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The function of a spindle checkpoint gene bub-1 in C. elegans development.

Wang X, Liu M, Li W, Suh CD, Zhu Z, Jin Y, Fan Q - PLoS ONE (2009)

Bottom Line: In addition, bub-1(fw5 and fw8) mutants showed widespread effects on postembryonic development in many cell lineages.We found that bub-1 functioned maternally in several developmental lineages at the embryonic stage in C. elegans.Our results demonstrate a conserved role of bub-1 in cell-cycle regulation and reveal that C. elegans bub-1 is required both maternally and zygotically.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Peking University, Beijing, China.

ABSTRACT

Background: The serine/threonine kinase BUB1 (Budding Uninhibited by Benzimidazole 1) was originally identified in yeast as a checkpoint protein, based on its mutant's incapacity of delaying the cell cycle in response to loss of microtubules. Our understanding of its function is primarily from studies carried out in yeast S. cerevisiae. It has been shown that it is a component of the mitotic spindle checkpoint and regulates the separation of sister chromatids through its downstream molecules. However, its roles in multi-cellular organisms remain unclear.

Methods and findings: In nematode C. elegans, rapid cell divisions primarily occur in embryos and in germline of postembryonic larvae and adults. In addition, a select set of cells undergo a few rounds of cell division postembryonically. One common phenotype associated with impaired cell division is described as Stu (Sterile and Uncoordinated) [1], [2]. We conducted a genetic screen for zygotic mutants that displayed Stu phenotype in C. elegans. We isolated seven Stu mutants that fell into five complementation groups. We report here that two mutations, FanWang5 (fw5) and FanWang8 (fw8) affect the bub-1 gene, a homolog of yeast BUB1. Both mutant alleles of fw5 and fw8 exhibited variable behavioral defects, including developmental arrest, uncoordination and sterility. The number of postembryonically born neurons in the ventral cord decreased and their axon morphology was abnormal. Also, the decrease of neurons in the ventral cord phenotype could not be suppressed by a caspase-3 loss-of-function mutant. In addition, bub-1(fw5 and fw8) mutants showed widespread effects on postembryonic development in many cell lineages. We found that bub-1 functioned maternally in several developmental lineages at the embryonic stage in C. elegans. Studies in yeast have shown that BUB1 functions as a spindle checkpoint protein by regulating the anaphase promoting complex/cyclosome (APC/C). We performed double mutant analysis and observed that bub-1 genetically interacted with several downstream genes, including fzy-1/CDC20, mat-2/APC1 and emb-27/APC6.

Conclusions: Our results demonstrate a conserved role of bub-1 in cell-cycle regulation and reveal that C. elegans bub-1 is required both maternally and zygotically. Further, our genetic analysis is consistent with that the function of bub-1 in C. elegans is likely similar to its yeast and mammalian homologs.

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Intestinal Ploidy Measurement of bub-1 Mutants.Body wall muscle nuclei were used as an internal 2 n standard. White bar indicates the average DNA content±s.d. of 10 body wall muscle nuclei in three independent animals. Black bar indicates the average DNA content±s.d of 30 intestinal nuclei in three independent animals.
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pone-0005912-g006: Intestinal Ploidy Measurement of bub-1 Mutants.Body wall muscle nuclei were used as an internal 2 n standard. White bar indicates the average DNA content±s.d. of 10 body wall muscle nuclei in three independent animals. Black bar indicates the average DNA content±s.d of 30 intestinal nuclei in three independent animals.

Mentions: The transgenic GFP line rrIs1 was used to visualize the nuclei of the intestine cells (Figure 4A). In wide type late L1 animals, the intestine cells have 30 to 34 diploid nuclei. All intestinal nuclei endoreduplicate their DNA prior to each of the four molts, thereby producing the 32 n DNA content nuclei in the adult intestine [39]. We found, however, about 24 intestinal nuclei in the bub-1(fw8) mutant L4 larvae (n = 17) (Figure 4B). In addition, some of the intestinal nuclei were elongated and showed a thread structure, suggesting a defect in chromosomal segregation [25]. This observation was consistent with the DAPI staining experiment (Figure 5B). Furthermore, we checked the DNA content of the intestinal nuclei in the bub-1(fw8) L4 or adults. Using body wall muscle nuclei as an internal 2 n control, we determined that the amount of DNA in the intestinal lineages was 24.3 n in the bub-1(fw8) mutant, while 28.4 n in the WT (Figure 6). If the arrest of cell division prior to L4 stage and the lack of the last DNA replication before L4 to adult molt are taken into account, we tend to believe that the intestinal nuclei endoreduplication might not be affected by the loss of bub-1 function. However, the cell division may be affected.


The function of a spindle checkpoint gene bub-1 in C. elegans development.

Wang X, Liu M, Li W, Suh CD, Zhu Z, Jin Y, Fan Q - PLoS ONE (2009)

Intestinal Ploidy Measurement of bub-1 Mutants.Body wall muscle nuclei were used as an internal 2 n standard. White bar indicates the average DNA content±s.d. of 10 body wall muscle nuclei in three independent animals. Black bar indicates the average DNA content±s.d of 30 intestinal nuclei in three independent animals.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2691579&req=5

pone-0005912-g006: Intestinal Ploidy Measurement of bub-1 Mutants.Body wall muscle nuclei were used as an internal 2 n standard. White bar indicates the average DNA content±s.d. of 10 body wall muscle nuclei in three independent animals. Black bar indicates the average DNA content±s.d of 30 intestinal nuclei in three independent animals.
Mentions: The transgenic GFP line rrIs1 was used to visualize the nuclei of the intestine cells (Figure 4A). In wide type late L1 animals, the intestine cells have 30 to 34 diploid nuclei. All intestinal nuclei endoreduplicate their DNA prior to each of the four molts, thereby producing the 32 n DNA content nuclei in the adult intestine [39]. We found, however, about 24 intestinal nuclei in the bub-1(fw8) mutant L4 larvae (n = 17) (Figure 4B). In addition, some of the intestinal nuclei were elongated and showed a thread structure, suggesting a defect in chromosomal segregation [25]. This observation was consistent with the DAPI staining experiment (Figure 5B). Furthermore, we checked the DNA content of the intestinal nuclei in the bub-1(fw8) L4 or adults. Using body wall muscle nuclei as an internal 2 n control, we determined that the amount of DNA in the intestinal lineages was 24.3 n in the bub-1(fw8) mutant, while 28.4 n in the WT (Figure 6). If the arrest of cell division prior to L4 stage and the lack of the last DNA replication before L4 to adult molt are taken into account, we tend to believe that the intestinal nuclei endoreduplication might not be affected by the loss of bub-1 function. However, the cell division may be affected.

Bottom Line: In addition, bub-1(fw5 and fw8) mutants showed widespread effects on postembryonic development in many cell lineages.We found that bub-1 functioned maternally in several developmental lineages at the embryonic stage in C. elegans.Our results demonstrate a conserved role of bub-1 in cell-cycle regulation and reveal that C. elegans bub-1 is required both maternally and zygotically.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, Peking University, Beijing, China.

ABSTRACT

Background: The serine/threonine kinase BUB1 (Budding Uninhibited by Benzimidazole 1) was originally identified in yeast as a checkpoint protein, based on its mutant's incapacity of delaying the cell cycle in response to loss of microtubules. Our understanding of its function is primarily from studies carried out in yeast S. cerevisiae. It has been shown that it is a component of the mitotic spindle checkpoint and regulates the separation of sister chromatids through its downstream molecules. However, its roles in multi-cellular organisms remain unclear.

Methods and findings: In nematode C. elegans, rapid cell divisions primarily occur in embryos and in germline of postembryonic larvae and adults. In addition, a select set of cells undergo a few rounds of cell division postembryonically. One common phenotype associated with impaired cell division is described as Stu (Sterile and Uncoordinated) [1], [2]. We conducted a genetic screen for zygotic mutants that displayed Stu phenotype in C. elegans. We isolated seven Stu mutants that fell into five complementation groups. We report here that two mutations, FanWang5 (fw5) and FanWang8 (fw8) affect the bub-1 gene, a homolog of yeast BUB1. Both mutant alleles of fw5 and fw8 exhibited variable behavioral defects, including developmental arrest, uncoordination and sterility. The number of postembryonically born neurons in the ventral cord decreased and their axon morphology was abnormal. Also, the decrease of neurons in the ventral cord phenotype could not be suppressed by a caspase-3 loss-of-function mutant. In addition, bub-1(fw5 and fw8) mutants showed widespread effects on postembryonic development in many cell lineages. We found that bub-1 functioned maternally in several developmental lineages at the embryonic stage in C. elegans. Studies in yeast have shown that BUB1 functions as a spindle checkpoint protein by regulating the anaphase promoting complex/cyclosome (APC/C). We performed double mutant analysis and observed that bub-1 genetically interacted with several downstream genes, including fzy-1/CDC20, mat-2/APC1 and emb-27/APC6.

Conclusions: Our results demonstrate a conserved role of bub-1 in cell-cycle regulation and reveal that C. elegans bub-1 is required both maternally and zygotically. Further, our genetic analysis is consistent with that the function of bub-1 in C. elegans is likely similar to its yeast and mammalian homologs.

Show MeSH
Related in: MedlinePlus