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Deconstructing Survivin: comprehensive genetic analysis of Survivin function by conditional knockout in a vertebrate cell line.

Yue Z, Carvalho A, Xu Z, Yuan X, Cardinale S, Ribeiro S, Lai F, Ogawa H, Gudmundsdottir E, Gassmann R, Morrison CG, Ruchaud S, Earnshaw WC - J. Cell Biol. (2008)

Bottom Line: However, these cells show normal sensitivity to the chemotherapeutic agent etoposide.Mutations in the nuclear export sequence or dimerization interface render cells temperature sensitive for growth.As an important caveat for other studies in which protein function is studied by transient transfection, three of the Survivin mutants fail to localize in the presence of the wild-type protein but do localize and indeed support life in its absence.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh, Scotland, UK.

ABSTRACT
Survivin is a key cellular protein thought to function in apoptotic regulation, mitotic progression, or possibly both. In this study, we describe the isolation of two conditional knockouts of the survivin gene in chicken DT40 cells. DT40 cells lacking Survivin die in interphase after failing to complete cytokinesis. However, these cells show normal sensitivity to the chemotherapeutic agent etoposide. Expression of Survivin mutants against a background to reassess the role of several key residues reveals that DT40 cells can grow normally if their sole Survivin is missing a widely studied cyclin-dependent kinase phosphorylation site or sites reportedly essential for binding to Smac or aurora B. Mutations in the nuclear export sequence or dimerization interface render cells temperature sensitive for growth. As an important caveat for other studies in which protein function is studied by transient transfection, three of the Survivin mutants fail to localize in the presence of the wild-type protein but do localize and indeed support life in its absence.

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A Survivin linker mutant localizes aberrantly. (A) SurvivinL98A/V100A-GFP fails to localize at 39°C in the presence of wild-type rescue Survivin (a, c, and e). In the absence of wild-type rescue Survivin, a subset of SurvivinL98A/V100A-GFP localizes normally (b, d, and f). INCENP localizes normally in all cells. (B) SurvivinL98A/V100A-GFP fails to localize at 41°C in the presence or absence of wild-type rescue Survivin. INCENP localizes normally only in the presence of wild-type rescue Survivin (a, c, and e). (C) Summary of the mutational analysis of Survivin. Bars, 5 μm.
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fig9: A Survivin linker mutant localizes aberrantly. (A) SurvivinL98A/V100A-GFP fails to localize at 39°C in the presence of wild-type rescue Survivin (a, c, and e). In the absence of wild-type rescue Survivin, a subset of SurvivinL98A/V100A-GFP localizes normally (b, d, and f). INCENP localizes normally in all cells. (B) SurvivinL98A/V100A-GFP fails to localize at 41°C in the presence or absence of wild-type rescue Survivin. INCENP localizes normally only in the presence of wild-type rescue Survivin (a, c, and e). (C) Summary of the mutational analysis of Survivin. Bars, 5 μm.

Mentions: Interestingly, neither Survivin mutant could compete with the wild-type protein for its localization in mitosis. In SurvivinON cells growing at 39°C, SurvivinL98A/V100A-GFP localization was diffuse in all stages of mitosis (Fig. 9 A, a, c, and e). These data are consistent with the analysis of a comparable human Survivin mutant (Knauer et al., 2006). Remarkably, SurvivinL98A/V100A-GFP did target to centromeres, the spindle midzone, and the midbody in SurvivinOFF cells grown with doxycycline at 39°C (Fig. 9 A, b, d, and f). Similar phenotypes were observed for SurvivinL104A/L106A-GFP at 37°C (Fig. S4 A).


Deconstructing Survivin: comprehensive genetic analysis of Survivin function by conditional knockout in a vertebrate cell line.

Yue Z, Carvalho A, Xu Z, Yuan X, Cardinale S, Ribeiro S, Lai F, Ogawa H, Gudmundsdottir E, Gassmann R, Morrison CG, Ruchaud S, Earnshaw WC - J. Cell Biol. (2008)

A Survivin linker mutant localizes aberrantly. (A) SurvivinL98A/V100A-GFP fails to localize at 39°C in the presence of wild-type rescue Survivin (a, c, and e). In the absence of wild-type rescue Survivin, a subset of SurvivinL98A/V100A-GFP localizes normally (b, d, and f). INCENP localizes normally in all cells. (B) SurvivinL98A/V100A-GFP fails to localize at 41°C in the presence or absence of wild-type rescue Survivin. INCENP localizes normally only in the presence of wild-type rescue Survivin (a, c, and e). (C) Summary of the mutational analysis of Survivin. Bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2568024&req=5

fig9: A Survivin linker mutant localizes aberrantly. (A) SurvivinL98A/V100A-GFP fails to localize at 39°C in the presence of wild-type rescue Survivin (a, c, and e). In the absence of wild-type rescue Survivin, a subset of SurvivinL98A/V100A-GFP localizes normally (b, d, and f). INCENP localizes normally in all cells. (B) SurvivinL98A/V100A-GFP fails to localize at 41°C in the presence or absence of wild-type rescue Survivin. INCENP localizes normally only in the presence of wild-type rescue Survivin (a, c, and e). (C) Summary of the mutational analysis of Survivin. Bars, 5 μm.
Mentions: Interestingly, neither Survivin mutant could compete with the wild-type protein for its localization in mitosis. In SurvivinON cells growing at 39°C, SurvivinL98A/V100A-GFP localization was diffuse in all stages of mitosis (Fig. 9 A, a, c, and e). These data are consistent with the analysis of a comparable human Survivin mutant (Knauer et al., 2006). Remarkably, SurvivinL98A/V100A-GFP did target to centromeres, the spindle midzone, and the midbody in SurvivinOFF cells grown with doxycycline at 39°C (Fig. 9 A, b, d, and f). Similar phenotypes were observed for SurvivinL104A/L106A-GFP at 37°C (Fig. S4 A).

Bottom Line: However, these cells show normal sensitivity to the chemotherapeutic agent etoposide.Mutations in the nuclear export sequence or dimerization interface render cells temperature sensitive for growth.As an important caveat for other studies in which protein function is studied by transient transfection, three of the Survivin mutants fail to localize in the presence of the wild-type protein but do localize and indeed support life in its absence.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh, Scotland, UK.

ABSTRACT
Survivin is a key cellular protein thought to function in apoptotic regulation, mitotic progression, or possibly both. In this study, we describe the isolation of two conditional knockouts of the survivin gene in chicken DT40 cells. DT40 cells lacking Survivin die in interphase after failing to complete cytokinesis. However, these cells show normal sensitivity to the chemotherapeutic agent etoposide. Expression of Survivin mutants against a background to reassess the role of several key residues reveals that DT40 cells can grow normally if their sole Survivin is missing a widely studied cyclin-dependent kinase phosphorylation site or sites reportedly essential for binding to Smac or aurora B. Mutations in the nuclear export sequence or dimerization interface render cells temperature sensitive for growth. As an important caveat for other studies in which protein function is studied by transient transfection, three of the Survivin mutants fail to localize in the presence of the wild-type protein but do localize and indeed support life in its absence.

Show MeSH
Related in: MedlinePlus