Fig. 2. Nucleostemin binds and retains MDM2 in the nucleoplasm of living cells under nucleolar stress. (A1) In vivo interaction between nucleostemin and MDM2 was shown by the bimolecular fluorescence complementation (BiFC) approach. The Flag-tagged N-terminal (Yn) and Myc-tagged C-terminal (Yc) fragments of Venus YFP were fused to MDM2 and nucleostemin, respectively. (A2) Yn-fused MDM2 (wild-type or mutant) and Yc-fused nucleostemin (wild-type or mutant) were coexpressed in HeLa cells with a nucleolar CFP (noCFP) marker. The percentages of YFP⁺ cells in the CFP⁺ population measured by FACS are indicated in the histogram. BC and A domains of nucleostemin were deleted in NS-GI-Yc. The AZ domain and the I1 and AZ domains were deleted in MDM2M2-dAZ-Yn and M2-dIAZ-Yn, respectively. (B1) The nucleoplasmic retention time of MDM2 was measured by FLIP in HeLa cells, in which the nucleolus was bleached and the nucleoplasmic fluorescence intensity was measured. Time-sequenced images with labels indicating the bleached areas in the nucleolus (yellow circles), the measured areas in the nucleoplasm (red rectangles) and intervals between image acquisition and the first bleaching pulse (in seconds) are shown. Scale bar: 5 µm. (B2) The average FLIP rates of MDM2 were calculated from 20 cells from 2-3 independent experiments. Coexpression of wild-type or nucleoplasmic mutants of nucleostemin increased the nucleoplasmic retention time of MDM2 (P<0.0001, by Repeated Measures ANOVA). Error bars represent s.e.m. and are shown on one side (indicated by arrows) of the control and dB(256) curves. Y-axis represents the relative fluorescence index (RFI), and arrows along the top indicate bleaching pulses. (C) The role of endogenous nucleostemin in regulating the dynamic distribution of MDM2 is revealed by doxorubicin (ADR) and mycophenolic (MPA) treatment, which mobilize nucleostemin from the nucleolus to the nucleoplasm. When exposed to ADR (2 µM, 4 hours) (left panel) or MPA (40 µM, 4 hours) (right panel), the retention time of MDM2 in the nucleoplasm was increased (blue) compared to the mock-treated cells (Ctrl, black, P<0.0001). Knocking down the endogenous expression of nucleostemin (siNS) was able to reverse a significant portion of the drug-induced retention of MDM2 in the nucleoplasm (red, P<0.0001).