1. Volume 46, Issue 3, Pages 274-286 (May 2012)
Structural and Functional Organization of the Ska Complex, a Key Component of the Kinetochore-Microtubule Interface 
A. Arockia Jeyaprakash, Anna Santamaria, Uma Jayachandran, Ying Wai Chan, Christian Benda, Erich A. Nigg, Elena Conti 
Molecular Cell 
Volume 46, Issue 3, Pages (May 2012)
DOI: /j.molcel
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Figure 1
The Ska Complex Is a Dimer with an Elongated Structure and Its Structural Core Is Formed by Ska11–91, Ska2, and Ska31–101
(A) A schematic representation of structural features of the Ska components (filled boxes represent regions that are predicted to be structured).
(B) Biophysical characterization of the Ska1-Ska2, Ska1-Ska2-Ska3, and Ska1ΔC-Ska2-Ska3ΔC complexes. Ska1-Ska2 is a monomer while Ska1-Ska2-Ska3 and Ska1ΔC-Ska2-Ska3ΔC are dimers. The Ska1-Ska2-Ska3 complex is elongated in shape with a maximum interatomic distance of about 350 Å.
(C) Overall structure of the monomeric Ska core complex (Ska1, green; Ska2, blue; Ska3, orange). This figure and other cartoon representations of the structure are prepared using Pymol (
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

3. Figure 2
Conserved Interactions Maintain the Coiled-Coil Structure of the Ska Complex with Ska1 Being a Structural Scaffold Bridging the Ska2-Ska3 Interaction
(A) The relative orientation of the N- and C-terminal helical bundles is determined by intramolecular interaction within Ska3.
(B–D) Close-up views of the intermolecular interactions stabilizing the coiled-coil structure of the Ska complex. Extensive hydrophobic and electrostatic contacts are present throughout the helical bundles.
(E) Amino acid conservation and interactions in the Ska core complex. The secondary structure elements are shown below the aligned sequences. The alignments include orthologs from H. sapiens (Hs), M. musculus (Mm), X. tropicalis (Xt), and D. rerio (Dr). Amino acid conservation is highlighted in gray. Colored circles indicate residue involved in interactions with Ska1 (green), Ska2 (blue), and Ska3 (orange).
(F) Ska1 provides the structural scaffold for Ska2-Ska3 interaction. Disrupting the interaction of Ska1-Ska2 results in the loss of Ska complex formation. Myc-tagged empty vector, Ska2, or Ska2I30R/L60E/V67E/I53E were expressed in HEK293T cells, and cells were harvested after 36 hr. Myc-Ska2 was then immunoprecipitated using anti-Myc antibodies and resolved by SDS-PAGE. Coimmunoprecipitated Ska1 and 3 were detected by western blotting.
(G) Table summarizing information from the live cell experiments with the mutant discussed in panel (F).
(H) Disrupting the central coiled-coil interaction results in prolonged delay in the onset of anaphase. Box-and-whisker plot showing the elapsed time (min) between NEBD and anaphase onset/death for individual cells. The total number of cells (n) from one experiment is given above each box. Lower and upper whiskers represent 10th and 90th percentiles, respectively.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

4. Figure 3
The Coiled-Coil Core of the Ska Complex Forms a W-Shaped Dimer
(A) Cartoon representation of the structure of the Ska core complex as seen in the asymmetric unit of the crystal. Ten copies of the Ska complex interact to form an oligomeric assembly of molecular weight 364 kDa. Two different dimeric arrangements are seen in the oligomeric assembly, dimer 1 (W-shape, highlighted in color) and dimer 2 (stretched W-shape, described in Figure S3). See text for details.
(B) W-shaped dimer with a close-up view of the dimeric interface. N-terminal helical bundle intercalates with its dimeric counterpart to form a W-shaped dimer.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

5. Figure 4
The C Terminus of Both Ska1 and Ska3 Are Required for Normal Mitotic Progression
(A) Schematic representation of Ska rescue protocol used to assess mitotic progression and timing. Cells were depleted of Ska members for 72 hr whereas plasmids were only transfected for 30 hr after a first thymidine arrest/release protocol. Six hours after transfection, cells were arrested with thymidine a second time and released (i) for 6 hr before starting filming or (ii) for 12 hr before fixation, in the presence of MG132 for the last 2 hr to collect a higher number of mitotic cells.
(B) Representative stills from time-lapse video-microscopy experiments illustrating mitotic progression of HeLa S3 cells stably expressing histone H2B-GFP treated as in (A). Time in hr:min is indicated. T = 0 was defined as the time point at which NEBD became evident. The schematic representation of the complexes are shown on the left, with Ska1 C terminus as a globular domain and Ska3 C terminus with an elongated shape to reflect secondary structure predictions of these regions.
(C) Box-and-whisker plot showing the elapsed time (min) between NEBD and anaphase onset/death for individual cells treated as described in the schematic below. The total number of cells (n) from five independent experiments is given above each box. Lower and upper whiskers represent 10th and 90th percentiles, respectively.
(D) Table summarizing information from the live cell experiments shown in (C) regarding the percentage of cells showing aligned/misaligned chromosomes and the percentage of cells dying with aligned/misaligned chromosomes for each condition (third and fourth column, respectively).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

6. Figure 5 Microtubule-Binding Determinants of the Ska Complex
(A) Negative stain electron micrographs show that the Ska1-Ska2-Ska3 complex can decorate (continuous fine meshwork-like pattern) microtubules, while removal of C-terminal domain of either Ska1 (Ska1ΔC-Ska2-Ska3) or Ska3 (Ska1-Ska2-Ska3ΔC) abrogates the decoration. Scale bar = 50 nm.
(B) Fluorescent light microscopy images of 4 μM rhodamine-labeled microtubules incubated with 6 μM Ska1-Ska2-Ska3, Ska1ΔC-Ska2-Ska3, Ska1-Ska2-Ska3ΔC, and Ska1ΔC-Ska2-Ska3Δ. The Ska1-Ska2-Ska3 complex shows robust microtubule bundling activity. While Ska1ΔC-Ska2-Ska3 and Ska1ΔC-Ska2-Ska3ΔC completely abolished the activity, Ska1-Ska2-Ska3ΔC shows reduced activity.
(C) Microtubule cosedimentation assays with Ska1-Ska2-Ska3 (lanes 1–4), Ska1ΔC-Ska2-Ska3ΔC (lanes 5–8), Ska1ΔC-Ska2-Ska3 (lanes 8–11), and Ska1-Ska2-Ska3ΔC (lanes 12–15). While deletion of the C-terminal domain of Ska1 results in the complete loss of MT-binding, deletion of the C-terminal domain of Ska3 significantly reduced MT-binding.
(D) Microtubule cosedimentation assays with Ska1ΔN (lanes 1–4), Ska3ΔN (lanes5–8), and equimolar mix of Ska1ΔN and Ska3ΔN (lane 9–12). C-terminal domains of Ska1 and Ska3 bind microtubules neither alone nor together.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

7. Figure 6
Dimerization Interface of the Ska Complex Is Essential for Its Function
(A) Representative stills from time-lapse video-microscopy experiments illustrating mitotic progression of HeLa S3 cells stably expressing histone H2B-GFP transfected with the indicated siRNA and Myc/mCherry-tagged siRNA-resistant constructs following the protocol as for Figure 4A. Time in hr:min is indicated. T = 0 was defined as the time point at which NEBD became evident.
(B) Box-and-whisker plot showing the elapsed time (min) between NEBD and anaphase onset/death. 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.
(C) Table summarizing information from the live cell experiments shown in (B) regarding the percentage of cells showing aligned/misaligned chromosomes and the percentage of cells dying with aligned/misaligned chromosomes for each condition.
(D) Schematic models recapitulating the proposed functions of the Ska and DAM1/DASH complexes in the stabilization of stable kinetochore-microtubule attachments in humans (left) and in S.cerevisiae (right), respectively. The structures of the Ndc80, DAM1/DASH, and Ska complexes are represented based on the structural information in Ciferri et al., 2008; Wang et al., 2007, and in this manuscript.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions