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First published online 16 September 2003
doi: 10.1242/jcs.00758

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Size, position and dynamic behavior of PML nuclear bodies following cell stress as a paradigm for supramolecular trafficking and assembly

¹ Programme in Cell Biology, The Hospital for Sick Children, Toronto, Canada
² Departments of Microbiology and Immunology, Physiology and Pharmacology, and Oncology, The University of Western Ontario, London Regional Cancer Centre, London, Canada

View larger version (44K): [in a new window]   Fig. 1. Immunofluorescence of stressed cells and western blot analysis of protein extracts. (A) Unstressed (Control) cells, cells heat shocked (+HS) at 42°C for 60 minutes, cells treated with cadmium 50µM (+Cd) for 2 hours and cells transfected with E1A (E1A) for 12 hours, were fixed and immunolabeled for PML. Some microstructures are indicated by arrows. (B) Unstressed cells (–HS) and cells that were heat shocked at 43°C for 30 minutes (+HS) were immunolabeled for PML and HSP70. Note the increased levels of HSP70 and the presence of microstructures in cells that were heat shocked. (C) Western blot analysis of protein extracts made from control (–HS) cells and cells heat shocked at 43°C for 30 minutes (+HS). Arrow indicates the specific bands corresponding to HSP70. Lower band is of non-specific activity, indicating that the increase in HSP70 is not due to unequal loading. Full image widths of micrographs correspond to 15 µm.

View larger version (27K): [in a new window]   Fig. 2. Microstructures form as a result of fission events from PML bodies. (A) HEp-2 cells stably expressing GFP-PML were used to measure the mean fluorescence signal intensity (arbitrary units) of GFP in PML bodies prior to heat shock (pre), and in PML body remnants immediately following heat shock at 43°C for 30 minutes (post), and up to 30 minutes into the recovery phase following the heat shock. Measurements from single optical slices (not deconvolved) for five PML bodies and nucleoplasmic background (x) are shown. Error bars indicate the measurement error, defined as the range obtained from three separate measurements of each object after subtraction of its corresponding background. Between 30% and 70% of the GFP-PML signal is lost as a result of the stress. Recovery of the signal begins to occur following the stress. (B) 3-D reconstruction of a PML-containing microstructure budding from a parental PML body. U2OS cells stably expressing GFP-PML IV were heat shocked in an environmental chamber on the stage of the microscope. Z stacks of images were collected consecutively (10 seconds/stack capture time). I is a low magnification image of the entire nucleus prior to budding of a microstructure (full field width corresponds to 12 µm). The PML body that will bud is indicated (arrow). Panels II-IV are high magnification images of the PML body shown in I, at time points through the budding process (see Movie 1, http://jcs.biologists.org/supplemental/).

View larger version (37K): [in a new window]   Fig. 3. Microstructures are mobile within stressed nuclei. (A) The movement of a PML-containing microstructure was characterized in a nucleus of a HEp-2 cell stably expressing GFP-PML I after 1 hour recovery period following heat shock at 43°C for 30 minutes (a movie showing the movement of this microstructure, which is contrast enhanced in A can be seen in Movie 2, http://jcs.biologists.org/supplemental/). (B) Its position was plotted at each time interval (18 seconds, every third frame of the movie), indicated by white diamonds. (C) The cumulated distance of its travel is plotted as a function of time. The total time recorded was 24x18=432 seconds. Three putative domains or regions (I-III) are indicated in both (B) and (C). (D) Mean squared displacement was calculated (Chubb et al., 2002) for slow moving (circles, n=37) and fast moving microstructures (squares, n=10) and plotted against t. The initial slope of the curve was used to determine diffusion constants. Both curves plateau indicating that the diffusion of both sets of microstructures is constrained. 1 pixel2 corresponds to 0.03 µm2.

View larger version (118K): [in a new window]   Fig. 4. Correlative fluorescence and electron spectroscopic imaging microscopy of heat shocked cells. (A) Fluorescence images of six serial physical sections were obtained by ultramicrotomy (thickness approximately 60 nm) of cells labeled by immunofluorescence against PML protein (yellow). The sections are numbered 1-6. The PML body remnant indicated by the arrow in the low magnification image of section 1 (left) is also represented in the zoomed and rotated image of the same section (right) (the zoomed image was rotated approximately 230°). The light blue structures are nucleoli. A single microstructure (a) is detected primarily in section 2 only. (B) Nitrogen map obtained by electron spectroscopic imaging of section 2 (in A). The upper PML body remnant indicated by the arrow is the same remnant indicated in A. Two nucleolar domains can be seen at the left and right of the field (upper panel). A second PML body remnant, below the one indicated by the arrow, is in the center of the box region. This region is shown at higher magnification in the lower panel, where the remnant is indicated by an arrowhead. (C) High magnification phosphorus (P) and nitrogen maps (N) of the PML body indicated by the arrowhead in the lower section of B. Comparison of the directions of the arrowheads in B and C indicate the relative rotation of the images in these panels. A microstructure labeled `a' in the merged P and N maps is the same microstructure referred to in the fluorescence image of section 2 in A. Microstructures were identified by triangulation by center-to-center measurements of PML-containing structures in the fluorescence images, relative to other fiduciary objects, such as nucleoli, with comparative measurements made with low, medium and high magnification electron spectroscopic images. Outlines of large open channels devoid of chromatin are shown in the phosphorus map, and a more concentrated region of chromatin fibers is indicated by am asterisk. A microstructure in the open region would be expected to be more mobile than one trapped in the region of higher chromatin fiber concentration. Scale bar (shown in B) represents 1100 (upper) and 700 (lower) nm in B, and 400 nm in C.

View larger version (48K): [in a new window]   Fig. 5. PML-containing microstructures fuse with each other and PML body remnants during recovery from cell stress. (A) Optical sections of cells were obtained by deconvolution and projected into a single plane. Cells were imaged before heat shock (Pre), immediately following a 30-minute heat shock at 43°C (Post) and at 5-minute intervals for 30 minutes. In the nucleus shown, arrows indicate PML bodies, and their remnants, before and after heat shock, respectively. The squares and rectangles indicate regions shown at higher magnification in B to demonstrate the movement and fusion of microstructures. (B) High magnification images the specified time points (P represents post-heat shock, 5 represents 5 minutes, etc.). Arrowheads indicate pairs of microstructures that eventually fuse during this time course (15 minutes (square), 20 minutes (rectangle)). (C) Microstructures fuse with parental PML bodies in cells recovering from heat shock. U2OS cells stably expressing GFP-PML IV were heat shocked as previously described and allowed to recover for 30 minutes at 37°C. Z-stacks were collected consecutively (13 second/stack capture time). (I) A recovering nucleus with a mobile microstructure (arrow). The following high magnification images demonstrate the microstructure approaching the surface of the PML body (II), making contact with the surface (III) and disappearing into the core of the body (IV). Multiple events identical to this example were observed. In each event, the fusion was observed from many angles and the microstructure is never seen again in the surrounding nucleoplasm, demonstrating that the microstructure did merge with the PML body remnant. (D) PML body position and relative size are conserved. A HEp-2 nucleus of a cell expressing GFP-PML was imaged before heat shock (pre-HS), immediately following heat shock for 30 minutes at 43°C (post-HS) and at hourly intervals. The relative sizes of the PML bodies, as determined by measurements of the integrated signal intensity of the bodies or remnants (1-11) were determined. The images were exposed so that the GFP signal did not saturate the detector; under these conditions, most PML-containing microstructures are not visible in the images. The rank order of size of the bodies from largest to smallest are indicated. At 3 hours, the original rank order (Pre) has been re-established, except the eighth largest does not fully recover, dropping to the tenth position.

View larger version (102K): [in a new window]   Fig. 6. Co-localization of PML and SUMO-1. (A) PML, and SUMO-1 were immunolabeled in control cells (–HS), cells heat shocked for 30 minutes at 43°C (0H), after a recovery period of 4 hours (4H) or immediately following Cd+2 stress (50 µM for 2 hours). PML bodies or PML body remnants are indicated by arrows, and PML-containing microstructures are indicated by arrowheads. (B) All 8 PML bodies in –HS were analyzed. Quantification of these structures is presented in Tables 1 and 2; the bodies, remnants or microstructures described in the tables correspond to the indicated structures, numbered in order from top to bottom in the respective image.

View larger version (57K): [in a new window]   Fig. 7. GFP-SUMO-1 expression in heat stressed and Cd+2 stressed cells. SK-N-SH cells transiently expressing GFP-SUMO-1 were heat shocked for 30 minutes at 43°C or U2OS cells stably expressing GFP-PML IV treated with 50 µM Cd+2 were immunolabeled with antibodies against PML. Cells expressing GFP-SUMO-1 are indicated with large arrows. PML bodies or PML body remnants are indicated with small arrows, and microstructures with arrowheads.

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