JCS00758 Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Microstructures can move up to 0.5 mm/second. (A) Pairs of single optical section images of consecutive 1-second time points (1 and 2, 2 and 3, 3 and 4) are overlaid to show the motion of the microstructure (inset) in 1-second intervals and the arrow acts as a fiduciary marker. (B) Higher magnification of the inset region of A. Arrows indicate the direction of motion of the microstructure. Scale bar represents 1.5 mm.
• Supplemental Figure 2 - Lines scans of PML body remnants and microstructures following stress. (A) The SUMO-1 and PML signal intensities from the immunolabeling shown in Fig. 6 were measured by line scans in control cells and cells stressed by heat shock. The microstructure (M) is not detected by SUMO-1 accumulation immediately following heat shock (0 hour) but is detected after 4 hours of recovery. The position of the body remnants is indicated by ?R?. (B) Line scans similar to (A), following exposure to cadmium. The remnants are visible by both the SUMO-1 and PML signals, whereas the microstructure is not detected by SUMO-1 following exposure to cadmium.
• Movie 1 - Microstructures ‘bleb’ from parental PML bodies. Data sets were collected and processed as described for Fig. 2. The movie begins at low magnification and the arrow in Fig. 2 indicates the structure that will generate a bleb on its surface, which is released into the surrounding nucleoplasm. The movie then zooms into the region of the blebbing event and proceeds to a 45º turn on the X-Y axis and a 45º turn on the Z axis to show that the bleb is being produced from the structure and not arising from the aggregation of molecular PML.
• Movie 2 - Mobile microstructures escape from sub-nuclear compartments to become trapped in other sub-nuclear domains. HEp-2 cells stably expressing GFP-PMLI were heat shocked for 30 minutes at 43ºC and single optical sections were collected at 6 second intervals. This movie contains 80 frames, playing at a rate of 8 frames per second and shows the irregular oscillatory motion of PML microstructures (arrow, Fig. 3) within a confined compartment, and its escape to other domains
• Movie 3 - Microstructures are mobile in the nucleoplasm of heat shocked cells. 3D data sets were acquired as previously described and compiled into movies. Each data set in this example is 10 seconds apart and depicts the movement of microstructures within the nuclear volume.
• Movie 4 - Mobile microstructures are present in Cd⁺² stress cells. U2OS cells stably expressing GFP-PML IV were stressed by the addition of 50 mM Cd⁺² for 2 hours. 2-D data sets of live cells were collected at 6 second intervals. Frame rate = 8 frames/second.
• Movie 5 - Mobile microstructures are present in cells expressing viral protein E1A. SK-N-SH cells were dual transfected with DSred-PML IV and GFP-E1A. 2-D data sets were collected of cells expressing both constructs, 14 hours post-transfection. Images represent the localization of DSred-PML IV. Frame rate = 8 frames/second.
• Movie 6 - Microstructure fusion to PML body remnant. SK-N-SH cells transiently expressing GFP-PML were heat shocked for 30 minutes at 43ºC and allowed to recover for 2 hours at 37ºC. Cells were again imaged at 1-second intervals and the images were than used to generate a movie. In this example, a microstructure fuses with a PML body remnant, first seen at the 6 o’clock position relative to the PML body remnant (arrow).