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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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APC/C activation is not required for changes in microtubule stability. (A) CSF extracts containing Cy3-tubulin and in vitro translated exogenously added securin were preincubated for 10 min in the presence of Ran-GTP to preassemble microtubules. Reactions were treated with buffer (Meta), calcium and cyclinBΔ90 (Ana), calcium (Inter), XErpND, or calcium and XErpND (XErpND + Ca2+). Microtubule structures were visualized by direct fluorescence of Cy3-tubulin. (B) Quantification of microtubule assemblies counted at the 40-min time point imaged in A. Error bars represent SD from three independent experiments; metaphase was set to 100%. (C) Amounts of cyclin B (endogenous, X.l. cyclin B; exogenously added [Δ90], H.s. cyclin B) were determined by immunoblotting, and amounts of exogenously added in vitro translated (IVT) securin were determined by autoradiography; Cdk1 activities were measured by histone H1 phosphorylation (pHistone H1). Meta, metaphase; Ana, anaphase; Inter, interphase. Bar, 5 μm.
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fig3: APC/C activation is not required for changes in microtubule stability. (A) CSF extracts containing Cy3-tubulin and in vitro translated exogenously added securin were preincubated for 10 min in the presence of Ran-GTP to preassemble microtubules. Reactions were treated with buffer (Meta), calcium and cyclinBΔ90 (Ana), calcium (Inter), XErpND, or calcium and XErpND (XErpND + Ca2+). Microtubule structures were visualized by direct fluorescence of Cy3-tubulin. (B) Quantification of microtubule assemblies counted at the 40-min time point imaged in A. Error bars represent SD from three independent experiments; metaphase was set to 100%. (C) Amounts of cyclin B (endogenous, X.l. cyclin B; exogenously added [Δ90], H.s. cyclin B) were determined by immunoblotting, and amounts of exogenously added in vitro translated (IVT) securin were determined by autoradiography; Cdk1 activities were measured by histone H1 phosphorylation (pHistone H1). Meta, metaphase; Ana, anaphase; Inter, interphase. Bar, 5 μm.

Mentions: As cyclin B degradation is mediated by APC/C activity (but according to our experiments is not required to destabilize microtubules), we aimed to further determine the overall role of APC/C activation in changing microtubule stability in anaphase. We assayed Ran-GTP–induced microtubule assembly by direct fluorescence and monitored cyclin B and securin levels as well as Cdk1 activity to determine APC/C activation in metaphase, anaphase, and interphase. To inhibit APC/C activation despite calcium-induced anaphase onset and therefore uncoupling APC/C activation from other calcium-dependent processes, we used a nondegradable variant of XErp (XErpND; Fig. 3; Rauh et al., 2005). As expected, Ran-GTP–induced microtubule assemblies were stable in CSF-arrested extracts, and high levels of cyclin B, securin, and Cdk1 activity were maintained (Fig. 3, A–C; Meta). Calcium induced the destabilization of microtubule assemblies as well as cyclin B and securin degradation, but high Cdk1 activity was maintained in the presence of cyclinBΔ90 (Fig. 3, A–C; Ana). Likewise, microtubule destabilization was observed in the absence of cyclinBΔ90 but was followed by mitotic exit (Fig. 3 A, Inter; note the appearance of interphasic microtubules after 60 min). Although XErpND alone had no effect in metaphase (Fig. 3, A–C; XErpND), interestingly, it allowed microtubule disassembly upon calcium addition (Fig. 3, A and B; XErpND + Ca2+) despite efficient inhibition of APC/C activation as indicated by stable cyclin B and securin (Fig. 3 C, XErpND + Ca2+). These results strongly suggest that the depolymerization of microtubules in anaphase of Xenopus egg extracts is triggered by calcium but does not require the activation of APC/C and, therefore, the degradation of its metaphase substrates securin and cyclin B.


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

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

APC/C activation is not required for changes in microtubule stability. (A) CSF extracts containing Cy3-tubulin and in vitro translated exogenously added securin were preincubated for 10 min in the presence of Ran-GTP to preassemble microtubules. Reactions were treated with buffer (Meta), calcium and cyclinBΔ90 (Ana), calcium (Inter), XErpND, or calcium and XErpND (XErpND + Ca2+). Microtubule structures were visualized by direct fluorescence of Cy3-tubulin. (B) Quantification of microtubule assemblies counted at the 40-min time point imaged in A. Error bars represent SD from three independent experiments; metaphase was set to 100%. (C) Amounts of cyclin B (endogenous, X.l. cyclin B; exogenously added [Δ90], H.s. cyclin B) were determined by immunoblotting, and amounts of exogenously added in vitro translated (IVT) securin were determined by autoradiography; Cdk1 activities were measured by histone H1 phosphorylation (pHistone H1). Meta, metaphase; Ana, anaphase; Inter, interphase. Bar, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2600749&req=5

fig3: APC/C activation is not required for changes in microtubule stability. (A) CSF extracts containing Cy3-tubulin and in vitro translated exogenously added securin were preincubated for 10 min in the presence of Ran-GTP to preassemble microtubules. Reactions were treated with buffer (Meta), calcium and cyclinBΔ90 (Ana), calcium (Inter), XErpND, or calcium and XErpND (XErpND + Ca2+). Microtubule structures were visualized by direct fluorescence of Cy3-tubulin. (B) Quantification of microtubule assemblies counted at the 40-min time point imaged in A. Error bars represent SD from three independent experiments; metaphase was set to 100%. (C) Amounts of cyclin B (endogenous, X.l. cyclin B; exogenously added [Δ90], H.s. cyclin B) were determined by immunoblotting, and amounts of exogenously added in vitro translated (IVT) securin were determined by autoradiography; Cdk1 activities were measured by histone H1 phosphorylation (pHistone H1). Meta, metaphase; Ana, anaphase; Inter, interphase. Bar, 5 μm.
Mentions: As cyclin B degradation is mediated by APC/C activity (but according to our experiments is not required to destabilize microtubules), we aimed to further determine the overall role of APC/C activation in changing microtubule stability in anaphase. We assayed Ran-GTP–induced microtubule assembly by direct fluorescence and monitored cyclin B and securin levels as well as Cdk1 activity to determine APC/C activation in metaphase, anaphase, and interphase. To inhibit APC/C activation despite calcium-induced anaphase onset and therefore uncoupling APC/C activation from other calcium-dependent processes, we used a nondegradable variant of XErp (XErpND; Fig. 3; Rauh et al., 2005). As expected, Ran-GTP–induced microtubule assemblies were stable in CSF-arrested extracts, and high levels of cyclin B, securin, and Cdk1 activity were maintained (Fig. 3, A–C; Meta). Calcium induced the destabilization of microtubule assemblies as well as cyclin B and securin degradation, but high Cdk1 activity was maintained in the presence of cyclinBΔ90 (Fig. 3, A–C; Ana). Likewise, microtubule destabilization was observed in the absence of cyclinBΔ90 but was followed by mitotic exit (Fig. 3 A, Inter; note the appearance of interphasic microtubules after 60 min). Although XErpND alone had no effect in metaphase (Fig. 3, A–C; XErpND), interestingly, it allowed microtubule disassembly upon calcium addition (Fig. 3, A and B; XErpND + Ca2+) despite efficient inhibition of APC/C activation as indicated by stable cyclin B and securin (Fig. 3 C, XErpND + Ca2+). These results strongly suggest that the depolymerization of microtubules in anaphase of Xenopus egg extracts is triggered by calcium but does not require the activation of APC/C and, therefore, the degradation of its metaphase substrates securin and cyclin B.

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