Congressing kinetochores progressively load Ska complexes to prevent force-dependent detachment

Auckland, Philip; Clarke, Nicholas I.; Royle, Stephen; McAinsh, Andrew D.

doi:10.1083/jcb.201607096

Cited by 53 publications

(62 citation statements)

References 48 publications

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“…Again, these results support that Thr358 and Thr360 are the major sites contributing to Ska functions. Of note, our results also suggest that the Ska complex may be dispensable for chromosome congression to metaphase plates from initial attachments (Figures S4A and S4B), consistent with the previous findings (Auckland et al, 2017;Daum et al, 2009). The significant delay in our experiments was derived from metaphase-to-anaphase transition.…”

Section: Cdk1 Phosphorylation On the Six Sites In Ska3 Is Required Fosupporting

confidence: 92%

“…However, how this is achieved is not fully understood. In cells, Ska starts to accumulate at kinetochores in prometaphase and peaks at metaphase (Auckland et al, 2017;Hanisch et al, 2006). The dynamic localization pattern coordinates well with the process of establishing proper kinetochoremicrotubule interactions from prophase to metaphase, suggesting that Ska binding to kinetochores could be important for its functions.…”

Section: Introductionmentioning

confidence: 64%

“…Why is multisite phosphorylation needed? The Ska complex starts to accumulate at kinetochores in prophase and peaks at metaphase (Auckland et al, 2017;Hanisch et al, 2006), suggesting that the binding affinity of Ska to kinetochores varies with the mitotic progression.…”

Section: Discussionmentioning

confidence: 99%

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In vitro Reconstitution Unveils the Phospho-regulated Ska-Ndc80 Macro-complex

Chen

Tian

et al. 2019

Preprint

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The human Ska (spindle and kinetochore associated) complex is essential for proper kinetochoremicrotubule interactions. Its loading onto the kinetochore complex Ndc80 (Ndc80C) plays a critical role in such function; however, the key determinant that docks Ska onto Ndc80C still remain elusive. Here, we address this question using biochemical reconstitution followed by functional analysis. We identified six Cdk1 sites in Ska3 distributed in three conserved regions. In vitro, Cdk1 phosphorylation on the Ska complex enhanced WT, not phospho-deficient 6A, binding to Ndc80C.Strikingly, the phospho-mimetic Ska 6D complex formed a stable macro-complex with Ndc80C, but Ska WT failed to do so, suggesting that Cdk1 phosphorylation is necessary and sufficient for the formation of the Ska-Ndc80 macro-complex. In cells, Ska3 6A completely lost its localization to kinetochores and its functions in chromosome segregation; whereas Ska3 6D partially restored them. Altogether, our findings not only for the first time reveal the key phospho-regulated Ska-Ndc80 macro-complex essential for faithful chromosome segregation, but also pave the way for mechanistic analyses of kinetochore-microtubule interactions.

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Section: Cdk1 Phosphorylation On the Six Sites In Ska3 Is Required Fosupporting

confidence: 92%

Section: Introductionmentioning

confidence: 64%

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In vitro Reconstitution Unveils the Phospho-regulated Ska-Ndc80 Macro-complex

Chen

Tian

et al. 2019

Preprint

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“…In contrast, both MTBD-microtubule interactions support force generation, which suggests that kinetochores utilise diffusion-based and curved-protofilament coupled mechanisms to produce tension. It is well established that the latter is mediated by the Ska complex (Schmidt et al, 2012), which is crucial for providing load-bearing capability to kinetochores in vivo (Auckland et al, 2017) and in vitro (Helgeson et al, 2018). However, the CENP-F MTBDs do not appear to contribute as they are dispensable for chromosome congression.…”

Section: Discussionmentioning

confidence: 99%

CENP-F controls force generation and the dynein-dependent stripping of CENP-E at kinetochores

Auckland

McAinsh

2019

Preprint

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Accurate chromosome segregation demands efficient capture of microtubules by kinetochores and their conversion to stable bi-oriented attachments that can congress and then segregate chromosomes. An early event is the shedding of the outermost fibrous corona layer of the kinetochore following microtubule attachment. Centromere protein F (CENP-F) is part of the corona, contains two microtubule-binding domains and physically associates with dynein motor regulators. Here, we have combined CRISPR gene editing and engineered separation-of-function mutants to define how CENP-F contributes to kinetochore function. We show here that the two microtubulebinding domains make distinct contributions to attachment stability and force generation that are required to minimise errors in anaphase, but are dispensable for congression. We further identify a specialised domain that functions to inhibit the dynein mediated stripping of CENP-E motors. We show how this "dynein-brake" is crucial for ensuring kinetochores contain the right number of CENP-E motors at the right time during mitosis, with loss of brake function delaying congression.

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“…These findings suggest that the Ska complex is important for establishing and/or maintaining end‐on kinetochore‐microtubule attachments. Recently, detailed analysis of kinetochore attachments using live imaging demonstrated that the Ska complex functions to prevent force‐dependent detachment of kinetochores from microtubules in the process of chromosome congression, which helps to stabilize the end‐on kinetochore attachments and promote proper chromosome alignment …”

Section: Ska Promotes End‐on Kinetochore Attachmentsmentioning

confidence: 99%

Multitasking Ska in Chromosome Segregation: Its Distinct Pools Might Specify Various Functions

2018

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The human spindle and kinetochore associated (Ska) complex is required for proper mitotic progression. Extensive studies have demonstrated its important functions in both stable kinetochore-microtubule interactions and spindle checkpoint silencing. We suggest a model to explain how various Ska functions might be fulfilled by distinct pools of Ska at kinetochores. The Ndc80-loop pool of Ska is recruited by the Ndc80 loop, or together with some of its flanking sequences, and the recruitment is also dependent on Cdk1-mediated Ska3 phosphorylation. This pool seems to play a more important role in silencing the spindle checkpoint than stabilizing kinetochore-microtubule interactions. In contrast, the Ndc80-N-terminus pool of Ska is recruited by the N-terminal domains of Ndc80 and appears to be more important for stabilizing kinetochore-microtubule interactions. Here, we review and discuss the evidence that supports this model and suggest further experiments to test the functioning mechanisms of the Ska complex.

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Congressing kinetochores progressively load Ska complexes to prevent force-dependent detachment

Abstract: Auckland et al. describe a previously overlooked step during chromosome congression that provides insight into how kinetochore maturation is linked to attachment load-bearing, microtubule occupancy, and spindle assembly checkpoint signaling.

Cited by 53 publications

References 48 publications

In vitro Reconstitution Unveils the Phospho-regulated Ska-Ndc80 Macro-complex

In vitro Reconstitution Unveils the Phospho-regulated Ska-Ndc80 Macro-complex

CENP-F controls force generation and the dynein-dependent stripping of CENP-E at kinetochores

Multitasking Ska in Chromosome Segregation: Its Distinct Pools Might Specify Various Functions

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