Summary
The centrosome consists of two centrioles surrounded by the pericentriolar material (PCM). In late G2 phase, centrosomes enlarge by recruiting extra PCM, and concomitantly its microtubule nucleation activity increases dramatically. The regulatory mechanisms of this dynamic change of centrosomes are not well understood. Protein phosphatase 4 (PP4) is known to localize to mitotic centrosomes in mammals and Drosophila. An involvement of PP4 in the mitotic spindle assembly has been implicated in Drosophila, but in vivo functions of PP4 in other organisms are largely unknown. Here we characterize two Caenorhabditis elegans PP4 genes, named pph-4.1 and pph-4.2. Inhibition of the function of each gene by RNA-mediated interference (RNAi) revealed that PPH-4.1 was essential for embryogenesis but PPH-4.2 was not. More specifically, PPH-4.1 was required for the formation of spindles in mitosis and sperm meiosis. However, this phosphatase was apparently dispensable for female meiotic divisions, which do not depend on centrosomes. In the cell depleted of pph-4.1 activity, localization of γ-tubulin and a Polo-like kinase homologue to the centrosome was severely disturbed. Immunofluorescence staining revealed that PPH-4.1 was present at centrosomes from prophase to telophase, but not during interphase. These results indicate that PPH-4.1 is a centrosomal protein involved in the recruitment of PCM components to the centrosome, and is essential for the activation of microtubule nucleation potential of the centrosome. Furthermore, chiasmata between homologous chromosomes were often absent in oocytes that lacked pph-4.1 activity. Thus, besides promoting spindle formation, PPH-4.1 appears to play a role in either the establishment or the maintenance of chiasmata during meiotic prophase I.
Introduction
The centrosome is a major microtubule organizing center in eukaryotic cells. It is composed of a pair of centrioles surrounded by the pericentriolar material (PCM), which provides a nucleation center for microtubules. The amount of PCM increases gradually as cells progress toward mitosis, and microtubule nucleation activity of centrosomes increases dramatically in the process called `centrosome maturation' at the G2/M transition ( Palazzo et al., 2000). As cells exit mitosis and come back into G1, PCM components are abruptly lost ( Dictenberg et al., 1998). Genetic and biochemical studies in a number of systems have shown thatγ -tubulin is a key component of PCM for the nucleation of microtubules ( Moritz et al., 1995; Stearns et al., 1991; Zheng et al., 1991).γ -Tubulin associates with several other components to form a ∼25 nm diameter ring complex, the γ-TuRC, which is competent at microtubule nucleation ( Moritz et al., 1995; Zheng et al., 1995). It is suggested that the increase in the microtubule nucleation potential of centrosomes is a result of incorporation of additional PCM components, including γ-tubulin. However, the steady recruitment alone cannot fully explain the sudden increase in the microtubule nucleation potential that occurs at the onset of mitosis. Therefore, it is proposed that protein modifications such as phosphorylation may have roles in the centrosome maturation. Indeed, several protein kinases, such as Polo-like kinases (PLKs) ( Lane and Nigg, 1996; Sunkel and Glover, 1988) and Cdk1 ( Buendia et al., 1992; Ohta et al., 1993; Verde et al., 1990), have been localized to the centrosome, and their function in the centrosome maturation has been implicated.
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 γ-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.
Materials and Methods
Strains
Maintenance and genetic manipulation of C. elegans were carried out as described ( Brenner, 1974). Strains used were wildtype C. elegans var. Bristol strain N2, rde-1 (ne219) (LGV), fem-1 (hc17ts) (LGIV) and fem-2 (b245ts) (LGIII). N2 and rde-1 strains were maintained at 20°C. fem-1 and fem-2 strains were maintained at 15°C and grown at 25°C to obtain hermaphrodites without sperm.
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 10×His-PPH-4.1 protein as follows. To create a 10×His-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 10×His-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-α-tubulin antibody DM1A (Sigma), anti-γ-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).
Results and Discussion
Identification of two genes encoding PP4 in C. elegans
We noticed two predicted ORFs, Y75B8A.30 and Y49E10.3, in the C. elegans genome sequence, which encode proteins similar to the human PP4 catalytic subunit. The corresponding genes are called hereafter pph-4.1 and pph-4.2, respectively. cDNA clones of pph-4.1 and pph-4.2 were obtained from the C. elegans expressed sequence tag project ( Fields et al., 1999). Sequencing of the cDNAs confirmed the exon structures predicted by the genome sequence project. Both genes encoded proteins that possessed all conserved motifs of the PPP family of serine/threonine phosphatases ( Cohen, 1997) ( Fig. 1). The predicted amino acid sequence of PPH-4.1 was most closely related to mammalian PP4, showing 74% identity and 80% similarity to a human PP4. The amino acid sequence of PPH-4.2 was most closely related to PPH-4.1, showing 71% identity and 80% similarity.
Proteins encoded by two PP4 genes of C. elegans. (A) Alignment of the amino acid sequences of C. elegans PPH-4.1 and PPH-4.2 with other PP4 families (human PPP4, Drosophila PP4 and Arabidopsis PPX1). Black shadowing indicates identical residues. (B) A phylogenic tree of PPP family protein phosphatses.
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.
Phenotypes of pph-4.1(RNAi) embryos at the one-cell stage. All embryos are shown with the anterior end to the left. (A) A wild-type one-cell embryo showing a male pronucleus (black arrowhead) and a female pronucleus. (B) A wild-type embryo with a bipolar spindle. (C) A pph-4.1(RNAi) embryo with two male pronuclei (black arrowheads). (D) A pph-4.1(RNAi) embryo showing a tetra-polar spindle. (E) A pph-4.1(m-/p+) embryo with a tetra-polar spindle. Three spindle poles are visible in this focal plane. Spindle poles in B, D and E are indicated by white arrowheads. Bar, 10 μm.
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.
PPH-4.1 is required for proper chromosome segregation during sperm meiosis. Hoechst staining (blue) is overlaid on the Nomarski image. (A) Wild-type spermatids. Each spermatid contains one nucleus. (B) pph-4.1(RNAi) spermatids. White arrowheads indicate anucleated spermatids. Black arrowheads indicate binucleated spermatids. (C-F) Secondary spermatocytes with spermatids (arrowheads) budding from the residual body (arrows). (C,E) Wild-type. (D,F) pph-4.1(RNAi). Chromosomes are not properly segregated in pph-4.1(RNAi) spermatocytes. Bar, 10 μm (A,B); 5 μm (C-F).
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.
The first mitotic cycle is delayed in both pph-4.1(RNAi) and pph-4.1(M-/p+) embryos. Each symbol represents a single embryo and indicates the time elapsed from pronuclear meeting to the beginning of anaphase B. Open square, wild-type; closed triangle, pph-4.1(RNAi); closed square, pph-4.1(m-/p+).
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-α-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.
PPH-4.1 is required for aster formation and recruitment of γ-tubulin to centrosomes during mitosis and sperm meiosis. (A,F,K,N) DAPI staining showing chromosomes. (B,G,I,O) Anti-α-tubulin antibody staining. (C,H,M,P) Anti-γ-tubulin antibody staining. (A-C) Wild-type embryo at pronuclear meeting. (F-H) pph-4.1(RNAi) embryo at pronuclear meeting. (D,E,I,J) Magnified view of the indicated area in B, C, G and H, respectively. (K-M) Wild-type primary spermatocyte showing a meiotic spindle. (N-P) pph-4.1(RNAi) primary spermatocyte showing condensed chromosomes similar to those in (K) and a spindle not properly organized. Note that astral microtubules were poorly organized from the centrosomes (G,I,O) and thatγ -tubulin on the centrosomes was dispersed (H,J,P). Each embryo is aligned with the anterior end to the left. White arrowheads indicate centrosomal localization of γ-tubulin. Bar, 10 μm (A-C, F-H); 2 μm (D,E,I,J); 5 μm (K-P).
Loss of PPH-4.1 function affects the localization of PLK-1 during mitosis. (A-C) Wild-type one-cell embryo at prometaphase. (D-F) pph-4.1(RNAi) one-cell embryo after pronuclear meeting. (A,D) DAPI staining showing chromosomes. (B,E) Anti-α-tubulin antibody staining. (C,F) Anti-PLK-1 antibody staining. White arrowheads indicate centrosomal staining of PLK-1. Black arrowheads indicate perinuclear PLK-1 staining. Black arrows indicate cytoplasmic granules. In pph-4.1(RNAi) embryos, PLK-1 did not localize to centrosomes even though chromosomes are condensed. Embryos are aligned with the anterior end to the left. Bar, 10 μm.
Depletion of PPH-4.1 resulted in mislocalization of some of the centrosomal proteins. γ-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 γ-tubulin foci were poorly organized at centrosomes ( Fig. 5H,J). The localization of γ-tubulin was also affected during sperm meiosis in pph-4.1(RNAi) animals. Althoughγ -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 γ-tubulin focus ( Fig. 5P). Mislocalization of γ-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 γ-tubulin and PLK-1, during the maturation of centrosomes when cells enter mitosis.
In mammals and Drosophila, PLK is required for the recruitment ofγ -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 γ-tubulin to centrosomes. However, unlike some organisms in which γ-tubulin and PLKs play essential roles for spindle formation, C. elegans does not require γ-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 ofγ -tubulin and PLK-1. We speculate that PPH-4.1 recruits not onlyγ -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).
PPH-4.1 localizes to centrosomes during mitosis. (A) Western blotting against a wild-type worm extract. Lane 1: detection by PPH-4.1 antibodies. Arrows indicate doublet bands corresponding to the predicted molecular mass of PPH-4.1 (37 kDa). Lane 2: detection by the antibodies pretreated with 10×His-PPH-4.1 protein. (B) Localization of PPH-4.1 during early cleavages. (a,d,g,j,m,p) Anti-α-tubulin antibody staining. (b,e,h,k,n,q) Anti-PPH-4.1 antibody staining. (c,f,i,l,o,r) Merged images. (a-o) Wild-type embryos. PPH-4.1 was localized to centrosomes from prophase to anaphase and only weakly during telophase (a-1; PPH-4.1 staining on centrosomes is indicated by white arrows). During interphase, centrosomal localization of PPH-4.1 was not observed (m-o; interphase cells are indicated with white arrowheads). (p-r) pph-4.1(RNAi) embryo at prophase. No signal was detected in severely affected pph-4.1(RNAi) embryos. Bar, 10μ m.
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 γ-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 [2×(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.
PPH-4.1 may be involved in chiasma formation during meiotic prophase I. (A,B) Oocytes in adult hermaphrodite gonads were stained with DAPI. The distal end is to the left. (A) Wild-type oocytes at the diakinesis stage contain six bivalent chromosomes. (B) In a pph-4.1(RNAi) gonad, oocytes in the distal region contain six bivalents, but oocytes in the proximal region contain twelve univalent chromosomes. (C,D) Magnified view of the indicated areas in (A) and (B), respectively. (A,B) Bar, 10 μm. (C,D) Bar, 2μ m.
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.
Concluding remarks
We have shown in this study that PPH-4.1 plays an essential role in spindle formation in both mitosis and sperm meiosis in C. elegans. Becauseγ -tubulin and PLK-1 are mislocalized during mitosis in pph-4.1(RNAi) embryos, PPH-4.1 appears to be necessary for the recruitment of some of the components of PCM during centrosome maturation. For female meiotic divisions, which do not require centrosomes, PPH-4.1 appears to be dispensable. This observation strongly argues for the PP4 function in centrosome maturation rather than spindle assembly per se. Consistent with the proposed function, immunofluorescence using anti-PPH-4.1 antibody has shown that PPH-4.1 predominantly localizes to centrosomes from prophase to telophase. We speculate that the involvement in centrosome maturation may be the common role of PP4 family members. As an unexpected additional function of PPH-4.1, we have found that it is required for the formation of functional chiasmata.
Acknowledgements
We thank Yuichi Iino for much helpful discussion. We are grateful to the following people for providing materials used in this work: Yuji Kohara for cDNA clones, Yves Bobinnec and Eisuke Nishida for anti-γ-tubulin antibody, and Andy Golden for anti-PLK-1 antibody. Some of the strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the National Center for Research Resources of the National Institutes of Health. This work was supported by grants from the Japan Science and Technology Corporation (A.S.) and a Grant-in-Aid for Scientific Research C (A.S.) and one for Specially Promoted Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (M.Y.).
- Accepted January 6, 2002.
- © The Company of Biologists Limited 2002
References
Article navigation
Related articles
Cited by...
More in this TOC section
Similar articles
Other journals from The Company of Biologists
Cell scientist to watch: Janet Iwasa
Read our interview with molecular animator Janet Iwasa, where she talks about her transition from the wet lab, explains how animation can facilitate research and discusses the challenges of the field.
New funding scheme supports sustainable events
As part of our Sustainable Conferencing Initiative, we are pleased to announce funding for organisers that seek to reduce the environmental footprint of their event. The next deadline to apply for a Scientific Meeting grant is 26 March 2021.
Mole – The Corona files
“Despite everything, it's just incredible that we get to do science.”
Mole continues to offer his wise words to researchers on how to manage during the COVID-19 pandemic.
JCS and COVID-19
For more information on measures Journal of Cell Science is taking to support the community during the COVID-19 pandemic, please see here.
If you have any questions or concerns, please do not hestiate to contact the Editorial Office.