Timely anaphase onset requires a novel spindle and kinetochore complex comprising Ska1 and Ska2

Abstract

Chromosome segregation during mitosis requires chromosomes to undergo bipolar attachment on spindle microtubules (MTs) and subsequent silencing of the spindle checkpoint. Here, we describe the identification and characterisation of a novel spindle and kinetochore (KT)-associated complex that is required for timely anaphase onset. The complex comprises at least two proteins, termed Ska1 (Spindle and KT Associated 1) and Ska2. Ska1 associates with KTs following MT attachment during prometaphase. Ska1 and Ska2 interact with each other and Ska1 is required for Ska2 stability in vivo. Depletion of either Ska1 or Ska2 by small interfering RNA results in the loss of both proteins from the KT. The absence of Ska proteins does not disrupt overall KT structure, but KT fibres show an increased cold-sensitivity. Most strikingly, Ska-depleted cells undergo a prolonged checkpoint-dependent delay in a metaphase-like state. This delay is characterised by the recruitment of Mad2 protein to a few KTs and the occasional loss of individual chromosomes from the metaphase plate. These data suggest that the Ska1/2 complex plays a critical role in the maintenance of the metaphase plate and/or spindle checkpoint silencing.

Figure 1

Mitotic spindle and outer KT localisation of Ska1. (A) HeLa S3 cells were transfected with a myc-tagged Ska1 construct and then fixed with PTEMF. Cells were stained with anti-myc 9E10 antibody (red), DAPI (DNA, blue) and with either anti-α-tubulin antibody (green) (upper panel) or CREST serum (green) (bottom panel). (B) HeLa S3 cells were fixed with PTEMF and then stained with anti-Ska1 antibody (red), anti-α-tubulin antibody (green) and DAPI (DNA, blue). (C) Same as in (B) except that cells were stained with anti-Ska1 antibody, anti-CENP-E antibody, CREST serum and DAPI followed by imaging with a Deltavision microscope. Merged pictures are single, deconvolved focal planes and show CREST (blue), CENP-E (green) and Ska1 (red). Right panels in the bottom row are magnifications of the above marked areas with scale bar indicating 1 μm. (D) An interphase cell is shown which was fixed with paraformaldehyde, permeabilised with Triton X–100 and stained as in (B). Scale bars=10 μm.

Figure 2

Ska1 KT localisation requires KT–MT attachment at physiological temperatures. (A) HeLa S3 cells were either left untreated as a control or treated for 14 h with nocodazole and taxol, respectively. After PTEMF fixation, cells were stained with anti-Ska1 antibody (red), CREST serum (green) and DAPI (DNA, blue). (B) HeLa S3 cells were fixed with PTEMF either directly (control) or after incubation in ice-cold medium for another 2 h. Cells were stained with anti-Ska1 antibody (red), CREST serum (green) and DAPI (DNA, blue). (C) HeLa S3 cells were treated with nocodazole for 14 h and then either fixed directly or incubated in ice-cold medium for another 2 h in the presence of nocodazole. Cells were fixed and stained as in (B). (D) HeLa S3 cells were treated as in (C) but stained with anti-Mad2 antibody (red), CREST serum (green) and DAPI (DNA, blue). Scale bars=10 μm.

Figure 3

Ska1 KT localisation requires the presence of Hec1 even in the cold. (A) HeLa S3 cells were treated for 48 h with control (GL2) and Hec1-specific siRNAs, respectively, then fixed with PTEMF and stained with anti-Ska1 (red), anti-Hec1 (green) and DAPI (DNA, blue). (B) HeLa S3 cells were treated for 48 h with control (GL2) and Hec1-specific siRNAs, respectively, before half of the samples were incubated for 2 h in ice-cold medium. Cells were fixed as in (A) but stained with anti-Ska1 antibody (red), CREST serum (green) and DAPI (DNA, blue). Scale bars=10 μm.

Figure 4

Ska1 interacts with another novel protein termed Ska2 (FAM33A). (A) Yeast two-hybrid interaction between Ska1 expressed as a binding domain (BD) fusion and Ska2 expressed as an activation domain (AD) fusion (top panel). In the bottom panel, Ska1 is expressed from the AD vector and Ska2 from the BD vector. As negative controls, the empty AD or BD (−) vectors were used. Interactions were reflected by growth on selective medium (−WLAdH, right panels). For control, growth on nonselective plates is also shown (−WL, left panels). (B) Myc- and FLAG-tagged versions of Ska1, Ska2 and Plk1 (negative control) were produced in different combinations by IVT in the presence of ³⁵S-labelled methionine. Myc-tagged Ska1 (left panels) or Ska2 (right panels) were subsequently immunoprecipitated and IVT input and myc-immunoprecipitates were analysed by SDS–PAGE followed by autoradiography. (C) Myc-tagged Ska2 was transiently expressed in HeLa S3 cells for 48 h. After PTEMF fixation cells were stained with anti-myc 9E10 antibody (red), DAPI (DNA, blue) and with either anti α-tubulin antibody (upper panel), CREST serum (middle panel) or anti-Ska1 antibody (bottom panel) (all in green). (D) As in (C) after staining with anti-myc 9E10 antibody (red), anti-Ska1 antibody (green) and DAPI (DNA, blue), cells were imaged with a Deltavision microscope. Pictures are single, deconvolved focal planes. Right panel is a magnification of the marked area in the merged picture with scale bar indicating 1 μm. (E) Lysates were prepared from mitotic HeLa S3 cells. They were then used for immunoprecipitations with anti-Ska1 antibody, anti-Ska2 antibody and rabbit IgGs (negative control), respectively. Lysate and immune complexes were separated by SDS–PAGE and probed by Western blotting with anti-Ska1 and anti-Ska2 antibodies, as indicated. (F) HeLa S3 cells were treated for 48 h with control (GL2) and Ska1 and Ska2-specific siRNAs, respectively. Equal amounts of cell extracts were separated by SDS–PAGE and probed by Western blotting with anti-Ska1 and anti-Ska2 antibodies. Detection of α-tubulin was used as a loading control. (G) HeLa S3 cells were treated for 48 h with control (GL2), Ska1- and Ska2-specific siRNAs, respectively, then fixed with PTEMF and stained with anti-Ska1 antibody (red), CREST serum (green) and DAPI (DNA, blue). Scale bars=10 μm.

Figure 5

Depletion of Ska1 and Ska2 results in a metaphase delay. (A) HeLa S3 cells were treated for 48 h with control (GL2), or Ska1- and Ska2-specific siRNAs, respectively. The mitotic indexes were determined by light microscopy. (B) The same cells as in (A) were fixed and stained with DAPI (DNA). Scale bar=20 μm. (C) Live-cell imaging of H2B-GFP expressing HeLa S3 cells. Selected images show H2B-GFP stained chromosomes of Hela S3 cells progressing through mitosis. Cells were treated with GL2 (control), or Ska1- and Ska2-specific siRNAs, respectively, for 30 h before filming. T=0 was defined as the time point at which chromosome condensation became evident (prophase). Time points are indicated in minutes. Arrows point to chromosomes that have transiently moved out of the metaphase plate. Scale bar=10 μm. (D) The durations of different periods from prophase to anaphase onset were calculated from time-lapse movies (C) of at least 37 control (GL2), and at least 76 Ska1 and Ska2 siRNA-treated cells. T=0 was defined as in (C). Complete alignment was defined as the duration from T=0 to the first time point at which a perfect metaphase plate was observed and anaphase onset was calculated from the first frame at which chromosome segregation was visible. Histograms show the percentages of mitotic cells that had progressed within the indicated time frames from prophase to the first complete metaphase plate (left panel) and from first metaphase plate to anaphase onset (right panel).

Figure 6

Characterisation of Ska-depletion phenotype. (A) HeLa S3 cells were treated for 48 h with control (GL2), Ska1-, Ska2- and Nuf2-specific siRNAs, respectively. They were fixed directly (left column, 37°C) or after incubation for 10 min at 4°C (right column, 4°C) before staining with anti-α-tubulin antibody (green), CREST serum (red) and DAPI (DNA, blue). (B) HeLa S3 cells were treated for 48 h with control (GL2), or Ska1- and Ska2-specific siRNAs, respectively, then fixed with PTEMF and stained with anti-Mad2 antibody (red), CREST serum (green) and DAPI (DNA, blue). Scale bars=10 μm. (C) Quantification of the mitotic indices (300 cells each) after 48 h of Ska1 and Ska2 siRNA together with control GL2 siRNA or after simultaneous depletion of Mad2.