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Control of the pericentrosomal H2O2 level by peroxiredoxin I is critical for mitotic progression.

Lim JM, Lee KS, Woo HA, Kang D, Rhee SG - J. Cell Biol. (2015)

Bottom Line: The intracellular concentration of H2O2 increases as the cell cycle progresses.Whereas the centrosome is shielded from H2O2 through its association with the H2O2-eliminating enzyme peroxiredoxin I (PrxI) during interphase, the centrosome-associated PrxI is selectively inactivated through phosphorylation by Cdk1 during early mitosis, thereby exposing the centrosome to H2O2 and facilitating inactivation of centrosome-bound phosphatases.Dephosphorylation of PrxI by okadaic acid-sensitive phosphatases during late mitosis again shields the centrosome from H2O2 and thereby allows the reactivation of Cdk1-opposing phosphatases at the organelle.

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Affiliation: Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, South Korea.

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Effects of centrosome-targeted catalase on mitotic entry and on the abundance of centrosome-associated mitotic regulatory proteins. (A) Schematic representation of Cat-PACT (in which the centrosome-targeting PACT sequence replaces the C-terminal peroxisome-targeting sequence KANL of catalase) and Cat-delPACT (in which the sequence K594 to W675 of Cat-PACT containing two domains [black boxes] essential for centrosome targeting is deleted). (B) HeLa cells infected with a retroviral vector (pMIN) encoding Cat-PACT or Cat-delPACT were subjected to confocal immunofluorescence microscopy with antibodies to catalase (green) and to γ-tubulin (red). DNA was also stained with DAPI (blue). The area indicated by the arrows (centrosome) is shown at higher magnification in each inset. (C–G) HeLa cells stably expressing GFP-tagged histone H2B were infected with empty, Cat-PACT, or Cat-delPACT retroviral vectors for 10 h in the presence of 6 µg/ml polybrene, treated with (2 mM thymidine) for 18 h, released in fresh medium for 3 h, and then treated with 100 ng/ml nocodazole for 10 h (C). The percentage of mitotic cells was then evaluated on the basis of cell rounding (D) or chromosome condensation (E). Data are means ± SD from three independent experiments (n = 50 cells examined for each experiment). *, P < 0.02; **, P < 0.01 (Student’s t test). The cells were also subjected to immunoblot analysis with antibodies to the indicated proteins (F). The relative immunoblot intensities of Cdk1, pY15-Cdk1, and pHH3 normalized by those of actin were also determined as means ± SD from three independent experiments (G). *, P < 0.05; **, P < 0.005 (Student’s t test). (H) HeLa cells expressing histone H2B-GFP were infected with Cat-PACT or Cat-delPACT retroviral vectors, synchronized at G1–S, and released in fresh medium for live-cell imaging. The proportion of cells exhibiting mitotic entry was estimated from Videos 1 and 2 that were acquired over 20 h at 30-min intervals. Data are means ± SD from three independent experiments (n = 90 or 70 cells examined in each experiment for Cat-PACT or Cat-delPACT, respectively). (I–K) HeLa cells infected with empty, Cat-PACT, or Cat-delPACT retroviral vectors were treated as in C and then subjected to confocal microscopy with antibodies to cyclin B1 (green) and to pericentrin (red; I), to Plk1 (green) and to pericentrin (red; J), or to Aurora A (green) and to γ-tubulin (red; K). The relative fluorescence intensity ratios of cyclin B1 to pericentrin (I), of Plk1 to pericentrin (J), and of Aurora A to γ-tubulin (K) were also evaluated at the centrosome. The areas indicated by the arrows are shown at higher magnification in the insets. Data are means ± SD from three independent experiments (n = 50 cells examined for each experiment). *, P < 0.02 (Student’s t test).
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fig2: Effects of centrosome-targeted catalase on mitotic entry and on the abundance of centrosome-associated mitotic regulatory proteins. (A) Schematic representation of Cat-PACT (in which the centrosome-targeting PACT sequence replaces the C-terminal peroxisome-targeting sequence KANL of catalase) and Cat-delPACT (in which the sequence K594 to W675 of Cat-PACT containing two domains [black boxes] essential for centrosome targeting is deleted). (B) HeLa cells infected with a retroviral vector (pMIN) encoding Cat-PACT or Cat-delPACT were subjected to confocal immunofluorescence microscopy with antibodies to catalase (green) and to γ-tubulin (red). DNA was also stained with DAPI (blue). The area indicated by the arrows (centrosome) is shown at higher magnification in each inset. (C–G) HeLa cells stably expressing GFP-tagged histone H2B were infected with empty, Cat-PACT, or Cat-delPACT retroviral vectors for 10 h in the presence of 6 µg/ml polybrene, treated with (2 mM thymidine) for 18 h, released in fresh medium for 3 h, and then treated with 100 ng/ml nocodazole for 10 h (C). The percentage of mitotic cells was then evaluated on the basis of cell rounding (D) or chromosome condensation (E). Data are means ± SD from three independent experiments (n = 50 cells examined for each experiment). *, P < 0.02; **, P < 0.01 (Student’s t test). The cells were also subjected to immunoblot analysis with antibodies to the indicated proteins (F). The relative immunoblot intensities of Cdk1, pY15-Cdk1, and pHH3 normalized by those of actin were also determined as means ± SD from three independent experiments (G). *, P < 0.05; **, P < 0.005 (Student’s t test). (H) HeLa cells expressing histone H2B-GFP were infected with Cat-PACT or Cat-delPACT retroviral vectors, synchronized at G1–S, and released in fresh medium for live-cell imaging. The proportion of cells exhibiting mitotic entry was estimated from Videos 1 and 2 that were acquired over 20 h at 30-min intervals. Data are means ± SD from three independent experiments (n = 90 or 70 cells examined in each experiment for Cat-PACT or Cat-delPACT, respectively). (I–K) HeLa cells infected with empty, Cat-PACT, or Cat-delPACT retroviral vectors were treated as in C and then subjected to confocal microscopy with antibodies to cyclin B1 (green) and to pericentrin (red; I), to Plk1 (green) and to pericentrin (red; J), or to Aurora A (green) and to γ-tubulin (red; K). The relative fluorescence intensity ratios of cyclin B1 to pericentrin (I), of Plk1 to pericentrin (J), and of Aurora A to γ-tubulin (K) were also evaluated at the centrosome. The areas indicated by the arrows are shown at higher magnification in the insets. Data are means ± SD from three independent experiments (n = 50 cells examined for each experiment). *, P < 0.02 (Student’s t test).

Mentions: To examine the effect of exposure of the centrosome to cytoplasmic H2O2, which would be expected to occur as a result of PrxI phosphorylation (inactivation), we infected HeLa cells with a retroviral vector encoding a modified form of catalase either with a centrosome-targeting pericentrin-AKAP450 centrosomal targeting (PACT) sequence (Cat-PACT) or with the corresponding sequence lacking the centrosome-targeting core (Cat-delPACT; Fig. 2 A; Gillingham and Munro, 2000). The presence of Cat-PACT and the absence of Cat-delPACT at the centrosome were verified by confocal immunofluorescence microscopy (Fig. 2 B). HeLa cells that had been infected with the empty, Cat-PACT, or Cat-delPACT vectors were synchronized at G1–S by thymidine treatment and released in the presence of nocodazole for 10 h (Fig. 2 C). Early mitotic cells were scored on the basis of cell rounding (Fig. 2 D) and chromosome condensation (Fig. 2 E). The number of mitotic cells (mean of values from Fig. 2, D and E) for cells expressing Cat-PACT was reduced by ∼45% and ∼35% compared with that for cells infected with the empty vector or the vector encoding Cat-delPACT, respectively. Immunoblot analysis of the infected HeLa cells showed that the level of inactive (Tyr15 phosphorylated) Cdk1 in Cat-PACT cells was about twice that in Cat-delPACT cells and that the level of pHH3 in Cat-PACT cells was ∼50% of that in Cat-delPACT cells (Fig. 2, F and G). Such analysis also revealed that Cat-PACT and Cat-delPACT were expressed stably at low levels relative to endogenous peroxisomal catalase, suggesting that any effect of Cat-PACT present in the cytosol instead of at the centrosome as a result of excessive expression was minimal (Fig. 2 F). We also monitored mitotic entry by fluorescence imaging of live cells after the release of Cat-PACT and Cat-delPACT cells from G1–S arrest. Mitotic entry of Cat-PACT cells was delayed by a mean of ∼2–3 h compared with that of Cat-delPACT cells (Fig. 2 H; and Videos 1 and 2). These results thus supported the notion that exposure of the centrosome to H2O2 is required for normal mitotic entry.


Control of the pericentrosomal H2O2 level by peroxiredoxin I is critical for mitotic progression.

Lim JM, Lee KS, Woo HA, Kang D, Rhee SG - J. Cell Biol. (2015)

Effects of centrosome-targeted catalase on mitotic entry and on the abundance of centrosome-associated mitotic regulatory proteins. (A) Schematic representation of Cat-PACT (in which the centrosome-targeting PACT sequence replaces the C-terminal peroxisome-targeting sequence KANL of catalase) and Cat-delPACT (in which the sequence K594 to W675 of Cat-PACT containing two domains [black boxes] essential for centrosome targeting is deleted). (B) HeLa cells infected with a retroviral vector (pMIN) encoding Cat-PACT or Cat-delPACT were subjected to confocal immunofluorescence microscopy with antibodies to catalase (green) and to γ-tubulin (red). DNA was also stained with DAPI (blue). The area indicated by the arrows (centrosome) is shown at higher magnification in each inset. (C–G) HeLa cells stably expressing GFP-tagged histone H2B were infected with empty, Cat-PACT, or Cat-delPACT retroviral vectors for 10 h in the presence of 6 µg/ml polybrene, treated with (2 mM thymidine) for 18 h, released in fresh medium for 3 h, and then treated with 100 ng/ml nocodazole for 10 h (C). The percentage of mitotic cells was then evaluated on the basis of cell rounding (D) or chromosome condensation (E). Data are means ± SD from three independent experiments (n = 50 cells examined for each experiment). *, P < 0.02; **, P < 0.01 (Student’s t test). The cells were also subjected to immunoblot analysis with antibodies to the indicated proteins (F). The relative immunoblot intensities of Cdk1, pY15-Cdk1, and pHH3 normalized by those of actin were also determined as means ± SD from three independent experiments (G). *, P < 0.05; **, P < 0.005 (Student’s t test). (H) HeLa cells expressing histone H2B-GFP were infected with Cat-PACT or Cat-delPACT retroviral vectors, synchronized at G1–S, and released in fresh medium for live-cell imaging. The proportion of cells exhibiting mitotic entry was estimated from Videos 1 and 2 that were acquired over 20 h at 30-min intervals. Data are means ± SD from three independent experiments (n = 90 or 70 cells examined in each experiment for Cat-PACT or Cat-delPACT, respectively). (I–K) HeLa cells infected with empty, Cat-PACT, or Cat-delPACT retroviral vectors were treated as in C and then subjected to confocal microscopy with antibodies to cyclin B1 (green) and to pericentrin (red; I), to Plk1 (green) and to pericentrin (red; J), or to Aurora A (green) and to γ-tubulin (red; K). The relative fluorescence intensity ratios of cyclin B1 to pericentrin (I), of Plk1 to pericentrin (J), and of Aurora A to γ-tubulin (K) were also evaluated at the centrosome. The areas indicated by the arrows are shown at higher magnification in the insets. Data are means ± SD from three independent experiments (n = 50 cells examined for each experiment). *, P < 0.02 (Student’s t test).
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fig2: Effects of centrosome-targeted catalase on mitotic entry and on the abundance of centrosome-associated mitotic regulatory proteins. (A) Schematic representation of Cat-PACT (in which the centrosome-targeting PACT sequence replaces the C-terminal peroxisome-targeting sequence KANL of catalase) and Cat-delPACT (in which the sequence K594 to W675 of Cat-PACT containing two domains [black boxes] essential for centrosome targeting is deleted). (B) HeLa cells infected with a retroviral vector (pMIN) encoding Cat-PACT or Cat-delPACT were subjected to confocal immunofluorescence microscopy with antibodies to catalase (green) and to γ-tubulin (red). DNA was also stained with DAPI (blue). The area indicated by the arrows (centrosome) is shown at higher magnification in each inset. (C–G) HeLa cells stably expressing GFP-tagged histone H2B were infected with empty, Cat-PACT, or Cat-delPACT retroviral vectors for 10 h in the presence of 6 µg/ml polybrene, treated with (2 mM thymidine) for 18 h, released in fresh medium for 3 h, and then treated with 100 ng/ml nocodazole for 10 h (C). The percentage of mitotic cells was then evaluated on the basis of cell rounding (D) or chromosome condensation (E). Data are means ± SD from three independent experiments (n = 50 cells examined for each experiment). *, P < 0.02; **, P < 0.01 (Student’s t test). The cells were also subjected to immunoblot analysis with antibodies to the indicated proteins (F). The relative immunoblot intensities of Cdk1, pY15-Cdk1, and pHH3 normalized by those of actin were also determined as means ± SD from three independent experiments (G). *, P < 0.05; **, P < 0.005 (Student’s t test). (H) HeLa cells expressing histone H2B-GFP were infected with Cat-PACT or Cat-delPACT retroviral vectors, synchronized at G1–S, and released in fresh medium for live-cell imaging. The proportion of cells exhibiting mitotic entry was estimated from Videos 1 and 2 that were acquired over 20 h at 30-min intervals. Data are means ± SD from three independent experiments (n = 90 or 70 cells examined in each experiment for Cat-PACT or Cat-delPACT, respectively). (I–K) HeLa cells infected with empty, Cat-PACT, or Cat-delPACT retroviral vectors were treated as in C and then subjected to confocal microscopy with antibodies to cyclin B1 (green) and to pericentrin (red; I), to Plk1 (green) and to pericentrin (red; J), or to Aurora A (green) and to γ-tubulin (red; K). The relative fluorescence intensity ratios of cyclin B1 to pericentrin (I), of Plk1 to pericentrin (J), and of Aurora A to γ-tubulin (K) were also evaluated at the centrosome. The areas indicated by the arrows are shown at higher magnification in the insets. Data are means ± SD from three independent experiments (n = 50 cells examined for each experiment). *, P < 0.02 (Student’s t test).
Mentions: To examine the effect of exposure of the centrosome to cytoplasmic H2O2, which would be expected to occur as a result of PrxI phosphorylation (inactivation), we infected HeLa cells with a retroviral vector encoding a modified form of catalase either with a centrosome-targeting pericentrin-AKAP450 centrosomal targeting (PACT) sequence (Cat-PACT) or with the corresponding sequence lacking the centrosome-targeting core (Cat-delPACT; Fig. 2 A; Gillingham and Munro, 2000). The presence of Cat-PACT and the absence of Cat-delPACT at the centrosome were verified by confocal immunofluorescence microscopy (Fig. 2 B). HeLa cells that had been infected with the empty, Cat-PACT, or Cat-delPACT vectors were synchronized at G1–S by thymidine treatment and released in the presence of nocodazole for 10 h (Fig. 2 C). Early mitotic cells were scored on the basis of cell rounding (Fig. 2 D) and chromosome condensation (Fig. 2 E). The number of mitotic cells (mean of values from Fig. 2, D and E) for cells expressing Cat-PACT was reduced by ∼45% and ∼35% compared with that for cells infected with the empty vector or the vector encoding Cat-delPACT, respectively. Immunoblot analysis of the infected HeLa cells showed that the level of inactive (Tyr15 phosphorylated) Cdk1 in Cat-PACT cells was about twice that in Cat-delPACT cells and that the level of pHH3 in Cat-PACT cells was ∼50% of that in Cat-delPACT cells (Fig. 2, F and G). Such analysis also revealed that Cat-PACT and Cat-delPACT were expressed stably at low levels relative to endogenous peroxisomal catalase, suggesting that any effect of Cat-PACT present in the cytosol instead of at the centrosome as a result of excessive expression was minimal (Fig. 2 F). We also monitored mitotic entry by fluorescence imaging of live cells after the release of Cat-PACT and Cat-delPACT cells from G1–S arrest. Mitotic entry of Cat-PACT cells was delayed by a mean of ∼2–3 h compared with that of Cat-delPACT cells (Fig. 2 H; and Videos 1 and 2). These results thus supported the notion that exposure of the centrosome to H2O2 is required for normal mitotic entry.

Bottom Line: The intracellular concentration of H2O2 increases as the cell cycle progresses.Whereas the centrosome is shielded from H2O2 through its association with the H2O2-eliminating enzyme peroxiredoxin I (PrxI) during interphase, the centrosome-associated PrxI is selectively inactivated through phosphorylation by Cdk1 during early mitosis, thereby exposing the centrosome to H2O2 and facilitating inactivation of centrosome-bound phosphatases.Dephosphorylation of PrxI by okadaic acid-sensitive phosphatases during late mitosis again shields the centrosome from H2O2 and thereby allows the reactivation of Cdk1-opposing phosphatases at the organelle.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Life and Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, South Korea.

Show MeSH
Related in: MedlinePlus