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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 triggers spindle disassembly in anaphase independently of APC/C activation. Spindles were preassembled in CSF extracts (Meta), and anaphase was induced by the addition of calcium or CaMKII* in the absence or presence of XErpND. (A) Spindles were visualized by direct fluorescence of Cy3-tubulin (red) and DAPI (blue) after 20 min. Out of n imaged spindles, the mean overall fluorescence intensity was determined, and an average image was generated using the Matlab macro. The pole (P) to pole distance (spindle size) was measured as well as the corresponding mean fluorescence distribution plotted along the pole to pole axis in a 1.5-μm-wide area (intensity distribution; see blue lines in average images). Intensity and spindle size are indicated in relative terms (percentage). (B) Immunoblots were used to determine the amounts of cyclin B, an autoradiograph was used to monitor amounts of exogenously added in vitro translated (IVT) securin, and an autoradiograph displaying a histone H1 kinase assay was used to determine Cdk1 activity under the conditions used in A. The black line indicates that intervening lanes have been spliced out. (C) Anaphase was induced in preassembled spindles by low doses of CaMKII* in the presence of cyclinBΔ90, and samples were fixed after 40 min. Spindles were analyzed as in A. (D) Determination of cyclin B by immunoblotting under the conditions used in C. (E) Spindles were first treated with CaMKII* and XErpND and mixed with extracts treated without (−) or with (+) CaMKII* to activate APC/C. (top) Immunoblotting was used to determine the amounts of cyclin B and histone H1 kinase assay to measure the Cdk1 activity. (bottom) Representative fluorescence images of structures after 40 min. Tubulin, red; DAPI (DNA), blue. MT, microtubules; Meta, extracts after spindle assembly. Bars, 5 μm.
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fig6: CaMKII triggers spindle disassembly in anaphase independently of APC/C activation. Spindles were preassembled in CSF extracts (Meta), and anaphase was induced by the addition of calcium or CaMKII* in the absence or presence of XErpND. (A) Spindles were visualized by direct fluorescence of Cy3-tubulin (red) and DAPI (blue) after 20 min. Out of n imaged spindles, the mean overall fluorescence intensity was determined, and an average image was generated using the Matlab macro. The pole (P) to pole distance (spindle size) was measured as well as the corresponding mean fluorescence distribution plotted along the pole to pole axis in a 1.5-μm-wide area (intensity distribution; see blue lines in average images). Intensity and spindle size are indicated in relative terms (percentage). (B) Immunoblots were used to determine the amounts of cyclin B, an autoradiograph was used to monitor amounts of exogenously added in vitro translated (IVT) securin, and an autoradiograph displaying a histone H1 kinase assay was used to determine Cdk1 activity under the conditions used in A. The black line indicates that intervening lanes have been spliced out. (C) Anaphase was induced in preassembled spindles by low doses of CaMKII* in the presence of cyclinBΔ90, and samples were fixed after 40 min. Spindles were analyzed as in A. (D) Determination of cyclin B by immunoblotting under the conditions used in C. (E) Spindles were first treated with CaMKII* and XErpND and mixed with extracts treated without (−) or with (+) CaMKII* to activate APC/C. (top) Immunoblotting was used to determine the amounts of cyclin B and histone H1 kinase assay to measure the Cdk1 activity. (bottom) Representative fluorescence images of structures after 40 min. Tubulin, red; DAPI (DNA), blue. MT, microtubules; Meta, extracts after spindle assembly. Bars, 5 μm.

Mentions: Our experiments on Ran-GTP–induced microtubule assemblies and centrosomal asters suggested a novel CaMKII-induced mechanism for anaphase microtubule destabilization. Therefore, we intended to analyze microtubule stability at the metaphase to anaphase transition in complete bipolar spindles assembled around sperm nuclei in Xenopus egg extracts. We visualized and quantified spindle microtubule densities by direct fluorescence of Cy3-labeled tubulin before and after anaphase onset by calcium or CaMKII*. For each condition, we imaged 40–80 spindle structures. Within all acquired images, the two poles were defined manually, and the mean fluorescence was determined using ImageJ to assay for the overall microtubule stability of the spindles (Fig. 6 A, intensity). A custom Matlab macro vertically aligned the spindles along the pole to pole axis, determined the mean pole to pole distance as a measure for spindle size (Fig. 6 A), and subsequently rescaled all spindles to the same size. Matlab was used to quantify and plot the fluorescence (i.e., microtubule) intensity distributions in a 1.5-μm-wide area along the pole to pole axis (Fig. 6 A, blue line). Metaphase spindles typically showed aligned chromosomes and a high local microtubule density in the central part close to the chromosomes (Fig. 6 A, Meta). Entry into anaphase after calcium addition led to a reduction of the intensity (33%) and to a slight (21%) decrease in spindle size (pole to pole distance; Fig. 6 A, calcium). Anaphase onset triggered by CaMKII* promoted a two-third decrease in intensity and an almost twofold reduction of spindle size as compared with metaphase (Fig. 6 A, CaMKII*). CaMKII-induced depolymerization was therefore more efficient than what was observed upon the addition of calcium (Fig. 6 A, CaMKII* and calcium; see average and intensity). However, under either condition we observed a characteristic local decrease in microtubule density in the central part of the spindle, which is consistent with previously published data on anaphase spindles (Murray et al., 1996). Calcium or CaMKII* addition led to the quick degradation of securin and cyclin B and reduced Cdk1 activity (Fig. 6 B). Importantly, the CaMKII*-induced reduction in spindle size and the characteristic drop of microtubule density in the central part of the spindle were also observed upon supplementing the extract with XErpND. Under these conditions, APC/C activation was inhibited, and, therefore, securin and cyclin B stayed stable (Fig. 6, A and B; CaMKII* + XErpND). Those partially depolymerized spindle structures were stable for >60 min (unpublished data). Even after elevated times, we did not visualize any spindle structures in which chromosomes had commenced to segregate (Fig. 6 A, CaMKII* + XErpND). As observed for Ran-GTP–mediated microtubule assemblies, anaphase induction by low CaMKII* activity in the presence of cyclinBΔ90 was insufficient to destabilize microtubules and thus revealed spindles with a similar microtubule density distribution but an even higher overall fluorescence than metaphase spindles (Fig. 6 C, compare Meta with low CaMKII*). Still, upon adding low CaMKII* activity, we observed chromosome segregation and degradation of cyclin B after 40 min (Fig. 6 D and not depicted).


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 triggers spindle disassembly in anaphase independently of APC/C activation. Spindles were preassembled in CSF extracts (Meta), and anaphase was induced by the addition of calcium or CaMKII* in the absence or presence of XErpND. (A) Spindles were visualized by direct fluorescence of Cy3-tubulin (red) and DAPI (blue) after 20 min. Out of n imaged spindles, the mean overall fluorescence intensity was determined, and an average image was generated using the Matlab macro. The pole (P) to pole distance (spindle size) was measured as well as the corresponding mean fluorescence distribution plotted along the pole to pole axis in a 1.5-μm-wide area (intensity distribution; see blue lines in average images). Intensity and spindle size are indicated in relative terms (percentage). (B) Immunoblots were used to determine the amounts of cyclin B, an autoradiograph was used to monitor amounts of exogenously added in vitro translated (IVT) securin, and an autoradiograph displaying a histone H1 kinase assay was used to determine Cdk1 activity under the conditions used in A. The black line indicates that intervening lanes have been spliced out. (C) Anaphase was induced in preassembled spindles by low doses of CaMKII* in the presence of cyclinBΔ90, and samples were fixed after 40 min. Spindles were analyzed as in A. (D) Determination of cyclin B by immunoblotting under the conditions used in C. (E) Spindles were first treated with CaMKII* and XErpND and mixed with extracts treated without (−) or with (+) CaMKII* to activate APC/C. (top) Immunoblotting was used to determine the amounts of cyclin B and histone H1 kinase assay to measure the Cdk1 activity. (bottom) Representative fluorescence images of structures after 40 min. Tubulin, red; DAPI (DNA), blue. MT, microtubules; Meta, extracts after spindle assembly. Bars, 5 μm.
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fig6: CaMKII triggers spindle disassembly in anaphase independently of APC/C activation. Spindles were preassembled in CSF extracts (Meta), and anaphase was induced by the addition of calcium or CaMKII* in the absence or presence of XErpND. (A) Spindles were visualized by direct fluorescence of Cy3-tubulin (red) and DAPI (blue) after 20 min. Out of n imaged spindles, the mean overall fluorescence intensity was determined, and an average image was generated using the Matlab macro. The pole (P) to pole distance (spindle size) was measured as well as the corresponding mean fluorescence distribution plotted along the pole to pole axis in a 1.5-μm-wide area (intensity distribution; see blue lines in average images). Intensity and spindle size are indicated in relative terms (percentage). (B) Immunoblots were used to determine the amounts of cyclin B, an autoradiograph was used to monitor amounts of exogenously added in vitro translated (IVT) securin, and an autoradiograph displaying a histone H1 kinase assay was used to determine Cdk1 activity under the conditions used in A. The black line indicates that intervening lanes have been spliced out. (C) Anaphase was induced in preassembled spindles by low doses of CaMKII* in the presence of cyclinBΔ90, and samples were fixed after 40 min. Spindles were analyzed as in A. (D) Determination of cyclin B by immunoblotting under the conditions used in C. (E) Spindles were first treated with CaMKII* and XErpND and mixed with extracts treated without (−) or with (+) CaMKII* to activate APC/C. (top) Immunoblotting was used to determine the amounts of cyclin B and histone H1 kinase assay to measure the Cdk1 activity. (bottom) Representative fluorescence images of structures after 40 min. Tubulin, red; DAPI (DNA), blue. MT, microtubules; Meta, extracts after spindle assembly. Bars, 5 μm.
Mentions: Our experiments on Ran-GTP–induced microtubule assemblies and centrosomal asters suggested a novel CaMKII-induced mechanism for anaphase microtubule destabilization. Therefore, we intended to analyze microtubule stability at the metaphase to anaphase transition in complete bipolar spindles assembled around sperm nuclei in Xenopus egg extracts. We visualized and quantified spindle microtubule densities by direct fluorescence of Cy3-labeled tubulin before and after anaphase onset by calcium or CaMKII*. For each condition, we imaged 40–80 spindle structures. Within all acquired images, the two poles were defined manually, and the mean fluorescence was determined using ImageJ to assay for the overall microtubule stability of the spindles (Fig. 6 A, intensity). A custom Matlab macro vertically aligned the spindles along the pole to pole axis, determined the mean pole to pole distance as a measure for spindle size (Fig. 6 A), and subsequently rescaled all spindles to the same size. Matlab was used to quantify and plot the fluorescence (i.e., microtubule) intensity distributions in a 1.5-μm-wide area along the pole to pole axis (Fig. 6 A, blue line). Metaphase spindles typically showed aligned chromosomes and a high local microtubule density in the central part close to the chromosomes (Fig. 6 A, Meta). Entry into anaphase after calcium addition led to a reduction of the intensity (33%) and to a slight (21%) decrease in spindle size (pole to pole distance; Fig. 6 A, calcium). Anaphase onset triggered by CaMKII* promoted a two-third decrease in intensity and an almost twofold reduction of spindle size as compared with metaphase (Fig. 6 A, CaMKII*). CaMKII-induced depolymerization was therefore more efficient than what was observed upon the addition of calcium (Fig. 6 A, CaMKII* and calcium; see average and intensity). However, under either condition we observed a characteristic local decrease in microtubule density in the central part of the spindle, which is consistent with previously published data on anaphase spindles (Murray et al., 1996). Calcium or CaMKII* addition led to the quick degradation of securin and cyclin B and reduced Cdk1 activity (Fig. 6 B). Importantly, the CaMKII*-induced reduction in spindle size and the characteristic drop of microtubule density in the central part of the spindle were also observed upon supplementing the extract with XErpND. Under these conditions, APC/C activation was inhibited, and, therefore, securin and cyclin B stayed stable (Fig. 6, A and B; CaMKII* + XErpND). Those partially depolymerized spindle structures were stable for >60 min (unpublished data). Even after elevated times, we did not visualize any spindle structures in which chromosomes had commenced to segregate (Fig. 6 A, CaMKII* + XErpND). As observed for Ran-GTP–mediated microtubule assemblies, anaphase induction by low CaMKII* activity in the presence of cyclinBΔ90 was insufficient to destabilize microtubules and thus revealed spindles with a similar microtubule density distribution but an even higher overall fluorescence than metaphase spindles (Fig. 6 C, compare Meta with low CaMKII*). Still, upon adding low CaMKII* activity, we observed chromosome segregation and degradation of cyclin B after 40 min (Fig. 6 D and not depicted).

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