Three-Dimensional organization of promyelocytic leukemia nuclear bodies

Division of High Resolution Optical Microscopy, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany.
Journal of Cell Science (Impact Factor: 5.43). 02/2010; 123(Pt 3):392-400. DOI: 10.1242/jcs.053496
Source: PubMed


Promyelocytic leukemia nuclear bodies (PML-NBs) are mobile subnuclear organelles formed by PML and Sp100 protein. They have been reported to have a role in transcription, DNA replication and repair, telomere lengthening, cell cycle control and tumor suppression. We have conducted high-resolution 4Pi fluorescence laser-scanning microscopy studies complemented with correlative electron microscopy and investigations of the accessibility of the PML-NB subcompartment. During interphase PML-NBs adopt a spherical organization characterized by the assembly of PML and Sp100 proteins into patches within a 50- to 100-nm-thick shell. This spherical shell of PML and Sp100 imposes little constraint to the exchange of components between the PML-NB interior and the nucleoplasm. Post-translational SUMO modifications, telomere repeats and heterochromatin protein 1 were found to localize in characteristic patterns with respect to PML and Sp100. From our findings, we derived a model that explains how the three-dimensional organization of PML-NBs serves to concentrate different biological activities while allowing for an efficient exchange of components.

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Available from: Thibaud Jegou, Jun 19, 2014
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    • "Thus, there are approximately twice as many PML-NBs in G2 as in G1 (12). Giant PML-NBs may appear under specific disease conditions, and they are shaped as spherical shells more than 1 μm in diameter in G2 phase (13). Promyelocytic leukemia nuclear bodies partition during mitosis and are reestablished in G1 phase (14, 15). "
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    ABSTRACT: In progressive multifocal leukoencephalopathy, JC virus-infected oligodendroglia display 2 distinct patterns of intranuclear viral inclusions: full inclusions in which progeny virions are present throughout enlarged nuclei and dot-shaped inclusions in which virions are clustered in subnuclear domains termed "promyelocytic leukemia nuclear bodies" (PML-NBs). Promyelocytic leukemia nuclear bodies may serve a scaffolding role in viral progeny production. We analyzed the formation process of intranuclear viral inclusions by morphometry and assessed PML-NB alterations in the brains of 2 patients with progressive multifocal leukoencephalopathy. By immunohistochemistry, proliferating cell nuclear antigen was most frequently detected in smaller nuclei; cyclin A was detected in larger nuclei. This suggests an S-to-G2 cell cycle transition in infected cells associated with nuclear enlargement. Sizes of PML-NBs were variable, but they were usually either small speckles 200 to 400 nm in diameter or distinct spherical shells with a diameter of 1 μm or more. By confocal microscopy, JC virus capsid proteins were associated with both small and large PML-NBs, but disruption of large PML-NBs was observed by ground-state depletion fluorescence nanoscopy. Clusters of progeny virions were also detected by electron microscopy. Our data suggest that, in progressive multifocal leukoencephalopathy, JC virus produces progeny virions in enlarging oligodendrocyte nuclei in association with growing PML-NBs and with cell cycle transition through an S-to-G2-like state.This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License, where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially.
    04/2014; 73(5). DOI:10.1097/NEN.0000000000000066
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    • "As demonstrated in Fig. 2 and reported previously (Cuchet et al., 2011), the individually expressed PML isoforms formed nucleate punctate structures that were associated with SUMO modification. Previous electron microscopy studies with immunolabelling revealed that PML bodies are ring shaped (Boisvert et al., 2000; Koken et al., 1994; Lallemand-Breitenbach et al., 2001) and more recent work using high resolution microscopy (Hattersley et al., 2011; Lang et al., 2010) confirmed that PML bodies consist of a spherical shell of PML, with SUMO1 and SUMO2/3 found predominantly interspersed within the PML shell and in the central core of the structure, respectively. We investigated the structure of the PML bodies formed by the individual isoforms and the changes in structure induced by arsenic treatment using three-dimensional structured illumination microscopy (3D SIM) to obtain super-resolution images. "
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    ABSTRACT: Arsenic is a clinically effective treatment for acute promyelocytic leukaemia (APL) in which the promyelocytic leukaemia (PML) protein is fused to retinoic receptor alpha (RARα). PML-RARα is degraded by the proteasome via a SUMO-dependent, ubiquitin-mediated pathway in response to arsenic treatment, curing the disease. Due to alternative splicing, six major PML isoforms are expressed, each of which encodes a unique C-terminal region. Using a system in which only a single EYFP-linked PML isoform is expressed, we demonstrate that PML I, II and VI accumulate in the cytoplasm following arsenic treatment, whereas PML III, IV and V do not. 3D structured illumination was employed to obtain super-resolution images of PML bodies, revealing spherical shells of PML along with associated SUMO. Arsenic treatment results in dramatic and isoform specific changes to PML body ultrastructure. After extended arsenic treatment most PML isoforms are degraded, leaving SUMO at the core of the nuclear bodies. A high content imaging assay identifies PML V as the isoform most readily degraded following arsenic treatment, and PML IV as relatively resistant to degradation. Immunoprecipitation analysis demonstrates that all PML isoforms are SUMO and ubiquitin modified after arsenic treatment and by using siRNA we demonstrate that arsenic induced degradation of all PML isoforms is dependent on the ubiquitin E3 ligase RNF4. Intriguingly, depletion of RNF4 results in marked accumulation of PML V, suggesting this isoform is an optimal substrate for RNF4. Thus the variable C-terminal domain influences the rate and location of degradation of PML isoforms following arsenic treatment.
    Journal of Cell Science 11/2013; 127(2). DOI:10.1242/jcs.132290 · 5.43 Impact Factor
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    • "These modifications occur hierarchically as SUMOylation on Lys160 is a prerequisite for SUMOylation on Lys65. High-resolution microscopy enabled fascinating insights into the architecture of PML-NBs and allowed the detection of SUMO1 within aggregated spots in the PML-Sp100 protein shell (Lang et al., 2010). The SUMO1-containing patches also showed intrusions and extrusions from the PML shell. "
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    ABSTRACT: Post-translational modifications (PTMs) regulate multiple biological functions of the promyelocytic leukemia (PML) protein and also the fission, disassembly, and rebuilding of PML nuclear bodies (PML-NBs) during the cell cycle. Pathway-specific PML modification patterns ensure proper signal output from PML-NBs that suit the specific functional requirements. Here we comprehensively review the signaling pathways and enzymes that modify PML and also the oncogenic PML-RARα fusion protein. Many PTMs occur in a hierarchical and timely organized fashion. Phosphorylation or acetylation constitutes typical starting points for many PML modifying events, while degradative ubiquitination is an irreversible end point of the modification cascade. As this hierarchical organization of PTMs frequently turns phosphorylation events as primordial events, kinases or phosphatases regulating PML phosphorylation may be interesting drug targets to manipulate the downstream modifications and thus the stability and function of PML or PML-RARα.
    Frontiers in Oncology 12/2012; 2:204. DOI:10.3389/fonc.2012.00204
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