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First published online September 18, 2007
doi: 10.1242/10.1242/jcs.007492

Journal of Cell Science 120, 3219-3227 (2007)
Published by The Company of Biologists 2007
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A nucleolar targeting signal in PML-I addresses PML to nucleolar caps in stressed or senescent cells

Wilfried Condemine*, Yuki Takahashi*, Morgane Le Bras and Hugues de Thé{ddagger}

CNRS/Université de Paris 7 UMR7151, Equipe labellisée par la Ligue Contre le Cancer, Hôpital St. Louis, 1 Av. C. Vellefaux 75475, Paris Cedex 10, France


Figure 1
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  		Fig. 1. (A,B) PML is targeted to the nucleolus upon cellular stresses. Confocal images of MRC5 human fibroblasts treated with 35 J/m2 UVC radiation (1 hour), 2 µg/ml doxorubicin (Doxo) for 24 hours, 5 nM actinomycin D (ActD) for 16 hours, 10 µM MG132 for 16 hours, or 10 Gy ionizing radiation {gamma} (IR-{gamma}) (1 hour). Immunofluorescence was performed using antibodies against PML, B23, fibrillarin or UBF, as indicated. (B) Summary of the localization patterns observed in Pml–/– MEFs expressing PML isoforms -I, -II, -III, -IV or -V, and treated as indicated. NBs, nuclear bodies. (C) PML-I targets nucleolar caps upon doxorubicin treatment. MRC5 cells were transfected with PML-I specific or control siRNA and treated with doxorubicin for 24 hours. Immunofluorescence was then performed using Pan-PML or PML-I-specific sera, as indicated. Nuclei were stained with DAPI.

 

Figure 2
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  		Fig. 2. Specific PML isoform C-terminus encodes a nucleolar targeting domain. (A) Plasmids encoding the various nls-GFP fusion proteins (top) were transfected into human osteosarcoma cells, SaOS, which were observed 24 hours later. GFP-PML fusion proteins appear in green, nucleolar markers UBF, fibrillarin, B23 and RNA (revealed by BET), in red. (B) The presence of a inhibitor domain SIM in the C-terminus of PML-I. Different PML-I C-terminal fragments are fused to nls-GFP to estimate their nucleolar targeting property. Results are reported in the diagram. (C,D) Characterization of the targeting of the PML-IV-specific terminus (C) and the PML-V-specific terminus (D). The PML sequence is indicated by the positions of the residues on the original isoforms. Staining as in A. (E) Amino acid sequences of the regions required for nucleolar association in PML-I (607-619 and 865-882), PML-IV (607-633) and PML-V (571-611).

 

Figure 3
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  		Fig. 3. Characterization of nucleolus-associated PML structures. (A-H) Senescence-associated nuclear bodies. (A,B) Phase contrast images of WI-38 cells transduced with GFP-PML-I (see Movie 1 in supplementary material). (C-H) Untransfected WI-38 cells labelled for the following proteins: PML (green) or Sp100, DAXX, SUMO-1 and 2, Brca1 and Nbs1 (red). (I-P) Nucleolar components in SANB. (I-L) PML (green). RNA, UBF, Fibrillarin and B23 appear in red. (M,N) RNA pol I associated transcriptional activity in SANB. PML appears red and BrU green. (O,P) PML budding structures from the nucleolus. PML appears green and the nucleolus (fibrillarin) red. (Q) Biopsies from skin cancers were analysed with PML antisera. PML localises in the nucleolus (white arrow). (R,S) Senescent Idh4 cells exhibit SANB-like bodies. PML (green) and B23 or ubiquitin (red). White filled arrowhead, SANB-containing nucleolar components; open and green arrowheads, nucleolar-free SANB not in contact with nucleolus. All the images were obtained using photon microscopy, except images M-N and S, which were obtained with confocal microscopy.

 

Figure 4
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  		Fig. 4. SANB composition varies with the advancement of senescence. (A) Progression of senescence [dividing cells (A) to late senescent cells (E)] is associated with a decrease in the nucleolar component, fibrillarin, but an increase in poly-ubiquitin conjugates. Fibrillarin is red, ubiquitin appears green and PML blue. (B) Bar graph representing the percentage of WI-38 cells with SANB depending on the state (dividing/senescent) of the cells. The columns represent the average of three independent experiments.

 

Figure 5
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  		Fig. 5. Inhibitors link PML bodies to protein degradation and trafficking. Top four panels: Primary WI-38 cells were treated with either 10 µM MG132 or 1 µM LMB or both for 2 hours, allowing co-localization of ubiquitin (green) and PML (red). LMB panel is a Z-projection of a confocal stack acquisition. Bottom panel: 3D reconstruction of deconvoluted images.

 




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