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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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Dynamic measurements of centrosomally nucleated microtubules in metaphase or anaphase. CSF-arrested egg extracts were supplemented with Cy3-tubulin and purified human centrosomes and were triggered to enter anaphase as indicated. (A) Microtubules were visualized by time-lapse microscopy in 2-s intervals, and microtubules were tracked manually. Still images (after 15 min) are shown. (B) Parameters of dynamic instability were determined from data gained by time-lapse imaging using a Matlab macro. MT, microtubules; vg, growth rate; vs, shrinkage rate; fcat, catastrophe frequency; fres, rescue frequency. Bar, 10 μm.
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fig5: Dynamic measurements of centrosomally nucleated microtubules in metaphase or anaphase. CSF-arrested egg extracts were supplemented with Cy3-tubulin and purified human centrosomes and were triggered to enter anaphase as indicated. (A) Microtubules were visualized by time-lapse microscopy in 2-s intervals, and microtubules were tracked manually. Still images (after 15 min) are shown. (B) Parameters of dynamic instability were determined from data gained by time-lapse imaging using a Matlab macro. MT, microtubules; vg, growth rate; vs, shrinkage rate; fcat, catastrophe frequency; fres, rescue frequency. Bar, 10 μm.

Mentions: The apparent changes in the stability of the aforementioned microtubules were likely caused by changes in dynamic instability parameters from metaphase to anaphase in the Xenopus system. To visualize and quantify these changes, we assembled Cy3-labeled microtubules from centrosomes and tracked them by time-lapse microscopy in metaphase or anaphase extracts (Fig. 5). It is known that in Xenopus metaphase extracts, the relatively short mean length of microtubules is largely determined by their high catastrophe frequency, which elevates at the entry into M phase to two to three catastrophes per minute (Niethammer et al., 2007). Consistent with that, microtubules in CSF-arrested extracts in our hands showed catastrophes with a frequency of 2.94 min−1 (Fig. 5, A and B; and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200807006/DC1).


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

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

Dynamic measurements of centrosomally nucleated microtubules in metaphase or anaphase. CSF-arrested egg extracts were supplemented with Cy3-tubulin and purified human centrosomes and were triggered to enter anaphase as indicated. (A) Microtubules were visualized by time-lapse microscopy in 2-s intervals, and microtubules were tracked manually. Still images (after 15 min) are shown. (B) Parameters of dynamic instability were determined from data gained by time-lapse imaging using a Matlab macro. MT, microtubules; vg, growth rate; vs, shrinkage rate; fcat, catastrophe frequency; fres, rescue frequency. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Dynamic measurements of centrosomally nucleated microtubules in metaphase or anaphase. CSF-arrested egg extracts were supplemented with Cy3-tubulin and purified human centrosomes and were triggered to enter anaphase as indicated. (A) Microtubules were visualized by time-lapse microscopy in 2-s intervals, and microtubules were tracked manually. Still images (after 15 min) are shown. (B) Parameters of dynamic instability were determined from data gained by time-lapse imaging using a Matlab macro. MT, microtubules; vg, growth rate; vs, shrinkage rate; fcat, catastrophe frequency; fres, rescue frequency. Bar, 10 μm.
Mentions: The apparent changes in the stability of the aforementioned microtubules were likely caused by changes in dynamic instability parameters from metaphase to anaphase in the Xenopus system. To visualize and quantify these changes, we assembled Cy3-labeled microtubules from centrosomes and tracked them by time-lapse microscopy in metaphase or anaphase extracts (Fig. 5). It is known that in Xenopus metaphase extracts, the relatively short mean length of microtubules is largely determined by their high catastrophe frequency, which elevates at the entry into M phase to two to three catastrophes per minute (Niethammer et al., 2007). Consistent with that, microtubules in CSF-arrested extracts in our hands showed catastrophes with a frequency of 2.94 min−1 (Fig. 5, A and B; and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200807006/DC1).

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