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. 2010 Mar 5;3(1):3.
doi: 10.1186/1757-5036-3-3.

Assembly dynamics of PML nuclear bodies in living cells

Peter Brand  1 , Thorsten Lenser, Peter Hemmerich
Affiliations

Assembly dynamics of PML nuclear bodies in living cells

Peter Brand et al. PMC Biophys. .

Abstract

The mammalian cell nucleus contains a variety of organelles or nuclear bodies which contribute to key nuclear functions. Promyelocytic leukemia nuclear bodies (PML NBs) are involved in the regulation of apoptosis, antiviral responses, the DNA damage response and chromatin structure, but their precise biochemical function in these nuclear pathways is unknown. One strategy to tackle this problem is to assess the biophysical properties of the component parts of these macromolecular assemblies in living cells. In this study we determined PML NB assembly dynamics by live cell imaging, combined with mathematical modeling. For the first time, dynamics of PML body formation were measured in cells lacking endogenous PML. We show that all six human nuclear PML isoforms are able to form nuclear bodies in PML negative cells. All isoforms exhibit individual exchange rates at NBs in PML positive cells but PML I, II, III and IV are static at nuclear bodies in PML negative cells, suggesting that these isoforms require additional protein partners for efficient exchange. PML V turns over at PML Nbs very slowly supporting the idea of a structural function for this isoform. We also demonstrate that SUMOylation of PML at Lysine positions K160 and/or K490 are required for nuclear body formation in vivo.We propose a model in which the isoform specific residence times of PML provide both, structural stability to function as a scaffold and flexibility to attract specific nuclear proteins for efficient biochemical reactions at the surface of nuclear bodies.MCS code: 92C37.

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Figures

Figure 1
Figure 1
All PML protein isoforms form nuclear bodies in cells lacking endogenous PML. (A) Schematic depiction of the domain structure of PML isoforms (data taken from [16]). All PML isoforms share a common N terminus (exons 1 to 6) but differ in their C termini due to alternative spilicing of exons 7 to 9. Numbers indicate exons. Stars indicate retained intron sequences. The postion of three SUMO-modifyable Lysin (K) residues are indicated. Note that PML VI does not contain and the SUMO-interacting motiv (SIM) present in the other isoforms. (B) Mouse 3T3 cells with (3T3-PML+/+) or without (3T3-PML-/-) endogenous PML expression were transfested with expression vectors encoding human PML isoforms I to VI as GFP fusion proteins. Cells on coverslips were fixed and processed for immunofluorescence staining to detect endogenous mouse PML protein (red) and the exogenous GFP-tagged human PML isoforms (green). Images show mid-nuclear confocal sections. Note that the anti-mouse-PML antibody does not cross-react with human PML. Bar, 5 μm.
Figure 2
Figure 2
FRAP to determine exchange of PML isoforms at nuclear bodies. (A) FRAP experiment of GFP-tagged PML II in 3T3-PML+/+ cells. Fluorescence was bleached in circled areas containing a nuclear body as indicated. Image stacks of the whole nucleus were taken before (pre) and after (post) the bleach pulse and at different time points thereafter. The upper row shows the projection of the whole nucleus while the bottom row shows an enlarged view of the bleached nuclear body indicated by a white box. (B and C) Same experiment as in (A) using 3T3-PML-/- cells. In one cell population fluorescence recovery was observed with kinetics similar to PML wild type cells (B) while in another fraction of cells no recovery of GFP-PML II at nuclear bodies was oberved (C). Bar, 5 μm.
Figure 3
Figure 3
Quantitation of FRAP experiments. FRAP experiments as described in Figure 2 were performed for all GFP-PML isoforms in PML positive (black curves) and PML negative cells (red curves) as indicated. A second mobility population of PML negative cells was observed for GFP-PML II and IV (blue curves). The graphs show the mean values from at least 20 FRAP evaluations as relative fluorescence intensity (RFI) after normalization to prebleach levels. Standart deviations (not shown) ranged between 10 to 15 percent of the mean values.
Figure 4
Figure 4
Kinetic model for molecule exchange at PML nuclear bodies. (A) Molecules with the potential to interact with PML nuclear bodies move by diffusion in the nucleoplasm outside nuclear bodies. Upon stochastic encounter, molecules associate and dissociate from the periphery of the nuclear body (kon and koff, respectively) and penetrate into and out of the shell of the nuclear body (kin and kout, respectively). (B-O) Fitting of the measured FRAP curves (blue dotted line) with the binding-diffusion model shown in (A) results in good fits (red lines).
Figure 5
Figure 5
PML SUMOylation sites K160 and K490 are required for PML body formation. The upper part shows a FRAP experiment of GFP-tagged mutant PML IV (K160/490R) in 3T3-PML-/- cells. Fluorescence was bleached in a circled area within the nucleoplasm (red circle). Confocal images were taken before (pre) and after (post) the bleach pulse and at different time points thereafter. Bottom part: FRAP experiments of GFP-tagged PML IV (K160/490R) were performed in 3T3-PML+/+ (black) and 3T3-PML-/- cells (red) and quantified. FRAP curves show mean values of at least ten measurements.
Figure 6
Figure 6
Assembly dynamics of subnuclear domains. The residence times of components of the indicated subnuclear domains are depicted on a logarithmic scale between 1 second and 1 hour. Fast or slow exchanging components provide flexibility or stabilty, respectively, probably in terms of both structue and function of subnuclear domains. Residence times of speckle, nucleolus and Cajal body compoents were assessed from FRAP experiments published previously [32-39]. Residence times of PML nuclear body components were taken from [25].
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