Imaging techniques for studying nuclear body function

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Imaging Techniques for studying Nuclear Body Function Observing changes in biochemical composition of nuclear domains/ bodies Wide field or laser confocal immunofluorescence microscopy Observing Nuclear Dynamics 4D live cell analysis of fluorescent protein tagged nuclear proteins - Wide field and spinning disk confocal microscopy Fluorescence recovery after photo-bleaching (FRAP) - Diffusion of single proteins or sub-resolution complexes Correlating changes in biochemical composition or dynamics with ultrastructural changes in the nucleus (macromolecule resolution) Correlative fluorescence and electron microscopy (Electron Spectroscopic Imaging) Eskiw et al. 2003 J. Cell Science 116 :4455-4466; Eskiw et al. 2004 JBC 279 : 9577-9585; Dellaire et al. 2006 J.Cell Science 119:1026-33.
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Models of PML Nuclear Body Function Figure 1 PML NBs may be platforms for assembly of multi-subunit complexes, Where factors may become activated, such as through post-translational modification (e.g. acetylation, phosphorylation of p53). Activated factors move to the nucleoplasm where they function. PML NBs may be platforms (left) but are situated in specific chromosome environments. Their location and size may depend on interactions with specific gene loci. Representative factors and interactions at PML NBs are shown in C. Compare with the electron spectroscopic image (right), described in Figure 2A. A B C
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Chromatin contacts the surface of the protein core of PML bodies P map N map P and N
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Structure of PML NB in U2OS cell stably expressing PMLIV isoform 150 nm
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PML bodies become mobile and fuse when neighbouring chromatin is damaged 2 photon irradiation (780 nm), followed by imaging beginning 5 minutes after damage for 22 minutes (2.5 minutes between frames). Hoechst 33258 in media.
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PML body integrity and stress DNase Treatment Loss of PML body integrity also occurs during: apoptosis, transcriptional inhibition, and during DNA damage Eskiw et al. (2004) JCS, JBC U-2 OS GFP-PML IV cells Heat Shock
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Number of PML NBs increases following IR through a fission process Graham Dellaire
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Conclusion: PML body positional and structural stability is related to chromatin integrity. Question: What happens to PML bodies when DNA is replicated in S-phase?
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In SK-N-SH cells, PML body number increases as cells progress into S-phase based on cyclin-A staining
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