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First published online 19 July 2005
doi: 10.1242/jcs.02450

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Functional interactions with Pit-1 reorganize co-repressor complexes in the living cell nucleus

Departments of Medicine and Cell Biology, University of Virginia, Charlottesville, VA 22908, USA

View larger version (86K): [in a new window]   Fig. 1. NCoR is localized to distinct subnuclear compartments. 3T3-L1 cells were fixed and subjected to ICC for detection of endogenous NCoR in (A) mock-transfected cells or (B-D) total NCoR in cells transfected with GFP-NCoR. Cells were stained with the H33342 chromatin dye. The profile plots (right) quantify the relative intensity of the fluorophores at the position along the yellow line in each overlay image. (B) The direct fluorescence signal from GFP-NCoR colocalized with anti-NCoR/Texas-Red immunofluorescence signal. (C,D) Representative images are labeled with the relative NCoR expression level based on the mean secondary-antibody/Texas-Red fluorescence intensity per nucleus. The computerized image-analysis algorithm automatically selected the foci (outlined in white) in each Texas-Red image. The mean area of the automatically selected foci is shown for each cell.

View larger version (72K): [in a new window]   Fig. 2. Co-repressors form specific intranuclear focal bodies. WFM images show living GHFT1-5 cells expressing: (A) both YFP-NCoR and RFP-PML, and stained briefly with the cell-permeant chromatin stain H33342; (B) both GFP-NCoR and mRFP-SMRT, and stained briefly with H33342; (C) CFP-HDAC-5 alone; or (D) YFP-NCoR and CFP-HDAC5 together. Each fluorescence channel is displayed separately and together in the overlay panel. Notice the different scale. Scale bars, 10 µm. (E) Living GHFT1-5 cells producing GFP-SMRT were subjected to FRAP analysis. The images show the nucleus of a cell taken at the same focal plane before selective photobleaching and at the indicated time points after. Fluorescence intensity in four foci was measured as indicated by the white square ROIs. The recovery plot shows the mean change in relative fluorescence intensity over a 300 second time frame, normalized to the prebleaching level for each ROI. Error bars denote s.e.m.

View larger version (50K): [in a new window]   Fig. 3. Co-repressors regulate Pit-1-dependent transcription. Luciferase reporter-gene transcription assays were performed using (A-C) GH4ZR7 pituitary cells or (D-F) non-pituitary HeLa cells. Cells were transfected with the indicated reporter vectors and expression vectors. The panels show the relative luciferase activity, corrected for total cellular protein in the lysates. Results are the means of measurements from at least three cultures and error bars denote the s.e.m. (B,D) Statistical analysis was performed with P<0.05 considered to be significant. The lower-case letters indicate sets of measurements that are statistically different. (D, insert) Western-blot analysis of HeLa cells transfected with the indicated expression vectors. (G) Lysates were prepared from cells transfected with the indicated expression vectors. Immunoprecipitations were performed using an anti-HA agarose conjugate and anti-GFP antibody was used for western blot analysis.

View larger version (62K): [in a new window]   Fig. 4. NCoR and Pit-1 are co-localized within the nucleus of mouse pituitary GHFT1-5 cells. Immunocytochemical staining was used to compare the subnuclear distribution of the endogenous NCoR and Pit-1 in fixed GHFT1-5 cells. GHFT1-5 cells probed with anti-Pit1 antibody followed by Texas-Red-conjugated anti-rabbit antibody, and anti-NCoR antibody followed by FITC-conjugated anti-goat antibody. The profile analysis shows the degree of co-localization of the immunostained proteins. The specificity of staining was confirmed by analysis of cells stained with both secondary antibodies alone, each individual primary antibody with the opposite secondary antibody, or each primary with both secondary antibodies.

View larger version (57K): [in a new window]   Fig. 5. Pit-1 redistributes NCoR in the nucleus. (A) GHFT1-5 cells were transfected with GFP-Pit-1 and PML-RFP, and the living cells were stained briefly with H33342 DNA dye immediately before imaging. (B) GHFT1-5 cells were co-transfected with GFP-NCoR, BFP-Pit-1 and PML-RFP, and the living cells were imaged using WFM. The square ROI in the overlay image is enlarged to highlight the subnuclear protein organization. Cyan in the overlay images indicates colocalization of the green and blue channels. The profile plots display the relative intensity of the fluorophores at the positions along the yellow line in the overlay images. The labeled bars indicate scale.

View larger version (50K): [in a new window]   Fig. 6. Pit-1 disperses NCoR foci in cell populations. Living cells were selected for WFM imaging using the mRFP signal, and subcellular ROIs were quantified using an automated computer algorithm to select ROI. The area and fluorescence intensity of each selected ROI were automatically measured, and the ratio of the surrounding region defines the enrichment factor (EF). (A) Overlay images of the YFP-NCoR and BFP-Pit-1 in three cells. The profile plots show the relative intensities of the fluorophores along the yellow line in each image. (B,D) Numerical results for 61 cells in the control population (open circles) and 48 cells in the experimental population (gray squares). Each point represents the mean data from a single cell. The relationships between YFP-NCoR expression level and (B) co-repressor focus size or (D) EF are shown. The black best-fit lines and 95% confidence intervals (surrounding gray regions) were calculated by linear regression for each population. The mean values of (C) focus size and (E) EF are shown normalized for expression level in each cell population.

View larger version (41K): [in a new window]   Fig. 7. Pit-1 redistributes SMRT and HDAC to chromatin-containing compartments. WFM images of living GHFT1-5 cells were captured 24 hours after transfection with vectors encoding the indicated fusion proteins. The profile plots display the relative intensity of the fluorophores at the positions along the yellow line in the overlay images.

View larger version (45K): [in a new window]   Fig. 8. The Pit-1 DNA-binding domain is required to disperse SMRT. GHFT1-5 cells were co-transfected with expression vectors encoding YFP-SMRT and the indicated BFP fusion protein. (A,B) The profile plots display the relative intensity of the fluorophores at the positions along the yellow line in the overlay images. (C,D) Cells were selected using co-transfected mRFP and subcellular features were quantified using integrated image analysis. The focus size and enrichment factor were normalized for the relative level of YFP-SMRT expression in each cell. The data are shown as the means of each cell population with error bars denoting s.e.m.