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First published online April 1, 2009
doi: 10.1242/10.1242/jcs.040840

Journal of Cell Science 122, 1201-1210 (2009)
Published by The Company of Biologists 2009
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Cell-cycle regulation and dynamics of cytoplasmic compartments containing the promyelocytic leukemia protein and nucleoporins

Åsne Jul-Larsen1,*, Amra Grudic1,*, Rolf Bjerkvig1 and Stig Ove Bøe1,2,{ddagger}

1 Department of Biomedicine, University of Bergen, Bergen, Norway
2 Institute of Clinical Biochemistry, University of Oslo, Rikshospitalet Medical Centre, Oslo, Norway


Figure 1
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  		Fig. 1. PML colocalizes with nucleoporins in the cytoplasm. (A) HaCaT cells were labeled with anti-PML antibodies (shown in green) in combination with one of the indicated nucleoporin specific antibodies (red). Merged images reveal colocalization (yellow) between PML and nucleoporins exclusively within cytoplasmic subdomains. (B) Immunofluorescence labeling of a primary human fibroblast cell using anti-PML (red) and the nucleoporin-specific Mab414 antibody (green). PML is detected in a subset of nucleoporin-positive cytoplasmic domains.

 

Figure 2
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  		Fig. 2. Cell-cycle-dependent formation of CyPNs. (A) HaCaT cells were grown in serum-free medium for 3 days and subsequently stimulated into the cell cycle by adding serum. Cell-cycle stages were determined by laser-scanning cytometry of TO-PRO-3-labeled cells (upper panels). The percentage of cells containing CyPNs and the average number of CyPNs per cell was determined by scoring cytoplasmic foci that stained positive for PML and the nucleoporin Nup153 (bottom panels). (B) HaCaT cells were synchronized by a nocodazole block and subsequently released from the block by drug removal. At the indicated time points, cells were analyzed as in A. All results are mean±s.d. (C) Analysis of HaCaT cells by confocal laser-scanning microscopy at different stages of mitosis and early G1 phase. Nucleoporins stained by the Mab414 antibody are shown in green, PML in red and DAPI nuclear staining is blue.

 

Figure 3
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  		Fig. 3. Dynamics of CyPNs in living cells. (A) Immunofluorescence labeling of U2OS cells transiently expressing YFP-PML-III (shown in green). The nucleoporins labeled with the Mab414 antibody are shown in red. (B) Selected still images of living U2OS cells transiently expressing YFP-PML-III. Z-stacks were collected at 30 second intervals for a total period of 40 minutes. The cytoplasmic particles indicated by arrows become stably associated with the nuclear membrane once they come into contact with the nuclear surface. The complete movie is presented as supplementary material Movie 2. (C) The motility of cytoplasmic PML is inhibited by the microtubule-disrupting drug nocodazole. U2OS cells transiently expressing YFP-PML-III (shown in yellow) and mCherry (shown in gray to visualize nuclear and cytoplasmic boundaries) were subjected to live-cell imaging. Z-stacks were collected at 30 second intervals for a total period of 40 minutes. A number was assigned to each of the cytoplasmic particles in the two cells, and for each time point the shortest distance for each of these particles to the nuclear membrane was determined. The data demonstrate decreased velocity of CyPNs upon nocodazole treatment. These data were derived from supplementary material Movies 2 and 4. (D) U2OS cells transiently coexpressing CFP-PML-III and YFP-{alpha}-tubulin were recorded at 1 frame per second for 2 minutes. The selected still pictures show a CFP-PML-III-containing particle that moves along microtubule filaments. The top panels show the merged images (CFP-PML-III in green and YFP-{alpha}-tubulin in blue). The bottom panels show YFP-{alpha}-tubulin only (arrow indicates the filament attachment sites).

 

Figure 4
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  		Fig. 4. The nuclear-targeting sequence and the RING-finger domain of PML are required for a functional interaction with cytoplasmic nucleoporins. (A) Schematics of PML isoforms and mutants. The upper bar indicates the functional domains, including the RING finger (R), the two B boxes (B1 and B2), the coiled-coil domain (CC) and the nuclear-targeting sequence (N). SUMOylation sites (S) are also indicated. Mutated residues are shown in red. (B) Transient expression of His-tagged PML isoforms in U2OS cells. Cells were fixed 20 hours following transfection and subsequently processed for immunofluorescence labeling by anti-His and anti-Nup153 antibodies. 100 cells that showed positive staining of the His tag were examined for the presence of CyPNs, and the percentage of successfully transfected cells containing CyPNs is indicated below the panels. These numbers represent the average of two independent experiments ± s.d. (C) Transient expression of the different mutated forms of PML-III in U2OS cells. Cells were labeled and examined as in B. (D) Living U2OS cells expressing YFP-PML-III or YFP-PML-IIIringS. The dotted circle indicates the nuclear boundary.

 

Figure 5
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  		Fig. 5. Loss of PML causes subcellular redistribution of nucleoporins. U2OS cells were transfected with the indicated siRNAs and fixed 3 days later. PML and nucleoporins were visualized in transfected cells by immunofluorescence labeling using anti-PML and the nucleoporin-specific Mab414 antibody. Panels to the right show cell-cycle distribution of the transfected cells as determined by laser-scanning cytometry of TO-PRO-3-stained nuclei.

 

Figure 6
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  		Fig. 6. CBP and PML colocalize with MAPPs and CyPNs in mitotic and interphase HaCaT cells, respectively. Asynchronous HaCaT cells were fixed in paraformaldeyhyde and subsequently fluorescently labeled using antibodies against PML (red) and CBP (green).

 

Figure 7
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  		Fig. 7. PML-RAR{alpha} targets CyPNs. (A) U2OS cells were transfected with a PML-RAR{alpha}-expressing plasmid and fixed in paraformaldehyde 20 hours later. Immunofluorescence was then detected using the antibodies indicated. An expanded view of these images showing untransfected neighboring cells is presented in supplementary material Fig. S1. (B) NB4 cells were fixed in paraformaldehyde and fluorescently labeled using anti-PML (red) and the nucleoporin-specific antibody Mab414 (green) at the indicated time points following addition of ATRA to the cell culture medium. Some CyPNs are indicated by white arrows. The fraction of cells containing CyPNs and the average number of CyPNs per cell was determined (lower panels). Results are means±s.d.

 

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