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First published online 21 February 2006
doi: 10.1242/jcs.02816

Journal of Cell Science 119, 1026-1033 (2006)
Published by The Company of Biologists 2006
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The number of PML nuclear bodies increases in early S phase by a fission mechanism

Graham Dellaire*, Reagan W. Ching*, Hesam Dehghani{ddagger}, Ying Ren and David P. Bazett-Jones§

Programme in Cell Biology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8 Canada


Figure 1
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  		Fig. 1. PML nuclear bodies increase in number during S phase. (A) Asynchronous human SK-N-SH cells were followed by DIC to determine relative cell cycle position after mitosis over an 18 hour period prior to immunolabelling for cyclin A. (B) Human U-2 OS cells stably expressing GFP-PML IV were synchronised at the G1-S border with aphidicolin and released into S phase for 2-6 hours. PML nuclear body number versus time is shown. (C) Asynchronous U-2 OS cells expressing diHcRed-PCNA (PCNA) and GFP-PML IV (PML) were observed for 2 hours by spinning-disk confocal microscopy. Images shown are compressed z-stacks. Newly replicated PML nuclear bodies are indicated by yellow arrowheads. (D) PML nuclear body doublets are present in SK-N-SH cells in mid S phase (arrowheads). Bars, 5 µm.

 

Figure 2
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  		Fig. 2. Dynamics of PML nuclear bodies and PML protein in S phase. (A) U-2 OS cells expressing GFP-PML IV were imaged for 2 hours in early S phase, during which several fission events were observed. Two fission events are shown in the bottom panel (1 and 2). The resulting microbodies are labelled 1' and 2'. (B) During a similar period supramolecular fusion events were also observed (labelled 1), shown in more detail in the bottom panel. The microbody that fuses with the larger PML nuclear body is indicated as 1' in the left panel. (C) Relative movement of several PML nuclear bodies is shown (top panel, nuclear bodies 1-4). A mean square displacement (MSD) plot (middle panel) and a log(MSD/{Delta}t) versus log({Delta}t) plot (bottom panel) describes the PML nuclear body movement (mean ± s.e.; n=57, over 45 minutes). Bar, 5 µm.

 

Figure 3
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  		Fig. 3. Ultrastructural analysis of PML nuclear bodies in S phase by correlative LM/ESI. (A) Correlative fluorescence and electron microscopy of a G2 cell, fluorescently labelled for PML protein. Elemental maps of nitrogen (N) and phosphorus (P), and the merged maps of a PML nuclear body and its surrounding nucleoplasm reveal protein-based (green) and nucleic-acid-based (red) components. Chromatin appears yellow in the merged image because of its high N and P content. A field is shown at higher magnification. The imaged PML nuclear body (right hand panel, red) makes extensive contacts (white arrowheads) with the surrounding chromatin (yellow) directly and through non-chromosomal protein (blue) contacts. (B) Electron micrographs of PML nuclear bodies in an early S phase cell. PML microbodies are indicated by white asterisks and chromatin contacts by white arrowheads. (C) Electron micrographs of PML nuclear bodies in a mid S phase cell. Chromatin contacts are indicated by white arrowheads. Bars, 500 nm.

 

Figure 4
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  		Fig. 4. Model for PML nuclear body (NB) replication in S phase. In early S phase, chromatin pulls PML nuclear bodies apart as changes occur in chromatin topology, related to replication. Some microbodies may fuse with each other or with the parental bodies, with a net increase in PML nuclear body number of up to twofold.

 

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