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First published online 28 February 2006
doi: 10.1242/jcs.02822

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IFN-type-I-mediated signaling is regulated by modulation of STAT2 nuclear export

Department of Gene Regulation and Differentiation, GBF-German Research Centre for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany

View larger version (44K): [in a new window]   Fig. 1. Effect of CRM1 inhibition on STAT2 nucleocytoplasmic distribution in untreated and IFN-ß-stimulated 2fTGH cells. (A) 2fTGH cells were either not treated or were treated with IFN-ß for the indicated time periods in the absence (upper panel) or presence of LMB (10 ng/ml) (lower panel). The cells were fixed and stained for STAT2 by indirect immunofluorescence. Fluorescence micrographs were obtained by confocal laser-scanning microscopy. (B) 2fTGH cells were treated as described in A. In addition, LMB alone was added to the cells for 4 hours. Whole-cell extracts were resolved by SDS-PAGE and activated STAT2 was detected with a phosphospecific STAT2-Tyr689 antibody (top). Loading of equal amounts of STAT2 was confirmed by reprobing with STAT2 antiserum (bottom). (C) 2fTGH cells were either left untreated or were treated with LMB (10 ng/ml) for the indicated time periods. Cells were stained for STAT2 and indirect immunofluorescence was analyzed by confocal laser-scanning microscopy. Staining profiles (line-scan analysis) of STAT2 are shown in the lower panel. (D) U2A cells were either left untreated or treated with LMB (10 ng/ml) for the indicated time periods. Cells were stained for STAT2 and analyzed by confocal laser-scanning microscopy. Staining profiles of STAT2 are shown in the lower panel. (E) U2A cells were transiently transfected with STAT2-EGFP expression plasmid. Fourty hours after transfection, subcellular localization of STAT2-EGFP was determined by fluorescence microscopy before and after 2 and 4 hours of LMB treatment.

View larger version (76K): [in a new window]   Fig. 2. Kinetics of nuclear import of STAT2-EGFP. (A) LMB induces nuclear accumulation of STAT2-EGFP. NIH3T3 cells stably expressing STAT2-EGFP (top) or STAT1-EGFP (bottom) were treated with LMB (10 ng/ml). Subcellular distribution of STAT2-EGFP and STAT1-EGFP was examined before and at the indicated time points after LMB treatment by fluorescence microscopy. (B) NIH3T3 cells expressing STAT2-EGFP were treated for 2 hours with LMB (10 ng/ml) and subjected to selective FRAP. The indicated area in the nucleus was bleached for 40 seconds. Subsequently, recovery of unbleached STAT2-EGFP into the nucleus was monitored at low-power every 100 seconds. Fluorescence intensity is shown in false color code (intensity increases from blue to red). (C) NIH3T3 were treated as described in B; cells expressing STAT1-EGFP were subjected to selective FRAP.

View larger version (65K): [in a new window]   Fig. 3. CRM1-independent nucleocytoplasmic shuttling of STAT2-EGFP. (A) NIH3T3 cells stably expressing STAT2-EGFP (left) or STAT1-EGFP (right) were treated for 4 hours with LMB (10 ng/ml) and subsequently subjected to cytoplasmic FLIP analysis. A cytoplasmic area was bleached with maximum laser intensity by scanning up to nine consecutive periods of 35-43 seconds. The bleached regions are indicated with white rectangles in the first post-bleach images. The total fluorescence of the bleached cells and that of the neighboring cells was monitored between bleaching. The representative image series shows the fluorescence intensities in false color codes (intensity increases from blue to red) before (0 sec) and after the indicated bleaching periods. (B) The nuclear fluorescence intensities of the bleached cells were measured in different experiments, normalized to the total fluorescence of the respective unbleached cells and plotted over time. The relative fluorescence intensities of STAT2-EGFP (left) and STAT1-EGFP (right) in the nucleus are shown during a period of 300 seconds. Colors of graphs reflect individual experiments.

View larger version (27K): [in a new window]   Fig. 4. Stimulation of IFN-ß blocks nuclear export of STAT2-EGFP. (A) U6A cells stably expressing STAT2-EGFP were treated with IFN-ß. At different times after treatment cytoplasmic FLIP analysis was performed using the same settings as described in Fig. 3. The fluorescence intensities of STAT2-EGFP in the nucleus of the bleached cells were measured, normalized to the total fluorescence of the unbleached cells and plotted over time. Relative fluorescence intensities at the indicated time periods after IFN-ß stimulation are shown. The localization of STAT2-EGFP during IFN-ß stimulation was documented by showing representative cells for the indicated time points. (B) 2fTGH cells expressing p50-GNES were subjected to cytoplasmic FLIP analysis. Relative nuclear fluorescence intensities are shown for untreated, IFN-ß-stimulated and LMB-treated cells. Insets show confocal images of representative cells. Colors of graphs reflect individual experiments.

View larger version (48K): [in a new window]   Fig. 5. STAT2 contains a strong constitutive nuclear export signal. (A) C243 cells were transfected with either STAT1-GNLS or STAT2-GNLS as indicated. Localization of the fluorescent proteins was determined in untreated cells by fluorescence microscopy. The effect of LMB was monitored in the same cells on STAT2-GNLS 15 and 30 minutes after treatment. (B) C243 cells transfected with STAT2-GNLS or STAT1-GNLS were subjected to cytoplasmic FLIP analysis using the same settings as described in Fig. 3A. The nuclear fluorescence intensities of the bleached cells were determined in different experiments, normalized to the total fluorescence of the unbleached cells and plotted over time. The relative nuclear fluorescence intensities of STAT2-GNLS (solid lines) and STAT1-GNLS (broken lines) are shown during a time period of 300 seconds. Colors of graphs reflect individual experiments.

View larger version (56K): [in a new window]   Fig. 6. Characterization of a CRM1 specific STAT2 NES element. (A) Sequence of human STAT2 between aa position 690 and 759. Positions of STAT2 C-terminal deletion mutants are marked by arrows and aa mutated to alanine are underlined. (B) Subcellular localization of STAT2-754-EYFP (a), STAT2-703-EYFP (b), STAT2-L733A-EGFP (c), STAT2-L747A-EGFP (d) and STAT2-LL737/741AA-EYFP (e) was monitored in untreated C243 cells by confocal analysis after transient transfection. Nucleocytoplasmic distribution (f) of STAT2-LL737/741AA-EYFP was compared in C243 cells (left) before and (right) after LMB treatment for 2 hours. The nuclear region is enclosed by the blue and red lines inside the graph of the staining profile. (g) Localization STAT2-LL737/741AA-EYFP was monitored after its transfection into U2A cells. (C) NIH3T3 cells stably expressing STAT1-CFP were transiently transfected with STAT2-LL737/741AA-EYFP expression plasmid. Subcellular localization of both STATs was determined by fluorescence microscopy in untreated cells and after stimulation with IFN-ß for 90 minutes. IFN was removed and nucleocytoplasmic distribution of STAT2-LL737/741AA-EYFP and STAT1-CFP was monitored 120 min later.

View larger version (73K): [in a new window]   Fig. 7. Characterization of CRM1-independent shuttling of the STAT2 NES mutants STAT2-LL737/741AA. (A) C243 cells were transfected with STAT2-LL737/741AA-EYFP and subjected to cytoplasmic FLIP analysis. The bleach area is indicated with a white square in the first post-bleach image. The representative image series shows the fluorescence intensity in false color code (intensity increases from blue to red) before (0 sec) and after the consecutive bleaching periods. After the last bleach pulse subcellular distribution of STAT2-LL737/741AA-EYFP was monitored after another 300 seconds (+300 sec). (B) The nuclear fluorescence intensities of the bleached cells from A were measured in different experiments, normalized to the total fluorescence of the unbleached cells and plotted over time. Relative fluorescence intensities of STAT2-LL737/741AA-EYFP in the nucleus are shown. Colors of graphs reflect individual experiments. (C) C243 cells were transfected with either STAT2-LL737/741AA-YNES or STAT2-LL737/741AA-GNLS as indicated. Localization was determined in untreated cells by confocal analysis. The effect of LMB was monitored on STAT2-LL737/741AA-YNES localization 1 hour after treatment. (D) C243 cells expressing STAT2-LL737/741AA-YNES were treated for 4 hours with LMB (10 ng/ml) and subsequently subjected to FLIP analysis as described in A. Relative nuclear fluorescence intensities of STAT2-LL737/741AA-YNES from different experiments are shown. (E) C243 cells expressing STAT2-LL737/741AA-GNLS (left) or STAT1-GNLS (right) were pre-treated with cytochalasin D to induce bi-nuclear cells. The indicated area in one nucleus of a single cell was bleached until complete loss of fluorescence. Subsequently, recovery of unbleached STAT2-LL737/741AA-GNLS and STAT1-GNLS molecules into the nucleus was monitored for 10 minutes. Representative image-series show fluorescence intensities in false color code (increasing from blue to red) for the bleached cells (left) and neighboring cells (right) prior to bleaching and after the indicated post-bleach time periods.

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