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CaM kinase II initiates meiotic spindle depolymerization independently of APC/C activation.

Reber S, Over S, Kronja I, Gruss OJ - J. Cell Biol. (2008)

Bottom Line: Using Ran-guanosine triphosphate-mediated microtubule assemblies and quantitative analysis of complete spindles, we demonstrate that CaMKII triggers anaphase microtubule depolymerization.A CaMKII-induced twofold increase in microtubule catastrophe rates can explain reduced microtubule stability.Therefore, our data demonstrate that CaMKII turns on parallel pathways to activate the APC/C and to induce microtubule depolymerization at meiotic anaphase onset.

View Article: PubMed Central - PubMed

Affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg, Deutsches Krebsforschungszentrum und Zentrum für Molekulare Biologie Heidelberg Allianz (DKFZ-ZMBH Alliance), 69120 Heidelberg, Germany.

ABSTRACT
Altered spindle microtubule dynamics at anaphase onset are the basis for chromosome segregation. In Xenopus laevis egg extracts, increasing free calcium levels and subsequently rising calcium-calmodulin-dependent kinase II (CaMKII) activity promote a release from meiosis II arrest and reentry into anaphase. CaMKII induces the activation of the anaphase-promoting complex/cyclosome (APC/C), which destines securin and cyclin B for degradation to allow chromosome separation and mitotic exit. In this study, we investigated the calcium-dependent signal responsible for microtubule depolymerization at anaphase onset after release from meiotic arrest in Xenopus egg extracts. Using Ran-guanosine triphosphate-mediated microtubule assemblies and quantitative analysis of complete spindles, we demonstrate that CaMKII triggers anaphase microtubule depolymerization. A CaMKII-induced twofold increase in microtubule catastrophe rates can explain reduced microtubule stability. However, calcium or constitutively active CaMKII promotes microtubule destabilization even upon APC/C inhibition and in the presence of high cyclin-dependent kinase 1 activity. Therefore, our data demonstrate that CaMKII turns on parallel pathways to activate the APC/C and to induce microtubule depolymerization at meiotic anaphase onset.

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Correlation of Cdk1 activity and changes in microtubule stability at metaphase to anaphase transition. (A) CSF-arrested egg extracts were incubated with calcium and cyclinBΔ90 (Ana) or CGP74514A (CGP). Ran-GTP–mediated microtubule assemblies were visualized by direct fluorescence after the addition of Cy3-labeled tubulin (top), and Cdk1 activities were determined by a histone H1 kinase activity assay (bottom). (B–D) CSF-arrested egg extracts (Meta or M) were triggered to go into anaphase by calcium addition in the presence of increasing cyclinBΔ90 concentrations. Ran-GTP–induced metaphase microtubule assemblies were visualized (B) and quantified relative to metaphase as indicated (C, black bars). In parallel, the Cdk1 kinase activity was measured using histone H1 phosphorylation (C, gray bars). (D) Anaphase was first induced with calcium in the presence of 80 nM cyclinBΔ90, increasing amounts of cyclinBΔ90 were added, and the capacity of Ran-GTP to still induce microtubule assembly was tested. These assemblies were quantified relative to metaphase (black bars), and the Cdk1 kinase activity was measured using histone H1 phosphorylation (gray bars). Error bars represent SD from three independent experiments. Ana, anaphase; Meta, metaphase. Bars, 5 μm.
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fig2: Correlation of Cdk1 activity and changes in microtubule stability at metaphase to anaphase transition. (A) CSF-arrested egg extracts were incubated with calcium and cyclinBΔ90 (Ana) or CGP74514A (CGP). Ran-GTP–mediated microtubule assemblies were visualized by direct fluorescence after the addition of Cy3-labeled tubulin (top), and Cdk1 activities were determined by a histone H1 kinase activity assay (bottom). (B–D) CSF-arrested egg extracts (Meta or M) were triggered to go into anaphase by calcium addition in the presence of increasing cyclinBΔ90 concentrations. Ran-GTP–induced metaphase microtubule assemblies were visualized (B) and quantified relative to metaphase as indicated (C, black bars). In parallel, the Cdk1 kinase activity was measured using histone H1 phosphorylation (C, gray bars). (D) Anaphase was first induced with calcium in the presence of 80 nM cyclinBΔ90, increasing amounts of cyclinBΔ90 were added, and the capacity of Ran-GTP to still induce microtubule assembly was tested. These assemblies were quantified relative to metaphase (black bars), and the Cdk1 kinase activity was measured using histone H1 phosphorylation (gray bars). Error bars represent SD from three independent experiments. Ana, anaphase; Meta, metaphase. Bars, 5 μm.

Mentions: A possible explanation for our observations is that activation of the APC/C, degradation of cyclin B, and a following drop in Cdk1 activity led to a changed phosphorylation pattern of MAPs and motors and consequently to the destabilization of microtubules. To test this, we assembled microtubule structures by the addition of Ran-GTP and subsequently observed their stability upon Cdk1 inhibition using CGP74514A (CGP), a selective chemical inhibitor of Cdk1 kinase (Fig. 2 A; Skoufias et al., 2007). Surprisingly, Ran-induced structures did not disassemble upon Cdk1 inhibition before interphasic microtubules were detectable (Fig. 2 A, CGP; see 15 min for coexisting M phase and interphase microtubule structures). In contrast, metaphase microtubule structures disassembled as expected upon the addition of calcium (Fig. 2 A, top) despite high Cdk1 activity (Fig. 2 A, bottom; Ana). This suggested that the sole reduction of Cdk1 activity is not sufficient for the observed changes in anaphase microtubule stability. To analyze the correlation between Cdk1 activity and microtubule stability more quantitatively, we determined Ran-GTP–mediated microtubule assemblies at different concentrations of nondegradable cyclin B. When cylinBΔ90 was added to only 40 nM and anaphase onset was induced by calcium addition, the system exited mitosis as judged by long and stable interphasic microtubules. Upon the addition of cyclinBΔ90 to endogenous levels (80 nM; Stemmann et al., 2001), no microtubule structures were observed (Fig. 1). This indicates that anaphase is faithfully established and maintained upon calcium addition and physiological cyclinBΔ90 levels. The addition of >150 nM cyclinBΔ90 allowed some but still significantly less microtubule assemblies than in metaphase (Fig. 2, B and C). Moreover, no microtubule assembly was observed if anaphase was initially established at 80 nM cyclinBΔ90, and the concentration only subsequently increased even up to 1 μM (Fig. 2 D).


CaM kinase II initiates meiotic spindle depolymerization independently of APC/C activation.

Reber S, Over S, Kronja I, Gruss OJ - J. Cell Biol. (2008)

Correlation of Cdk1 activity and changes in microtubule stability at metaphase to anaphase transition. (A) CSF-arrested egg extracts were incubated with calcium and cyclinBΔ90 (Ana) or CGP74514A (CGP). Ran-GTP–mediated microtubule assemblies were visualized by direct fluorescence after the addition of Cy3-labeled tubulin (top), and Cdk1 activities were determined by a histone H1 kinase activity assay (bottom). (B–D) CSF-arrested egg extracts (Meta or M) were triggered to go into anaphase by calcium addition in the presence of increasing cyclinBΔ90 concentrations. Ran-GTP–induced metaphase microtubule assemblies were visualized (B) and quantified relative to metaphase as indicated (C, black bars). In parallel, the Cdk1 kinase activity was measured using histone H1 phosphorylation (C, gray bars). (D) Anaphase was first induced with calcium in the presence of 80 nM cyclinBΔ90, increasing amounts of cyclinBΔ90 were added, and the capacity of Ran-GTP to still induce microtubule assembly was tested. These assemblies were quantified relative to metaphase (black bars), and the Cdk1 kinase activity was measured using histone H1 phosphorylation (gray bars). Error bars represent SD from three independent experiments. Ana, anaphase; Meta, metaphase. Bars, 5 μm.
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Related In: Results  -  Collection

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fig2: Correlation of Cdk1 activity and changes in microtubule stability at metaphase to anaphase transition. (A) CSF-arrested egg extracts were incubated with calcium and cyclinBΔ90 (Ana) or CGP74514A (CGP). Ran-GTP–mediated microtubule assemblies were visualized by direct fluorescence after the addition of Cy3-labeled tubulin (top), and Cdk1 activities were determined by a histone H1 kinase activity assay (bottom). (B–D) CSF-arrested egg extracts (Meta or M) were triggered to go into anaphase by calcium addition in the presence of increasing cyclinBΔ90 concentrations. Ran-GTP–induced metaphase microtubule assemblies were visualized (B) and quantified relative to metaphase as indicated (C, black bars). In parallel, the Cdk1 kinase activity was measured using histone H1 phosphorylation (C, gray bars). (D) Anaphase was first induced with calcium in the presence of 80 nM cyclinBΔ90, increasing amounts of cyclinBΔ90 were added, and the capacity of Ran-GTP to still induce microtubule assembly was tested. These assemblies were quantified relative to metaphase (black bars), and the Cdk1 kinase activity was measured using histone H1 phosphorylation (gray bars). Error bars represent SD from three independent experiments. Ana, anaphase; Meta, metaphase. Bars, 5 μm.
Mentions: A possible explanation for our observations is that activation of the APC/C, degradation of cyclin B, and a following drop in Cdk1 activity led to a changed phosphorylation pattern of MAPs and motors and consequently to the destabilization of microtubules. To test this, we assembled microtubule structures by the addition of Ran-GTP and subsequently observed their stability upon Cdk1 inhibition using CGP74514A (CGP), a selective chemical inhibitor of Cdk1 kinase (Fig. 2 A; Skoufias et al., 2007). Surprisingly, Ran-induced structures did not disassemble upon Cdk1 inhibition before interphasic microtubules were detectable (Fig. 2 A, CGP; see 15 min for coexisting M phase and interphase microtubule structures). In contrast, metaphase microtubule structures disassembled as expected upon the addition of calcium (Fig. 2 A, top) despite high Cdk1 activity (Fig. 2 A, bottom; Ana). This suggested that the sole reduction of Cdk1 activity is not sufficient for the observed changes in anaphase microtubule stability. To analyze the correlation between Cdk1 activity and microtubule stability more quantitatively, we determined Ran-GTP–mediated microtubule assemblies at different concentrations of nondegradable cyclin B. When cylinBΔ90 was added to only 40 nM and anaphase onset was induced by calcium addition, the system exited mitosis as judged by long and stable interphasic microtubules. Upon the addition of cyclinBΔ90 to endogenous levels (80 nM; Stemmann et al., 2001), no microtubule structures were observed (Fig. 1). This indicates that anaphase is faithfully established and maintained upon calcium addition and physiological cyclinBΔ90 levels. The addition of >150 nM cyclinBΔ90 allowed some but still significantly less microtubule assemblies than in metaphase (Fig. 2, B and C). Moreover, no microtubule assembly was observed if anaphase was initially established at 80 nM cyclinBΔ90, and the concentration only subsequently increased even up to 1 μM (Fig. 2 D).

Bottom Line: Using Ran-guanosine triphosphate-mediated microtubule assemblies and quantitative analysis of complete spindles, we demonstrate that CaMKII triggers anaphase microtubule depolymerization.A CaMKII-induced twofold increase in microtubule catastrophe rates can explain reduced microtubule stability.Therefore, our data demonstrate that CaMKII turns on parallel pathways to activate the APC/C and to induce microtubule depolymerization at meiotic anaphase onset.

View Article: PubMed Central - PubMed

Affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg, Deutsches Krebsforschungszentrum und Zentrum für Molekulare Biologie Heidelberg Allianz (DKFZ-ZMBH Alliance), 69120 Heidelberg, Germany.

ABSTRACT
Altered spindle microtubule dynamics at anaphase onset are the basis for chromosome segregation. In Xenopus laevis egg extracts, increasing free calcium levels and subsequently rising calcium-calmodulin-dependent kinase II (CaMKII) activity promote a release from meiosis II arrest and reentry into anaphase. CaMKII induces the activation of the anaphase-promoting complex/cyclosome (APC/C), which destines securin and cyclin B for degradation to allow chromosome separation and mitotic exit. In this study, we investigated the calcium-dependent signal responsible for microtubule depolymerization at anaphase onset after release from meiotic arrest in Xenopus egg extracts. Using Ran-guanosine triphosphate-mediated microtubule assemblies and quantitative analysis of complete spindles, we demonstrate that CaMKII triggers anaphase microtubule depolymerization. A CaMKII-induced twofold increase in microtubule catastrophe rates can explain reduced microtubule stability. However, calcium or constitutively active CaMKII promotes microtubule destabilization even upon APC/C inhibition and in the presence of high cyclin-dependent kinase 1 activity. Therefore, our data demonstrate that CaMKII turns on parallel pathways to activate the APC/C and to induce microtubule depolymerization at meiotic anaphase onset.

Show MeSH
Related in: MedlinePlus