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Cell Cycle Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kccy20
Versatile Kinetochore Components Control Central Spindle Assembly a
a
Frances Edwards , Gilliane Maton & Julien Dumont
a
a
Institut Jacques Monod, CNRS, UMR 7592, University Paris Diderot, Sorbonne Paris Cité, Paris, France Accepted author version posted online: 17 Jun 2015.
Click for updates To cite this article: Frances Edwards, Gilliane Maton & Julien Dumont (2015): Versatile Kinetochore Components Control Central Spindle Assembly, Cell Cycle, DOI: 10.1080/15384101.2015.1062329 To link to this article: http://dx.doi.org/10.1080/15384101.2015.1062329
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Versatile Kinetochore Components Control Central Spindle Assembly
Frances Edwards, Gilliane Maton, and Julien Dumont
Institut Jacques Monod, CNRS, UMR 7592, University Paris Diderot, Sorbonne
Downloaded by [UQ Library] at 07:54 21 June 2015
Paris Cité, Paris, France
Correspondence: [email protected]
Comment on: Maton G, et al. Kinetochore components are required for central spindle assembly. Nat Cell Biol 2015; 17(5):697-705; PMID: 25866924; http://dx.doi.org/10.1038/ncb3150
1
Defects in cell division can lead to genomic instability and aneuploidy, both hallmarks of many cancers. During mitosis, sister chromatids are segregated on a microtubule-based spindle. Concomitant to spindle assembly, proteinaceous structures termed kinetochores are assembled on sister chromatids. In metaphase, kinetochores allow chromosome alignment and biorientation by interacting with microtubules coming from opposite spindle poles.
At anaphase onset, microtubules organize between the separating
chromosomes to form an array of antiparallel microtubules with their plus-ends overlapping at the center, called the central spindle. Once formed, the central spindle acts as a localization hub for key proteins required for cytokinesis, the
Downloaded by [UQ Library] at 07:54 21 June 2015
physical division of one cell into two.
Importantly, the mechanisms that
coordinate central spindle assembly with anaphase onset and chromosome segregation are unclear. In HeLa cells, de novo microtubule nucleation by the Augmin complex participates in central spindle assembly1. However, this complex is missing in some species that assemble central spindles in anaphase, including yeast and Caenorhabditis elegans. In most species, central spindle organization relies on the microtubule bundling activity of the conserved Ase1/PRC1 family of microtubule-associated proteins (MAPs), as well as on the microtubule motor of the kinesin-6 family MKLP12.
Microtubule motors of the kinesin-4 and -5
families also regulate central spindle integrity. However, none of these proteins display microtubule assembly or nucleation activities. Therefore, the relative contribution of pre-existing and/or de novo-assembled microtubules is unclear. To study the mechanisms of central spindle assembly, we combined RNAi-mediated depletion of key proteins with quantitative fluorescence live-cell microscopy3. We capitalized on two transgenic C. elegans strains expressing fluorescent markers for chromosomes and microtubules or the kinase AIR2AuroraB, a component of the Chromosomal Passenger Complex that specifically localizes to the central spindle.
We then developed an assay to track and
quantify central spindle intensity over time, and extract the chromosome segregation profile, which is a read-out of central spindle mechanical integrity. In 2
the one-cell C. elegans embryo, chromosome segregation is primarily driven by astral microtubule pulling forces generated by the cortically anchored microtubule motor dynein. The anaphase central spindle acts as a brake that counteracts these dynein-driven astral pulling forces, limiting the extent and velocity of sister chromatid segregation4. Thus we can quantify the mechanical integrity of the central spindle by monitoring the rate and extent of chromosome segregation with and without astral pulling forces.
We first confirmed the
contribution of SPD-1PRC1 and ZEN-4MKLP1 in central spindle integrity. However, while these two proteins are often presented in the literature as being required for central spindle assembly, our analysis suggested that their primary function
Downloaded by [UQ Library] at 07:54 21 June 2015
is to provide mechanical integrity to the central spindle during its elongation but not to promote de novo central spindle microtubule assembly. Interestingly, reducing astral pulling forces (by preventing cortical targeting of dynein) restored central spindle integrity in the absence of SPD-1PRC1 or ZEN-4MKLP1. These results show that these two proteins are dispensible for initial central spindle assembly but are essential for mechanical integrity during central spindle elongation, when normal cortical pulling forces are exerted. We next set out to identify components involved in initial central spindle microtubule assembly. We focused on the potential contribution of the kinetochore, which participates in pre-anaphase spindle microtubule formation in most systems and is associated with microtubule assembly promoting activities5. We identified a kinetochore sub-branch essential for central spindle formation and comprised of the conserved kinase BUB-1, the two redundant CENP-F orthologs HCP-1 and -2, and the CLASP ortholog CLS-2. RNAi-mediated depletion of any one of these components led to impaired central spindle assembly. Interestingly, a role for CLASP in central spindle formation had already been described in Drosophila spermatocytes6. We then followed the dynamic localization of these kinetochore components and showed that during metaphase HCP-1/2CENP-F and CLS-2CLASP are recruited to kinetochores by BUB-1 and then translocate to the central spindle region upon anaphase onset. By analyzing two mutants of the upstream kinetochore scaffold protein KNL-1 that led to decreased or excess BUB-1 at the metaphase kinetochore, we 3
demonstrated that the level of kinetochore BUB-1 directly controls central spindle microtubule density.
Further, pre-anaphase levels of BUB-1 at the
kinetochores control overall central spindle mechanical robustness by modulating the levels of HCP-1/2CENP-F and CLS-2CLASP that translocate between the separating chromosomes in anaphase. CLASP proteins contain TOGL (Tumor Overexpressed Gene Like) domains, which are characteristic of proteins with microtubule assembling activity7. Secondary structure analysis of CLS-2CLASP revealed three potential TOGL domains. We thus purified recombinant CLS-2CLASP and tested its potential
Downloaded by [UQ Library] at 07:54 21 June 2015
activity as a microtubule polymerase. We showed that in vitro recombinant CLS2CLASP led to an increase in the rate of microtubule polymerization and in the total amount of assembled microtubules. Furthermore, replacing endogenous CLS-2CLASP with a mutant version that has impaired microtubule polymerase activity completely disrupted central spindle assembly in vivo. Altogether our results led us to propose a new model for central spindle formation and elongation based on a two-step mechanism3. In the first step, metaphase kinetochore recruitment of BUB-1, HCP-1/2CENP-F and CLS-2CLASP is essential to trigger their anaphase translocation between the separating chromosomes, which then promotes central spindle microtubule assembly through CLS-2CLASP activity. During the second step, central spindle elongation, anti-parallel microtubules are organized and cross-linked by SPD-1PRC1 and ZEN4MKLP1, which provides additional mechanical integrity. Overall, we propose that by promoting central spindle assembly, kinetochores coordinate the two critical events of cell division: chromosome segregation and cytokinesis. As kinetochore components are conserved across metazoans, this previously unknown function of the kinetochore is also likely to be conserved.
4
REFERENCES Uehara R, et al. J. Cell Biol., 2010; 202, 623-636
2.
Lee K-Y, et al. J. Cell Sci. 2012; 125:3495-500.
3.
Maton G, Edwards F, et al. Nat Cell Biol 2015; 17:697-705.
4.
Grill SW, et al. Nature 2001; 409:630-3.
5.
Cheeseman IM, at al. Nat Rev Mol Cell Biol 2008; 9:33-46.
6.
Máthé E, et al. Development 2003; 130:901-15.
7.
Al-Bassam J, et al. Trends Cell Biol. 2011; 21:604-14.
Downloaded by [UQ Library] at 07:54 21 June 2015
1.
5
Downloaded by [UQ Library] at 07:54 21 June 2015
A two-step model for anaphase central spindle organization. During metaphase, BUB-1, HCP-1/2Cenp-F and CLS-2CLASP are localized at the kinetochore. At anaphase onset, their translocation between the segregating sister chromatids promotes initiation of central spindle microtubule assembly through CLS-2CLASP activity (step 1). Microtubules are then cross-linked and organized by SPD-1PRC1 and ZEN-4MKLP1, which allows central spindle elongation (step 2). Please provide figure label and call out
6
Cell Cycle Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kccy20
Versatile Kinetochore Components Control Central Spindle Assembly a
a
Frances Edwards , Gilliane Maton & Julien Dumont
a
a
Institut Jacques Monod, CNRS, UMR 7592, University Paris Diderot, Sorbonne Paris Cité, Paris, France Accepted author version posted online: 17 Jun 2015.
Click for updates To cite this article: Frances Edwards, Gilliane Maton & Julien Dumont (2015): Versatile Kinetochore Components Control Central Spindle Assembly, Cell Cycle, DOI: 10.1080/15384101.2015.1062329 To link to this article: http://dx.doi.org/10.1080/15384101.2015.1062329
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Versatile Kinetochore Components Control Central Spindle Assembly
Frances Edwards, Gilliane Maton, and Julien Dumont
Institut Jacques Monod, CNRS, UMR 7592, University Paris Diderot, Sorbonne
Downloaded by [UQ Library] at 07:54 21 June 2015
Paris Cité, Paris, France
Correspondence: [email protected]
Comment on: Maton G, et al. Kinetochore components are required for central spindle assembly. Nat Cell Biol 2015; 17(5):697-705; PMID: 25866924; http://dx.doi.org/10.1038/ncb3150
1
Defects in cell division can lead to genomic instability and aneuploidy, both hallmarks of many cancers. During mitosis, sister chromatids are segregated on a microtubule-based spindle. Concomitant to spindle assembly, proteinaceous structures termed kinetochores are assembled on sister chromatids. In metaphase, kinetochores allow chromosome alignment and biorientation by interacting with microtubules coming from opposite spindle poles.
At anaphase onset, microtubules organize between the separating
chromosomes to form an array of antiparallel microtubules with their plus-ends overlapping at the center, called the central spindle. Once formed, the central spindle acts as a localization hub for key proteins required for cytokinesis, the
Downloaded by [UQ Library] at 07:54 21 June 2015
physical division of one cell into two.
Importantly, the mechanisms that
coordinate central spindle assembly with anaphase onset and chromosome segregation are unclear. In HeLa cells, de novo microtubule nucleation by the Augmin complex participates in central spindle assembly1. However, this complex is missing in some species that assemble central spindles in anaphase, including yeast and Caenorhabditis elegans. In most species, central spindle organization relies on the microtubule bundling activity of the conserved Ase1/PRC1 family of microtubule-associated proteins (MAPs), as well as on the microtubule motor of the kinesin-6 family MKLP12.
Microtubule motors of the kinesin-4 and -5
families also regulate central spindle integrity. However, none of these proteins display microtubule assembly or nucleation activities. Therefore, the relative contribution of pre-existing and/or de novo-assembled microtubules is unclear. To study the mechanisms of central spindle assembly, we combined RNAi-mediated depletion of key proteins with quantitative fluorescence live-cell microscopy3. We capitalized on two transgenic C. elegans strains expressing fluorescent markers for chromosomes and microtubules or the kinase AIR2AuroraB, a component of the Chromosomal Passenger Complex that specifically localizes to the central spindle.
We then developed an assay to track and
quantify central spindle intensity over time, and extract the chromosome segregation profile, which is a read-out of central spindle mechanical integrity. In 2
the one-cell C. elegans embryo, chromosome segregation is primarily driven by astral microtubule pulling forces generated by the cortically anchored microtubule motor dynein. The anaphase central spindle acts as a brake that counteracts these dynein-driven astral pulling forces, limiting the extent and velocity of sister chromatid segregation4. Thus we can quantify the mechanical integrity of the central spindle by monitoring the rate and extent of chromosome segregation with and without astral pulling forces.
We first confirmed the
contribution of SPD-1PRC1 and ZEN-4MKLP1 in central spindle integrity. However, while these two proteins are often presented in the literature as being required for central spindle assembly, our analysis suggested that their primary function
Downloaded by [UQ Library] at 07:54 21 June 2015
is to provide mechanical integrity to the central spindle during its elongation but not to promote de novo central spindle microtubule assembly. Interestingly, reducing astral pulling forces (by preventing cortical targeting of dynein) restored central spindle integrity in the absence of SPD-1PRC1 or ZEN-4MKLP1. These results show that these two proteins are dispensible for initial central spindle assembly but are essential for mechanical integrity during central spindle elongation, when normal cortical pulling forces are exerted. We next set out to identify components involved in initial central spindle microtubule assembly. We focused on the potential contribution of the kinetochore, which participates in pre-anaphase spindle microtubule formation in most systems and is associated with microtubule assembly promoting activities5. We identified a kinetochore sub-branch essential for central spindle formation and comprised of the conserved kinase BUB-1, the two redundant CENP-F orthologs HCP-1 and -2, and the CLASP ortholog CLS-2. RNAi-mediated depletion of any one of these components led to impaired central spindle assembly. Interestingly, a role for CLASP in central spindle formation had already been described in Drosophila spermatocytes6. We then followed the dynamic localization of these kinetochore components and showed that during metaphase HCP-1/2CENP-F and CLS-2CLASP are recruited to kinetochores by BUB-1 and then translocate to the central spindle region upon anaphase onset. By analyzing two mutants of the upstream kinetochore scaffold protein KNL-1 that led to decreased or excess BUB-1 at the metaphase kinetochore, we 3
demonstrated that the level of kinetochore BUB-1 directly controls central spindle microtubule density.
Further, pre-anaphase levels of BUB-1 at the
kinetochores control overall central spindle mechanical robustness by modulating the levels of HCP-1/2CENP-F and CLS-2CLASP that translocate between the separating chromosomes in anaphase. CLASP proteins contain TOGL (Tumor Overexpressed Gene Like) domains, which are characteristic of proteins with microtubule assembling activity7. Secondary structure analysis of CLS-2CLASP revealed three potential TOGL domains. We thus purified recombinant CLS-2CLASP and tested its potential
Downloaded by [UQ Library] at 07:54 21 June 2015
activity as a microtubule polymerase. We showed that in vitro recombinant CLS2CLASP led to an increase in the rate of microtubule polymerization and in the total amount of assembled microtubules. Furthermore, replacing endogenous CLS-2CLASP with a mutant version that has impaired microtubule polymerase activity completely disrupted central spindle assembly in vivo. Altogether our results led us to propose a new model for central spindle formation and elongation based on a two-step mechanism3. In the first step, metaphase kinetochore recruitment of BUB-1, HCP-1/2CENP-F and CLS-2CLASP is essential to trigger their anaphase translocation between the separating chromosomes, which then promotes central spindle microtubule assembly through CLS-2CLASP activity. During the second step, central spindle elongation, anti-parallel microtubules are organized and cross-linked by SPD-1PRC1 and ZEN4MKLP1, which provides additional mechanical integrity. Overall, we propose that by promoting central spindle assembly, kinetochores coordinate the two critical events of cell division: chromosome segregation and cytokinesis. As kinetochore components are conserved across metazoans, this previously unknown function of the kinetochore is also likely to be conserved.
4
REFERENCES Uehara R, et al. J. Cell Biol., 2010; 202, 623-636
2.
Lee K-Y, et al. J. Cell Sci. 2012; 125:3495-500.
3.
Maton G, Edwards F, et al. Nat Cell Biol 2015; 17:697-705.
4.
Grill SW, et al. Nature 2001; 409:630-3.
5.
Cheeseman IM, at al. Nat Rev Mol Cell Biol 2008; 9:33-46.
6.
Máthé E, et al. Development 2003; 130:901-15.
7.
Al-Bassam J, et al. Trends Cell Biol. 2011; 21:604-14.
Downloaded by [UQ Library] at 07:54 21 June 2015
1.
5
Downloaded by [UQ Library] at 07:54 21 June 2015
A two-step model for anaphase central spindle organization. During metaphase, BUB-1, HCP-1/2Cenp-F and CLS-2CLASP are localized at the kinetochore. At anaphase onset, their translocation between the segregating sister chromatids promotes initiation of central spindle microtubule assembly through CLS-2CLASP activity (step 1). Microtubules are then cross-linked and organized by SPD-1PRC1 and ZEN-4MKLP1, which allows central spindle elongation (step 2). Please provide figure label and call out
6
