Molecular determinants of the Ska-Ndc80 interaction and their influence on microtubule tracking and force-coupling

Veld, Pim J Huis in 't; Stender, Isabelle D; Musacchio, Andrea; Dogterom, Marileen

doi:10.1101/675363

Cited by 5 publications

(2 citation statements)

References 99 publications

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“…6E). Ndc80 has been previously shown to track the ends of dynamic microtubules together with Ska1 [9,17], and in our hand, we also observed the formation of weak tip associated complexes with Ndc80 and Ska1 (Sup. Fig.…”

Section: The Tripartite Cdt1-ska1-ndc80 Super-complex Tracks the Ends...supporting

confidence: 73%

The Ndc80-Cdt1-Ska1 complex constitute a minimal processive kinetochore-microtubule coupling unit

Rahi

Chakraborty

Agarwal

et al. 2022

Preprint

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The Ndc80 kinetochore complex is essential for robust kinetochore-microtubule (k-MT) attachments during mitosis. Ndc80 has been shown to recruit the Ska1 complex to kinetochores, where Ska1 is thought to aid in k-MT coupling by Ndc80. Our previous work has shown that Cdt1, a DNA replication licensing factor, is a novel mitotic spindle-associated protein that is also recruited to kinetochores via Ndc80 and is required for stabilizing k-MT attachments. In this study, we developed auxin-induced degron (AID)-tagging to validate the previously demonstrated mitotic role of Cdt1. We demonstrate a direct interaction between Cdt1 and Ska1 that is essential for proper recruitment of Cdt1 to kinetochores and spindle microtubules. We find that Cdk1-dependent phosphorylation of Cdt1 during mitosis is critical for Ska1-binding, consequently regulating the stabilization of metaphase k-MT attachments and normal mitotic progression. Total internal reflection fluorescence microscopy (TIR-FM) experiments reveal that Cdt1 synergizes with the Ndc80 and the Ska1 complexes for microtubule-binding. Further, we show that single Cdt1 molecules form diffusive tripartite complexes with Ndc80 and Ska1 that can processively track the ends of dynamic microtubules in vitro. Taken together our data identifies a minimal molecular unit responsible for bidirectional processive tip tracking of kinetochores.

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Section: The Tripartite Cdt1-ska1-ndc80 Super-complex Tracks the Ends...supporting

confidence: 73%

The Ndc80-Cdt1-Ska1 complex constitute a minimal processive kinetochore-microtubule coupling unit

Rahi

Chakraborty

Agarwal

et al. 2022

Preprint

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“…To test whether CDC20 expression was directly linked to SACi-induced CIN, we characterized the effect of CDC20 levels on the overall rate and severity of mitotic errors in mouse and human cells using live cell imaging. To this end, we quantified mitotic aberrations in control and CDC20 knockout cells, with or without SAC inhibition, and scored the mitotic aberrations on a severity scale of 0-5, with 0 (green) representing normal division and increasingly higher scores (warmer colors) representing more severe mitotic aberrations, as has been done in previous works (Crozier et al, 2022;Thu et al, 2018;Huis In 'T Veld et al, 2019; see Methods section). Both in NIH-3T3 and HCT116 cells subjected to SAC inhibition, CDC20 knockout significantly reduced the prevalence and the severity of reversine-induced mitotic aberrations, thus alleviating SAC-inhibition-induced CIN (Figure 4C and Figure 4D, respectively).…”

Section: Increased Cdc20 Expression Is Associated With the Preferenti...mentioning

confidence: 99%

CDC20 determines the sensitivity to spindle assembly checkpoint (SAC) inhibitors

Zheng,

Raz,

Zhou

et al. 2023

Preprint

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Spindle assembly checkpoint (SAC) inhibitors are a recently developed class of drugs that perturb the regulation of chromosome segregation during division, induce chromosomal instability (CIN), and eventually lead to cell death. While they are currently in clinical trials for solid cancers, biomarkers to predict the response to SAC inhibitors are still lacking. We recently reported that aneuploid cancer cells are preferentially sensitive to SAC inhibition. Here, we investigated the molecular determinants of the response to SAC inhibition that underlies the differential sensitivity of aneuploid cells to these drugs. We found that this response was largely driven by the expression of CDC20, a main activator of the anaphase-promoting complex (APC/C), rather than by APC/C itself. Mechanistically, we discovered that CDC20 depletion prolonged metaphase duration, diminished mitotic errors, and reduced sensitivity to SAC inhibition. Aneuploid cells expressed high levels of CDC20 and experienced shorter metaphases and multiple mitotic errors, resulting in increased long-term sensitivity to SAC inhibition. Our findings propose high CDC20 expression as a favorable biomarker for SAC inhibition therapy and as an aneuploidy-induced therapeutic vulnerability.

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SKAP binding to microtubules reduces friction at the kinetochore-microtubule interface and increases attachment stability under force

Rosas-Salvans,

Rux,

Das

et al. 2024

Preprint

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The kinetochore links chromosomes to spindle microtubules to drive chromosome segregation at cell division. We recently uncovered that the kinetochore complex Astrin-SKAP, which binds microtubules, reduces rather than increases friction at the mammalian kinetochore-microtubule interface. How it does so is not known. Astrin-SKAP could affect how other kinetochore complexes bind microtubules, reducing their friction along microtubules, or it could itself bind microtubules with similar affinity but lower friction than other attachment factors. Using SKAP mutants unable to bind microtubules, live imaging and laser ablation, we show that SKAP’s microtubule binding is essential for sister kinetochore coordination, force dissipation at the interface and attachment responsiveness to force changes. Further, we show that SKAP’s microtubule binding is essential to prevent chromosome detachment under both spindle forces and microneedle-generated forces. Together, our findings indicate that SKAP’s microtubule binding reduces kinetochore friction and increases attachment responsiveness and stability under force. We propose that having complexes with both high and low sliding friction on microtubules, making a mechanically heterogeneous interface, is key to maintaining robust attachments under force and thus accurate segregation.

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Molecular determinants of the Ska-Ndc80 interaction and their influence on microtubule tracking and force-coupling

Cited by 5 publications

References 99 publications

The Ndc80-Cdt1-Ska1 complex constitute a minimal processive kinetochore-microtubule coupling unit

The Ndc80-Cdt1-Ska1 complex constitute a minimal processive kinetochore-microtubule coupling unit

CDC20 determines the sensitivity to spindle assembly checkpoint (SAC) inhibitors

SKAP binding to microtubules reduces friction at the kinetochore-microtubule interface and increases attachment stability under force

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