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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.

No MeSH data available.


Ska3 directly interacts with tubulin monomers and Ska1-MTBD.(a) Representative SDS-PAGE of 6 μM Ska complex cross-linked to 10 μM microtubules with EDC. The band analyzed in this study is marked with an asterisk. (b) Cartoon representation of tubulin dimer (pink: β-tubulin, blue: α-tubulin) where residues involved in cross-linking with Ska3 (orange) and Ska1-MTBD (green) are shown in stick representation. Green, blue and purple lines indicate cross-links observed between Ska3 K199/K202 and β-tubulin, Ska3 S394/K399 and α- and β- tubulin, and Ska3 D321/E323/D326 and Ska1, respectively. (c) Microtubule-binding curves of the wt Ska complex (grey) and Ska3 microtubule-binding mutants (K199/202A and S394/K399A; green and blue respectively). Kd values were calculated using 1 μM Ska and 0–15 μM MTs (mean ± s.d., n ≥ 4). (d) Microtubule-binding curve of the wt Ska complex (grey) and Ska1-binding inefficient Ska3 mutant (D321/E323/D326K, purple). Kd values were calculated using 1 μM Ska and 0–15 μM MTs (mean ± s.d., n ≥ 4).
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f3: Ska3 directly interacts with tubulin monomers and Ska1-MTBD.(a) Representative SDS-PAGE of 6 μM Ska complex cross-linked to 10 μM microtubules with EDC. The band analyzed in this study is marked with an asterisk. (b) Cartoon representation of tubulin dimer (pink: β-tubulin, blue: α-tubulin) where residues involved in cross-linking with Ska3 (orange) and Ska1-MTBD (green) are shown in stick representation. Green, blue and purple lines indicate cross-links observed between Ska3 K199/K202 and β-tubulin, Ska3 S394/K399 and α- and β- tubulin, and Ska3 D321/E323/D326 and Ska1, respectively. (c) Microtubule-binding curves of the wt Ska complex (grey) and Ska3 microtubule-binding mutants (K199/202A and S394/K399A; green and blue respectively). Kd values were calculated using 1 μM Ska and 0–15 μM MTs (mean ± s.d., n ≥ 4). (d) Microtubule-binding curve of the wt Ska complex (grey) and Ska1-binding inefficient Ska3 mutant (D321/E323/D326K, purple). Kd values were calculated using 1 μM Ska and 0–15 μM MTs (mean ± s.d., n ≥ 4).

Mentions: To better understand the molecular basis for the contribution of Ska3C-term microtubule binding, we re-examined the CLMS experiments of our previous study17, which used a zero-length cross-linker EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) and dissected the Ska1-microtubule binding interface through the identification of specific intermolecular crosslinks involving Lys (and less favorably Ser, Thr and Tyr) and Asp/Glu. We have now analysed the higher molecular weight cross-linked products containing all the subunits of the Ska complex and microtubules. This revealed cross-links involving the Ska3C-term residues: (i) Lys199/Lys202/Ser394/Lys399/Lys410 and β tubulin; (ii) Lys399 and α tubulin and (iii) Asp321/Glu323/Asp326 and Ska1-MTBD residue Lys183, part of a Lys cluster (Lys183/Lys184/Lys203/Lys206) previously identified as important for microtubule binding17 (Fig. 3a,b, Supplementary Fig. S3a). We note that the Ska3 residues identified by CLMS are either conserved or flanked by highly conserved regions (Fig. 1c). Interestingly, Ska3C-term contacts tubulin monomers at the same sites where we had previously shown Ska1-MTBD to engage with microtubules (H3 and H4 of β-tubulin and H12 of α-tubulin) (Fig. 3b). These observations suggested that Ska3C-term contributes to the Ska complex microtubule binding by directly contacting both the tubulin monomers and Ska1-MTBD. To validate this, we mutated the Ska3C-term residues contacting microtubules (Ska3 K199/202A and Ska3 S394/K399A) and Ska1-MTBD (Ska3 D321/E323/D326K) and tested them separately in microtubule cosedimentation assays. Importantly, all Ska complexes containing the above mentioned Ska3 mutants bound microtubules less efficiently as compared to the wt Ska complex (Fig. 3c,d, Supplementary Fig. S3b). The size exclusion chromatography profiles of the Ska3 mutant Ska complexes were almost identical (Supplementary Fig. S3c), suggesting that the mutations do not perturb the overall architecture of the Ska complex. Thus, we conclude that the effects of these mutations on microtubule binding activity are due to the perturbation of Ska3C-term-microtubule and Ska3C-term-Ska1-MTBD interactions.


Ska3 Ensures Timely Mitotic Progression by Interacting Directly With Microtubules and Ska1 Microtubule Binding Domain
Ska3 directly interacts with tubulin monomers and Ska1-MTBD.(a) Representative SDS-PAGE of 6 μM Ska complex cross-linked to 10 μM microtubules with EDC. The band analyzed in this study is marked with an asterisk. (b) Cartoon representation of tubulin dimer (pink: β-tubulin, blue: α-tubulin) where residues involved in cross-linking with Ska3 (orange) and Ska1-MTBD (green) are shown in stick representation. Green, blue and purple lines indicate cross-links observed between Ska3 K199/K202 and β-tubulin, Ska3 S394/K399 and α- and β- tubulin, and Ska3 D321/E323/D326 and Ska1, respectively. (c) Microtubule-binding curves of the wt Ska complex (grey) and Ska3 microtubule-binding mutants (K199/202A and S394/K399A; green and blue respectively). Kd values were calculated using 1 μM Ska and 0–15 μM MTs (mean ± s.d., n ≥ 4). (d) Microtubule-binding curve of the wt Ska complex (grey) and Ska1-binding inefficient Ska3 mutant (D321/E323/D326K, purple). Kd values were calculated using 1 μM Ska and 0–15 μM MTs (mean ± s.d., n ≥ 4).
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Related In: Results  -  Collection

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f3: Ska3 directly interacts with tubulin monomers and Ska1-MTBD.(a) Representative SDS-PAGE of 6 μM Ska complex cross-linked to 10 μM microtubules with EDC. The band analyzed in this study is marked with an asterisk. (b) Cartoon representation of tubulin dimer (pink: β-tubulin, blue: α-tubulin) where residues involved in cross-linking with Ska3 (orange) and Ska1-MTBD (green) are shown in stick representation. Green, blue and purple lines indicate cross-links observed between Ska3 K199/K202 and β-tubulin, Ska3 S394/K399 and α- and β- tubulin, and Ska3 D321/E323/D326 and Ska1, respectively. (c) Microtubule-binding curves of the wt Ska complex (grey) and Ska3 microtubule-binding mutants (K199/202A and S394/K399A; green and blue respectively). Kd values were calculated using 1 μM Ska and 0–15 μM MTs (mean ± s.d., n ≥ 4). (d) Microtubule-binding curve of the wt Ska complex (grey) and Ska1-binding inefficient Ska3 mutant (D321/E323/D326K, purple). Kd values were calculated using 1 μM Ska and 0–15 μM MTs (mean ± s.d., n ≥ 4).
Mentions: To better understand the molecular basis for the contribution of Ska3C-term microtubule binding, we re-examined the CLMS experiments of our previous study17, which used a zero-length cross-linker EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) and dissected the Ska1-microtubule binding interface through the identification of specific intermolecular crosslinks involving Lys (and less favorably Ser, Thr and Tyr) and Asp/Glu. We have now analysed the higher molecular weight cross-linked products containing all the subunits of the Ska complex and microtubules. This revealed cross-links involving the Ska3C-term residues: (i) Lys199/Lys202/Ser394/Lys399/Lys410 and β tubulin; (ii) Lys399 and α tubulin and (iii) Asp321/Glu323/Asp326 and Ska1-MTBD residue Lys183, part of a Lys cluster (Lys183/Lys184/Lys203/Lys206) previously identified as important for microtubule binding17 (Fig. 3a,b, Supplementary Fig. S3a). We note that the Ska3 residues identified by CLMS are either conserved or flanked by highly conserved regions (Fig. 1c). Interestingly, Ska3C-term contacts tubulin monomers at the same sites where we had previously shown Ska1-MTBD to engage with microtubules (H3 and H4 of β-tubulin and H12 of α-tubulin) (Fig. 3b). These observations suggested that Ska3C-term contributes to the Ska complex microtubule binding by directly contacting both the tubulin monomers and Ska1-MTBD. To validate this, we mutated the Ska3C-term residues contacting microtubules (Ska3 K199/202A and Ska3 S394/K399A) and Ska1-MTBD (Ska3 D321/E323/D326K) and tested them separately in microtubule cosedimentation assays. Importantly, all Ska complexes containing the above mentioned Ska3 mutants bound microtubules less efficiently as compared to the wt Ska complex (Fig. 3c,d, Supplementary Fig. S3b). The size exclusion chromatography profiles of the Ska3 mutant Ska complexes were almost identical (Supplementary Fig. S3c), suggesting that the mutations do not perturb the overall architecture of the Ska complex. Thus, we conclude that the effects of these mutations on microtubule binding activity are due to the perturbation of Ska3C-term-microtubule and Ska3C-term-Ska1-MTBD interactions.

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.