activated Aurora A at the mitotic spindle. Consistent with the In contrast, cells depleted of PPP6C failed to recruit Eg5 and biochemical changes, PPP6C-depleted cells showed altered NuMa to spindle poles in prophase (Fig. 9 A, siPPP6C). Promet-a staining at the spindle for activated Aurora A pT288 and the phase spindle poles were also highly fragmented and disordered Aurora A activator TPX2 (Fig. S4, siPPP6C, arrowheads). (Fig. 9 A, siPPP6C), which was consistent with the results In control cells, activated Aurora A pT288 and TPX2 staining obtained by live-cell imaging, which showed delayed spindle was most predominant at the spindle poles with a faint staining formation and defective spindle integrity (Fig. 5 B). To allow a spread along spindle microtubules F(ig. 8 A and Fig. S4, better measurement of this effect, cells were arrested in pro- siControl). In PPP6C-depleted cells, the spindle microtubule metaphase using the Eg5 inhibitor S-trityl-l-cysteine, and bipolar staining of both activated Aurora A and TPX2 was increased spindle formation followed after washout of the drug. In this (Fig. 8 A and Fig. S4, siPPP6C), and this could be reversed by a assay, control cells rapidly formed a bipolar spindle within 15 min combined PPP6C and TPX2 depletion (Fig. S4, siPPP6C + of drug washout, aligned their chromosomes, and entered ana- TPX2). It was not possible to completely deplete TPX2, and a phase by 60 min (Fig. 10, A and B). In contrast, PPP6C-depleted small amount always remained at centrosomes, where it ove-r cells had a pronounced delay in both bipolar spindle formation lapped with a population of activated Aurora A that was u n- and chromosome alignment (Fig. 10, A and B). This suggested altered by PPP6C depletion. that the spindle-associated activity of Eg5 and other spindle Highly specific small molecule inhibitors for Aurora fa-m assembly factors may be limiting in PPP6C-depleted cells. ily kinases have recently been developed (Manfredi et al., 2007; In accordance with this idea, the spindle-associated level of Karthigeyan et al., 2010), and this suggested a rescue strategy both Eg5 and NuMa was reduced compared with control cells for the PPP6C depletion phenotype. Low doses of Aurora A in- (Fig. 9 A and measured in Fig. S5). If this reduction was hibitors were therefore tested to see whether they reversed the caused by unchecked Aurora A activity, Aurora A inhibition increased Aurora A activation and T-loop phosphorylation seen should reverse this effect. When PPP6C-depleted cells were upon PPP6C depletion. Limiting titration of the Aurora A in- treated with MLN8237 for 10 min to inhibit Aurora A, Eg5 local- hibitor MLN8237 did not block cell growth but reversed the ization to the spindle recovered to the levels seen in control effect of PPP6C depletion on Aurora A T-loop phosphorylation, cells, and focused NuMa staining around the spindle poles was with 10 nM MLN8237 resulting in the same ratio of activated to reinstated (Fig. 9 B, compare siPPP6C with siPPP6C + 10 µM inactivated Aurora A seen in control cells in the absence of the MLN8237 conditions). Collectively, these findings support a drug (Fig. 8 A, gel image on bottom). Depletion of PPP6C re- model in which PP6 regulates spindle formation by limiting the sulted in a twofold increase in the pT288 form of Aurora A at activity of the Aurora A–TPX2 complex toward key spindle a-s prometaphase and metaphase spindles compared with control sembly factors, including Eg5 and NuMa. cells, and addition of 10 nM MLN8237 reversed this increase (Fig. 8 A). Importantly, this partial Aurora A inhibition with Discussion 10 nM MLN8237 also reduced the micronucleation seen in PPP6C-depleted cells from 40 to 5% (Fig. 8 B). Reduction of PP6 is the Aurora A T-loop phosphatase Aurora A activity using TPX2 depletion also rescued the PPP6C The findings reported here suggest that PP6 plays an important depletion nuclear morphology defect (Fig. 8 C), which was co-n role during mitotic spindle formation. Specifically, PP6 is the sistent with the idea that TPX2 and PP6 have opposing effects T-loop phosphatase regulating Aurora A activity at the mitotic on Aurora A activity. These results suggest that PPP6C deple- spindle. This may explain recent findings inC. elegans in which tion results in unchecked activation of Aurora A caused by loss mutations in PP6 were found to cause mitotic defects in spin- of the T-loop phosphatase activity and, thus, disturbs the fine dle positioning and cortical contractility of the early embryo balance of Aurora A–TPX2 complexes at the spindle required (Afshar et al., 2010). These findings add to the previously r-e for chromosome capture and alignment. The reversal of the ported functions of PP6 in S phase and nuclear factor–B sig- PPP6C phenotype by reduction of Aurora A activity using TPX2 naling (Stefansson and Brautigan, 2006; Mi et al., 2009; Douglas depletion or MLN8237 also indicates that Aurora A is a major et al., 2010) and negative regulation of the Mis12 kinetochore substrate of PP6. protein in fission yeast (Goshima et al., 2003). Other PPP fam- ily phosphatases have many functions at different stages of the PP6 opposes Aurora A to control cell cycle and in a variety of processes in nondividing cells, spindle formation which can be explained by their many different isoforms com- Aurora A, together with the Ran–importin pathway, is part of a prised of a series of closely related subunits. It is reasonable to complex regulatory system controlling the action of spindle-as believe that this is also true for PP6 (Stefansson et al., 2008) and sembly factors, such as NuMa and TACC, and kinesin motor that specific isoforms are likely to have functions in S phase and proteins, such as Eg5/KIF11, required to form a bipolar mitotic M phase. The results presented here suggest that the ANKRD28 spindle (Giet et al., 1999, 2002; Tsai et al., 2003; Clarke and and 44 subunits are required for PP6 function in mitosis, Zhang, 2008). The effect of PP6 inactivation by PPP6C deple- whereas ANKRD52 is not. Interestingly, ANKRD28 and 44 are tion on these spindle assembly factors was therefore invest-i most closely related, whereas ANKRD52 is less similar. This gated. In control cells undergoing unperturbed mitosis, Eg5 and is intriguing because the MYPT1 ankyrin repeat subunit of PP1 NuMa were recruited to the forming spindle poles in prophase is important in substrate recognition (Tóth et al., 2000; Terrak and remained there until the onset of anaphase (Fig. 9A, siControl). et al., 2004). Therefore, the forms of PP6 important in mitosis Protein phosphates 6 regulates Aurora A • Zeng et al. 1325