br Figure A Heterozygous P R
Figure 2. A Heterozygous P179R Mutation in PP2A-Aa Decreases PP2AB56 Assembly and Targeting
(A) Schematic of PPP2R1A gene-targeting strategy. Exons, rectangles; loxP sites, triangles; ITR, AAV-speciÞc inverted tandem repeats; homologous sequences, green and red dashed lines.
(B) Sanger sequencing the modiÞed region of PPP2R1A in a mutant clone.
(D and E) (D) Western blot analysis of lysates (lanes 1Ð2) and immunoprecipitations (IPs) of control IgG (lane 3) or PP2A-Aa IgG (lane 4Ð5) in indicated cell lines. (E) Plotted is the normalized mean G SEM of the experiment in (D) performed three times.
(FÐG) WT (+/+) and PP2A-AaP179R/+ (P179R/+) Relebactam in nocodazole were Þxed and processed for immunoßuorescence. (F) Maximum intensity projection. Scale bar, 5 mm. (G) Normalized centromere or kinetochore signal is plotted. Circle, cell; line, mean.
See also Figure S1.
Given that PP2A-AaP179R/+ cells have a modest delay in mitosis (22 min in clone a and 21 min in clone b versus 18 min in WT cells) (Figure S2A) and increased time spent in mitosis suppresses multipolar mitoses (Kwon et al., 2008), we next compared the mitotic duration of binucleate cells that underwent
a bipolar cell division. Both WT and PP2A-AaP179R/+ cells completed mitosis with indistinguishable kinetics (Figure 3G). We also examined if the cell division phenotype was caused by centrosome inacti-
vation as observed in Drosophila (Basto et al., 2008). However, microtubule nucleation was observed at all centrosomes in WT and PP2A-AaP179R/+ cells (Figure S2B). Moreover, in PP2A-AaP179R/+ cells, cen-trosomes were always associated with spindle poles (Figure S2C) and were more likely to be clustered (Figure S2D). Our data suggest that enhanced bipolar cell division after WGD in PP2A-AaP179R/+ cells is likely due to more efÞcient centrosome clustering and not due to centrosome inactivation or length-ening of mitosis.
PP2A-AaP179R/+ Cells Cluster Supernumerary Centrosomes More Efficiently
To test the hypothesis that PP2A-AaP179R/+ cells may cluster supernumerary centrosomes more efÞciently than WT cells, centrosome ampliÞcation was induced by over-expression of Plk4 kinase
Figure 3. The P179R Mutation in PP2A-Aa Suppresses Multipolar Cell Division after Cytokinesis Failure
(A) Schematic of cytokinesis failure assay.
(P179R/+) cells after cytochalasin D treatment. Time (minutes) relative to nuclear envelope breakdown is indicated.
(C) QuantiÞcation of cell division outcome of Tp53+/+ cells. (D) QuantiÞcation of multipolar cell divisions in Tp53 / cells treated as in (A).
(E and F) Cell lines of the indicated genotype expressing GFP-PP2A-Aa-WT or GFP-PP2A-Aa-P179R were (E) analyzed by western blot, where * shows non-speciÞc band and ** shows GFP-PP2A-Aa truncation product, and (F) treated with cytochalasin D as in (A), imaged, and cell division outcome quantiÞed.
See also Figure S2.
(Kleylein-Sohn et al., 2007; Peel et al., 2007) (Figure 4A). This increases centrosome number without altering ploidy. We introduced a doxycycline-inducible Plk4 into Tp53 / WT and PP2A-AaP179R/+ cells. After doxy-cycline induction, immunoßuorescence analyses of C-Nap1, a marker of functional centrioles (Wang et al., 2011), indicated that 64% of WT cells and 68% of PP2A-AaP179R/+ cells had centrosome ampliÞcation (Fig-ures S2E and S2F). Immunoßuorescence analysis of anaphase cells with extra centrosomes revealed that PP2A-AaP179R/+ cells clustered centrosomes into a bipolar spindle more efÞciently than WT cells, with a 6-fold reduction in multipolar anaphases (Figures 4B and 4C). A similar result was observed by live imaging (Figures 4D and 4E), with no delay in mitosis (Figure 4F).