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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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CaMKII induces microtubule instability in anaphase. CSF extracts containing Cy3-tubulin and in vitro translated securin were preincubated for 10 min in the presence of RanQ69L to preassemble microtubules. (A) Reactions were treated with constitutively active CaMKII (CaMKII*), the catalytically inactive mutant (CaMKII*K42A), or CaMKII* together with cyclinBΔ90 (CaMKII* + Δ90) or XErpND (CamKII* + XErpND) as indicated. Microtubule assemblies were visualized by direct fluorescence. (B) Quantification of microtubule assemblies counted in A after 40 min. Error bars represent SD from three independent experiments; metaphase was set to 100%. (C) Reactions were performed as in A; the amounts of endogenous cyclin B (X.l. cyclin B) were determined by immunoblotting, and the amounts of exogenously added in vitro translated (IVT) securin were determined by autoradiography; Cdk1 activities were measured by histone H1 phosphorylation (pHistone H1). (D) Inhibition of endogenous CaMKII activity; CSF extracts containing Cy3-tubulin and RanQ69L were left untreated (Meta) or incubated with calcium in the absence (−) or presence (+) of the CaMKII inhibitor IINtide. (left) Microtubule structures were visualized by direct fluorescence of Cy3-tubulin. (right) Cyclin B (X.l. cyclin B) and Cdk1 activity (pHistone H1) were determined. (D, bottom) Reactions were chased using CaMKII* for the indicated time points and were visualized. Bars, 5 μm.
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fig4: CaMKII induces microtubule instability in anaphase. CSF extracts containing Cy3-tubulin and in vitro translated securin were preincubated for 10 min in the presence of RanQ69L to preassemble microtubules. (A) Reactions were treated with constitutively active CaMKII (CaMKII*), the catalytically inactive mutant (CaMKII*K42A), or CaMKII* together with cyclinBΔ90 (CaMKII* + Δ90) or XErpND (CamKII* + XErpND) as indicated. Microtubule assemblies were visualized by direct fluorescence. (B) Quantification of microtubule assemblies counted in A after 40 min. Error bars represent SD from three independent experiments; metaphase was set to 100%. (C) Reactions were performed as in A; the amounts of endogenous cyclin B (X.l. cyclin B) were determined by immunoblotting, and the amounts of exogenously added in vitro translated (IVT) securin were determined by autoradiography; Cdk1 activities were measured by histone H1 phosphorylation (pHistone H1). (D) Inhibition of endogenous CaMKII activity; CSF extracts containing Cy3-tubulin and RanQ69L were left untreated (Meta) or incubated with calcium in the absence (−) or presence (+) of the CaMKII inhibitor IINtide. (left) Microtubule structures were visualized by direct fluorescence of Cy3-tubulin. (right) Cyclin B (X.l. cyclin B) and Cdk1 activity (pHistone H1) were determined. (D, bottom) Reactions were chased using CaMKII* for the indicated time points and were visualized. Bars, 5 μm.

Mentions: Increasing calcium levels and subsequent CaMKII activation initiate metaphase to anaphase transition in CSF-arrested egg extracts. This conclusion was initially based on the observation that constitutively active CaMKII promotes anaphase entry also in the absence of free calcium (Lorca et al., 1993). Therefore, we aimed to test the stability of Ran-GTP–induced microtubule assemblies after anaphase induction by constitutively active CaMKII (CaMKII 1–290 [Lorca et al., 1993] and CaMKII* [Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200807006/DC1]). We recombinantly expressed and purified CaMKII*, and, as a control, a corresponding mutant in which a lysine in position 42 essential for nucleotide binding was replaced by an alanine (CaMKII*K42A; Hanks and Quinn, 1991). The mutant protein displayed no detectable activity on myelin basic protein, whereas the activity of the wild-type kinase could be readily detected and quantified (see Materials and methods and Fig. S1). When added to Xenopus egg extract, CaMKII* triggered the destabilization of preassembled microtubule structures and promoted entry into interphase as judged by typically long interphasic microtubules (Fig. 4, A and B; CaMKII*). Consistent with this, it induced the degradation of endogenous cyclin B (Fig. 4 C, X.l. cyclin B) and securin as well as decreased Cdk1 activities (Fig. 4 C, CaMKII*). In contrast, the mutant kinase had no effect on the system (Fig. 4, A and C; CaMKII*K42A). Microtubules were also destabilized by CaMKII*-induced anaphase entry in the presence of cyclinBΔ90 (Fig. 4, A and B; CaMKII* + Δ90), which rescued high Cdk1 activity, although APC/C was activated (Fig. 4 C, CaMKII* + Δ90; see degradation of endogenous securin and cyclin B). Likewise, inhibition of APC/C activation by XErpND (Fig. 4 C, CaMKII* + XErpND; note that cyclin B and securin are stable) did not interfere with the CaMKII*-induced reduction in microtubule stability (Fig. 4, A and B; CaMKII* + XErpND).


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

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

CaMKII induces microtubule instability in anaphase. CSF extracts containing Cy3-tubulin and in vitro translated securin were preincubated for 10 min in the presence of RanQ69L to preassemble microtubules. (A) Reactions were treated with constitutively active CaMKII (CaMKII*), the catalytically inactive mutant (CaMKII*K42A), or CaMKII* together with cyclinBΔ90 (CaMKII* + Δ90) or XErpND (CamKII* + XErpND) as indicated. Microtubule assemblies were visualized by direct fluorescence. (B) Quantification of microtubule assemblies counted in A after 40 min. Error bars represent SD from three independent experiments; metaphase was set to 100%. (C) Reactions were performed as in A; the amounts of endogenous cyclin B (X.l. cyclin B) were determined by immunoblotting, and the amounts of exogenously added in vitro translated (IVT) securin were determined by autoradiography; Cdk1 activities were measured by histone H1 phosphorylation (pHistone H1). (D) Inhibition of endogenous CaMKII activity; CSF extracts containing Cy3-tubulin and RanQ69L were left untreated (Meta) or incubated with calcium in the absence (−) or presence (+) of the CaMKII inhibitor IINtide. (left) Microtubule structures were visualized by direct fluorescence of Cy3-tubulin. (right) Cyclin B (X.l. cyclin B) and Cdk1 activity (pHistone H1) were determined. (D, bottom) Reactions were chased using CaMKII* for the indicated time points and were visualized. Bars, 5 μm.
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fig4: CaMKII induces microtubule instability in anaphase. CSF extracts containing Cy3-tubulin and in vitro translated securin were preincubated for 10 min in the presence of RanQ69L to preassemble microtubules. (A) Reactions were treated with constitutively active CaMKII (CaMKII*), the catalytically inactive mutant (CaMKII*K42A), or CaMKII* together with cyclinBΔ90 (CaMKII* + Δ90) or XErpND (CamKII* + XErpND) as indicated. Microtubule assemblies were visualized by direct fluorescence. (B) Quantification of microtubule assemblies counted in A after 40 min. Error bars represent SD from three independent experiments; metaphase was set to 100%. (C) Reactions were performed as in A; the amounts of endogenous cyclin B (X.l. cyclin B) were determined by immunoblotting, and the amounts of exogenously added in vitro translated (IVT) securin were determined by autoradiography; Cdk1 activities were measured by histone H1 phosphorylation (pHistone H1). (D) Inhibition of endogenous CaMKII activity; CSF extracts containing Cy3-tubulin and RanQ69L were left untreated (Meta) or incubated with calcium in the absence (−) or presence (+) of the CaMKII inhibitor IINtide. (left) Microtubule structures were visualized by direct fluorescence of Cy3-tubulin. (right) Cyclin B (X.l. cyclin B) and Cdk1 activity (pHistone H1) were determined. (D, bottom) Reactions were chased using CaMKII* for the indicated time points and were visualized. Bars, 5 μm.
Mentions: Increasing calcium levels and subsequent CaMKII activation initiate metaphase to anaphase transition in CSF-arrested egg extracts. This conclusion was initially based on the observation that constitutively active CaMKII promotes anaphase entry also in the absence of free calcium (Lorca et al., 1993). Therefore, we aimed to test the stability of Ran-GTP–induced microtubule assemblies after anaphase induction by constitutively active CaMKII (CaMKII 1–290 [Lorca et al., 1993] and CaMKII* [Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200807006/DC1]). We recombinantly expressed and purified CaMKII*, and, as a control, a corresponding mutant in which a lysine in position 42 essential for nucleotide binding was replaced by an alanine (CaMKII*K42A; Hanks and Quinn, 1991). The mutant protein displayed no detectable activity on myelin basic protein, whereas the activity of the wild-type kinase could be readily detected and quantified (see Materials and methods and Fig. S1). When added to Xenopus egg extract, CaMKII* triggered the destabilization of preassembled microtubule structures and promoted entry into interphase as judged by typically long interphasic microtubules (Fig. 4, A and B; CaMKII*). Consistent with this, it induced the degradation of endogenous cyclin B (Fig. 4 C, X.l. cyclin B) and securin as well as decreased Cdk1 activities (Fig. 4 C, CaMKII*). In contrast, the mutant kinase had no effect on the system (Fig. 4, A and C; CaMKII*K42A). Microtubules were also destabilized by CaMKII*-induced anaphase entry in the presence of cyclinBΔ90 (Fig. 4, A and B; CaMKII* + Δ90), which rescued high Cdk1 activity, although APC/C was activated (Fig. 4 C, CaMKII* + Δ90; see degradation of endogenous securin and cyclin B). Likewise, inhibition of APC/C activation by XErpND (Fig. 4 C, CaMKII* + XErpND; note that cyclin B and securin are stable) did not interfere with the CaMKII*-induced reduction in microtubule stability (Fig. 4, A and B; CaMKII* + XErpND).

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