PMC

Limited exchange of ubiquitin between nuclear and cytosolic compartments. (A) GFP-Ub cells were photobleached in either the complete cytosol (top) or the complete nucleus (bottom) and recovery was measured in both compartments. (B) Quantification of the photobleaching experiments with GFP-Ub cells. The ratios of nuclear fluorescence to cytosolic fluorescence are plotted for cytosolic bleaching (shaded circles) and nuclear bleaching (open circles). (C) GFP-Ub^K0,G76V cells were photobleached in either the complete cytosol (top) or the complete nucleus (bottom) and recovery was measured in both compartments. (D) Quantification of the photobleaching experiments with GFP-Ub^K0,G76V cells. The relative ratios of nuclear fluorescence to cytosolic fluorescence are plotted for cytosolic bleaching (shaded circles) and nuclear bleaching (open circles). Bars, 20 μm.

Dynamics of ubiquitin in nucleus and cytosol. (A) FRAP curves for GFP-Ub in the nucleus (black line) and cytoplasm (gray line). Mobile fractions (R) are indicated for both nuclear (dark gray) and cytoplasmic bleached area (light gray). The interpolations for the t _1/2 are indicated with dashed lines in the same respective gray values. (B) Confocal images of a FRAP experiment in the nucleus (top) or in the cytoplasm (bottom) before, immediately after, and 25 s after a 2-s photobleaching. Bars, 10 μm. (C) Mobile fractions (R) of GFP-Ub^K0,G76V, GFP-Ub, and proteasome α3-GFP in stably transfected Mel JuSo cells. Diffusion was measured in both the nucleus (black bars) and the cytoplasm (gray bars). Error bars are SD (n > 10). (D) Diffusion rates of GFP-Ub^K0,G76V, GFP-Ub, and α3-GFP in stably transfected Mel JuSo cells. Diffusion was measured in both the nucleus (black bars) and the cytoplasm (gray bars). Error bars are SD (n > 10).

Generation and characterization of cell lines for in vivo monitoring of ubiquitin. (A) Western blot analysis of cell lysates of parental Mel JuSo cells and Mel JuSo cells stably expressing GFP-Ub or GFP-Ub^K0,G76V. The samples were separated under reducing and nonreducing conditions and probed with an anti-GFP antibody (left) and an antiubiquitin antibody (right). The blots were reprobed with an anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody to check for protein loading. (B) Micrographs of living GFP-Ub and GFP-Ub^K0,G76V cells. (C) Fluorescence micrographs of live GFP-Ub cells stained with LysoTracker red. GFP fluorescence (left), LysoTracker fluorescence (middle), and merged images (right) are shown. (bottom) Magnification of the boxed regions. (D) Mel JuSo cells expressing GFP-Ub were stained with the ubiquitin-specific antibody FK2. Native GFP fluorescence, antiubiquitin staining, a DAPI nuclear staining, and the merge of the three images are shown. Bars, 10 μm.

Competition for free ubiquitin causes depletion of uH2A during proteotoxic stress. (A) Confocal images of PAGFP-Ub in Mel JuSo cells before photoactivation (Pre) and three time points after photoactivation (0, 200, and 1,200 s). In the image before activation (Pre), the contours of the cell (C), the nucleus (N), and the region to be activated (A) are indicated. The look-up table is provided on the right (see also Video 1). (B) Quantification of fluorescence in an activated nuclear region visualizes the decay of PAGFP-Ub before and after the addition of MG132. PAGFP fluorescence is depicted with the blue line, whereas trend lines derived from similar experiments with untreated or MG132-treated cells are shown in red and green, respectively. (C) Confocal images of GFP-Ub cells injected with dextran–Texas red and anti-GFP antibody or (D) injected with dextran–Texas red and anti-mCD27 antibody. (E) Quantifications of the relative fluorescence ratio between the nucleus and the cytoplasm in control, anti-mCD27–injected, anti-GFP–injected, or MG132-treated cells. Error bars are standard deviations (n > 50). (F) Schematic representation of the dynamic ubiquitin equilibrium in the cell. Video 1 is available at http://www.jcb.org/cgi/content/full/jcb.200510071/DC1. Bars: (A) 10 μm; (C and D) 20 μm.

Accumulation of polyubiquitylated proteins coincides with depletion of uH2A and chromatin remodeling. (A) Fluorescence images of living GFP-Ub cells before and after 2 h incubation with 25 μM MG132. Bar, 10 μm. (B) Quantification of GFP-Ub levels in the cytoplasm (open circles) and nucleus (closed squares) during proteasome inhibition with 25 μM MG132. Black line is the ratio of the nucleus to the cytoplasm as plotted on the left y axis. The relative fluorescence of the nucleus and cytoplasm is plotted on the right y axis. (C) Diffusion rates of GFP-Ub in nucleus and cytoplasm without treatment (black bars) and after 2 h of MG132 treatment (gray bars). P values are indicated (unpaired t tests). Error bars represent the mean and SD of 15 independent cells in one representative experiment. (D) Western blot analysis of nuclear (N) and cytosolic (C) fractions of GFP-Ub cells. Cells were left untreated or treated for 2 h with DMSO, MG132, or a heat shock, and nuclear (N) and cytosolic (C) fractions were isolated. Nuclear fractions contained, on average, two times less protein than the cytosolic fractions. For analysis, 15- and 30-μg proteins were loaded for the nuclear and cytosolic fractions, respectively. Membranes were probed with an anti-GFP antibody. Molecular mass markers are indicated. (E) FRAP curves for GFP-Ub in the nucleus of untreated cells (black line) and after 2 h incubation with MG132 (gray line). (F) Western blot analysis of uH2A in GFP-Ub cells left untreated or exposed for 2 h to DMSO, MG132, or a heat shock. The membranes were probed with an anti-uH2A antibody. Molecular mass markers are indicated. (G) Lysates of Mel JuSo cells expressing GFP-Ub that were treated for various time periods with MG132 were probed with an anti-GFP antibody (top) and uH2A antibody (bottom). (H) Quantification of two independent experiments as shown in G. The values of experiment 1 (circles), experiment 2 (diamonds), and the mean of the two experiments (bars) shown. The values were standardized to the intensities of the corresponding band in untreated cells. Deubiquitylation of GFP-Ub–histone and uH2A followed similar kinetics.