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First published online 29 July 2008
doi: 10.1242/jcs.031922

Journal of Cell Science 121, 2731-2743 (2008)
Published by The Company of Biologists 2008
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Dynamics of component exchange at PML nuclear bodies

Stefanie Weidtkamp-Peters1,*, Thorsten Lenser2,*, Dmitri Negorev3, Norman Gerstner1, Thomas G. Hofmann4, Georg Schwanitz1, Christian Hoischen1, Gerd Maul3, Peter Dittrich2 and Peter Hemmerich1,{ddagger}

1 Leibniz-Institute of Age Research, Fritz-Lipman-Institute, Beutenbergstr. 11, 07745 Jena, Germany
2 Institute of Computer Science, Friedrich-Schiller-University, 07743 Jena, Germany
3 The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA
4 German Cancer Research Center, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany


Figure 1
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  		Fig. 1. GFP-PML isoforms are biologically active as growth suppressors. (A) Schematic depiction of the domain structure of PML isoforms (Jensen et al., 2001Go). All PML isoforms share a common N terminus but differ in their C termini, attributable to the alternative splicing of exons 7 to 9. Protein domains of all PML isoforms include the RING finger (R), the B1 and B2 boxes, the coiled-coil motif (CC), a nuclear localization signal (NLS) and three SUMOylation sites (S). PML VI does not contain the SUMO interacting motif (SIM) within exon 7a. (B) U-2 OS cells were transiently transfected with equal amounts of plasmids expressing GFP, or GFP–PML-I, GFP–PML-II, GFP–PML-III or GFP–PML-VI. Four days after transfection the number of living cells was determined by Trypan-Blue exclusion and normalized to GFP-expressing cells. The diagram shows mean values ± s.d. of three independent experiments.

 

Figure 2
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  		Fig. 2. Exchange of PML isoforms at NBs. (A) FRAP experiments were performed on U-2 OS cells that express the indicated GFP-tagged PML isoforms by bleaching circled areas that contain an NB (pre, before bleach pulse; post, immediately after the bleach pulse) and monitoring fluorescence recovery for 20 minutes. Scale bars, 5 µm. (B) Quantification of FRAP experiments for each isoform. The graphs show mean values (± s.d. from at least 20 FRAP experiments each) as relative fluorescence intensity (RFI) after normalization to prebleach levels.

 

Figure 3
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  		Fig. 3. Diffusion behavior of PML protein isoforms outside NBs. (A) U-2 OS cell expressing GFP–PML-IV (green) merged with the respective DIC image before FCS measurement. The inset shows only the GFP signals within the nucleus (indicated by dotted line) of this cell. +, position of the FCS laser beam. Scale bar, 5 µm. (B) Mean autocorrelation data obtained from FCS-count-rate traces for GFP–PML-IV-expressing cells (solid black line). Data were fitted using an anomalous diffusion model (dashed red line). The inset graph displays a residual plot from the fit. (C) Kinetics modeling of PML NB assembly according to a diffusion-binding model. Molecules with the potential to accumulate at PML NBs move by diffusion (D) in the nucleoplasm outside NBs. Upon stochastic encounter, molecules associate and dissociate from the periphery of the NB (kon and koff, respectively) and penetrate into and out of the core of the NB (kin and kout, respectively). Dashed circle, ROI for bleaching and recovery measurements employed in FRAP experiments.

 

Figure 4
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  		Fig. 4. (A-O) Fitting of FRAP data with the diffusion-binding model. The mean values of FRAP curves for the indicated GFP-tagged proteins (blue dots) were fitted using the diffusion-binding model depicted in Fig. 3C. Fit curves are shown as solid red lines. Note that these fits also consider the individual D values of PML isoforms derived from FCS measurements.

 

Figure 5
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  		Fig. 5. Exchange of PML protein variants at NBs. (A) Schematic depiction of PML-fusion proteins used for FRAP experiments. The domain structure is as described in Fig. 1. SUMO-modifiable lysine residues at positions 65, 160 and 490 in PML are also shown. (B-F) U-2 OS cells transfected with the indicated GFP-tagged PML protein constructs were used in FRAP analyses of areas that contain NBs and fluorescence recovery was monitored for various times as indicated. Scale bars, 5 µm. In C and F, cells were co-transfected with PML IV tagged to mRFP in order to analyze the impact of wild-type PML on the exchange dynamics of PML-IV–SUMO-2K (C) and PML-RAR{alpha} (F). (G-I) Graphs show mean values (± s.d.) from at least 20 FRAP experiments each, of the indicated proteins or protein combinations as relative fluorescence intensity (RFI) after normalization to pre-bleach levels.

 

Figure 6
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  		Fig. 6. Exchange dynamics of SP100 and SP100 mutant constructs at NBs. (A) Schematic depiction of SP100 protein and mutants used for FRAP analyses. The oligomerization and PML NB targeting resides within a region spanning amino acids 29-152 of SP100 (Negorev et al., 2001Go). SP100 has a SUMOylation site at Lys297 (S) within the HP1-binding motif and a nuclear localization signal at the C-terminus (NLS). Note, that SP100 variants that lack the endogenous NLS were engineered to contain one at their N-termini (Negorev et al., 2001Go). (B) FRAP experiments were performed in U-2 OS cells that express GFP-tagged SP100 wild-type (aa 1-480) and the indicated variants, by bleaching areas that contain NBs and by monitoring fluorescence recovery for various intervals as indicated. Scale bars, 5 µm. (C,D) Quantification of FRAP experiments as shown in B. Graphs show mean values (± s.d.) from at least 20 FRAP experiments. For comparison, the FRAP curve of GFP–PML-IV at NBs was included in C.

 

Figure 7
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  		Fig. 7. Exchange dynamics of HIPK2, DAXX and BLM at PML NBs. FRAP experiments were performed in U-2 OS cells that express (A) GFP-tagged wild-type HIPK2, (B) GFP-HIPK2 in combination with mRFP–PML-IV (red), (C) the kinase-defective GFP-HIPK2(K221A) mutant in combination with mRFP–PML-IV (red), GFP-DAXX (D), and GFP-BLM (E). Areas containing HIPK2 accumulations were bleached and fluorescence recovery was recorded for various times as indicated. Scale bars, 5 µm. (F-I) Quantification of FRAP experiments as shown in A-E. Graphs show mean values (± s.d.) from at least 20 FRAP experiments each.

 

Figure 8
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  		Fig. 8. Protein exchange at PML NBs is dependent on ATP. (A) FRAP experiments were performed on the same GFP–PML-I-expressing U-2 OS cells before (37°C) or after treatment with 10 mM sodium azide/50 mM 2-deoxyglucose for 1 hour (37°C/{Delta} ATP). Similar experiments were performed at ambient temperature, without or in the presence of ATP-depleting drugs (22°C or 22°C/{Delta} ATP, respectively). Images represent maximum intensity projections from 3D time-lapse FRAP data sets. Arrows indicate bleached regions that contain a PML NB; these areas are also shown as enlarged insets. Scale bar, 5 µm. (B) FRAP experiments as described in A were performed on cells after the indicated times of ATP depletion. Fluorescence recovery after 10 minutes of the bleach pulse was quantified from 30 PML NBs (3 NBs x10 cells each) and displayed as mean ± s.d. (C) FRAP experiments as described in A were performed on cells that express the indicated GFP fusion protein after 60 minutes of ATP depletion. Fluorescence recovery at NBs after 10 minutes of the bleach pulse was quantified from ten cells each and displayed as the mean ± s.d.

 

Figure 9
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  		Fig. 9. Dynamics of component exchange at PML NBs. The Rt values of the indicated PML NB components as deduced from our kinetics-modeling approach is shown on a logarithmic seconds scale.

 

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© The Company of Biologists Ltd 2008