Regular articleRole of Caenorhabditis elegans protein phosphatase type 1, CeGLC-7β,in metaphase to anaphase transition during embryonic development
Introduction
In the early stages of the embryonic development of a multicellular organism, accurate chromosome segregation is essential. Phosphorylation and dephosphorylation events are critical to the cytoskeletal and chromosome changes that occur during mitosis [1]. In Caenorhabditis elegans, several genes that are essential to chromosome segregation during embryonic development have recently been identified, including air-1 [2], [3], air-2 [4], [5], [6], and bir-1 [7]. It has been shown, through double-stranded (ds)RNA-mediated interference (RNAi) experiments, that the elimination of both the maternal and zygotic expression of these genes results in embryonic lethality. Further detailed analysis has revealed that phosphorylations by Aurora/Ip11p-related kinases are correlated to the dissolution of the nuclear membrane, the condensation of chromosomes, duplication of the spindle poles, and cytokinesis [8], [9]. The localization of these kinases to particular sites may play an important role in positioning them close to their substrates.
Dephosphorylation events in embryogenesis were also studied, mainly by RNAi experiments [4], [10]. These experiments demonstrated that an injection of Ceglc-7α and Ceglc-7β dsRNA results in embryonic lethality. The embryos of animals injected with Ceglc-7α/β arrest at a stage similar to that found with air-2 alone.
To further investigate the details of dephosphorylation during embryogenesis, we isolated a deletion mutant of Ceglc-7β gene. Here, we present genetic and biochemical evidence that Ceglc-7β is essential to mitosis from the first embryonic cell cycle onward and is required for chromosome segregation. By means of immunohistochemical analysis, we revealed that CeGLC-7β localizes to chromosomes during mitotic progression. A Ceglc-7β deletion mutation or dsRNAi (Ceglc-7β) leads to phenotypic defects in embryogenesis. Our results suggest that the Ceglc-7β gene function is critical to C. elegans embryogenesis for dephosphorylation at anaphase.
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C. elegans strains
The general methods used for maintaining the C. elegans strains were as described by Brenner [11]. Bristol strain N2 was used as the standard wild-type strain. All the strains were cultured at 20°C. The AZ212 strain, which is carried on an integrated H2B-green fluorescent protein (GFP) transgene under the control of the PIE-1 promoter [12], was obtained from the Caenorhabditis Genetics Center, Minnesota.
Isolation and sequencing of Ceglc-7β cDNA clones
Five λZAP II yk clones, namely yk112g3, yk150g8, yk230h1, yk264d6, and yk522e10, were
Identification and cloning of Ceglc-7β
The C. elegans genomic DNA containing the deduced open reading frame with significance to the human and rat protein phosphatase 1 (PP1) which was found in cosmid F56C9 (Accession No. U00063) on chromosome III was designated Ceglc-7β (designated F56C9.1 by the C. elegans Sequencing Consortium [23] (Fig. 1A). Hsu et al. [4] originally noticed the presence of two protein phosphatases, CeGLC-7β and CeGLC-7α. Two putative PP1 isoforms, CeGLC-7β and −7α, are 85% identical in amino acid sequences. The
Discussion
Previous reports have shown that two PP1 phosphatases, CeGLC-7α and -7β, regulated chromosome condensation/segregation in meiosis and mitosis by double-stranded RNA interference [4], [10]. These two PP1 isoforms are 85% identical in amino acid sequence. In this report, we have described the isolation and characterization of a Ceglc-7β (tm301) deletion mutant. The Ceglc-7β homozygous progeny derived from heterozygotes (tm301/eT1;+/eT1) lay eggs. However, the brood size is approximately one-third
Acknowledgements
This work was supported by a grant from the Japan Society for the Promotion of Science Research for the Future, contract Grant 97L00401. We thank T. Stiernagle of the Caenorhabditis Genetics Center (St. Paul, MN), which is funded by the NIH-NCRR, for providing the nematode strains used in this study. We also thank Y. Kohara (National Institute of Genetics, Shizuoka, Japan) for the cDNA clones, A. Coulson of the Sanger Centre (Cambridge, UK) for providing cosmids, and S. Mitani and K.
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