Assembly dynamics of PML nuclear bodies in living cells
© Brand et al 2010
Received: 15 September 2009
Accepted: 5 March 2010
Published: 5 March 2010
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
The cell nucleus is functionally devoted to the realization and protection of the genetic material it contains in the form of chromosome territories . RNA transcription and processing, DNA replication and DNA repair occur in a spatio-temporal coordinated fashion in small, usually less than 100 nm large foci scattered throughout the nuclear volume [2–4]. In addition, the mammalian cell nucleus contains a variety of internal structures, also termed domains or bodies . These macromolecular assemblies include nucleoli, speckles, Cajal bodies, and promyelocytic leukemia nuclear bodies (PML NBs) [6, 7]. While the structure and function of nucleoli, which is mainly ribosomal RNA synthesis and ribosome biogenesis, is very well understood, the precise biochemical function of speckles, Cajal bodies or PML nuclear bodies is not known . With the exception, again, of the nucleolus which builds on ribosomal RNA genes, it also remains elusive if and how the other nuclear domains are spatially and functionally related to sites of transcritpion, replication, DNA repair, or how they relate to specific genomic regions .
PML nuclear bodies, also known as nuclear domain 10 (ND10) are macromolecular protein assemblies in the nucleus of mammalian cells. They have been implicated in key cellular functions including cell cycle progression, the DNA damage response, transcriptional regulation, viral infection, and apoptosis, however the precise biochemical functions of PML NBs in these processes is not known [8, 9]. PML NBs range in size from 0.2 μm to 1.2 μm in diameter . The number and distribution of PML NBs varies considerably depending on cell type, cell cycle and cell condition, but typically between 10 and 20 PML NBs can be found per nucleus . Electron and 4Pi-microscopy revealed a ring-like shape of PML NBs under normal growth conditions with an 50 to 100 nm thick proteinacous outer shell . The core of PML NBs was found either free of protein, DNA, or RNA accumulations [10–12], or to contain specific SUMO isoforms or specific chromatin subregions . Chromatin threads and RNA in direct contact with the surface of the bodies might help to stabilize nuclear body structure [13, 14].
The formation of PML nuclear bodies relies primarily on the self-assembly abilities of the N-terminal RBCC domain in PML, and its SUMOylation status [15, 19, 20]. PML as well as other PML NB components, such as Sp100 and Daxx contain a SUMO interacting motif (SIM) with which these proteins can bind SUMO noncovalently . Binding of proteins to PML nuclear bodies can therefore be modulated by noncovalent interactions between the SUMO moieties and SIMs of PML-interacting components [22, 23].
PML nuclear bodies may be directly involved in biochemical reactions in the cell nucleus by modulating chromatin structure, regulating transcription of specific genes, sequestering of specific nuclear proteins, and/or mediating posttranslational modifications of specific target proteins . Inherent to all these models is the question if PML NB components function directly within this structure or somewhere outside at different intra-nuclear sites, or both. A regulated network traffic between these sites may constitute a potential control mechanism with PML at its core, as suggested very early .
In order to reveal and study such mechanisms we have previously assessed the biophysical properties of PML nuclear body components and the assembly dynamics of these macromolecular domains in nuclei of living human cells using fluorescence correlation spectroscopy, fluorescence recovery after photobleaching and mathematic modeling . These analyses uncovered a kinetic model for factor exchange at PML nuclear bodies and highlighted potential mechanisms to regulate intra-nuclear trafficking of PML NB components. To further characterize the assembly of PML nuclear bodies we now performed biophysical analyses in living mouse cells lacking endogenous PML proteins. This allowed us to study the nuclear body formation abilities of individual PML isoforms in a live cell setting.
Cell Culture and Transfection
Mouse 3T3-PML+/+ and 3T3-PML-/- cells , kindly provided by T. Hofmann (DKFZ Heidelberg) were cultured in Dulbecco' modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum in a 10% CO2 atmosphere at 37°C. For live cell imaging experiments, cells were seeded on 42 mm glass dishes (Saur Laborbedarf, Reutlingen, Germany) and transfected with plasmid DNA one to two days before observation using FuGENE-HD Transfection reagent (Roche, Basel, Switzerland) according to the manufacturer' protocol.
The GFP-PML expression constructs have been described in detail previously .
Whole cell extracts were produced from transiently or stably transfected cell lines, electrophoresed on SDS-PAGE and transferred to Protran nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany). The membrane was incubated with primary antibodies (in PBS-T) and developed with a peroxidase conjugated secondary species-specific antibody (Jackson Immunoresearch, West Grove, PA, USA). Signal was detected using the ECL reagent (Amersham, Uppsala, Sweden) on imaging film (Biomax, Kodak, Stuttgart, Germany). Anti-GFP monoclonal antibody was from Santa-Cruz Biotechnology (Heidelberg, Germany). Anti-mouse PML monoclonal antibody (# 05-718), non-cross reactive with human PML protein, was purchased from Upstate.
Immunocytochemistry and Microscopy
Cells grown on 15 mm diameter coverslips were fixed with 4% formaldehyde for 10 minutes and permeabilized with 0.25% Triton-X100 for 3 minutes. Diluted anti-mouse PML mAB was incubated on cells for 45 minutes. After 3 washing steps with PBS, an anti-mouse secondary antibody coupled to Cy3 (Jackson Immunosearch, West Grove, USA) was incubated on cells for 45 minutes, followed by a DNA-staining step using ToPro3 or DAPI (Invitrogen, Carlsbad, USA) for 10 minutes and mounting with Prolong Gold antifade mounting medium (Invitrogen, Carlsbad, USA). For microscopy, a LSM 510Meta or LSM710 laser scanning confocal microscope (Carl Zeiss, Jena, Germany) was used.
Fluorescence Correlation Spectroscopy Measurements
Fluorescence correlation spectroscopy (FCS) measurements were performed at 37°C on a LSM 510Meta/ConfoCor2 combi system using a C-Apochromat infinity-corrected 1.2 NA 40× water objective (Carl Zeiss, Jena, Germany) as described in detail elsewhere . Briefly, GFP-tagged proteins were spot-illuminated with the 488 nm line of a 20 mW Argon laser at 5.5 Ampere tube current attenuated by an acousto-optical tunable filter (AOTF) to 0.1%. The detection pinhole had a diameter of 70 μm and emission was recorded through a 505-530 nm band-path filter. For the measurments, 10 × 30 time series of 10 s each were recorded with a time resolution of 1 μs and then superimposed for fitting to an anomalous diffusion model in three dimensions with triplett function  using Origin Software (OriginLab, Northhampton, MA, USA). The diffusion coefficients and anomaly parameters were extracted from fit curves as previously described .
Fluorescence Recovery after Photobleaching
Fluorescence Recovery after Photobleaching (FRAP) experiments were carried out on a Zeiss LSM 510Meta confocal microscope (Carl Zeiss, Jena, Germany). One or two image stacks were taken before the bleach pulse and 50-70 image stacks after bleaching of "regions of interest" (ROIs) containing one nuclear body each at 0.05% laser transmission to minimize scan bleaching. Image aquisition frequency was adapted to the recovery rate of the respective GFP fusion protein, usually a 20 second interval was applied. The pinhole was adjusted to 1 airy unit. The image stacks were maximum-projected into a single plane from which relative fluorescence intensities within the ROIs were quantitated according to  using Excel (Microsoft, Redmond, WA, USA) and Origin software (OriginLab, Northhampton, MA, USA).
The differential equations were numerically solved using an explicit Runge-Kutta formula (method ode45 in MATLAB). To fit the parameters of the model, an Evolution Strategy with Covariance Matrix Adaptation (CMA-ES) was employed .
We determined the ratio p = 20 of steady state fluorescence in the body vs. the background by confocal microscopy of GFP-PML isoforms and pixel intensity evaluation using MetaMorph software (Molecular Devices, Sunnyvale, USA).
These values were used to normalize the concentrations used in the model. The mathematical model treats normalized concentrations of fluorescent molecules in free diffusion (x), loosely bound (y) and tightly bound (z) to the PML body.
This enabled us to remove one degree of freedom, kon, from the model.
3 Results and Discussion
3.1 All PML isoforms form nuclear bodies in PML-/- cells
We had previously analyzed the dynamics of component exchange at PML nuclear bodies in human cells expressing endogenous PML proteins . The objective of the current study was to study the formation of PML NBs in the absence of endogenous PML expression. All six human PML isoforms (Fig. 1A) were therefore expressed as GFP fusion proteins in mouse 3T3 control cells (3T3-PML+/+) or mouse 3T3 cells derived from PML knock-out mice (3T3-PML-/-). All GFP-PML constructs are functional in human cells  and were expressed as full-length proteins in 3T3 cells, as judged by western-blotting (data not shown).
Immunofluorescence analyses showed that all six human GFP-PML isoforms localized to enogenous PML nuclear bodies in 3T3-PML+/+ mouse cells (Fig. 1B). Importantly, in 3T3-PML-/- mouse cells, each individual human GFP-PML isoform was able to form nuclear bodies (Fig. 1B). This confirmed that the nuclear body formation ability of PML resides within sequences encoded by exons 1 to 6, represented by GFP-PML VI, and suggests that the C-terminal extensions of PML isoforms (exons 7 to 9) do not alter this function (Fig. 1B). Because GFP-PML VI which does not contain the SUMO-interacting motiv (SIM) (Fig. 1A) is still able to form nuclear bodies in the absence of endogenous PML bodies we conclude that a SIM is not essential for nuclear body formation by PML as previously suggested .
3.2 PML isoform specific binding properties at nuclear bodies
Human cells contain six nuclear PML isoforms, whereas for mouse cells only two PML transcripts have been described so far, and the corrsponding mouse PML sequences are more than 80% similar to human PML isoform I . It should therefore be pointed out that PML isoforms II to VI have no direct counterparts in the murine system with respect to alternative expression of exons 6 to 9. Nevertheless, the analysis of these human isoforms in mouse cells may still deliver valuable information on PML nuclear body formation, particularly in a PML-negative background. Based on the high sequence similarity the human GFP-PML I construct has the potential to functionally (and thus dynamically) act in a similar fashion as mouse PML in murine cells. Indeed, the exchange dynamics of human GFP-PML I protein at nuclear bodies of PML-positive murine cells was similar to human cells (Fig. 3A) . In contrast, GFP-PML I was alomst immobile at nuclear bodies in 3T3 cells lacking endogenous PML (Fig. 3A). The same phenomenon was observed for GFP-PML isoform III and in a subpopulation of cells expressing GFP-PML isoforms II or IV (Fig. 3, B-D). Thus, in the absence of endogenous PML proteins, GFP-PML I to IV form stable aggregates. These observations suggest that these isoforms require the presence of other PML isoforms or endogenous PML-binding proteins for efficient exchange at nuclear bodies. They also suggest that the capacity to contribute to nuclear body stability is inherent to all of these PML isoforms. We also observed a minor population (< 20%) of cells in which GFP-PML isoforms II and IV showed dynamic exchange at nuclear bodies (Fig. 3B, and 3D, red curves). These observations suggest a cell cycle dependent behavior of PML II and PML IV at nuclear bodies which likely originate from interaction of these isoforms with as yet unknown binding sites outside nuclear bodies. However, since human PML II and PML IV are not conserved in mouse cells, such interactions might be biologically non-relevant. In contrast to GFP-PML I to IV, the dynamics of GFP-PML V and VI were almost unaltered in PML negative cells (Fig. 3E, and 3F).
The presence of slow exchanging populations of GFP-PML I to IV is consistent with the idea that these isoforms are also able to provide nuclear body stability, as concluded for PML V. In human cells, GFP-PML I and III exhibit much higher exchange rates at nuclear bodies  than observed here in mouse cells (Fig. 3).
That GFP-PML I and III do not exchange with soluble nucleoplasmic populations in mouse cells may indicate that the human isoforms can not be dissociated from nuclear bodies through interaction with soluble mouse PML-binding proteins outside nuclear bodies. Since the SUMOylation status of PML also regulates the exchange rate at nuclear bodies , changing SUMO patterns on GFP-PML I to IV may also explain their changing exchange rate at NBs.
3.3 A kinetic model to quantitatively describe PML nuclear body assembly
Exchange dynamics of PML isoforms at nuclear bodies
Compared to PML-positive cells, the residence time at nuclear bodies of GFP-tagged PML isoforms I to IV was increased several-fold in PML-negative cells (Table 1). This result suggests that the exchange rate of overexpressed human PML isoforms is influenced by the dynamics of the endogenous mouse PML proteins. Thus, in the absence of endogenous mouse PML protein, the unrelated human isoforms tend to form more insoluble aggregates. Interestingly, this is not true for the shortest PML isoforms V and VI, the residence time of which is almost identical independent of the presence or absence of endogenous PML bodies (Table 1). This individual property of GFP-PML V and VI argues in favor of a more structural role for these isoforms at PML nuclear bodies.
3.4 SUMOylation of PML is required to form nuclear bodies
3.5 Assembly properties of PML bodies are different from other subnuclear domains
Four main mechanisms are known through which cellular scaffolds can modify signalling between active components . They can (i) tether enzymes close in space and enhance effective local concentrations, (ii) mediate assembly of signalling complexes in a combinatorial manner, (iii) dynamically regulate turnover or accessibility of specific factors, or (iv) modify the conformation of enzymes binding to them . All these potential functions are fully compatible with the biophysical properties of PML nuclear bodies assessed in this report and previously . The scaffold model for PML body function is also compatible with the biochemistry (phosphorylation, SUMOylation, acetylation) believed to occur on specific nuclear proteins at these macromolecular assemblies . Interestingly, although direct evidence is lacking so far, PML NBs have recently been suggested as scaffolds for caspase-2 mediated cell death . Future research should therefore aim to establish new experimental approaches with which the potential function of PML nuclear bodies as nuclear scaffolds can be tested in a more direct and functional way.
We thank S. Ohndorf and M. Koch for plasmid purification. We are grateful to Thomas Hofmann for providing the PML-positive and negative 3T3 mouse cells. T.L. thanks Bashar Ibrahim for fruitful discussions and the EU (ESIGNET, project no. 12789) for financial support. This work was supported by grant HE 2484/3-1 from the Deutsche Forschungsgemeinschaft.
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