penetration, but no embryonic lethality. Double knockout of pph-4.1 and pph-4.2 by RNAi did not enhance the embryonic lethal phenotype of pph-4.1(RNAi). These results suggest that both PPH-4.1 and PPH-4.2 may be required for the development of C. elegans, but PPH-4.1 apparently plays more significant roles, especially during embryogenesis. Therefore, we focused on PPH-4.1 and carried out further characterization.
PPH-4.1 is required for proper nuclear division during sperm
meiosis
Analyses with Nomarski microscopy and DAPI staining revealed that the
pph-4.1(RNAi) embryos arrested with various numbers of cells, showing
severe aneuploidy (data not shown). To determine the earliest defective event
in the pph-4.1(RNAi) embryos, early cell cycles were followed by
Nomarski microscopy (Fig. 2).
Polar bodies and the female pronucleus were always present, suggesting that
oocyte meiosis was completed. However, at the posterior end, where a male
pronucleus is normally produced (Albertson,
1984), either two pronuclei
(Fig. 2C) or no pronucleus was
often observed. In addition, the pph-4.1(RNAi) embryos often gave
rise to a tetrapolar spindle at the one-cell stage
(Fig. 2D). Generation of a
tetra-polar spindle did not necessarily correlate with the number of male
pronuclei, and was observed in embryos that had either one or two male
pronuclei. Normal bipolar spindles were observed in later divisions in these
embryos, suggesting that the duplication cycle of centrosomes was unaffected.
To examine whether the male pronuclei defect and the formation of tetra-polar
spindle were due to abnormality in sperm, pph-4.1(RNAi) males were
crossed with hermaphrodites in which the expression of PPH-4.1 is not
repressed. To exclude the effect of RNAi via sperm, we crossed
pph-4.1(RNAi) F1 males to rde-1(ne219) hermaphrodites, which
are not susceptible to RNAi (Tabara et
al., 1999
). Tetra-polar spindles were observed in 5/16 of the
cross progeny (Fig. 2E),
suggesting that loss of PPH-4.1 function results in aberrant sperm that
contain an abnormal number of nuclei or centrosomes, or both.
|
To characterize further the defect of the sperm, we directly observed
spermatids and spermatocytes isolated from pph-4.1(RNAi) animals
(Fig. 3). We found that a
significant portion of pph-4.1(RNAi) sperm contained an abnormal
number of nuclei (Fig. 3B): 30%
(33/109) of spermatids contained two or more nuclei, and 20% (22/109)
contained no nucleus. Every spermatid isolated from untreated wild-type worms
contained one nucleus. The aberrant sperm of pph-4.1(RNAi) appeared
to be produced by defective nuclear division during meiosis. In wild-type,
primary spermatocytes undergo the first meiotic division to form two secondary
spermatocytes that may or may not complete cytokinesis
(Ward et al., 1981). In
meiosis II, spermatids bud from the residual body and inherit one centrosome
and a haploid nucleus, together with other organelles and cytosolic
components, by asymmetric partitioning
(Fig. 3C,E)
(Ward et al., 1981
). In the
pph-4.1(RNAi) secondary spermatocytes, budding from residual bodies
and asymmetric segregation of cytoplasm appeared to occur normally. However,
in some spermatocytes, chromosomes were not properly separated by the time of
bud formation and remained in the residual body
(Fig. 3F). In some others,
multiple masses of chromosomes were segregated to one bud
(Fig. 3D). Thus, PPH-4.1
activity is apparently required for proper nuclear division during sperm
meiosis.
|
Loss of maternal PPH-4.1 causes delay in the progression of the first
mitotic division
In addition to the defective sperm formation described above, RNAi of
pph-4.1 caused delay in the progression of the first cell cycle of
the fertilized eggs. In wild-type, is takes about six minutes to proceed from
the meeting of pronuclei to the beginning of the anaphase B, which corresponds
to the completion of spindle formation. By contrast, it often took over 30
minutes in pph-4.1(RNAi) embryos
(Fig. 4). This delay was
observed irrespective of the number of spindle poles. We tested whether the
depletion of PPH-4.1 in sperm was responsible for the delay in spindle
formation. To obtain embryos that contained the paternal supply of PPH-4.1 but
not the maternal product, we crossed wild-type males with
pph-4.1(RNAi) F1 hermaphrodites. In the resulted cross-progeny
[pph-4.1(m-/p+)], spindle formation for the first cell division was
delayed as seen in pph-4.1(RNAi) embryos
(Fig. 4). Thus, we concluded
that maternal supply of PPH-4.1 is required for timely formation of the first
mitotic spindles.
|
Depletion of PPH-4.1 affect the spindle structure in both mitosis and
sperm meiosis
To examine the spindle structure in the absence of PPH-4.1, the
pph-4.1(RNAi) embryos were stained with an anti--tubulin
antibody (Figs 5,
6). In 26% (33/128) of the
one-cell pph-4.1(RNAi) embryos showing condensed chromosomes, astral
microtubules were either poorly organized
(Fig. 5G,I) or not detected at
all (Fig. 6E). In these
embryos, few astral microtubules were extended from the centrosomes, and
cytoplasmic microtubules appeared to be randomly oriented as in interphase
cells (Fig. 5G,I). We also
examined the spindle structure during sperm meiosis in pph-4.1(RNAi)
F1 animals. When the first meiotic asters are formed in the wild-type
spermatocytes, six masses of condensed chromosomes can be recognized
(Fig. 5K,L). However, 22/91 of
the pph-4.1(RNAi) spermatocytes showed similar condensed chromosomes
but they did not have astral microtubules
(Fig. 5N,O). Under the same
experimental conditions, only 2/60 wild-type spermatocytes showed no asters.
Disorganized bipolar spindles were also observed in the RNAi spermatocytes.
These results indicate that PPH-4.1 is required for the proper spindle
formation both in mitosis and in sperm meiosis.
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Depletion of PPH-4.1 resulted in mislocalization of some of the centrosomal
proteins. -Tubulin in C. elegans is known to accumulate at
centrosomes during M phase (Fig.
5C,E) and is required for the organization and function of
kinetochore and interpolar microtubules
(Bobinnec et al., 2000
;
Strome et al., 2001
). In
pph-4.1(RNAi) embryos at the one-cell stage that failed to form
asters after the pronuclei meeting, the
-tubulin foci were poorly
organized at centrosomes (Fig.
5H,J). The localization of
-tubulin was also affected
during sperm meiosis in pph-4.1(RNAi) animals. Although
-tubulin at centrosomes was detected in 58/60 of wild-type
spermatocytes undergoing meiosis (Fig.
5M), 18/72 of pph-4.1(RNAi) spermatocytes at the same
stage showed no
-tubulin focus (Fig.
5P). Mislocalization of
-tubulin was also observed at the
later stages of meiosis (data not shown).
Polo-like kinases localize to centrosomes in various organisms, including
C. elegans (Chase et al.,
2000; Golsteyn et al.,
1995
; Logarinho and Sunkel,
1998
). The role of PLK for the bipolar spindle formation has been
shown in some organisms (Lane and Nigg,
1996
; Sunkel and Glover,
1988
). In wild-type C. elegans embryos, PLK-1 localized
to the anterior cytoplasm and to the centrosomes during the first mitosis
(Fig. 6C), as reported
previously (Chase et al.,
2000
). In pph-4.1(RNAi) embryos at the one-cell stage
that failed to form spindles, PLK-1 did not localize to centrosomes but
instead accumulated around the nucleus
(Fig. 6F). In addition, PLK-1
did not show the anterior cytoplasmic localization but formed clumps dispersed
in the cytoplasm. This irregular localization of PLK-1 in
pph-4.1(RNAi) embryos appeared to be specific, because it was
abolished by simultaneous RNAi of the plk-1 gene. Thus, PPH-4.1
appears to be required for the proper localization of PLK-1. These
observations suggest that the PPH-4.1 activity may be necessary for the
recruitment of centrosomal components, including
-tubulin and PLK-1,
during the maturation of centrosomes when cells enter mitosis.
In mammals and Drosophila, PLK is required for the recruitment of
-tubulin to centrosomes (Donaldson
et al., 2001
; Lane and Nigg,
1996
). Therefore, it is possible that mislocalization of PLK-1 in
pph-4.1(RNAi) embryos caused a failure in recruiting
-tubulin
to centrosomes. However, unlike some organisms in which
-tubulin and
PLKs play essential roles for spindle formation, C. elegans does not
require
-tubulin or PLK-1 for microtubule nucleation
(Bobinnec et al., 2000
;
Chase et al., 2000
;
Strome et al., 2001
).
Therefore, the failure in microtubule nucleation and spindle formation caused
by depletion of PPH-4.1 cannot be fully explained by mislocalization of
-tubulin and PLK-1. We speculate that PPH-4.1 recruits not only
-tubulin and PLK-1 but also other components of PCM that are essential
for microtubule organization.
PPH-4.1 is a component of mitotic centrosomes
To analyze the distribution of PPH-4.1 in C. elegans, we raised
and affinity purified rabbit polyclonal antibodies against the amino-terminal
197 amino acids of PPH-4.1. In immunoblot analysis against a total C.
elegans lysate, the anti-PPH-4.1 antibodies recognized two major bands
corresponding to the predicted molecular mass of PPH-4.1 (37 kDa), and an
additional weak band of a higher molecular mass (67 kDa) with unknown identity
(Fig. 7A, lane 1). These
signals were abolished when the antibodies were preincubated with the antigen,
indicating that the antibodies specifically recognize the PPH-4.1 protein
(Fig. 7A, lane 2).
|
Immunostaining using the affinity-purified antibodies revealed that PPH-4.1 localized to centrosomes during mitosis (Fig. 7B). The signal was first detected as two small dots adjacent to the male pronucleus, corresponding to the centrosomes of sperm asters. The signal became more intense at metaphase through anaphase, concomitantly with enlargement of the asters. The signal at centrosomes then became weaker and dispersed in telophase, and was undetectable in interphase. PPH-4.1 was also present in the cytoplasm, throughout the cell cycle. This cell-cycle-dependent localization of PPH-4.1 was observed in later embryonic cell divisions too. Consistent with the results of RNAi showing that PPH-4.1 is dispensable for female meiotic divisions, PPH-4.1 was not detected at meiotic spindles in fertilized eggs. No signal was detected in pph-4.1(RNAi) embryos that did not form a spindle, further confirming the specificity of the antibody.
In Drosophila and mammalian cells, PP4 localizes to centrosome
throughout the cell cycle, except at telophase, and is also detectable in
nuclei at interphase (Brewis et al.,
1993; Helps et al.,
1998
). Thus, despite minor differences in the precise timing of
localization, the presence of PP4 at mitotic centrosomes appears to be widely
conserved among metazoans. Furthermore, the mitotic spindle defects observed
in pph-4.1(RNAi) embryos resemble to those in the Drosophila
cmm mutant, in which the amount of PP4 is reduced. In both cases,
localization of
-tubulin at the centrosome was reduced during M phase,
and centrosomes with asters not well defined were observed. Therefore, the
function of PP4 in mitotic spindle formation is likely to be conserved among
organisms, and its localization at mitotic centrosomes appears to be crucial
for its action.
PPH-4.1 may be involved in chiasma formation during meiotic prophase
I
To examine the function of pph-4.1 in oogenesis, we observed
oocyte nuclei in pph-4.1(RNAi) F1 adult hermaphrodites. In wild-type,
six bivalent chromosomes, each corresponding to a pair of homologous
chromosomes linked by a chiasma, were observed by DAPI staining
(Fig. 8A,C). By contrast, 65%
(41/63) of pph-4.1(RNAi) diakinesis nuclei contained multiple
univalent chromosomes (Fig.
8B,D). The average number of bivalent chromosomes per nucleus was
3.7 and that of univalent chromosomes was 4.1. The total number of chromosomes
[2x(number of bivalents)+(number of univalents)] was 11.9±1.3
(n=63), which corresponded well with the number of a chromosome set.
Therefore, we speculate that the presence of univalents were not the result of
mitotic nondisjunction, but of lack of chiasmata between homologous
chromosomes. Lack of chiasmata can result from a defect in the pairing of
homologous chromosomes (as in the chk-2 mutant)
(Higashitani et al., 2000;
MacQueen and Villeneuve, 2001
;
Oishi et al., 2001
), in
homologous recombination (as in the spo-11 or mre-11 mutant)
(Chin and Villeneuve, 2001
;
Dernburg et al., 1998
) and in
the establishment of stable chiasmata after crossing over. Pachytene
chromosomes are disorganized in the chk-2 mutant
(MacQueen and Villeneuve,
2001
). However, we did not detect any abnormality in pachytene
chromosomes in pph-4.1(RNAi) germ cells (data not shown). Moreover,
in a single gonad, univalent chromosomes tend to be observed more frequently
in proximal oocytes (Fig. 8B).
One possible explanation is that PPH-4.1 may be dispensable for the initial
pairing and crossing-over between homologous chromosomes, but may play an
important role in the formation of functional chiasmata.
|
Our results show that in C. elegans, PP4 has at least two
functions during gametogenesis. It is required for meiotic spindle formation
during spermatogenesis and for chiasmata formation during oogenesis. In rat,
PP4 is highly expressed in testis and ovary, as well as in various somatic
tissues (Kloeker et al.,
1997). Although yet to be confirmed, it is possible that PP4 has
conserved functions in gametogenesis in various organisms.
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Concluding remarks |
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Acknowledgments |
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