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

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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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The C-terminal domain of Ska3 is required for timely mitotic progression.(a) Quantification of MT-cosedimentation assays comparing the wt Ska complex and Ska1-Ska2-Ska3ΔC with Ska1-Ska2-Ska31–151 and Ska1-Ska2-Ska31–195. Kd values were calculated using 1 μM Ska and 0–12 μM MTs (mean ± s.d., n = 4). (b) Box-and-whisker plot showing the elapsed time (min) between nuclear envelope breakdown (NEBD) and anaphase onset/death for individual cells. The number of cells (n) from three independent time-lapse experiments is given above each box. Lower and upper whiskers represent 10th and 90th percentiles, respectively. Below, table summarizing information from the live cell experiments regarding the average time between NEBD to anaphase onset/death and the percentage of cells dying in mitosis. Myc-V and mCherry-V were used as transfection controls. (c) Representative stills from time-lapse video-microscopy experiments illustrating mitotic progression of HeLa S3 cells stably expressing histone H2B-GFP treated as in (b). Time in hr:min is indicated. T = 0 was defined as the time point where NEBD became evident. Scale bar, 10 μm.
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f2: The C-terminal domain of Ska3 is required for timely mitotic progression.(a) Quantification of MT-cosedimentation assays comparing the wt Ska complex and Ska1-Ska2-Ska3ΔC with Ska1-Ska2-Ska31–151 and Ska1-Ska2-Ska31–195. Kd values were calculated using 1 μM Ska and 0–12 μM MTs (mean ± s.d., n = 4). (b) Box-and-whisker plot showing the elapsed time (min) between nuclear envelope breakdown (NEBD) and anaphase onset/death for individual cells. The number of cells (n) from three independent time-lapse experiments is given above each box. Lower and upper whiskers represent 10th and 90th percentiles, respectively. Below, table summarizing information from the live cell experiments regarding the average time between NEBD to anaphase onset/death and the percentage of cells dying in mitosis. Myc-V and mCherry-V were used as transfection controls. (c) Representative stills from time-lapse video-microscopy experiments illustrating mitotic progression of HeLa S3 cells stably expressing histone H2B-GFP treated as in (b). Time in hr:min is indicated. T = 0 was defined as the time point where NEBD became evident. Scale bar, 10 μm.

Mentions: To narrow down the region of Ska3 that influences the microtubule binding properties of the Ska complex, we reconstituted Ska complexes containing two different C-terminal truncations of Ska3 that were designed based on the presence of conserved amino acid stretches (Ska1-Ska2-Ska31–151 ; Ska1-Ska2-Ska31–195). As expected, SEC-MALS analyses confirmed that the truncation of Ska3 C-terminal regions does not affect the overall oligomeric structure of these complexes (Supplementary Fig. S2a). Microtubule cosedimentation assays showed that these complexes bound microtubules with affinities similar to that of Ska1-Ska2-Ska3ΔC (Fig. 2a, Supplementary Fig. S2b; Kd = 13.9 μM and 14.3 μM, respectively), indicating that the Ska3 region between residues 196 and the C-terminal end is critical in modulating the microtubule binding of the Ska complex.


Ska3 Ensures Timely Mitotic Progression by Interacting Directly With Microtubules and Ska1 Microtubule Binding Domain
The C-terminal domain of Ska3 is required for timely mitotic progression.(a) Quantification of MT-cosedimentation assays comparing the wt Ska complex and Ska1-Ska2-Ska3ΔC with Ska1-Ska2-Ska31–151 and Ska1-Ska2-Ska31–195. Kd values were calculated using 1 μM Ska and 0–12 μM MTs (mean ± s.d., n = 4). (b) Box-and-whisker plot showing the elapsed time (min) between nuclear envelope breakdown (NEBD) and anaphase onset/death for individual cells. The number of cells (n) from three independent time-lapse experiments is given above each box. Lower and upper whiskers represent 10th and 90th percentiles, respectively. Below, table summarizing information from the live cell experiments regarding the average time between NEBD to anaphase onset/death and the percentage of cells dying in mitosis. Myc-V and mCherry-V were used as transfection controls. (c) Representative stills from time-lapse video-microscopy experiments illustrating mitotic progression of HeLa S3 cells stably expressing histone H2B-GFP treated as in (b). Time in hr:min is indicated. T = 0 was defined as the time point where NEBD became evident. Scale bar, 10 μm.
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f2: The C-terminal domain of Ska3 is required for timely mitotic progression.(a) Quantification of MT-cosedimentation assays comparing the wt Ska complex and Ska1-Ska2-Ska3ΔC with Ska1-Ska2-Ska31–151 and Ska1-Ska2-Ska31–195. Kd values were calculated using 1 μM Ska and 0–12 μM MTs (mean ± s.d., n = 4). (b) Box-and-whisker plot showing the elapsed time (min) between nuclear envelope breakdown (NEBD) and anaphase onset/death for individual cells. The number of cells (n) from three independent time-lapse experiments is given above each box. Lower and upper whiskers represent 10th and 90th percentiles, respectively. Below, table summarizing information from the live cell experiments regarding the average time between NEBD to anaphase onset/death and the percentage of cells dying in mitosis. Myc-V and mCherry-V were used as transfection controls. (c) Representative stills from time-lapse video-microscopy experiments illustrating mitotic progression of HeLa S3 cells stably expressing histone H2B-GFP treated as in (b). Time in hr:min is indicated. T = 0 was defined as the time point where NEBD became evident. Scale bar, 10 μm.
Mentions: To narrow down the region of Ska3 that influences the microtubule binding properties of the Ska complex, we reconstituted Ska complexes containing two different C-terminal truncations of Ska3 that were designed based on the presence of conserved amino acid stretches (Ska1-Ska2-Ska31–151 ; Ska1-Ska2-Ska31–195). As expected, SEC-MALS analyses confirmed that the truncation of Ska3 C-terminal regions does not affect the overall oligomeric structure of these complexes (Supplementary Fig. S2a). Microtubule cosedimentation assays showed that these complexes bound microtubules with affinities similar to that of Ska1-Ska2-Ska3ΔC (Fig. 2a, Supplementary Fig. S2b; Kd = 13.9 μM and 14.3 μM, respectively), indicating that the Ska3 region between residues 196 and the C-terminal end is critical in modulating the microtubule binding of the Ska complex.

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