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Dynamic targeting of protein phosphatase 1 within the nuclei of living mammalian cells

MSI/WTB Complex, University of Dundee, Dundee DD1 5EH, Scotland

View larger version (19K): [in a new window]   Fig. 1. Expression of FP-tagged PP1 isoforms in transiently transfected mammalian cells. (A) Cartoon depicting the plasmids constructed for expression of EGFP, EYFP or ECFP PP1, ß/ and . (B) Western blot showing the bands detected with anti-PP1 antibodies in lysates (30 µg total protein) from non-transfected HEK293 cells (lane 1), and from cells transiently expressing EYFP alone (lane 2), EYFP-PP1 (lane 3), EGFP-PP1ß/ (lane 4) or EYFP-PP1 (lane 5). Arrows point to expressed and endogenous PP1. (C) Western blot showing the bands detected with anti-GFP antibodies in the same lysates. (D) In vitro phosphorylase a phosphatase activity associated with EYFP, EYFP-PP1, EGFP-PP1ß/, EYFP-PP1 and EYFP-PP1(H125A) immunoprecipitated from these lysates using anti-GFP/YFP antibodies.

View larger version (97K): [in a new window]   Fig. 2. Localization of FP-tagged PP1 isoforms in transiently transfected HeLa cells. Panel A shows the localization of EYFP-PP1 in live HeLa cells, with an accompanying DIC (differential interference contrast) image in Panel B and the two images merged in Panel C. EGFP-PP1ß/ localization is shown the same way in Panels D-F, and EYFP-PP1 localization in Panels G-I. PP1 and PP1 maintain their specific localization patterns when co-expressed in the same cells as ECFP-PP1 (Panel J) and EYFP-PP1 (Panel K). Panel L shows the two patterns merged, with ECFP-PP1 pseudocolored green and EYFP-PP1 pseudocolored red. Arrowheads indicate nucleoli. Scale bars are 10 µM.

View larger version (63K): [in a new window]   Fig. 3. Relocalization of FP-PP1 and FP-PP1 by overexpression of a targeting subunit. Co-expression of EYFP-PP1 and ECFP-NIPP1 in transiently transfected HeLa cells leads to retargeting of both the isoform (A) and the isoform (E) to nuclear speckles, where NIPP1 accumulates (B and F). An ECFP fusion of the NIPP1(V201K/F203K) mutant that still accumulates at speckles (D and H), but does not interact strongly with PP1 in vitro (Trinkle-Mulcahy et al., 1999), was not able to retarget either isoform (C and G). Arrows indicate nuclear speckles, whereas arrowheads indicate nucleoli. Scale bars are 10 µM.

View larger version (54K): [in a new window]   Fig. 4. Dynamic relocalization of PP1 by a spatially distinct targeting subunit. Heterokaryons created by the fusion of transiently transfected mammalian cells in the presence of cyclohexamide demonstrate that ECFP-NIPP1 can retarget the nucleolar pool of EYFP-PP1 to nucleoplasmic speckles in both HEK293 (A and B) and HeLa (E and F) cells, whereas the NIPP1(V201K/F203K) mutant cannot (C and D, HEK293; G and H, HeLa). The dashed arrow in Panel E indicates a nucleus within the EYFP-PP1/ECFP-NIPP1 HeLa cell heterokaryon that has a lower level of NIPP1 than its neighbors and shows no significant retargeting of PP1, demonstrating that this is a concentration-dependent effect. Arrows indicate nuclear speckles, whereas arrowheads indicate nucleoli. Scale bars are 10 µM.

View larger version (48K): [in a new window]   Fig. 5. Photobleaching analyses of EYFP-PP1. EYFP-PP1 was transiently expressed in HeLa cells and imaged live on a confocal laser scanning microscope. Panel A shows a FRAP (fluorescence recovery after photobleaching) experiment in which a nucleolar pool of EYFP-PP1 in a cell was photobleached at full laser intensity (region indicated in dashed brackets) for a total of four seconds. The recovery of the signal was monitored by repeated scanning of the entire field of view at low laser power. An unbleached neighboring cell is included for comparison. Panel B shows a FLIP (fluorescence loss in photobleaching) experiment in which repeated photobleaching within a defined region (dashed box) was shown to deplete the fluorescent signal throughout the cell. The entire field of view was scanned at low laser power, and the boxed region then bleached with 2 scans at full laser intensity (4 seconds total). This sequence was repeated 15 times, with the bleached cell gradually becoming dimmer while the unbleached control cell remained bright. Times indicated are total photobleaching times at full laser intensity. Arrows indicate photobleached cells whereas arrowheads indicate nucleoli. The scale bar is 10 µM.

View larger version (58K): [in a new window]   Fig. 6. Measurement of the direct interaction between PP1 and a targeting subunit. EYFP-PP1 (A) and ECFP-NIPP1 (B) show a strong FRET signal (C) when co-expressed in transiently transfected HeLa cells, with the phosphatase retargeting from nucleoli to nuclear speckles. Mutant ECFP-NIPP1(V201K/F203K) (E) does not retarget EYFP-PP1 to speckles (D), and no significant FRET signal is observed (F). Although both EYFP-U1A (G) and ECFP-NIPP1 (H) are found to colocalize at nuclear speckles, the proteins are not known to interact and no FRET signal is observed between them (I). The FRET signal between PP1 and NIPP1 can also be measured when the fluorophores are exchanged (L), by co-expression of EYFP-NIPP1 (J) and ECFP-PP1 (K). In the cell shown here, a significant nucleoplasmic pool of EYFP-PP1 remains to be retargeted, and it can be seen that the FRET signal is found mainly in the nucleoplasm and at the speckles. All EYFP and ECFP signals are shown scaled from 0-4094 pixels, although FRET signals are scaled from 0-1000 pixels and intensity colored as shown. Arrows indicate nuclear speckles, whereas arrowheads indicate nucleoli. Scale bars are 10 µM.