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Cdc25B cooperates with Cdc25A to induce mitosis but has a unique role in activating cyclin B1-Cdk1 at the centrosome.

Lindqvist A, Källström H, Lundgren A, Barsoum E, Rosenthal CK - J. Cell Biol. (2005)

Bottom Line: Cdc25 phosphatases are essential for the activation of mitotic cyclin-Cdks, but the precise roles of the three mammalian isoforms (A, B, and C) are unclear.Using RNA interference to reduce the expression of each Cdc25 isoform in HeLa and HEK293 cells, we observed that Cdc25A and -B are both needed for mitotic entry, whereas Cdc25C alone cannot induce mitosis.We found that the G2 delay caused by small interfering RNA to Cdc25A or -B was accompanied by reduced activities of both cyclin B1-Cdk1 and cyclin A-Cdk2 complexes and a delayed accumulation of cyclin B1 protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Karolinska Institutet, S-171 77 Stockholm, Sweden.

ABSTRACT
Cdc25 phosphatases are essential for the activation of mitotic cyclin-Cdks, but the precise roles of the three mammalian isoforms (A, B, and C) are unclear. Using RNA interference to reduce the expression of each Cdc25 isoform in HeLa and HEK293 cells, we observed that Cdc25A and -B are both needed for mitotic entry, whereas Cdc25C alone cannot induce mitosis. We found that the G2 delay caused by small interfering RNA to Cdc25A or -B was accompanied by reduced activities of both cyclin B1-Cdk1 and cyclin A-Cdk2 complexes and a delayed accumulation of cyclin B1 protein. Further, three-dimensional time-lapse microscopy and quantification of Cdk1 phosphorylation versus cyclin B1 levels in individual cells revealed that Cdc25A and -B exert specific functions in the initiation of mitosis: Cdc25A may play a role in chromatin condensation, whereas Cdc25B specifically activates cyclin B1-Cdk1 on centrosomes.

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Decreased time between centrosome separation and DNA condensation in a fraction of cells injected with siRNA to Cdc25B. HeLa cells were microinjected with the marker plasmids pYFP-histone H2B and pdsRED-γ-tubulin alone or together with siRNA to Cdc25A or -B. After release from a thymidine block, injected cells were followed by 3D time-lapse microscopy. (A) Examples of the behavior of cells entering mitosis. Images show maximum intensity projections of YFP-histone H2B and dsRED-γ-tubulin fluorescence. The time between images is 12 min. (top) Normal mitotic progression of a cell injected with pYFP-histone H2B and pdsRED-γ-tubulin. (middle) Delay between centrosome separation and chromosome condensation in cell microinjected with siRNA to Cdc25A. (bottom) Less than 12 min between centrosome separation and chromosome condensation in cell injected with siRNA to Cdc25B. Bar, 10 μm. (B) Time between centrosome separation and DNA condensation in a larger number of siRNA-treated cells. The number of images between centrosome separation and DNA condensation is shown below. The distance between images is 12 min. (C) Centrosomes separate but reunite in a subset of cells injected with siRNA to Cdc25B. Example of two cells microinjected with siRNA to Cdc25B that do not enter mitosis in the time frame of the experiment. The time after release from a thymidine block is indicated below the figure. (D) Quantification of centrosome distances before entry into mitosis. Each graph presents the behavior of three single cells. The distance between centrosomes was measured in 3D and plotted against time. Time 0 is defined as first time point when DNA condensation is clearly visible.
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fig5: Decreased time between centrosome separation and DNA condensation in a fraction of cells injected with siRNA to Cdc25B. HeLa cells were microinjected with the marker plasmids pYFP-histone H2B and pdsRED-γ-tubulin alone or together with siRNA to Cdc25A or -B. After release from a thymidine block, injected cells were followed by 3D time-lapse microscopy. (A) Examples of the behavior of cells entering mitosis. Images show maximum intensity projections of YFP-histone H2B and dsRED-γ-tubulin fluorescence. The time between images is 12 min. (top) Normal mitotic progression of a cell injected with pYFP-histone H2B and pdsRED-γ-tubulin. (middle) Delay between centrosome separation and chromosome condensation in cell microinjected with siRNA to Cdc25A. (bottom) Less than 12 min between centrosome separation and chromosome condensation in cell injected with siRNA to Cdc25B. Bar, 10 μm. (B) Time between centrosome separation and DNA condensation in a larger number of siRNA-treated cells. The number of images between centrosome separation and DNA condensation is shown below. The distance between images is 12 min. (C) Centrosomes separate but reunite in a subset of cells injected with siRNA to Cdc25B. Example of two cells microinjected with siRNA to Cdc25B that do not enter mitosis in the time frame of the experiment. The time after release from a thymidine block is indicated below the figure. (D) Quantification of centrosome distances before entry into mitosis. Each graph presents the behavior of three single cells. The distance between centrosomes was measured in 3D and plotted against time. Time 0 is defined as first time point when DNA condensation is clearly visible.

Mentions: We microinjected siRNA together with plasmids coding for YFP-histone H2B and Discosoma red fluorescent protein (dsRED)-γ-tubulin in synchronized HeLa cells to be able to follow DNA condensation and centrosome separation with three-dimensional (3D) time-lapse microscopy. Images were collected every 12 min. In almost all cells microinjected with only the marker plasmids, the centrosomes separated at between one and four time points (corresponding to 0–60 min) before chromatin condensation was visible. The majority of cells microinjected with siRNA to Cdc25A or -B behaved in a manner similar to that of control cells. However, in a subset of cells microinjected with siRNA to Cdc25A, the time between centrosome separation and DNA condensation was prolonged. In these cells, chromosomes had not condensed 1 h or more after the start of centrosome separation (Fig. 5, A and B).


Cdc25B cooperates with Cdc25A to induce mitosis but has a unique role in activating cyclin B1-Cdk1 at the centrosome.

Lindqvist A, Källström H, Lundgren A, Barsoum E, Rosenthal CK - J. Cell Biol. (2005)

Decreased time between centrosome separation and DNA condensation in a fraction of cells injected with siRNA to Cdc25B. HeLa cells were microinjected with the marker plasmids pYFP-histone H2B and pdsRED-γ-tubulin alone or together with siRNA to Cdc25A or -B. After release from a thymidine block, injected cells were followed by 3D time-lapse microscopy. (A) Examples of the behavior of cells entering mitosis. Images show maximum intensity projections of YFP-histone H2B and dsRED-γ-tubulin fluorescence. The time between images is 12 min. (top) Normal mitotic progression of a cell injected with pYFP-histone H2B and pdsRED-γ-tubulin. (middle) Delay between centrosome separation and chromosome condensation in cell microinjected with siRNA to Cdc25A. (bottom) Less than 12 min between centrosome separation and chromosome condensation in cell injected with siRNA to Cdc25B. Bar, 10 μm. (B) Time between centrosome separation and DNA condensation in a larger number of siRNA-treated cells. The number of images between centrosome separation and DNA condensation is shown below. The distance between images is 12 min. (C) Centrosomes separate but reunite in a subset of cells injected with siRNA to Cdc25B. Example of two cells microinjected with siRNA to Cdc25B that do not enter mitosis in the time frame of the experiment. The time after release from a thymidine block is indicated below the figure. (D) Quantification of centrosome distances before entry into mitosis. Each graph presents the behavior of three single cells. The distance between centrosomes was measured in 3D and plotted against time. Time 0 is defined as first time point when DNA condensation is clearly visible.
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fig5: Decreased time between centrosome separation and DNA condensation in a fraction of cells injected with siRNA to Cdc25B. HeLa cells were microinjected with the marker plasmids pYFP-histone H2B and pdsRED-γ-tubulin alone or together with siRNA to Cdc25A or -B. After release from a thymidine block, injected cells were followed by 3D time-lapse microscopy. (A) Examples of the behavior of cells entering mitosis. Images show maximum intensity projections of YFP-histone H2B and dsRED-γ-tubulin fluorescence. The time between images is 12 min. (top) Normal mitotic progression of a cell injected with pYFP-histone H2B and pdsRED-γ-tubulin. (middle) Delay between centrosome separation and chromosome condensation in cell microinjected with siRNA to Cdc25A. (bottom) Less than 12 min between centrosome separation and chromosome condensation in cell injected with siRNA to Cdc25B. Bar, 10 μm. (B) Time between centrosome separation and DNA condensation in a larger number of siRNA-treated cells. The number of images between centrosome separation and DNA condensation is shown below. The distance between images is 12 min. (C) Centrosomes separate but reunite in a subset of cells injected with siRNA to Cdc25B. Example of two cells microinjected with siRNA to Cdc25B that do not enter mitosis in the time frame of the experiment. The time after release from a thymidine block is indicated below the figure. (D) Quantification of centrosome distances before entry into mitosis. Each graph presents the behavior of three single cells. The distance between centrosomes was measured in 3D and plotted against time. Time 0 is defined as first time point when DNA condensation is clearly visible.
Mentions: We microinjected siRNA together with plasmids coding for YFP-histone H2B and Discosoma red fluorescent protein (dsRED)-γ-tubulin in synchronized HeLa cells to be able to follow DNA condensation and centrosome separation with three-dimensional (3D) time-lapse microscopy. Images were collected every 12 min. In almost all cells microinjected with only the marker plasmids, the centrosomes separated at between one and four time points (corresponding to 0–60 min) before chromatin condensation was visible. The majority of cells microinjected with siRNA to Cdc25A or -B behaved in a manner similar to that of control cells. However, in a subset of cells microinjected with siRNA to Cdc25A, the time between centrosome separation and DNA condensation was prolonged. In these cells, chromosomes had not condensed 1 h or more after the start of centrosome separation (Fig. 5, A and B).

Bottom Line: Cdc25 phosphatases are essential for the activation of mitotic cyclin-Cdks, but the precise roles of the three mammalian isoforms (A, B, and C) are unclear.Using RNA interference to reduce the expression of each Cdc25 isoform in HeLa and HEK293 cells, we observed that Cdc25A and -B are both needed for mitotic entry, whereas Cdc25C alone cannot induce mitosis.We found that the G2 delay caused by small interfering RNA to Cdc25A or -B was accompanied by reduced activities of both cyclin B1-Cdk1 and cyclin A-Cdk2 complexes and a delayed accumulation of cyclin B1 protein.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Karolinska Institutet, S-171 77 Stockholm, Sweden.

ABSTRACT
Cdc25 phosphatases are essential for the activation of mitotic cyclin-Cdks, but the precise roles of the three mammalian isoforms (A, B, and C) are unclear. Using RNA interference to reduce the expression of each Cdc25 isoform in HeLa and HEK293 cells, we observed that Cdc25A and -B are both needed for mitotic entry, whereas Cdc25C alone cannot induce mitosis. We found that the G2 delay caused by small interfering RNA to Cdc25A or -B was accompanied by reduced activities of both cyclin B1-Cdk1 and cyclin A-Cdk2 complexes and a delayed accumulation of cyclin B1 protein. Further, three-dimensional time-lapse microscopy and quantification of Cdk1 phosphorylation versus cyclin B1 levels in individual cells revealed that Cdc25A and -B exert specific functions in the initiation of mitosis: Cdc25A may play a role in chromatin condensation, whereas Cdc25B specifically activates cyclin B1-Cdk1 on centrosomes.

Show MeSH
Related in: MedlinePlus