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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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Survivin-depleted cells fail to complete cytokinesis. (A) The mitotic index of SurvivinOFF cells is increased for both KO1 and KO2 relative to wild-type (WT) DT40. (B) Shutoff of Survivin expression leads to an increase in multinucleated (primarily binucleated) cells. Values for cells grown in doxycycline are shown as striped bars. Error bars indicate SD. (C) Selected frames from videos of SurvivinON and SurvivinOFF cells. Merged images show differential interference contrast (red) and histone H2B-mRFP (green), with H2B-mRFP also shown in grayscale. Time is given in hours/minutes. A control SurvivinON cell (top) completes mitosis normally. The SurvivinOFF cell achieves a metaphase alignment (0:25) but exhibits lagging chromosomes in anaphase, and cytokinesis ultimately fails. Bar, 5 μm. (D) Synchronization of DT40 cells by centrifugal elutriation. (E) Cells harvested later in the cell cycle die before those harvested earlier in the cell cycle. (F) Cells harvested later in the cell cycle fail cytokinesis before those harvested earlier in the cell cycle.
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fig3: Survivin-depleted cells fail to complete cytokinesis. (A) The mitotic index of SurvivinOFF cells is increased for both KO1 and KO2 relative to wild-type (WT) DT40. (B) Shutoff of Survivin expression leads to an increase in multinucleated (primarily binucleated) cells. Values for cells grown in doxycycline are shown as striped bars. Error bars indicate SD. (C) Selected frames from videos of SurvivinON and SurvivinOFF cells. Merged images show differential interference contrast (red) and histone H2B-mRFP (green), with H2B-mRFP also shown in grayscale. Time is given in hours/minutes. A control SurvivinON cell (top) completes mitosis normally. The SurvivinOFF cell achieves a metaphase alignment (0:25) but exhibits lagging chromosomes in anaphase, and cytokinesis ultimately fails. Bar, 5 μm. (D) Synchronization of DT40 cells by centrifugal elutriation. (E) Cells harvested later in the cell cycle die before those harvested earlier in the cell cycle. (F) Cells harvested later in the cell cycle fail cytokinesis before those harvested earlier in the cell cycle.

Mentions: Despite an earlier report that Survivin promotes entry of CD34+ cells into the cell cycle (Fukuda et al., 2002), our time-lapse imaging confirmed that cells lacking Survivin after 36 h in doxycycline continued to cycle through mitosis (see Figs. 2–4). The mitotic index of SurvivinOFF cultures was slightly elevated compared with wild-type DT40 or SurvivinON cells after 60 h in doxycycline (Fig. 3 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)

Survivin-depleted cells fail to complete cytokinesis. (A) The mitotic index of SurvivinOFF cells is increased for both KO1 and KO2 relative to wild-type (WT) DT40. (B) Shutoff of Survivin expression leads to an increase in multinucleated (primarily binucleated) cells. Values for cells grown in doxycycline are shown as striped bars. Error bars indicate SD. (C) Selected frames from videos of SurvivinON and SurvivinOFF cells. Merged images show differential interference contrast (red) and histone H2B-mRFP (green), with H2B-mRFP also shown in grayscale. Time is given in hours/minutes. A control SurvivinON cell (top) completes mitosis normally. The SurvivinOFF cell achieves a metaphase alignment (0:25) but exhibits lagging chromosomes in anaphase, and cytokinesis ultimately fails. Bar, 5 μm. (D) Synchronization of DT40 cells by centrifugal elutriation. (E) Cells harvested later in the cell cycle die before those harvested earlier in the cell cycle. (F) Cells harvested later in the cell cycle fail cytokinesis before those harvested earlier in the cell cycle.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Survivin-depleted cells fail to complete cytokinesis. (A) The mitotic index of SurvivinOFF cells is increased for both KO1 and KO2 relative to wild-type (WT) DT40. (B) Shutoff of Survivin expression leads to an increase in multinucleated (primarily binucleated) cells. Values for cells grown in doxycycline are shown as striped bars. Error bars indicate SD. (C) Selected frames from videos of SurvivinON and SurvivinOFF cells. Merged images show differential interference contrast (red) and histone H2B-mRFP (green), with H2B-mRFP also shown in grayscale. Time is given in hours/minutes. A control SurvivinON cell (top) completes mitosis normally. The SurvivinOFF cell achieves a metaphase alignment (0:25) but exhibits lagging chromosomes in anaphase, and cytokinesis ultimately fails. Bar, 5 μm. (D) Synchronization of DT40 cells by centrifugal elutriation. (E) Cells harvested later in the cell cycle die before those harvested earlier in the cell cycle. (F) Cells harvested later in the cell cycle fail cytokinesis before those harvested earlier in the cell cycle.
Mentions: Despite an earlier report that Survivin promotes entry of CD34+ cells into the cell cycle (Fukuda et al., 2002), our time-lapse imaging confirmed that cells lacking Survivin after 36 h in doxycycline continued to cycle through mitosis (see Figs. 2–4). The mitotic index of SurvivinOFF cultures was slightly elevated compared with wild-type DT40 or SurvivinON cells after 60 h in doxycycline (Fig. 3 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