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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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Phenotype of cells after Survivin shutoff. (A) Distribution of Survivin (red), α-tubulin (green), and DNA (blue) in SurvivinON (A and A′) and SurvivinOFF (A″ and A‴) mitotic cells. Bar, 5 μm. (B) Phase-contrast images of SurvivinON and SurvivinOFF cultures (the latter after 60-h growth in doxycycline). Bar, 10 μm. (C) Cells from both survivin knockouts KO1 and KO2 die after exposure to doxycycline. (D) Annexin V–positive (apoptotic) cells appear 36 h after shutoff of survivin transcription in KO1. (E) INCENP (red) is diffuse, but kinetochores (CENP-H–GFP, green) appear normal in SurvivinOFF cells (bottom). Top, SurvivinON control. Bar, 5 μm. (F) INCENP and H3S10ph levels decrease shortly after Survivin levels drop after addition of doxycycline to KO1 cells. Loading control, anti–α-tubulin. WT, wild type. (G) Micrograph showing decreased H3S10ph staining (green) in SurvivinOFF cells (bottom). Top, SurvivinON control. Bar, 5 μm.
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fig2: Phenotype of cells after Survivin shutoff. (A) Distribution of Survivin (red), α-tubulin (green), and DNA (blue) in SurvivinON (A and A′) and SurvivinOFF (A″ and A‴) mitotic cells. Bar, 5 μm. (B) Phase-contrast images of SurvivinON and SurvivinOFF cultures (the latter after 60-h growth in doxycycline). Bar, 10 μm. (C) Cells from both survivin knockouts KO1 and KO2 die after exposure to doxycycline. (D) Annexin V–positive (apoptotic) cells appear 36 h after shutoff of survivin transcription in KO1. (E) INCENP (red) is diffuse, but kinetochores (CENP-H–GFP, green) appear normal in SurvivinOFF cells (bottom). Top, SurvivinON control. Bar, 5 μm. (F) INCENP and H3S10ph levels decrease shortly after Survivin levels drop after addition of doxycycline to KO1 cells. Loading control, anti–α-tubulin. WT, wild type. (G) Micrograph showing decreased H3S10ph staining (green) in SurvivinOFF cells (bottom). Top, SurvivinON control. Bar, 5 μm.

Mentions: The level of Survivin protein in KO2 cultures growing without doxycycline was similar to or slightly below that in wild-type cells (Fig. 1 H, 0 h), whereas in KO1, Survivin was substantially overexpressed (Fig. 1 G). Remarkably, the growth of KO1 and KO2 cells appeared to be identical; thus, an excess of the canonical isoform of Survivin did not appear to confer a growth advantage on KO1 cells (Fig. 2 C). Doxycycline addition caused a rapid drop in Survivin levels, which became essentially undetectable by 36 h in KO2. Loss of Survivin was more variable for KO1, and in some experiments cells died before the protein was completely lost from cultures. Indirect immunofluorescence staining of KO2 cells using antibody against chicken Survivin showed no detectable Survivin signal during mitosis after 60 h in doxycycline (Fig. 2, A″ and 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)

Phenotype of cells after Survivin shutoff. (A) Distribution of Survivin (red), α-tubulin (green), and DNA (blue) in SurvivinON (A and A′) and SurvivinOFF (A″ and A‴) mitotic cells. Bar, 5 μm. (B) Phase-contrast images of SurvivinON and SurvivinOFF cultures (the latter after 60-h growth in doxycycline). Bar, 10 μm. (C) Cells from both survivin knockouts KO1 and KO2 die after exposure to doxycycline. (D) Annexin V–positive (apoptotic) cells appear 36 h after shutoff of survivin transcription in KO1. (E) INCENP (red) is diffuse, but kinetochores (CENP-H–GFP, green) appear normal in SurvivinOFF cells (bottom). Top, SurvivinON control. Bar, 5 μm. (F) INCENP and H3S10ph levels decrease shortly after Survivin levels drop after addition of doxycycline to KO1 cells. Loading control, anti–α-tubulin. WT, wild type. (G) Micrograph showing decreased H3S10ph staining (green) in SurvivinOFF cells (bottom). Top, SurvivinON control. Bar, 5 μm.
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fig2: Phenotype of cells after Survivin shutoff. (A) Distribution of Survivin (red), α-tubulin (green), and DNA (blue) in SurvivinON (A and A′) and SurvivinOFF (A″ and A‴) mitotic cells. Bar, 5 μm. (B) Phase-contrast images of SurvivinON and SurvivinOFF cultures (the latter after 60-h growth in doxycycline). Bar, 10 μm. (C) Cells from both survivin knockouts KO1 and KO2 die after exposure to doxycycline. (D) Annexin V–positive (apoptotic) cells appear 36 h after shutoff of survivin transcription in KO1. (E) INCENP (red) is diffuse, but kinetochores (CENP-H–GFP, green) appear normal in SurvivinOFF cells (bottom). Top, SurvivinON control. Bar, 5 μm. (F) INCENP and H3S10ph levels decrease shortly after Survivin levels drop after addition of doxycycline to KO1 cells. Loading control, anti–α-tubulin. WT, wild type. (G) Micrograph showing decreased H3S10ph staining (green) in SurvivinOFF cells (bottom). Top, SurvivinON control. Bar, 5 μm.
Mentions: The level of Survivin protein in KO2 cultures growing without doxycycline was similar to or slightly below that in wild-type cells (Fig. 1 H, 0 h), whereas in KO1, Survivin was substantially overexpressed (Fig. 1 G). Remarkably, the growth of KO1 and KO2 cells appeared to be identical; thus, an excess of the canonical isoform of Survivin did not appear to confer a growth advantage on KO1 cells (Fig. 2 C). Doxycycline addition caused a rapid drop in Survivin levels, which became essentially undetectable by 36 h in KO2. Loss of Survivin was more variable for KO1, and in some experiments cells died before the protein was completely lost from cultures. Indirect immunofluorescence staining of KO2 cells using antibody against chicken Survivin showed no detectable Survivin signal during mitosis after 60 h in doxycycline (Fig. 2, A″ and 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