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

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.

Figure 1. The intrinsically disordered C-terminal domain of Ska3 contributes to the microtubule binding activity of the Ska complex.

(a) Domain architecture of the Ska components where filled boxes represent structured regions. MTBD: MicroTubule Binding Domain of Ska1 (residues from 133 to 255). (b) Predicted disordered and protein-binding regions of Ska3 using Disopred (http://bioinf.cs.ucl.ac.uk/psipred). Blue: predicted disordered residues, Orange: predicted protein-binding residues. (c) Amino acid conservation of Ska3 (conservation score is mapped from red to cyan, where red corresponds to highly conserved and cyan to poorly conserved). The alignments include orthologs from H. sapiens (hs), Bos taurus (bt), Sus scrofa (ss), Mus musculus (mm), Echinops telfairi (et), Orcinus orca (oo). Ska3 residues evaluated in this study are marked with asterisks. Multiple sequence alignment was performed with MUSCLE (MUltiple Sequence Comparison by Log-Expectation, EMBL-EBI) and edited with Aline. (d) Quantification of MT-cosedimentation assay comparing the microtubule-binding activity of the wt Ska complex, Ska1ΔC and Ska3ΔC. Concentrations used in the assay: 1 μM protein, 9 μM MTs (mean ± s.d., n = 3, **P ≤0.01, ***P ≤0.001; t-test). (e) Left, Representative SDS-PAGE of MT-cosedimentation assays comparing the microtubule-binding activity of the wt Ska complex, Ska1ΔC and Ska3ΔC. Right, Microtubule-binding curve for the wt Ska complex, Ska1ΔC and Ska3ΔC. K_d values were calculated using 1 μM Ska and 0–15 μM MTs (mean ± s.d., n = 4).

Figure 2. 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-Ska3_1–151 and Ska1-Ska2-Ska3_1–195. K_d 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 10^th and 90^th 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.

Figure 3. 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). K_d 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). K_d values were calculated using 1 μM Ska and 0–15 μM MTs (mean ± s.d., n ≥ 4).

Figure 4. Direct binding of Ska3_C-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 10^th and 90^th 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.