Figure 3. PP6 catalytic activity is required for normal nuclear morphology. (A) A schematic depicting the PP6 holoenzyme, PPP6C catalytic subunit, posi- tions of siRNA duplexes used, and inactivating mutations. (B) HeLa cells stably expressing PPP6C si08–resistant wild-type (si08ResPPP6CWT) and phosphatase- dead mutant (si08ResPPP6CPD) forms of PPP6C tagged at the C terminus with a FLAG epitope were transfected with control (siCon) or si07 and si08 duplexes targeting PPP6C for 48 h. The cells were Western blotted or fixed and then stained with DAPI, tubulin (Tub), and CREST antibodies. Abnormal nuclear morphology was scored for each condition and is plotted in the graph (n = 3). Molecular mass is given in kilodaltons. Error bars indicate the standard error of the mean. Bars, 10 µm. form by si07 alone was verified by Western blotting (Fig. 3 B). caused similar patterns of altered nuclear morphology and the Interestingly, the levels of transgene-expressed PPP6C increased formation of micronuclei (Fig. 4, A–C). The micronuclei over- when the endogenous PPP6C was depleted with si08 (Fig. 3 B), lap with a single puncta of CREST centromeric marker auto- suggesting that the availability of the PP6 regulatory and a-n antiserum staining (Fig. 4, A and B). This indicates that they kyrin repeat subunits may be limiting for PPP6C stability under correspond to single chromosomes, possibly arising through d-e these conditions. fects in chromosome segregation. To test whether the abnormal nuclei in PPP6C-depleted cells arise because of defective ch-ro PP6 is required for timely mosome alignment and segregation, HeLa (Fig. 4 D) or MRC-5 spindle formation (Fig. 4 E) cells were examined in mitosis. Control cells showed Although the visual screen for defective nuclear morphology tightly aligned chromosomes in metaphase, which remain as implicates PP6 in mitotic progression, it does not define the pro- well-defined masses as they segregate in anaphase and give rise cess it functions in or, more importantly, what its substrates are. to single nuclei in telophase (Fig. 4, D and E). In both cell lines PP6 is localized to the cytoplasmF(ig. S2, A and B), giving little tested, efficient PPP6C depletion resulted in poorly organized clue to its function. PPP6C depletion in either the HeLa cervical chromosome segregation in anaphase and in the formation of carcinoma cell line or MRC-5 human diploid lung fibroblasts fragmented nuclei in telophase (Fig. 4, D and E). Similar effects Protein phosphates 6 regulates Aurora A • Zeng et al. 1319