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First published online 4 March 2003
doi: 10.1242/jcs.00370

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Accumulation of c-Myc and proteasomes at the nucleoli of cells containing elevated c-Myc protein levels

Azadeh Arabi^1,2,*,, Cecilia Rustum^1,3,*, Einar Hallberg¹ and Anthony P. H. Wright^1,2

¹ Natural Sciences Section, Södertörns University College, S-141 89 Huddinge, Sweden
² Department of Biosciences, Karolinska Institute, S-141 57 Huddinge, Sweden
³ Department of Neurochemistry and Neurotoxicology, Stockholms University, S-106 91 Stockholm, Sweden

View larger version (46K): [in a new window]   Fig. 1. Cellular distribution and nucleo-cytoplasmic shuttling of c-Myc. (A) c-Myc and c-Myc-GFP are predominantly nucleoplasmic. Both endogenous c-Myc (upper panels) and transiently transfected c-Myc-GFP (lower panels) were evenly distributed in the nucleoplasm while little or none was detected in the nucleoli of COS-7 cells. The cells (lower panel) were transfected with 1 µg of c-Myc-GFP expression plasmid. Bars, 20 µm. (B) FLIP analysis demonstrates that c-Myc-GFP shuttles between the nucleus and the cytoplasm. A COS-7 cell expressing c-Myc-GFP (outlined by the white dashed line) was repeatedly bleached, in a defined area in the cytoplasm (white circle). Confocal images were acquired before bleaching (pre-bleach) and between the bleach pulses. Images of the cell at the indicated times are shown. The lower cell, which was not bleached, serves as a control for unintentional bleaching during scanning. Bar, 20 µm. (C) Comparison of fluorescence loss in bleached and unbleached cells. The fluorescence loss curve indicates the mean value for relative fluorescence intensity measured in the nucleoplasm of cells subjected to FLIP, plotted as a function of time (filled squares). The error bars show standard deviation (n=5 cells). The filled triangles indicate the relative fluorescent intensity in the nucleoplasm of the neighbouring unbleached cell shown in B.

View larger version (48K): [in a new window]   Fig. 2. c-Myc-GFP localisation in cells expressing elevated levels of the protein. The c-Myc-GFP distribution pattern changes progressively as its expression level is increased. (A) COS-7 cells were transfected with 1, 2 or 4 µg of the c-Myc-GFP expression vector. The distribution of c-Myc-GFP (green), and fibrillarin, a nucleolar marker (red), in representative cells is shown. Transfected cells were classified into three groups based on the c-Myc-GFP expression pattern as follows: (1) cells with nucleoplasmic c-Myc-GFP (upper panel); (2) cells with c-Myc-GFP in the nucleoli (lower panel, yellow arrows); and (3) cells with nuclear inclusions, which had the highest levels of fluorescence intensity (lower panel, grey arrow). The total number of c-Myc-GFP expressing cells and the number of cells in group 2 and 3 increased as the amount of c-Myc-GFP expression vector was increased. (B) There is a positive correlation between the amount of transfected c-Myc-GFP expression vector, and the ratio of cells with nucleolar (2): nucleoplasmic (1) c-Myc-GFP. The ratios are mean values. Error bars indicate the s.d. (n=5).

View larger version (51K): [in a new window]   Fig. 3. Distribution of c-Myc in proteasome-inhibitor-treated cells. (A) c-Myc and c-Myc-GFP are nucleolar in proteasome-inhibitor-treated cells. COS-7 cells were cultured in the presence of 100 µM ALLN for 5 hours. Both c-Myc (upper panels) and c-Myc-GFP (lower panels) were localised to the nucleoli. The cells (lower panel) were transfected with 1 µg of c-Myc-GFP expression plasmid. Bar, 20 µm. (B) Time-lapse analysis shows redistribution of c-Myc-GFP in live cells after addition of 50 µM MG132. Confocal images were acquired every 120 seconds over a 3 hour period. The panel shows pictures selected at the times indicated. The arrows indicate accumulation of c-Myc-GFP in the vicinity of nucleoli. Nucleoli were identified by phase contrast microscopy as in A. Bar, 20 µm. (C) Proteasome inhibitor induced c-Myc redistribution is concurrent with its cellular accumulation. Whole cell extracts from COS-7 cells were analysed by SDS-PAGE and immunoblotting using anti-Myc C33 antibody. Lane 1 shows untreated cells. c-Myc protein levels increase after 3 and 6 hours of 100 µM ALLN treatment, lanes 2 and 3, respectively. ß-actin was used as an internal loading control.

View larger version (60K): [in a new window]   Fig. 4. Localisation of c-Myc in relation to fibrillarin. (A) c-Myc-GFP is predominantly located in the nucleoplasm of cells in the absence of proteasome inhibitors. Confocal images showing the distribution of c-Myc-GFP and fibrillarin in untreated COS-7 cells are shown in a and b, respectively. The distinct localisation of c-Myc-GFP and fibrillarin is shown in c-e. In c, the confocal images in a and b are overlaid. The relative fluorescence intensity (RFI) from c-Myc-GFP (green) and fibrillarin (red), along the white line in c is quantified in d. (e) Reconstitution of the stack of confocal xy plane images; e' and e'' show yz and xz sections through the stack of images, respectively. The positions of the sections are shown by the fine white lines in e. Bar, 10 µm. (B) Nucleolus-associated c-Myc-GFP accumulates in regions adjacent to but distinct from the fibrillarin marker for the dense fibrillar region of the nucleolus in proteasome-inhibitor-treated cells. Confocal images showing the distribution of c-Myc-GFP and fibrillarin in cells treated with proteasome inhibitor are shown in a and b, respectively. The distinct localisation of c-Myc-GFP and fibrillarin within the nucleolus is shown in c-e. In c the confocal images in a and b are overlayed. The relative fluorescence intensity (RFI) from c-Myc-GFP (green) and fibrillarin (red), along the white line in c is quantified (d). (e) Reconstitution of the stack of confocal xy plane images; e' and e'' show yz and xz sections through the stack of images, respectively. The positions of the sections are shown by the fine white lines in e. Bar, 10 µm.

View larger version (40K): [in a new window]   Fig. 5. Mobility of the nucleolar c-Myc-GFP in proteasome inhibitor treated cells. (A) FLIP analysis demonstrates that c-Myc-GFP shuttles between the nucleolus and the nucleoplasm/cytoplasm after proteasome inhibition. A cell expressing c-Myc-GFP (outlined by white dashed line) was treated with MG132 for 3 hours before being repeatedly bleached in a defined area in the cytoplasm (white circle). Confocal images of the cell were taken before bleaching and then at 2 second intervals. The panel shows images taken at the indicated time points. Bar, 20 µm. (B) FRAP analysis demonstrates a relatively stable interaction of c-Myc-GFP with a nucleolus after proteasome inhibition. A cell expressing c-Myc-GFP was treated with ALLN for 4 hours before being bleached in part of the nucleolus for 1 second (white circle). Confocal images were taken before bleaching and then every 120 seconds. The panel shows images taken at the indicated time points. Bar, 20 µm. (C) Quantitative measurements of the fluorescence recovery. The curve indicates the mean value for the relative fluorescence intensity plotted as a function of time. The error bars represent the standard deviation (n=5 cells). The fluorescence was recovered within 30 minutes and the mobile fraction was calculated to 84%.

View larger version (66K): [in a new window]   Fig. 6. Distribution of proteasomes in COS-7 cells in the presence and absence of proteasome inhibitors. (A) Proteasomes are recruited to the nucleolus in proteasome-inhibitor-treated cells. Cells were cultured in the presence or absence of 100 µM ALLN, and immunostained for proteasomes using a rabbit pAb (1:10,000) and a mouse mAb (1:500). Proteasomes were detected in the nucleus and the cytoplasm, but were not detected in the nucleoli of untreated cells (upper panels). In cells treated with ALLN for 5 hours, a subpopulation of proteasomes was detected in the nucleoli (lower panels). (B) Localisation of the proteasomes (red), and fibrillarin (green) in proteasome-inhibitor-treated cells is shown at higher magnification (left panels). Confocal images show the accumulation of proteasomes adjacent to the fibrillarin marker for the dense fibrillar region of the nucleolus in the nucleolus (right panels). Rabbit pAb was used for proteasomes immuno-staining.

View larger version (60K): [in a new window]   Fig. 7. Localisation of proteasomes in relation to c-Myc in proteasome inhibitor treated cells. (A) The distribution of c-Myc and proteasomes in a proteasome-inhibitor-treated cell is shown at high resolution by the confocal images in a and b, respectively. The colocalisation of c-Myc and proteasomes is shown in c-e. In c the confocal images in a and b are overlayed. The relative fluorescence intensity from c-Myc (green) and proteasomes (red), along the white line in c is quantified in d. (e) Reconstitution of the stack of confocal xy plane images; e' and e'' show yz and xz sections through the stack of images, respectively. The positions of the sections are shown by the fine white lines in e. Bar, 10 µm. (B) The distribution of c-Myc-GFP and proteasomes in cells expressing increased levels of c-Myc in the absence of proteasome inhibitor is shown by the confocal images a-d. Proteasomes accumulate specifically in the nucleoli that contain c-Myc (e-g, yellow arrows). Nucleoli lacking c-Myc remain devoid of nucleolar proteasome staining (e-g, white arrowheads)