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Securin is not required for chromosomal stability in human cells.

Pfleghaar K, Heubes S, Cox J, Stemmann O, Speicher MR - PLoS Biol. (2005)

Bottom Line: Here we show that, after hSecurin knockout through homologous recombination, chromosome losses are only a short, transient effect.This was unexpected, as the securin loss resulted in a persisting reduction of the sister-separating protease separase and inefficient cleavage of the cohesin subunit Scc1.We propose that human cells possess efficient mechanisms to compensate for the loss of genes involved in chromosome segregation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Human Genetics, Technical University Munich, Munich, Germany.

ABSTRACT
Abnormalities of chromosome number are frequently observed in cancers. The mechanisms regulating chromosome segregation in human cells are therefore of great interest. Recently it has been reported that human cells without an hSecurin gene lose chromosomes at a high frequency. Here we show that, after hSecurin knockout through homologous recombination, chromosome losses are only a short, transient effect. After a few passages hSecurin(-/-) cells became chromosomally stable and executed mitoses normally. This was unexpected, as the securin loss resulted in a persisting reduction of the sister-separating protease separase and inefficient cleavage of the cohesin subunit Scc1. Our data demonstrate that securin is dispensable for chromosomal stability in human cells. We propose that human cells possess efficient mechanisms to compensate for the loss of genes involved in chromosome segregation.

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Chromosomally Stable hSecurin−/− Cells of Passage 12 and Higher Show Reduction in Both the Level and the Activity of Separase(A) Quantitation of full-length separase and the N-terminal cleavage product in both hSecurin+/+ and hSecurin−/− cells. Lysates from nocodazole-arrested cells were analyzed by immunoblotting with an antibody against the N-terminus of separase. The chromosomally stable hSecurin−/− cells show reduced levels of both the full-length and the cleaved N-terminal form of separase. β-tubulin was used as a loading control.(B) Separase was immunoprecipitated from nocodazole-arrested hSecurin+/+ and hSecurin−/− cells, activated by incubation in Xenopus anaphase extracts, and incubated with 35S-hScc1 for 0, 20, or 90 min before analysis by SDS-PAGE and autoradiography. For these experiments we used four times as many hSecurin−/− cells as hSecurin+/+ cells. Note the absence of detectable Scc1 cleavage fragments in the hSecurin−/− samples.(C) Separase used for the activity assay in (B) was analyzed by Western blotting before (−) and after (+) exposure to Xenopus anaphase extracts. The hSecurin+/+ cells clearly demonstrate an increase in self-cleavage of separase upon activation in the extract. Occurrence of auto-cleavage in hSecurin−/− cells even before incubation in Xenopus extract suggests deregulation of separase, at least under the given conditions of this in vitro experiment.
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pbio-0030416-g005: Chromosomally Stable hSecurin−/− Cells of Passage 12 and Higher Show Reduction in Both the Level and the Activity of Separase(A) Quantitation of full-length separase and the N-terminal cleavage product in both hSecurin+/+ and hSecurin−/− cells. Lysates from nocodazole-arrested cells were analyzed by immunoblotting with an antibody against the N-terminus of separase. The chromosomally stable hSecurin−/− cells show reduced levels of both the full-length and the cleaved N-terminal form of separase. β-tubulin was used as a loading control.(B) Separase was immunoprecipitated from nocodazole-arrested hSecurin+/+ and hSecurin−/− cells, activated by incubation in Xenopus anaphase extracts, and incubated with 35S-hScc1 for 0, 20, or 90 min before analysis by SDS-PAGE and autoradiography. For these experiments we used four times as many hSecurin−/− cells as hSecurin+/+ cells. Note the absence of detectable Scc1 cleavage fragments in the hSecurin−/− samples.(C) Separase used for the activity assay in (B) was analyzed by Western blotting before (−) and after (+) exposure to Xenopus anaphase extracts. The hSecurin+/+ cells clearly demonstrate an increase in self-cleavage of separase upon activation in the extract. Occurrence of auto-cleavage in hSecurin−/− cells even before incubation in Xenopus extract suggests deregulation of separase, at least under the given conditions of this in vitro experiment.

Mentions: We analyzed separase levels in chromosomally stable hSecurin−/− cells synchronized by nocodazole. Lysates from hSecurin+/+ and hSecurin−/− cells were probed with antibodies to separase. For each cell line we detected both the full-length and the cleaved forms of separase (Figure 5A). However, both the full-length and the cleaved forms of separase were consistently 3- to 4-fold weaker in the chromosomally stable hSecurin−/− cells.


Securin is not required for chromosomal stability in human cells.

Pfleghaar K, Heubes S, Cox J, Stemmann O, Speicher MR - PLoS Biol. (2005)

Chromosomally Stable hSecurin−/− Cells of Passage 12 and Higher Show Reduction in Both the Level and the Activity of Separase(A) Quantitation of full-length separase and the N-terminal cleavage product in both hSecurin+/+ and hSecurin−/− cells. Lysates from nocodazole-arrested cells were analyzed by immunoblotting with an antibody against the N-terminus of separase. The chromosomally stable hSecurin−/− cells show reduced levels of both the full-length and the cleaved N-terminal form of separase. β-tubulin was used as a loading control.(B) Separase was immunoprecipitated from nocodazole-arrested hSecurin+/+ and hSecurin−/− cells, activated by incubation in Xenopus anaphase extracts, and incubated with 35S-hScc1 for 0, 20, or 90 min before analysis by SDS-PAGE and autoradiography. For these experiments we used four times as many hSecurin−/− cells as hSecurin+/+ cells. Note the absence of detectable Scc1 cleavage fragments in the hSecurin−/− samples.(C) Separase used for the activity assay in (B) was analyzed by Western blotting before (−) and after (+) exposure to Xenopus anaphase extracts. The hSecurin+/+ cells clearly demonstrate an increase in self-cleavage of separase upon activation in the extract. Occurrence of auto-cleavage in hSecurin−/− cells even before incubation in Xenopus extract suggests deregulation of separase, at least under the given conditions of this in vitro experiment.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1287505&req=5

pbio-0030416-g005: Chromosomally Stable hSecurin−/− Cells of Passage 12 and Higher Show Reduction in Both the Level and the Activity of Separase(A) Quantitation of full-length separase and the N-terminal cleavage product in both hSecurin+/+ and hSecurin−/− cells. Lysates from nocodazole-arrested cells were analyzed by immunoblotting with an antibody against the N-terminus of separase. The chromosomally stable hSecurin−/− cells show reduced levels of both the full-length and the cleaved N-terminal form of separase. β-tubulin was used as a loading control.(B) Separase was immunoprecipitated from nocodazole-arrested hSecurin+/+ and hSecurin−/− cells, activated by incubation in Xenopus anaphase extracts, and incubated with 35S-hScc1 for 0, 20, or 90 min before analysis by SDS-PAGE and autoradiography. For these experiments we used four times as many hSecurin−/− cells as hSecurin+/+ cells. Note the absence of detectable Scc1 cleavage fragments in the hSecurin−/− samples.(C) Separase used for the activity assay in (B) was analyzed by Western blotting before (−) and after (+) exposure to Xenopus anaphase extracts. The hSecurin+/+ cells clearly demonstrate an increase in self-cleavage of separase upon activation in the extract. Occurrence of auto-cleavage in hSecurin−/− cells even before incubation in Xenopus extract suggests deregulation of separase, at least under the given conditions of this in vitro experiment.
Mentions: We analyzed separase levels in chromosomally stable hSecurin−/− cells synchronized by nocodazole. Lysates from hSecurin+/+ and hSecurin−/− cells were probed with antibodies to separase. For each cell line we detected both the full-length and the cleaved forms of separase (Figure 5A). However, both the full-length and the cleaved forms of separase were consistently 3- to 4-fold weaker in the chromosomally stable hSecurin−/− cells.

Bottom Line: Here we show that, after hSecurin knockout through homologous recombination, chromosome losses are only a short, transient effect.This was unexpected, as the securin loss resulted in a persisting reduction of the sister-separating protease separase and inefficient cleavage of the cohesin subunit Scc1.We propose that human cells possess efficient mechanisms to compensate for the loss of genes involved in chromosome segregation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Human Genetics, Technical University Munich, Munich, Germany.

ABSTRACT
Abnormalities of chromosome number are frequently observed in cancers. The mechanisms regulating chromosome segregation in human cells are therefore of great interest. Recently it has been reported that human cells without an hSecurin gene lose chromosomes at a high frequency. Here we show that, after hSecurin knockout through homologous recombination, chromosome losses are only a short, transient effect. After a few passages hSecurin(-/-) cells became chromosomally stable and executed mitoses normally. This was unexpected, as the securin loss resulted in a persisting reduction of the sister-separating protease separase and inefficient cleavage of the cohesin subunit Scc1. Our data demonstrate that securin is dispensable for chromosomal stability in human cells. We propose that human cells possess efficient mechanisms to compensate for the loss of genes involved in chromosome segregation.

Show MeSH
Related in: MedlinePlus