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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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Changes in microtubule stability from metaphase to anaphase in Xenopus egg extracts. (A) Calcium was added to CSF-arrested Xenopus egg extracts (0 min) in the presence of cyclinBΔ90, and samples were withdrawn at the indicated times to determine the activity of Cdk1 toward histone H1 (pHistone H1, top) and the amounts of endogenous cyclin B (X.I. cyclin B, bottom). (B) Microtubules or spindles were assembled in metaphase-arrested extracts (left), calcium was added in the presence of cyclinBΔ90 (right), and microtubules were visualized by direct fluorescence of added Cy3-tubulin. Sperm nuclei, RanQ69L, or chromatin beads were used as a nucleating source. (C) Immunoprecipitation using anti-TPX2 antibodies from metaphase or anaphase extracts. Immunocomplexes were eluted with high salt, and aurora A/Eg2 was determined by immunoblotting. (D) The microtubule assembly assay is as described in B; purified human centrosomes were used as a nucleating source. Cycled, microtubule asters were preassembled in metaphase before calcium addition; Meta, metaphase; Ana, anaphase. Bars, 10 μm.
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fig1: Changes in microtubule stability from metaphase to anaphase in Xenopus egg extracts. (A) Calcium was added to CSF-arrested Xenopus egg extracts (0 min) in the presence of cyclinBΔ90, and samples were withdrawn at the indicated times to determine the activity of Cdk1 toward histone H1 (pHistone H1, top) and the amounts of endogenous cyclin B (X.I. cyclin B, bottom). (B) Microtubules or spindles were assembled in metaphase-arrested extracts (left), calcium was added in the presence of cyclinBΔ90 (right), and microtubules were visualized by direct fluorescence of added Cy3-tubulin. Sperm nuclei, RanQ69L, or chromatin beads were used as a nucleating source. (C) Immunoprecipitation using anti-TPX2 antibodies from metaphase or anaphase extracts. Immunocomplexes were eluted with high salt, and aurora A/Eg2 was determined by immunoblotting. (D) The microtubule assembly assay is as described in B; purified human centrosomes were used as a nucleating source. Cycled, microtubule asters were preassembled in metaphase before calcium addition; Meta, metaphase; Ana, anaphase. Bars, 10 μm.

Mentions: To investigate changes in microtubule stability from metaphase to anaphase, we assembled spindles from sperm nuclei in CSF-arrested Xenopus egg extracts supplemented with Cy3-labeled tubulin. We initiated anaphase by adding 0.6 mM calcium but blocked cell cycle progression in anaphase using nondegradable cyclin B (cyclinBΔ90) to maintain high Cdk1 activity throughout the experiment (Fig. 1 A). After the addition of calcium and thus anaphase initiation, the density of spindle microtubules in the central part of the spindle was decreased concomitant with chromosome segregation, but spindles did not completely depolymerize (Fig. 1 B, top right). Like sperm nuclei, Ran-GTP and chromatin beads readily assembled microtubule structures in CSF-arrested Xenopus egg extracts (Fig. 1 B, left). In contrast, neither Ran-GTP nor chromatin beads stabilized any microtubules in anaphase extracts in the presence of cyclinBΔ90, suggesting an overall reduced microtubule stability (Fig. 1 B, right).


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

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

Changes in microtubule stability from metaphase to anaphase in Xenopus egg extracts. (A) Calcium was added to CSF-arrested Xenopus egg extracts (0 min) in the presence of cyclinBΔ90, and samples were withdrawn at the indicated times to determine the activity of Cdk1 toward histone H1 (pHistone H1, top) and the amounts of endogenous cyclin B (X.I. cyclin B, bottom). (B) Microtubules or spindles were assembled in metaphase-arrested extracts (left), calcium was added in the presence of cyclinBΔ90 (right), and microtubules were visualized by direct fluorescence of added Cy3-tubulin. Sperm nuclei, RanQ69L, or chromatin beads were used as a nucleating source. (C) Immunoprecipitation using anti-TPX2 antibodies from metaphase or anaphase extracts. Immunocomplexes were eluted with high salt, and aurora A/Eg2 was determined by immunoblotting. (D) The microtubule assembly assay is as described in B; purified human centrosomes were used as a nucleating source. Cycled, microtubule asters were preassembled in metaphase before calcium addition; Meta, metaphase; Ana, anaphase. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2600749&req=5

fig1: Changes in microtubule stability from metaphase to anaphase in Xenopus egg extracts. (A) Calcium was added to CSF-arrested Xenopus egg extracts (0 min) in the presence of cyclinBΔ90, and samples were withdrawn at the indicated times to determine the activity of Cdk1 toward histone H1 (pHistone H1, top) and the amounts of endogenous cyclin B (X.I. cyclin B, bottom). (B) Microtubules or spindles were assembled in metaphase-arrested extracts (left), calcium was added in the presence of cyclinBΔ90 (right), and microtubules were visualized by direct fluorescence of added Cy3-tubulin. Sperm nuclei, RanQ69L, or chromatin beads were used as a nucleating source. (C) Immunoprecipitation using anti-TPX2 antibodies from metaphase or anaphase extracts. Immunocomplexes were eluted with high salt, and aurora A/Eg2 was determined by immunoblotting. (D) The microtubule assembly assay is as described in B; purified human centrosomes were used as a nucleating source. Cycled, microtubule asters were preassembled in metaphase before calcium addition; Meta, metaphase; Ana, anaphase. Bars, 10 μm.
Mentions: To investigate changes in microtubule stability from metaphase to anaphase, we assembled spindles from sperm nuclei in CSF-arrested Xenopus egg extracts supplemented with Cy3-labeled tubulin. We initiated anaphase by adding 0.6 mM calcium but blocked cell cycle progression in anaphase using nondegradable cyclin B (cyclinBΔ90) to maintain high Cdk1 activity throughout the experiment (Fig. 1 A). After the addition of calcium and thus anaphase initiation, the density of spindle microtubules in the central part of the spindle was decreased concomitant with chromosome segregation, but spindles did not completely depolymerize (Fig. 1 B, top right). Like sperm nuclei, Ran-GTP and chromatin beads readily assembled microtubule structures in CSF-arrested Xenopus egg extracts (Fig. 1 B, left). In contrast, neither Ran-GTP nor chromatin beads stabilized any microtubules in anaphase extracts in the presence of cyclinBΔ90, suggesting an overall reduced microtubule stability (Fig. 1 B, right).

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