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INCENP-aurora B interactions modulate kinase activity and chromosome passenger complex localization.

Xu Z, Ogawa H, Vagnarelli P, Bergmann JH, Hudson DF, Ruchaud S, Fukagawa T, Earnshaw WC, Samejima K - J. Cell Biol. (2009)

Bottom Line: Intermediate kinase activity levels obtained with an INCENP mutant that binds aurora B but cannot fully activate it are sufficient for a robust response against taxol, but cannot trigger CPC transfer from the chromosomes to the anaphase spindle midzone.This transfer requires significantly higher levels of aurora B activity.These experiments reveal that INCENP interactions with aurora B in vivo modulate the level of kinase activity, thus regulating CPC localization and functions during mitosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland, UK.

ABSTRACT
Dynamic localization of the chromosomal passenger complex (CPC) during mitosis is essential for its diverse functions. CPC targeting to centromeres involves interactions between Survivin, Borealin, and the inner centromere protein (CENP [INCENP]) N terminus. In this study, we investigate how interactions between the INCENP C terminus and aurora B set the level of kinase activity. Low levels of kinase activity, seen in INCENP-depleted cells or in cells expressing a mutant INCENP that cannot bind aurora B, are sufficient for a spindle checkpoint response when microtubules are absent but not against low dose taxol. Intermediate kinase activity levels obtained with an INCENP mutant that binds aurora B but cannot fully activate it are sufficient for a robust response against taxol, but cannot trigger CPC transfer from the chromosomes to the anaphase spindle midzone. This transfer requires significantly higher levels of aurora B activity. These experiments reveal that INCENP interactions with aurora B in vivo modulate the level of kinase activity, thus regulating CPC localization and functions during mitosis.

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Phenotypic analysis of conditional INCENP knockout cell line. (A) Immunofluorescence images showing INCENPON and INCENPOFF cells stained with specific antibodies recognizing endogenous INCENP (green; panels 3 and 7), α-tubulin (red; panels 2 and 6), and DAPI for DNA (blue; panels 4 and 8). After incubation with doxycycline for 26 h, endogenous INCENP was hardly detected (INCENPOFF; panel 7). Panels 9–16 show immunofluorescence images of H3S10ph (green; panels 10 and 14) and H3S28ph (red; panels 11 and 15) plus DAPI for DNA (blue; panels 12 and16) in INCENPON (panels 9–12) and INCENPOFF (panels 13–16) cells. Merged images are shown in panels 1, 5, 9, and 13. INCENPON and INCENPOFF cells were harvested and fixed at the same time. Images were acquired using the same microscope settings for all experiments. Bar, 5 µm. (B) Mitotic indices of wild-type, INCENPON, and INCENPOFF cells. (C) Scoring of mitotic cells in late prometaphase and metaphase where cells are bioriented and two spindle poles are on opposite sides. (D–G) Percentage of mitotic cells in prometaphase or metaphase (D), anaphase (E), telophase (F), or cytokinesis (G) is shown. (H–J) Percentage of binucleated cells (H), multinucleated cells (I), and mitotic cells with multipolar spindles (J) is shown. Error bars indicate SD.
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fig2: Phenotypic analysis of conditional INCENP knockout cell line. (A) Immunofluorescence images showing INCENPON and INCENPOFF cells stained with specific antibodies recognizing endogenous INCENP (green; panels 3 and 7), α-tubulin (red; panels 2 and 6), and DAPI for DNA (blue; panels 4 and 8). After incubation with doxycycline for 26 h, endogenous INCENP was hardly detected (INCENPOFF; panel 7). Panels 9–16 show immunofluorescence images of H3S10ph (green; panels 10 and 14) and H3S28ph (red; panels 11 and 15) plus DAPI for DNA (blue; panels 12 and16) in INCENPON (panels 9–12) and INCENPOFF (panels 13–16) cells. Merged images are shown in panels 1, 5, 9, and 13. INCENPON and INCENPOFF cells were harvested and fixed at the same time. Images were acquired using the same microscope settings for all experiments. Bar, 5 µm. (B) Mitotic indices of wild-type, INCENPON, and INCENPOFF cells. (C) Scoring of mitotic cells in late prometaphase and metaphase where cells are bioriented and two spindle poles are on opposite sides. (D–G) Percentage of mitotic cells in prometaphase or metaphase (D), anaphase (E), telophase (F), or cytokinesis (G) is shown. (H–J) Percentage of binucleated cells (H), multinucleated cells (I), and mitotic cells with multipolar spindles (J) is shown. Error bars indicate SD.

Mentions: INCENPON cells are phenotypically indistinguishable from wild-type DT40 clone 18 cells with regard to growth rate (Fig. 1 G), mitotic index (Fig. 2 B), and ratio of individual mitotic phases (Fig. 2, C–J). In contrast, INCENPOFF cultures cease proliferating within 12 h, and the majority of cells die within 48 h of doxycycline addition (Fig. 1 G). Levels of INCENP class I and II (two alternative splice variants of INCENP; Mackay et al., 1993) expressed from the hijacked allele dropped 12–20 h after the addition of doxycycline, becoming essentially undetectable by 24 h (Fig. 1 H). INCENP was undetectable on chromosomes by indirect immunofluorescence in INCENPOFF cells after 26 h in doxycycline (Fig. 2 A, panel 7; and Fig. S1 B), and H3 phospho-Ser10 (H3S10ph) and H3 phospho-Ser28 (H3S28ph) levels were also substantially reduced (Fig. 2 A, panels 14 and 15).


INCENP-aurora B interactions modulate kinase activity and chromosome passenger complex localization.

Xu Z, Ogawa H, Vagnarelli P, Bergmann JH, Hudson DF, Ruchaud S, Fukagawa T, Earnshaw WC, Samejima K - J. Cell Biol. (2009)

Phenotypic analysis of conditional INCENP knockout cell line. (A) Immunofluorescence images showing INCENPON and INCENPOFF cells stained with specific antibodies recognizing endogenous INCENP (green; panels 3 and 7), α-tubulin (red; panels 2 and 6), and DAPI for DNA (blue; panels 4 and 8). After incubation with doxycycline for 26 h, endogenous INCENP was hardly detected (INCENPOFF; panel 7). Panels 9–16 show immunofluorescence images of H3S10ph (green; panels 10 and 14) and H3S28ph (red; panels 11 and 15) plus DAPI for DNA (blue; panels 12 and16) in INCENPON (panels 9–12) and INCENPOFF (panels 13–16) cells. Merged images are shown in panels 1, 5, 9, and 13. INCENPON and INCENPOFF cells were harvested and fixed at the same time. Images were acquired using the same microscope settings for all experiments. Bar, 5 µm. (B) Mitotic indices of wild-type, INCENPON, and INCENPOFF cells. (C) Scoring of mitotic cells in late prometaphase and metaphase where cells are bioriented and two spindle poles are on opposite sides. (D–G) Percentage of mitotic cells in prometaphase or metaphase (D), anaphase (E), telophase (F), or cytokinesis (G) is shown. (H–J) Percentage of binucleated cells (H), multinucleated cells (I), and mitotic cells with multipolar spindles (J) is shown. Error bars indicate SD.
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Related In: Results  -  Collection

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fig2: Phenotypic analysis of conditional INCENP knockout cell line. (A) Immunofluorescence images showing INCENPON and INCENPOFF cells stained with specific antibodies recognizing endogenous INCENP (green; panels 3 and 7), α-tubulin (red; panels 2 and 6), and DAPI for DNA (blue; panels 4 and 8). After incubation with doxycycline for 26 h, endogenous INCENP was hardly detected (INCENPOFF; panel 7). Panels 9–16 show immunofluorescence images of H3S10ph (green; panels 10 and 14) and H3S28ph (red; panels 11 and 15) plus DAPI for DNA (blue; panels 12 and16) in INCENPON (panels 9–12) and INCENPOFF (panels 13–16) cells. Merged images are shown in panels 1, 5, 9, and 13. INCENPON and INCENPOFF cells were harvested and fixed at the same time. Images were acquired using the same microscope settings for all experiments. Bar, 5 µm. (B) Mitotic indices of wild-type, INCENPON, and INCENPOFF cells. (C) Scoring of mitotic cells in late prometaphase and metaphase where cells are bioriented and two spindle poles are on opposite sides. (D–G) Percentage of mitotic cells in prometaphase or metaphase (D), anaphase (E), telophase (F), or cytokinesis (G) is shown. (H–J) Percentage of binucleated cells (H), multinucleated cells (I), and mitotic cells with multipolar spindles (J) is shown. Error bars indicate SD.
Mentions: INCENPON cells are phenotypically indistinguishable from wild-type DT40 clone 18 cells with regard to growth rate (Fig. 1 G), mitotic index (Fig. 2 B), and ratio of individual mitotic phases (Fig. 2, C–J). In contrast, INCENPOFF cultures cease proliferating within 12 h, and the majority of cells die within 48 h of doxycycline addition (Fig. 1 G). Levels of INCENP class I and II (two alternative splice variants of INCENP; Mackay et al., 1993) expressed from the hijacked allele dropped 12–20 h after the addition of doxycycline, becoming essentially undetectable by 24 h (Fig. 1 H). INCENP was undetectable on chromosomes by indirect immunofluorescence in INCENPOFF cells after 26 h in doxycycline (Fig. 2 A, panel 7; and Fig. S1 B), and H3 phospho-Ser10 (H3S10ph) and H3 phospho-Ser28 (H3S28ph) levels were also substantially reduced (Fig. 2 A, panels 14 and 15).

Bottom Line: Intermediate kinase activity levels obtained with an INCENP mutant that binds aurora B but cannot fully activate it are sufficient for a robust response against taxol, but cannot trigger CPC transfer from the chromosomes to the anaphase spindle midzone.This transfer requires significantly higher levels of aurora B activity.These experiments reveal that INCENP interactions with aurora B in vivo modulate the level of kinase activity, thus regulating CPC localization and functions during mitosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland, UK.

ABSTRACT
Dynamic localization of the chromosomal passenger complex (CPC) during mitosis is essential for its diverse functions. CPC targeting to centromeres involves interactions between Survivin, Borealin, and the inner centromere protein (CENP [INCENP]) N terminus. In this study, we investigate how interactions between the INCENP C terminus and aurora B set the level of kinase activity. Low levels of kinase activity, seen in INCENP-depleted cells or in cells expressing a mutant INCENP that cannot bind aurora B, are sufficient for a spindle checkpoint response when microtubules are absent but not against low dose taxol. Intermediate kinase activity levels obtained with an INCENP mutant that binds aurora B but cannot fully activate it are sufficient for a robust response against taxol, but cannot trigger CPC transfer from the chromosomes to the anaphase spindle midzone. This transfer requires significantly higher levels of aurora B activity. These experiments reveal that INCENP interactions with aurora B in vivo modulate the level of kinase activity, thus regulating CPC localization and functions during mitosis.

Show MeSH