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Jennifer G Deluca - One of the best experts on this subject based on the ideXlab platform.
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The Hec1/Ndc80 tail domain is required for force generation at Kinetochores, but is dispensable for Kinetochore-Microtubule attachment formation and Ska complex recruitment.
Molecular biology of the cell, 2020Co-Authors: Robert T. Wimbish, Keith F. Deluca, Jeanne E. Mick, Jack Himes, A. Arockia Jeyaprakash, Ignacio Jiménez-sánchez, Jennifer G DelucaAbstract:The conserved Kinetochore-associated NDC80 complex (composed of Hec1/Ndc80, Nuf2, Spc24, and Spc25) has well-documented roles in mitosis including 1) connecting mitotic chromosomes to spindle Microtubules to establish force-transducing Kinetochore-Microtubule attachments and 2) regulating the binding strength between Kinetochores and Microtubules such that correct attachments are stabilized and erroneous attachments are released. Although the NDC80 complex plays a central role in forming and regulating attachments to Microtubules, additional factors support these processes as well, including the spindle and Kinetochore-associated (Ska) complex. Multiple lines of evidence suggest that Ska complexes strengthen attachments by increasing the ability of NDC80 complexes to bind Microtubules, especially to depolymerizing Microtubule plus ends, but how this is accomplished remains unclear. Using cell-based and in vitro assays, we demonstrate that the Hec1 tail domain is dispensable for Ska complex recruitment to Kinetochores and for generation of Kinetochore-Microtubule attachments in human cells. We further demonstrate that Hec1 tail phosphorylation regulates Kinetochore-Microtubule attachment stability independently of the Ska complex. Finally, we map the location of the Ska complex in cells to a region near the coiled-coil domain of the NDC80 complex and demonstrate that this region is required for Ska complex recruitment to the NDC80 complex--Microtubule interface.
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Hec1/Ndc80 Tail Domain Function at the Kinetochore-Microtubule Interface.
Frontiers in Cell and Developmental Biology, 2020Co-Authors: Robert T. Wimbish, Jennifer G DelucaAbstract:Successful mitotic cell division is critically dependent on the formation of correct attachments between chromosomes and spindle Microtubules. Microtubule attachments are mediated by Kinetochores, which are large proteinaceous structures assembled on centromeric chromatin of mitotic chromosomes. These attachments must be sufficiently stable to transduce force; however, the strength of these attachments are also tightly regulated to ensure timely, error-free progression through mitosis. The highly conserved, Kinetochore-associated NDC80 complex is a core component of the Kinetochore-Microtubule attachment machinery in eukaryotic cells. A small, disordered region within the Hec1 subunit of the NDC80 complex – the N-terminal “tail” domain – has been actively investigated during the last decade due to its roles in generating and regulating Kinetochore-Microtubule attachments. In this review, we discuss the role of the NDC80 complex, and specifically the Hec1 tail domain, at the Kinetochore-Microtubule interface, and how recent studies provide a more unified view of Hec1 tail domain function.
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Coordination of NDC80 and Ska complexes at the Kinetochore-Microtubule interface in human cells
2019Co-Authors: Robert T. Wimbish, Keith F. Deluca, Jeanne E. Mick, Jack Himes, Ignacio J. Sánchez, A. Arockia Jeyaprakash, Jennifer G DelucaAbstract:Abstract The conserved Kinetochore-associated NDC80 complex (comprised of Hec1/Ndc80, Nuf2, Spc24, and Spc25) has well-documented roles in mitosis including (1) connecting mitotic chromosomes to spindle Microtubules to establish force-transducing Kinetochore-Microtubule attachments, and (2) regulating the binding strength between Kinetochores and Microtubules such that correct attachments are stabilized and erroneous attachments are released. Although the NDC80 complex plays a central role in forming and regulating attachments to Microtubules, additional factors support these processes as well, including the spindle and Kinetochore-associated (Ska) complex. Multiple lines of evidence suggest that Ska complexes strengthen attachments by increasing the ability of NDC80 complexes to bind Microtubules, especially to depolymerizing Microtubule plus-ends, but how this is accomplished remains unclear. Using cell-based and in vitro assays, we demonstrate that the Hec1 tail domain is dispensable for Ska complex recruitment to Kinetochores and for generation of Kinetochore-Microtubule attachments in human cells. We further demonstrate that Hec1 tail phosphorylation regulates Kinetochore-Microtubule attachment stability independently of the Ska complex. Finally, we map the location of the Ska complex in cells to a region near the coiled-coil domain of the NDC80 complex, and demonstrate that this region is required for Ska complex recruitment to the NDC80 complex-Microtubule interface.
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aurora a kinase phosphorylates hec1 to regulate metaphase Kinetochore Microtubule dynamics
Journal of Cell Biology, 2018Co-Authors: Keith F. Deluca, Amanda Meppelink, Amanda J. Broad, Jeanne E. Mick, Olve B. Peersen, Sibel Pektas, Susanne M.a. Lens, Jennifer G DelucaAbstract:Precise regulation of Kinetochore–Microtubule attachments is essential for successful chromosome segregation. Central to this regulation is Aurora B kinase, which phosphorylates Kinetochore substrates to promote Microtubule turnover. A critical target of Aurora B is the N-terminal “tail” domain of Hec1, which is a component of the NDC80 complex, a force-transducing link between Kinetochores and Microtubules. Although Aurora B is regarded as the “master regulator” of Kinetochore–Microtubule attachment, other mitotic kinases likely contribute to Hec1 phosphorylation. In this study, we demonstrate that Aurora A kinase regulates Kinetochore–Microtubule dynamics of metaphase chromosomes, and we identify Hec1 S69, a previously uncharacterized phosphorylation target site in the Hec1 tail, as a critical Aurora A substrate for this regulation. Additionally, we demonstrate that Aurora A kinase associates with inner centromere protein (INCENP) during mitosis and that INCENP is competent to drive accumulation of the kinase to the centromere region of mitotic chromosomes. These findings reveal that both Aurora A and B contribute to Kinetochore–Microtubule attachment dynamics, and they uncover an unexpected role for Aurora A in late mitosis.
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Aurora A kinase phosphorylates Hec1 to regulate metaphase Kinetochore–Microtubule dynamics
The Journal of cell biology, 2017Co-Authors: Keith F. Deluca, Amanda Meppelink, Amanda J. Broad, Jeanne E. Mick, Olve B. Peersen, Sibel Pektas, Susanne M.a. Lens, Jennifer G DelucaAbstract:Precise regulation of Kinetochore–Microtubule attachments is essential for successful chromosome segregation. Central to this regulation is Aurora B kinase, which phosphorylates Kinetochore substrates to promote Microtubule turnover. A critical target of Aurora B is the N-terminal “tail” domain of Hec1, which is a component of the NDC80 complex, a force-transducing link between Kinetochores and Microtubules. Although Aurora B is regarded as the “master regulator” of Kinetochore–Microtubule attachment, other mitotic kinases likely contribute to Hec1 phosphorylation. In this study, we demonstrate that Aurora A kinase regulates Kinetochore–Microtubule dynamics of metaphase chromosomes, and we identify Hec1 S69, a previously uncharacterized phosphorylation target site in the Hec1 tail, as a critical Aurora A substrate for this regulation. Additionally, we demonstrate that Aurora A kinase associates with inner centromere protein (INCENP) during mitosis and that INCENP is competent to drive accumulation of the kinase to the centromere region of mitotic chromosomes. These findings reveal that both Aurora A and B contribute to Kinetochore–Microtubule attachment dynamics, and they uncover an unexpected role for Aurora A in late mitosis.
Charles L. Asbury - One of the best experts on this subject based on the ideXlab platform.
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Cdk1 Phosphorylation of the Dam1 Complex Strengthens Kinetochore-Microtubule Attachments
Current biology : CB, 2020Co-Authors: Abraham Gutierrez, Charles L. Asbury, Matthew P. Miller, Jae Ook Kim, Neil T. Umbreit, Trisha N. Davis, Sue BigginsAbstract:To ensure the faithful inheritance of DNA, a macromolecular protein complex called the Kinetochore sustains the connection between chromosomes and force-generating dynamic Microtubules during cell division. Defects in this process lead to aneuploidy, a common feature of cancer cells and the cause of many developmental diseases [1-4]. One of the major Microtubule-binding activities in the Kinetochore is mediated by the conserved Ndc80 complex (Ndc80c) [5-7]. In budding yeast, the retention of Kinetochores on dynamic Microtubule tips also depends on the essential heterodecameric Dam1 complex (Dam1c) [8-15], which binds to the Ndc80c and is proposed to be a functional ortholog of the metazoan Ska complex [16, 17]. The load-bearing activity of the Dam1c depends on its ability to oligomerize, and the purified complex spontaneously self-assembles into Microtubule-encircling oligomeric rings, which are proposed to function as collars that allow Kinetochores to processively track the plus-end tips of Microtubules and harness the forces generated by disassembling Microtubules [10-15, 18-22]. However, it is unknown whether there are specific regulatory events that promote Dam1c oligomerization to ensure accurate segregation. Here, we used a reconstitution system to discover that Cdk1, the major mitotic kinase that drives the cell cycle, phosphorylates the Ask1 component of the Dam1c to increase its residence time on Microtubules and enhance Kinetochore-Microtubule attachment strength. We propose that Cdk1 activity promotes Dam1c oligomerization to ensure that Kinetochore-Microtubule attachments are stabilized as Kinetochores come under tension in mitosis.
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human ska complex and ndc80 complex interact to form a load bearing assembly that strengthens Kinetochore Microtubule attachments
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Luke A Helgeson, Charles L. Asbury, Alex Zelter, Michael Riffle, Michael J. Maccoss, Trisha N. DavisAbstract:Accurate segregation of chromosomes relies on the force-bearing capabilities of the Kinetochore to robustly attach chromosomes to dynamic Microtubule tips. The human Ska complex and Ndc80 complex are outer-Kinetochore components that bind Microtubules and are required to fully stabilize Kinetochore–Microtubule attachments in vivo. While purified Ska complex tracks with disassembling Microtubule tips, it remains unclear whether the Ska complex–Microtubule interaction is sufficiently strong to make a significant contribution to Kinetochore–Microtubule coupling. Alternatively, Ska complex might affect Kinetochore coupling indirectly, through recruitment of phosphoregulatory factors. Using optical tweezers, we show that the Ska complex itself bears load on Microtubule tips, strengthens Ndc80 complex-based tip attachments, and increases the switching dynamics of the attached Microtubule tips. Cross-linking mass spectrometry suggests the Ska complex directly binds Ndc80 complex through interactions between the Ska3 unstructured C-terminal region and the coiled-coil regions of each Ndc80 complex subunit. Deletion of the Ska complex Microtubule-binding domain or the Ska3 C terminus prevents Ska complex from strengthening Ndc80 complex-based attachments. Together, our results indicate that the Ska complex can directly strengthen the Kinetochore–Microtubule interface and regulate Microtubule tip dynamics by forming an additional connection between the Ndc80 complex and the Microtubule.
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Human Ska complex and Ndc80 complex interact to form a load-bearing assembly that strengthens Kinetochore–Microtubule attachments
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Luke A Helgeson, Charles L. Asbury, Alex Zelter, Michael Riffle, Michael J. Maccoss, Trisha N. DavisAbstract:Accurate segregation of chromosomes relies on the force-bearing capabilities of the Kinetochore to robustly attach chromosomes to dynamic Microtubule tips. The human Ska complex and Ndc80 complex are outer-Kinetochore components that bind Microtubules and are required to fully stabilize Kinetochore–Microtubule attachments in vivo. While purified Ska complex tracks with disassembling Microtubule tips, it remains unclear whether the Ska complex–Microtubule interaction is sufficiently strong to make a significant contribution to Kinetochore–Microtubule coupling. Alternatively, Ska complex might affect Kinetochore coupling indirectly, through recruitment of phosphoregulatory factors. Using optical tweezers, we show that the Ska complex itself bears load on Microtubule tips, strengthens Ndc80 complex-based tip attachments, and increases the switching dynamics of the attached Microtubule tips. Cross-linking mass spectrometry suggests the Ska complex directly binds Ndc80 complex through interactions between the Ska3 unstructured C-terminal region and the coiled-coil regions of each Ndc80 complex subunit. Deletion of the Ska complex Microtubule-binding domain or the Ska3 C terminus prevents Ska complex from strengthening Ndc80 complex-based attachments. Together, our results indicate that the Ska complex can directly strengthen the Kinetochore–Microtubule interface and regulate Microtubule tip dynamics by forming an additional connection between the Ndc80 complex and the Microtubule.
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The human Ska complex and Ndc80 complex interact to form a load-bearing assembly that strengthens Kinetochore-Microtubule attachments
2018Co-Authors: Luke A Helgeson, Charles L. Asbury, Alex Zelter, Michael Riffle, Michael J. Maccoss, Trisha N. DavisAbstract:Accurate segregation of chromosomes relies on the force-bearing capabilities of the Kinetochore to robustly attach chromosomes to dynamic Microtubule tips. The human Ska complex and Ndc80 complex are outer-Kinetochore components that bind Microtubules and are required to fully stabilize Kinetochore-Microtubule attachments in vivo. While purified Ska complex tracks with disassembling Microtubule tips, it remains unclear whether the Ska complex-Microtubule interaction is sufficiently strong to make a significant contribution to Kinetochore-Microtubule coupling. Alternatively, Ska complex might affect Kinetochore coupling indirectly, through recruitment of phospho-regulatory factors. Using optical tweezers, we show that the Ska complex itself bears load on Microtubule tips, strengthens Ndc80 complex-based tip attachments, and increases the switching dynamics of the attached Microtubule tips. Crosslinking mass spectrometry suggests the Ska complex directly binds Ndc80 complex through interactions between the Ska3 unstructured C-terminal region and the coiled-coil regions of each Ndc80 complex subunit. Deletion of the Ska complex Microtubule-binding domain or the Ska3 C-terminus prevents Ska complex from strengthening Ndc80 complex-based attachments. Together our results indicate that the Ska complex can directly strengthen the Kinetochore Microtubule interface and regulate Microtubule tip dynamics by forming an additional connection between the Ndc80 complex and the Microtubule.
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A TOG Protein Confers Tension Sensitivity to Kinetochore-Microtubule Attachments
Cell, 2016Co-Authors: Matthew P. Miller, Charles L. Asbury, Sue BigginsAbstract:The development and survival of all organisms depends on equal partitioning of their genomes during cell division. Accurate chromosome segregation requires selective stabilization of Kinetochore-Microtubule attachments that come under tension due to opposing pulling forces exerted on sister Kinetochores by dynamic Microtubule tips. Here, we show that the XMAP215 family member, Stu2, makes a major contribution to Kinetochore-Microtubule coupling. Stu2 and its human ortholog, ch-TOG, exhibit a conserved interaction with the Ndc80 Kinetochore complex that strengthens its attachment to Microtubule tips. Strikingly, Stu2 can either stabilize or destabilize Kinetochore attachments, depending on the level of Kinetochore tension and whether the Microtubule tip is assembling or disassembling. These dichotomous effects of Stu2 are independent of its previously studied regulation of Microtubule dynamics. Altogether, our results demonstrate how a Kinetochore-associated factor can confer opposing, tension-dependent effects to selectively stabilize tension-bearing attachments, providing mechanistic insight into the basis for accuracy during chromosome segregation.
Ping Gui - One of the best experts on this subject based on the ideXlab platform.
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methylation of plk1 by set7 9 ensures accurate Kinetochore Microtubule dynamics
Journal of Molecular Cell Biology, 2020Co-Authors: Hazrat Ismail, Jun Cao, Jianyu Wang, Tarsha Ward, Fengrui Yang, Ping GuiAbstract:Faithful segregation of mitotic chromosomes requires bi-orientation of sister chromatids, which relies on the sensing of correct attachments between spindle Microtubules and Kinetochores. Although the mechanisms underlying PLK1 activation have been extensively studied, the regulatory mechanisms that couple PLK1 activity to accurate chromosome segregation are not well understood. In particular, PLK1 is implicated in stabilizing Kinetochore-Microtubule attachments, but how Kinetochore PLK1 activity is regulated to avoid hyperstabilized Kinetochore-Microtubules in mitosis remains elusive. Here, we show that Kinetochore PLK1 kinase activity is modulated by SET7/9 via lysine methylation during early mitosis. The SET7/9-elicited dimethylation occurs at the Lys191 of PLK1, which tunes down its activity by limiting ATP utilization. Overexpression of the non-methylatable PLK1 mutant or chemical inhibition of SET7/9 methyltransferase activity resulted in mitotic arrest due to destabilized Kinetochore-Microtubule attachments. These data suggest that Kinetochore PLK1 is essential for stable Kinetochore-Microtubule attachments and methylation by SET7/9 promotes dynamic Kinetochore-Microtubule attachments for accurate error correction. Our findings define a novel homeostatic regulation at the Kinetochore that integrates protein phosphorylation and methylation with accurate chromosome segregation for maintenance of genomic stability.
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Methylation of PLK1 by SET7/9 ensures accurate Kinetochore-Microtubule dynamics.
Journal of molecular cell biology, 2019Co-Authors: Hazrat Ismail, Jun Cao, Jianyu Wang, Tarsha Ward, Fengrui Yang, Ping Gui, Mahboob AliAbstract:Faithful segregation of mitotic chromosomes requires bi-orientation of sister chromatids, which relies on the sensing of correct attachments between spindle Microtubules and Kinetochores. Although the mechanisms underlying PLK1 activation have been extensively studied, the regulatory mechanisms that couple PLK1 activity to accurate chromosome segregation are not well understood. In particular, PLK1 is implicated in stabilizing Kinetochore-Microtubule attachments, but how Kinetochore PLK1 activity is regulated to avoid hyperstabilized Kinetochore-Microtubules in mitosis remains elusive. Here, we show that Kinetochore PLK1 kinase activity is modulated by SET7/9 via lysine methylation during early mitosis. The SET7/9-elicited dimethylation occurs at the Lys191 of PLK1, which tunes down its activity by limiting ATP utilization. Overexpression of the non-methylatable PLK1 mutant or chemical inhibition of SET7/9 methyltransferase activity resulted in mitotic arrest due to destabilized Kinetochore-Microtubule attachments. These data suggest that Kinetochore PLK1 is essential for stable Kinetochore-Microtubule attachments and methylation by SET7/9 promotes dynamic Kinetochore-Microtubule attachments for accurate error correction. Our findings define a novel homeostatic regulation at the Kinetochore that integrates protein phosphorylation and methylation with accurate chromosome segregation for maintenance of genomic stability.
Claudio E Sunkel - One of the best experts on this subject based on the ideXlab platform.
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polo regulates spindly to prevent premature stabilization of Kinetochore Microtubule attachments
The EMBO Journal, 2020Co-Authors: Claudio E Sunkel, João Barbosa, Torcato Martins, Tanja Bange, Li Tao, Carlos CondeAbstract:Accurate chromosome segregation in mitosis requires sister Kinetochores to bind to Microtubules from opposite spindle poles. The stability of Kinetochore-Microtubule attachments is fine-tuned to prevent or correct erroneous attachments while preserving amphitelic interactions. Polo kinase has been implicated in both stabilizing and destabilizing Kinetochore-Microtubule attachments. However, the mechanism underlying Polo-destabilizing activity remains elusive. Here, resorting to an RNAi screen in Drosophila for suppressors of a constitutively active Polo mutant, we identified a strong genetic interaction between Polo and the Rod-ZW10-Zwilch (RZZ) complex, whose Kinetochore accumulation has been shown to antagonize Microtubule stability. We find that Polo phosphorylates Spindly and impairs its ability to bind to Zwilch. This precludes dynein-mediated removal of the RZZ from Kinetochores and consequently delays the formation of stable end-on attachments. We propose that high Polo-kinase activity following mitotic entry directs the RZZ complex to minimize premature stabilization of erroneous attachments, whereas a decrease in active Polo in later mitotic stages allows the formation of stable amphitelic spindle attachments. Our findings demonstrate that Polo tightly regulates the RZZ-Spindly-dynein module during mitosis to ensure the fidelity of chromosome segregation.
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Polo regulates Spindly to prevent premature stabilization of Kinetochore–Microtubule attachments
The EMBO journal, 2019Co-Authors: João Barbosa, Torcato Martins, Tanja Bange, Li Tao, Carlos Conde, Claudio E SunkelAbstract:Accurate chromosome segregation in mitosis requires sister Kinetochores to bind to Microtubules from opposite spindle poles. The stability of Kinetochore-Microtubule attachments is fine-tuned to prevent or correct erroneous attachments while preserving amphitelic interactions. Polo kinase has been implicated in both stabilizing and destabilizing Kinetochore-Microtubule attachments. However, the mechanism underlying Polo-destabilizing activity remains elusive. Here, resorting to an RNAi screen in Drosophila for suppressors of a constitutively active Polo mutant, we identified a strong genetic interaction between Polo and the Rod-ZW10-Zwilch (RZZ) complex, whose Kinetochore accumulation has been shown to antagonize Microtubule stability. We find that Polo phosphorylates Spindly and impairs its ability to bind to Zwilch. This precludes dynein-mediated removal of the RZZ from Kinetochores and consequently delays the formation of stable end-on attachments. We propose that high Polo-kinase activity following mitotic entry directs the RZZ complex to minimize premature stabilization of erroneous attachments, whereas a decrease in active Polo in later mitotic stages allows the formation of stable amphitelic spindle attachments. Our findings demonstrate that Polo tightly regulates the RZZ-Spindly-dynein module during mitosis to ensure the fidelity of chromosome segregation.
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Stability of Kinetochore-Microtubule attachment and the role of different KMN network components in Drosophila
Cytoskeleton, 2013Co-Authors: Tália Feijão, André F. Maia, Olga Afonso, Claudio E SunkelAbstract:Kinetochores bind spindle Microtubules and also act as signaling centers that monitor this interaction. Defects in Kinetochore assembly lead to chromosome missegregation and aneuploidy. The interaction between Microtubules and chromosomes involves a conserved super-complex of proteins, known as the KNL1Mis12Ndc80 (KMN) network, composed by the KNL1 (Spc105), Mis12, and Ndc80 complexes. Previous studies indicate that all components of the network are required for Kinetochore-Microtubule attachment and all play relevant functions in chromosome congression, biorientation, and segregation. Here, we report a comparative study addressing the role of the different KMN components using dsRNA and in vivo fluorescence microscopy in Drosophila S2 cells allowing us to suggest that different KMN network components might perform different roles in chromosome segregation and the mitotic checkpoint signaling. Depletion of different components results in mostly lateral Kinetochore-Microtubule attachments that are relatively stable on depletion of Mis12 or Ndc80 but very unstable after Spc105 depletion. In vivo analysis on depletion of Mis12, Ndc80, and to some extent Spc105, shows that lateral Kinetochore-Microtubule interactions are still functional allowing poleward Kinetochore movement. We also find that different KMN network components affect differently the localization of spindle assembly checkpoint (SAC) proteins at Kinetochores. Depletion of Ndc80 and Spc105 abolishes the mitotic checkpoint, whereas depletion of Mis12 causes a delay in mitotic progression. Taken together, our results suggest that Mis12 and Ndc80 complexes help to properly orient Microtubule attachment, whereas Spc105 plays a predominant role in the Kinetochore-Microtubule attachment as well as in the poleward movement of chromosomes, SAC response, and cell viability. © 2013 Wiley Periodicals, Inc.
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Stability of Kinetochore-Microtubule attachment and the role of different KMN network components in Drosophila.
Cytoskeleton (Hoboken N.J.), 2013Co-Authors: Tália Feijão, André F. Maia, Olga Afonso, Claudio E SunkelAbstract:Kinetochores bind spindle Microtubules and also act as signaling centers that monitor this interaction. Defects in Kinetochore assembly lead to chromosome missegregation and aneuploidy. The interaction between Microtubules and chromosomes involves a conserved super-complex of proteins, known as the KNL1Mis12Ndc80 (KMN) network, composed by the KNL1 (Spc105), Mis12, and Ndc80 complexes. Previous studies indicate that all components of the network are required for Kinetochore-Microtubule attachment and all play relevant functions in chromosome congression, biorientation, and segregation. Here, we report a comparative study addressing the role of the different KMN components using dsRNA and in vivo fluorescence microscopy in Drosophila S2 cells allowing us to suggest that different KMN network components might perform different roles in chromosome segregation and the mitotic checkpoint signaling. Depletion of different components results in mostly lateral Kinetochore-Microtubule attachments that are relatively stable on depletion of Mis12 or Ndc80 but very unstable after Spc105 depletion. In vivo analysis on depletion of Mis12, Ndc80, and to some extent Spc105, shows that lateral Kinetochore-Microtubule interactions are still functional allowing poleward Kinetochore movement. We also find that different KMN network components affect differently the localization of spindle assembly checkpoint (SAC) proteins at Kinetochores. Depletion of Ndc80 and Spc105 abolishes the mitotic checkpoint, whereas depletion of Mis12 causes a delay in mitotic progression. Taken together, our results suggest that Mis12 and Ndc80 complexes help to properly orient Microtubule attachment, whereas Spc105 plays a predominant role in the Kinetochore-Microtubule attachment as well as in the poleward movement of chromosomes, SAC response, and cell viability.
Sue Biggins - One of the best experts on this subject based on the ideXlab platform.
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Cdk1 Phosphorylation of the Dam1 Complex Strengthens Kinetochore-Microtubule Attachments
Current biology : CB, 2020Co-Authors: Abraham Gutierrez, Charles L. Asbury, Matthew P. Miller, Jae Ook Kim, Neil T. Umbreit, Trisha N. Davis, Sue BigginsAbstract:To ensure the faithful inheritance of DNA, a macromolecular protein complex called the Kinetochore sustains the connection between chromosomes and force-generating dynamic Microtubules during cell division. Defects in this process lead to aneuploidy, a common feature of cancer cells and the cause of many developmental diseases [1-4]. One of the major Microtubule-binding activities in the Kinetochore is mediated by the conserved Ndc80 complex (Ndc80c) [5-7]. In budding yeast, the retention of Kinetochores on dynamic Microtubule tips also depends on the essential heterodecameric Dam1 complex (Dam1c) [8-15], which binds to the Ndc80c and is proposed to be a functional ortholog of the metazoan Ska complex [16, 17]. The load-bearing activity of the Dam1c depends on its ability to oligomerize, and the purified complex spontaneously self-assembles into Microtubule-encircling oligomeric rings, which are proposed to function as collars that allow Kinetochores to processively track the plus-end tips of Microtubules and harness the forces generated by disassembling Microtubules [10-15, 18-22]. However, it is unknown whether there are specific regulatory events that promote Dam1c oligomerization to ensure accurate segregation. Here, we used a reconstitution system to discover that Cdk1, the major mitotic kinase that drives the cell cycle, phosphorylates the Ask1 component of the Dam1c to increase its residence time on Microtubules and enhance Kinetochore-Microtubule attachment strength. We propose that Cdk1 activity promotes Dam1c oligomerization to ensure that Kinetochore-Microtubule attachments are stabilized as Kinetochores come under tension in mitosis.
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A TOG Protein Confers Tension Sensitivity to Kinetochore-Microtubule Attachments
Cell, 2016Co-Authors: Matthew P. Miller, Charles L. Asbury, Sue BigginsAbstract:The development and survival of all organisms depends on equal partitioning of their genomes during cell division. Accurate chromosome segregation requires selective stabilization of Kinetochore-Microtubule attachments that come under tension due to opposing pulling forces exerted on sister Kinetochores by dynamic Microtubule tips. Here, we show that the XMAP215 family member, Stu2, makes a major contribution to Kinetochore-Microtubule coupling. Stu2 and its human ortholog, ch-TOG, exhibit a conserved interaction with the Ndc80 Kinetochore complex that strengthens its attachment to Microtubule tips. Strikingly, Stu2 can either stabilize or destabilize Kinetochore attachments, depending on the level of Kinetochore tension and whether the Microtubule tip is assembling or disassembling. These dichotomous effects of Stu2 are independent of its previously studied regulation of Microtubule dynamics. Altogether, our results demonstrate how a Kinetochore-associated factor can confer opposing, tension-dependent effects to selectively stabilize tension-bearing attachments, providing mechanistic insight into the basis for accuracy during chromosome segregation.
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Measuring Kinetochore-Microtubule interaction in vitro.
Methods in enzymology, 2014Co-Authors: Jonathan W. Driver, Andrew F Powers, Krishna K Sarangapani, Sue Biggins, Charles L. AsburyAbstract:Abstract Many proteins and protein complexes perform sophisticated, regulated functions in vivo . Many of these functions can be recapitulated using in vitro reconstitution, which serves as a means to establish unambiguous cause–effect relationships, for example, between a protein and its phosphorylating kinase. Here, we describe a protocol to purify Kinetochores, the protein complexes that attach chromosomes to Microtubules during mitosis, and quantitatively assay their Microtubule-binding characteristics. Our assays, based on DIC imaging and laser trapping microscopy, are used to measure the attachment of Microtubules to Kinetochores and the load-bearing capabilities of those attachments. These assays provide a platform for studying kinase disruption of Kinetochore–Microtubule attachments, which is believed to be critical for correcting erroneous Kinetochore–spindle attachments and thereby avoiding chromosome missegregation. The principles of our approach should be extensible to studies of a wide range of force-bearing interactions in biology.
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Reconstituting the Kinetochore–Microtubule interface: what, why, and how
Chromosoma, 2012Co-Authors: Bungo Akiyoshi, Sue BigginsAbstract:The Kinetochore is the proteinaceous complex that governs the movement of duplicated chromosomes by interacting with spindle Microtubules during mitosis and meiosis. Faithful chromosome segregation requires that Kinetochores form robust load-bearing attachments to the tips of dynamic spindle Microtubules, correct Microtubule attachment errors, and delay the onset of anaphase until all chromosomes have made proper attachments. To understand how this macromolecular machine operates to segregate duplicated chromosomes with exquisite accuracy, it is critical to reconstitute and study Kinetochore–Microtubule interactions in vitro using defined components. Here, we review the current status of reconstitution as well as recent progress in understanding the Microtubule-binding functions of Kinetochores in vivo.
