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Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
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Abstract |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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The changes in the organization and dynamic properties of microtubules in early embryos are especially striking. In Caenorhabditis elegans, as in most animals, the sperm contributes to a pair of centrioles, which duplicates to form the new zygotic centrosomes and recruits the pericentriolar material components from the cytoplasm, allowing microtubules nucleation and growth. Prior to mitosis, growth of long astral microtubule is required for migration of the maternal pronucleus toward the paternal one, for migration of the pronuclear-centrosomal complex to the cell centre, and for rotation of the complex on to the anterior-posterior axis (Sulston et al. 1983; Albertson 1984; Hyman & White 1987; Gonczy et al. 1999b).
A pioneering work of Hyman's group reported a functional genomic screen to identify genes required for cell division in C. elegans (Gonczy et al. 2000). In that report, in the ‘pronuclear migration’ phenotypic class comprising six genes, migration of pronuclei is affected. Two of the corresponding genes encode ß-tubulin subunits. This is reasonable because pronuclear migration is a microtubule-dependent process (Strome & Wood 1983; Wright & Hunter 2003). One of the other genes encodes a dynein light chain, and one encodes a dynactin component p50/dynamitin. These are also reasonable because cytoplasmic dynein function is shown to be required for this process in C. elegans (Skop & White 1998; Gonczy et al. 1999a; Yoder & Han 2001). Moreover, the C. elegans homolog of lis-1, the human gene mutated in Miller-Dieker lissencephaly (Reiner et al. 1993; Lo Nigro et al. 1997), is also required for pronuclear migration (Dawe et al. 2001). The lis-1 homologue acts in concert with dynein to mediate the positioning of the nucleus in both Aspergillus and Drosophila (Swan et al. 1999; Willins et al. 1997). In contrast, the other one gene, C05D11.3, has not been characterized in detail. We found putative C05D11.3 homologues by screening genomic databases of many organisms.
In this study, we first describe characterization of APACD, a mammalian homologue of C05D11.3. Mouse APACD is present in both the nucleus and the cytoplasm, showing often localization to centrosomes and midbody. Then, we characterized C05D11.3 in C. elegans. C05D11.3 is expressed throughout development with higher levels of expression in most cells of the nervous system and in vulva. C05D11.3 RNAi-treated embryos show apparent defects in not only pronuclear migration but also nuclear-centrosome rotation, and exhibit little astral microtubules and defective small spindles. These results suggest that this gene family may be a conserved regulator of microtubule dynamics and function.
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Results |
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Genomic databases of many organisms were screened to identify members of C. elegans C05D11.3. The organisms include mouse, rat, human, Xenopus, Drosophila, Arabidopsis, Saccharomyces cerevisiae and other unicellular eukaryotes (Fig. 1A). Mouse and human homologues are called APACD (ATP binding protein associated with cell differentiation). The mouse and C. elegans proteins show 45% homology, and the mouse and human proteins show 90%. Mouse APACD comprises 226 amino acids with a thioredoxin-like domain in the middle portion (Fig. 1B). Thioredoxins are a family of proteins that have a conserved catalytic site (CGPC) that undergoes reversible oxidation to the cystine disulphide (Arner & Holmgren 2000). APACD does not include the conserved catalytic site. This indicates that APACD has no thioredoxin activity. Vertebrate (mouse, rat, human or Xenopus) APACD has in common an ATP/GTP-binding site motif A (Fig. 1A,B). An ATP/GTP-binding site motif A is the evolutionarily conserved motif (G/AxxxxGKT/S), that is present in many nucleotide-binding proteins including dynein heavy chain (Walker et al. 1982; Silvanovich et al. 2003), forming a typical loop, called the P-loop. P-loop is predicted to bind and/or hydrolyse ATP (Walker et al. 1982). It is possible that APACD may have an ATP binding and/or hydrolysis function. An S. cerevisiae homologue of C05D11.3 (30% homologous to mouse APACD) is called phosducin-like protein 1, Plp1, which binds to G protein ß
subunits (Flanary et al. 2000). A Dictyostelium homologue, PhLP3 (42% homologous to mouse APACD) is a member of a family of phosducin-like proteins, which might facilitate folding, localization or function of proteins, in addition to modulation of G-protein signalling (Blaauw et al. 2003).
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APACD localizes to the nucleus and the cytoplasm
We cloned mouse APACD cDNA and expressed myc-tagged or GFP-tagged APACD in HeLa cells. Cell extracts were immunoblotted with anti-myc or anti-GFP antibody. The protein was detected as a single band in each immunoblotting (Fig. 1D).
Indirect immunofluorescence with anti-myc antibody showed that myc-APACD was localized both in the cytoplasm and in the nucleus. GFP-APACD was also found in the nucleus and the cytoplasm. Furthermore, GFP-APACD often showed co-localization with a centrosomal marker -tubulin. APACD protein also concentrated on the midbody during cytokinesis (Fig. 1E). To examine the function of APACD, we tried to down-regulate endogenous APACD by siRNA (small interference RNA). But we were unable to reduce it significantly (data not shown).
C.elegans C05D11.3 is expressed in various stages and tissues
We then used a model animal, C. elegans, to investigate the function of C05D11.3. We first examined the expression of C05D11.3 at various developmental stages. C05D11.3 was expressed mainly in the young adult and adult stages (Fig. 2A).
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5.7 kb fragment that contained C05D11.3 and putative upstream regulatory sequences (Fig. 2B). The C05D11.3::GFP fusion construct was injected into the gonad of wild-type worms. The obtained transgenic animals started to express the construct during late embryogenesis. We could not find the GFP expression in early embryogenesis (data not shown and see Fig. 2E). A late embryo showed diffuse expression (Fig. 2C). In early larvae, a faint and diffuse staining was observed in all tissues (data not shown). Interestingly, the C05D11.3::GFP continued to be expressed in most cells of the nervous system, at all larval stages and in adult animals: the ventral nerve cord as well as dorsal and lateral cords (Fig. 3A). And it is expressed in the pharynx (Fig. 2D,E, arrowheads). Larval stages showed strong expression, in particular non-neuronal cells (data not shown). Furthermore, an oval of fluorescence in the middle of the L4 worm suggested that C05D11.3 was expressed in the vulva (Fig. 2D,E, arrows). After the L4 stage, C05D11.3::GFP expression was seen in the vulva in all life. A more detailed analysis of this expression is shown in Fig. 3A,B. These results indicate that C05D11.3 is highly expressed in the nervous system and vulva. This expression pattern might suggest its function in the nervous system and vulva, as can also be suggested from the Unc and Egl phenotypes noted in the previous reports of large-scale RNAi screens (Kamath et al. 2003; Simmer et al. 2003). We also examined the subcellular localization of C05D11.3. C05D11.3::GFP is partly co-localized with DAPI (Fig. 3C), suggesting that C05D11.3 is localized both in the nucleus and the cytoplasm.
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To examine the function of C05D11.3, feeding RNAi for C05D11.3 was carried out. The progeny of RNAi-targeted worms showed about 60% embryonic lethality (Fig. 4). This result is consistent with the previous reports (Gonczy et al. 2000; Kamath et al. 2003; Simmer et al. 2003). In the wild-type case, embryos from the 2-cell to 64-cell stage were arranged in a well-organized pattern showing normal development and cell divisions. On the other hand, the C05D11.3 RNAi-treated embryos showed a highly disorganized pattern with no sign of cell division. DAPI staining showed large clumps of nuclei, indicating that these embryos failed to progress through early cleavage. However, eggshells were observed around the embryos, and oval-shaped embryos were apparent, indicating that fertilization had occurred in these embryos. These results indicate that C05D11.3 is essential for proper cell division.
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To analyse the C05D11.3 RNAi-treated embryos in more detail, we observed C05D11.3 RNAi-treated embryos at one cell stage by time-lapse DIC microscopy. The C05D11.3 RNAi-treated embryos reproducibly fell into one of the three phenotypic types. About 40% of C05D11.3 RNAi-treated embryos showed nuclear-centrosome rotation defects (Fig. 5A, type I). Proper pronuclear migration and proper pronuclear meeting occurred as in wild-type embryos, but the nuclear-centrosome complex failed to move to the cell centre and did not undergo a 90° rotation. Nuclear envelope then breakdown occurred. Cytokinesis did not occur. Then, the successive emergence of several small nuclei was observed. In about 30% of C05D11.3 RNAi-treated embryos (Fig. 5B, type II), the male pronuclear migration did not occur. The female pronucleus migrated toward the male pronucleus, but the male pronucleus did not migrate. Then, the two pronuclei met at the posterior cortex. In about 30% of C05D11.3 RNAi-treated embryos (Fig. 5C, type III), appeared multiple pronuclei. Migration of the male and female pronuclei rarely took place. These results demonstrate that C05D11.3 has a pivotal role in pronuclear migration or nuclear-centrosome rotation.
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Since pronuclear migration and nuclear-centrosome rotation are dependent on microtubules (Strome & Wood 1983; Albertson 1984; Hyman & White 1987; Hyman 1989; Reinsch & Gonczy 1998), we examined the organization of microtubules in C05D11.3 RNAi-treated embryos during the cell cycle. We visualized microtubules in wild-type and C05D11.3 RNAi-treated embryos by immunofluorescence by using 
-tubulin antibody (Fig. 6A). In wild-type embryos, prior to mitotic prophase, long microtubules emanated from the two centrosomes that were associated with the sperm-derived pronucleus. In contrast, in C05D11.3 RNAi-treated embryos, microtubules were very short. This observation suggests that the phenotype of C05D11.3 RNAi-treated embryos might be caused by the poor and short microtubules.
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C05D11.3 RNAi treatment causes improper cytokinesis
In C05D11.3 RNAi-treated embryos, cytokinetic furrows were formed but regressed without completion of cytokinesis (data not shown and see Fig. 5A). In our -tubulin and -tubulin staining experiments (Fig. 6C), many centrosomes and multipolar spindles were found. Moreover, additional DNA was detected in these embryos, suggesting that C05D11.3 RNAi embryos underwent multiple rounds of nuclear division without completing cytokinesis.
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Discussion |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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Mouse APACD contains the conserved ATP binding motif, P-loop, like dynein heavy chain and other cytoskeletal proteins. Moreover, we have shown that mouse APACD is often localized at the centrosome and midbody. Thus, APACD might be a regulator of microtubule dynamics and function in mammalian cells.
Our results have demonstrated that C05D11.3 RNAi-treated embryos show marked defects in pronuclear migration and nuclear-centrosome rotation in C. elegans. A recent global RNAi study demonstrated that in C05D11.3 RNAi-treated embryos, male pronuclear migration defects and multiple nuclei were observed (Gonczy et al. 2000). Our results also detected these two types of phenotypes (type II and type III, in Fig. 5B,C). In addition, we have found nuclear-centrosome rotation defects (type I, in Fig. 5A). It has been proposed that both pronuclear migration and nuclear-centrosome rotation are dependent on microtubules (Strome & Wood 1983; Albertson 1984; Hyman & White 1987; Hyman 1989; Reinsch & Gonczy 1998). Our data have shown that astral microtubules become much less extensive and spindle microtubules are small and poorly organized in C05D11.3 RNAi-treated embryos. It is possible that this defect in microtubule architecture causes the defects in pronuclear migration and nuclear-centrosome rotation.
We speculate that this gene family may be a conserved regulator of microtubule dynamics and function. Molecular mechanisms by which this gene product regulates microtubule architecture should be elucidated in future studies.
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Experimental procedures |
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The 5.7 kb sequence comprising all C05D11.3 coding and upstream sequences (including the two genes C05D11.9 and C05D11.10) were amplified by PCR from cosmid C05D11 and transcriptionally fused to the green fluorescent protein (GFP) coding sequence in the pPD95.75 vector (Cassata et al. 1998). The open reading frame of mouse APACD was amplified by PCR from a mouse cDNA library, and PCR products were subcloned into pCDNA3 (Invitrogen)-myc, and pEGFP-C1 (Clontech) vectors.
Northern blot analysis
A 300 base pair fragment of mouse APACD cDNA was radiolabelled using the Rediprime DNA Labeling System (Amersham Biosciences). Mouse Multiple Tissue Northern Blots (Clontech) were hybridized with the probe in ExpressHyb hybridization solution (Clontech) at 68 °C according to the manufacturer's protocol. The bands were visualized by autoradiography.
Immunoblotting analysis
Cells were harvested by scraping in hot 1 x SDS sample buffer, and the lysates were separated by SDS-PAGE, blotted to Immobilon-P membrane (Millipore), and detected using anti-myc (Santa Cruz) or anti-GFP antibody (Clontech).
Immunofluorescence of mammalian cells
Cells were fixed in 3.7% formaldehyde and then permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 10 min. The coverslips were washed with PBS and incubated with PBS containing 3% bovine serum albumin (BSA) and 0.1% goat immunoglobulins (blocking buffer) for 30 min, and then stained with anti-myc antibody (Santa Cruz) and anti--tubulin antibody (Sigma) for overnight. After three washes with PBS, the cells were stained with the appropriate secondary antibody for 1 h at room temperature. Fluorescence microscopy was performed using a Zeiss Axiophot2 microscope.
Detection of the C05D11.3 mRNA
Total RNA was isolated from synchronized cultures by the guanidinium-acid-phenol-chloroform method. For RT-PCR using rTth DNA polymerase (TOYOBO), 1 mg RNA was used as the template. The oligo DNA primers used for C05D11.3 amplification were 5'-ATGGCCGCTAATATTCAACAGCAG-3' and 5'-CTACCAATCCTCTTCGTTATCGTAC-3', and those used for EF-1 (R03G5.1) were 5'-CATTGTCGTCATCGGACATGTCGACTCCGG-3' and 5'-TGGCTCGGTGGAGTCCATCTTGTTGCAAGC-3'. After reverse transcription for 30 min at 60 °C, the products were amplified with the following amplification profile for 40 cycles: denaturation for 1 min at 94 °C, annealing and extension for 1.5 min at 60 °C.
C.elegans culturing methods
Wild-type C. elegans strain N2 was cultured under standard conditions (Brenner 1974). To perform RNAi studies, the full-length cDNA of C05D11.3 was cloned into the vector L4440 (Timmons et al. 2001) and transformed into HT115 bacterial cells. Transformed bacteria were grown for 14 h at 37 °C in 2 mL LB supplemented with 50 µg/mL ampicillin. These cultures were plated on to standard worm plates containing 1 mM IPTG and 50 µg/mL ampicillin and allowed to grow overnight at room temperature. N2 larvae were then placed on to these plates and their offspring were examined after 48 h at 20 °C (Kamath et al. 2001). Embryos analysed by timelapse video microscopy were dissected in M9 medium from worms and placed on a 2% agarose pad with a coverslip mounted on top. Typically early development was analysed in wild-type and C05D11.3 RNAi-treated embryos from shortly after fertilization through two rounds of cell division (40 min).
Analysis of C05D11.3::GFP-expressing animals
Living animals were immobilized with 10 mM levamisole in M9 buffer, mounted on a 4% agar pad and analysed with a Zeiss Axiophot2 microscope.
Immunofluorescence of C. elegans embryos
Gravid N2 adults were cut in a drop of M9 buffer (Sulston & Hodgkin 1988) containing 10 mM levamisole to release embryos. A coverslip was gently applied and the slide was frozen in liquid nitrogen. The coverslip was then removed and the slide was immediately immersed in cold (–20 °C) methanol for 5 min. Slides were air-dried for 5 min. Embryos were rehydrated in PBS-BSA (1%) for 30 min, and incubated in PBS-BSA (3%) with primary antibodies for 1 h. After three 10 min washes with PBS, secondary antibodies were applied in PBS-BSA (3%) for 1 h. Slides were washed with PBS and mounted in Mowiol (Calbiochem, La Jolla, USA; prepared as described by Harlow & Lane 1988). Nuclei were stained with DAPI. Dilutions of antibodies were 1 : 500 for monoclonal anti--tubulin antibody, clone DM1A (Sigma); 1: 2000 for affinity-purified anti--tubulin (Bobinnec et al. 2000). Microscopy was performed with a Zeiss Axiophot2 microscope.
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Acknowledgements |
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Footnotes |
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* Correspondence: Email: L50174{at}sakura.kudpc.kyoto-u.ac.jp
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References |
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Received: 19 November 2003
Accepted: 5 December 2003
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