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Ska3 Ensures Timely Mitotic Progression by Interacting Directly With Microtubules and Ska1 Microtubule Binding Domain

View Article: PubMed Central - PubMed

ABSTRACT

The establishment of physical attachment between the kinetochore and dynamic spindle microtubules, which undergo cycles of polymerization and depolymerization generating straight and curved microtubule structures, is essential for accurate chromosome segregation. The Ndc80 and Ska complexes are the major microtubule-binding factors of the kinetochore responsible for maintaining chromosome-microtubule coupling during chromosome segregation. We previously showed that the Ska1 subunit of the Ska complex binds dynamic microtubules using multiple contact sites in a mode that allows conformation-independent binding. Here, we show that the Ska3 subunit is required to modulate the microtubule binding capability of the Ska complex (i) by directly interacting with tubulin monomers and (ii) indirectly by interacting with tubulin contacting regions of Ska1 suggesting an allosteric regulation. Perturbing either the Ska3-microtubule interaction or the Ska3-Ska1 interactions negatively influences microtubule binding by the Ska complex in vitro and affects the timely onset of anaphase in cells. Thus, Ska3 employs additional modulatory elements within the Ska complex to ensure robust kinetochore-microtubule attachments and timely progression of mitosis.

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Direct binding of Ska3C-term to Ska1 and microtubules is required for maintaining kinetochore-microtubule attachments and timely anaphase onset.(a,b) Box-and-whisker plot showing the elapsed time (min) between nuclear envelope breakdown (NEBD) and anaphase onset/death for individual cells. The total number of cells (n) from three independent experiments is given above each box. Lower and upper whiskers represent 10th and 90th percentiles, respectively. Table summarizing information from the live cell experiments shown below regarding the average time between NEBD to anaphase onset/death and the percentage of cells dying in mitosis. (c) Representative stills from time-lapse video microscopy experiments illustrating mitotic progression of HeLa S3 cells stably expressing H2B-GFP, treated as in (a,b). Time in hr:min is indicated. T = 0 was defined as the time point at which NEBD became evident. Scale bar, 10 μm.
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f4: Direct binding of Ska3C-term to Ska1 and microtubules is required for maintaining kinetochore-microtubule attachments and timely anaphase onset.(a,b) Box-and-whisker plot showing the elapsed time (min) between nuclear envelope breakdown (NEBD) and anaphase onset/death for individual cells. The total number of cells (n) from three independent experiments is given above each box. Lower and upper whiskers represent 10th and 90th percentiles, respectively. Table summarizing information from the live cell experiments shown below regarding the average time between NEBD to anaphase onset/death and the percentage of cells dying in mitosis. (c) Representative stills from time-lapse video microscopy experiments illustrating mitotic progression of HeLa S3 cells stably expressing H2B-GFP, treated as in (a,b). Time in hr:min is indicated. T = 0 was defined as the time point at which NEBD became evident. Scale bar, 10 μm.

Mentions: We next evaluated the consequences of disrupting Ska3C-term–microtubule and Ska3C-term-Ska1-MTBD interactions on mitotic progression, using siRNA rescue assays with the Ska3 mutants characterized above. In agreement with the in vitro results, rescues by the Ska3 K199/202A and K394/399A resulted in perturbed mitotic progression, characterized by about a 2 fold delay in anaphase onset and a significant increase in the number of dead cells (Fig. 4a,c). Similarly, the Ska3 D321/323/326K mutant, which is predicted not to interact with Ska1-MTBD, resulted in a 3 fold delay in anaphase onset with around 17% cells undergoing cell death (Fig. 4b,c), highlighting the requirement for Ska3C-term–microtubule and Ska3C-term-Ska1-MTBD interactions. All Ska3 mutants tested were able to form a complex with the other Ska subunits comparable to wt Ska3 (Supplementary Fig. S4; shown for Ska1), indicating that the observed mitotic defects are not due to disruption of complex formation. We also noted that the majority of cells expressing these mutants were able to align their chromosomes in a timely manner (data not shown), as seen for the Ska1 microtubule binding deficient mutants17, suggesting that the Ska3C-term contributes to the robustness of kinetochore-microtubule attachments, rather than to the establishment of the initial kinetochore-microtubule contacts.


Ska3 Ensures Timely Mitotic Progression by Interacting Directly With Microtubules and Ska1 Microtubule Binding Domain
Direct binding of Ska3C-term to Ska1 and microtubules is required for maintaining kinetochore-microtubule attachments and timely anaphase onset.(a,b) Box-and-whisker plot showing the elapsed time (min) between nuclear envelope breakdown (NEBD) and anaphase onset/death for individual cells. The total number of cells (n) from three independent experiments is given above each box. Lower and upper whiskers represent 10th and 90th percentiles, respectively. Table summarizing information from the live cell experiments shown below regarding the average time between NEBD to anaphase onset/death and the percentage of cells dying in mitosis. (c) Representative stills from time-lapse video microscopy experiments illustrating mitotic progression of HeLa S3 cells stably expressing H2B-GFP, treated as in (a,b). Time in hr:min is indicated. T = 0 was defined as the time point at which NEBD became evident. Scale bar, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5036024&req=5

f4: Direct binding of Ska3C-term to Ska1 and microtubules is required for maintaining kinetochore-microtubule attachments and timely anaphase onset.(a,b) Box-and-whisker plot showing the elapsed time (min) between nuclear envelope breakdown (NEBD) and anaphase onset/death for individual cells. The total number of cells (n) from three independent experiments is given above each box. Lower and upper whiskers represent 10th and 90th percentiles, respectively. Table summarizing information from the live cell experiments shown below regarding the average time between NEBD to anaphase onset/death and the percentage of cells dying in mitosis. (c) Representative stills from time-lapse video microscopy experiments illustrating mitotic progression of HeLa S3 cells stably expressing H2B-GFP, treated as in (a,b). Time in hr:min is indicated. T = 0 was defined as the time point at which NEBD became evident. Scale bar, 10 μm.
Mentions: We next evaluated the consequences of disrupting Ska3C-term–microtubule and Ska3C-term-Ska1-MTBD interactions on mitotic progression, using siRNA rescue assays with the Ska3 mutants characterized above. In agreement with the in vitro results, rescues by the Ska3 K199/202A and K394/399A resulted in perturbed mitotic progression, characterized by about a 2 fold delay in anaphase onset and a significant increase in the number of dead cells (Fig. 4a,c). Similarly, the Ska3 D321/323/326K mutant, which is predicted not to interact with Ska1-MTBD, resulted in a 3 fold delay in anaphase onset with around 17% cells undergoing cell death (Fig. 4b,c), highlighting the requirement for Ska3C-term–microtubule and Ska3C-term-Ska1-MTBD interactions. All Ska3 mutants tested were able to form a complex with the other Ska subunits comparable to wt Ska3 (Supplementary Fig. S4; shown for Ska1), indicating that the observed mitotic defects are not due to disruption of complex formation. We also noted that the majority of cells expressing these mutants were able to align their chromosomes in a timely manner (data not shown), as seen for the Ska1 microtubule binding deficient mutants17, suggesting that the Ska3C-term contributes to the robustness of kinetochore-microtubule attachments, rather than to the establishment of the initial kinetochore-microtubule contacts.

View Article: PubMed Central - PubMed

ABSTRACT

The establishment of physical attachment between the kinetochore and dynamic spindle microtubules, which undergo cycles of polymerization and depolymerization generating straight and curved microtubule structures, is essential for accurate chromosome segregation. The Ndc80 and Ska complexes are the major microtubule-binding factors of the kinetochore responsible for maintaining chromosome-microtubule coupling during chromosome segregation. We previously showed that the Ska1 subunit of the Ska complex binds dynamic microtubules using multiple contact sites in a mode that allows conformation-independent binding. Here, we show that the Ska3 subunit is required to modulate the microtubule binding capability of the Ska complex (i) by directly interacting with tubulin monomers and (ii) indirectly by interacting with tubulin contacting regions of Ska1 suggesting an allosteric regulation. Perturbing either the Ska3-microtubule interaction or the Ska3-Ska1 interactions negatively influences microtubule binding by the Ska complex in vitro and affects the timely onset of anaphase in cells. Thus, Ska3 employs additional modulatory elements within the Ska complex to ensure robust kinetochore-microtubule attachments and timely progression of mitosis.

No MeSH data available.


Related in: MedlinePlus