Figure 4. Abnormal chromosome segregation in PPP6C-depleted cells. (A and B) HeLa (A) and MRC-5 (B) cells were transfected with control and PPP6C siRNA duplexes for 24–72 h. The cells were fixed and then stained with DAPI, tubulin (green), and CREST antibodies. Bars, 10 µm. (C) The same samples were also Western blotted to test for PPP6C depletion. Molecular mass is given in kilodaltons. (D and E) Control and PPP6C-depleted HeLa (D) and MRC-5 (E) cells in anaphase and telophase are shown. (F) HeLa cells were transfected with all three SAPS domain siRNA duplexes for 72 h. The cells were fixed and then stained with DAPI, tubulin (green), and CREST antibodies. Cells in anaphase and telophase are shown. (D–F) Bars, 5 µm. In all panels, arrow- heads mark lagging chromosomes, and these regions are shown in the inset enlargements. were seen in cells depleted of all three SAPS domain proteins Cold-stable kinetochore fiber microtubule bundles, although -dis (Fig. 4 F). This supports the idea that the PP6 holoenzyme is re- ordered, were also still present in PPP6C-depleted cells but lost quired for normal mitotic progression. Live-cell imaging of in Nuf2-depleted cells (Fig. S3 B), in which the kinetochore cells stably expressing EGFP-tagged histone H2BF(ig. 5 A) or attachment site is disrupted (DeLuca et al., 2002). In contrast, mCherry-tagged histone H2B and EGFP-tagged -tubulin the centrosomes defined by pericentrin often failed to localize (Fig. 5 B) revealed that PPP6C-depleted cells did not form a -bi at the spindle poles in PPP6C-depleted cells (Fig. S3, B and C). polar spindle with normal kinetics and failed to efficiently align The defects in efficient chromosome capture and alignment seen their chromosomes at the metaphase plate.These cells also showed in the absence of PP6 function do not appear to be caused by a prolonged arrest in mitosis (Fig. 5, A–C), during which time the unstable kinetochore fibers or gross kinetochore defects and chromosomes continuously tumbled about before eventually could therefore be caused by misregulation of the centrosome- aligning to form a loose metaphase plate (Fig. 5, A and B; mediated spindle formation pathway. Video 1 and Video 2 show control and PPP6C-depleted cells, respectively). After an extended period, PPP6C-depleted cells PP6 is an Aurora A T-loop phosphatase formed bipolar spindles (Fig. 5 B). However, these were disordered To pursue the idea that PP6 is a mitotic regulator important for compared with control cells. Biochemical analysis of thymidine- spindle formation, stage 2 of the screen was focused on the key synchronized cells depleted of PPP6C passing through mitosis kinase regulators of spindle formation from the centrosome revealed elevated levels of pT288-phosphorylated Aurora A and Aurora A and Plk1 (Llamazares et al., 1991; Glover et al., 1995; delayed passage through mitosis, as judged by the timing of Lane and Nigg, 1996; Roghi et al., 1998). If the T-loop pho-s cyclin B degradation (Fig. 5 D). These observations hinted at a phatase acting on either kinase is absent, the phosphorylated defect in the pathway of spindle formation or chromosome cap- form will be stabilized under conditions in which dephosphor-y ture. Examination of the PPP6C-depleted cells revealed no lation is normally favored over phosphorylation (Ahonen et al., obvious changes in kinetochore or checkpoint proteins (Fig. S3 A). 2005; Daum and Gorbsky, 2006). This can be simply achieved 1320 JCB • VOLUME 191 • NUMBER 7 • 2010