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First published online 3 August 2005
doi: 10.1242/dev.01950

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Polycomb group protein complexes exchange rapidly in living Drosophila

¹ Max Planck Institute for Biophysical Chemistry, Department of Molecular Biology, 37070 Göttingen, Germany
² King's College London, Randall Division of Cell and Molecular Biophysics, New Hunt's House Guy's Campus, London SE1 1UL, UK

View larger version (50K): [in a new window]   Fig. 1. Distribution patterns of PhGFP and PcGFP in the embryos and larval wing discs. (A-C) Western blot analysis of Drosophila tissue extracts from wild-type and transgenic fly lines. (A) Extracts from wing discs from wild-type and PhGFP-expressing larvae (probed against Polyhomeotic). (B) Extracts from wild-type and PcGFP-expressing embryos (probed against Polycomb). (C) Extracts from salivary glands from PcGFP- and PhGFP-expressing larvae (probed against GFP). Antibodies against Poly-A-binding protein (PABP) (A), S6 Ribosomal protein (B) and eIF4A (C) were used for loading control. PhGFP expression was induced using the en:GAL4 driver in embryos (D) and the BXMS1096:Gal4 driver in wing imaginal discs (H). The corresponding distribution pattern of PhGFP in nuclei can be seen in E (embryos) and I (wing discs). PcGFP expression in embryos and wing imaginal discs is shown in F and J, and the respective nuclei in G and K. Scale bars: 5 µm.

View larger version (39K): [in a new window]   Fig. 2. Diffusion of PcGFP in nuclei of preblastoderm embryos. A square-shaped region (1.5x1.5 µm) in the center of a preblastoderm nucleus (A) of a PcGFP embryo was photobleached and the fluorescence recovery was measured over time. (B) Data points for 40 FRAP curves from similar nuclei as in (A) were averaged and fitted to a hyperbolic function (Eqn 2 in the Materials and methods). Scale bar: 5 µm.

View larger version (110K): [in a new window]   Fig. 3. Chromatin dynamics in diploid nuclei. (A) Inset: fluorescence image of a PhGFP larval wing disc. Intensity pseudo-colored magnified image of the region boxed in the inset. A stack of seven z-sections was imaged repeatedly at 5-second intervals for 120 seconds. Time traces of several loci are indicated by the colored tracks. (B) A single XY plane at time 0 from the stacks is displayed. Time XY tracks are superimposed for two loci (blue and green traces). Zero time (C) XZ planes and (D) ZY planes for the slices designated by the cross-hair in image B. Time traces for the positions of the two loci are superimposed showing the large movements.

View larger version (69K): [in a new window]   Fig. 4. 3D-iFRAP. (A) Overview of part of a wing disc with cross-hair on the fluorescent locus selected for iFRAP. Fluorescence depletion was calculated from a recorded series of z-stack images over time after bleaching and image registration. (B) Magnified XY confocal image of the nucleus before bleaching. Cross-hair set in the bleach region next to the unbleached locus. The images have been registered to correct for the movement of the chromatin, and the nucleus itself and the fluorescent locus is centered in the final analysis image time stack. (C,D) X-time and time-Y views, respectively, of the registered images at the slices in the XY image corresponding to the cross-hair in B. In C, dissociation of the fluorescent molecules from the spared locus can be seen over time. In D, a bleached region is shown over time. The transition from before bleaching to after bleaching is indicated by the arrows in the time planes.

View larger version (36K): [in a new window]   Fig. 5. Dissociation rate constants for PhGFP and PcGFP. Individual PcG protein loci in the diploid nuclei of the embryos and wing imaginal discs were subjected to 3D-iFRAP. Fluorescence decay curves were fitted to a single exponential function. (A) Histogram of dissociation rate constants of individual PhGFP loci obtained from 3D-iFRAP experiments in embryos and larval wing imaginal discs are shown in red and black, respectively. (B) A single dissociation rate constant could be fitted by averaging all 30 normalized data from PhGFP embryos (koff=0.051±0.004 second–1). (C) Average of all 32 normalized data from PhGFP wing discs (koff=0.032±0.002 second–1). (D) Average dissociation rate curve for data from 10 determinations of PcGFP loci in imaginal discs (koff=0.034±0.003 second–1).

View larger version (70K): [in a new window]   Fig. 6. Distribution of PhGFP and PcGFP in the larval salivary gland nuclei and FRAP curves for individual bands. Maximum intensity projections of the fluorescence in whole salivary gland nuclei are shown in A (PhGFP) and C (PcGFP). The amount of the background fluorescence (the unbound protein) differs. Typical FRAP curves from individual PcG loci are shown in B (PhGFP) and D (PcGFP). The amount of background fluorescence substantially changes the aspect of the recovery curves. Images from selected time points in a typical FRAP experiment (PhGFP in this case) are shown in E. Upper panels, overview of part of nucleus with bleach box indicated in the second image. Lower panels, magnified region used for analysis. (F) FRAP curves for sequential bleaching of a single PcGFP locus showing reproducible reassociation kinetics.

View larger version (34K): [in a new window]   Fig. 7. Segmentation of the bound fraction and fitting of the FRAP curves. A confocal image of PhGFP in a salivary gland nucleus is shown in A. Inset: confocal sections for PhGFP and PcGFP salivary gland nuclei. (B) Segmentation of the bleach box area into bound and unbound fluorescence regions. (C,E) Plot of the intensity for all pixels (red curve), the 30% highest pixels, bound fraction (blue curve) and the 30% lowest pixels (green curve). (C) PhGFP. (E) PcGFP. (D,F) Fit of the bound fraction of the protein to a single exponential (blue curve). The height of the green curve (unbound component) differs for PhGFP and PcGFP. (D) PhGFP. (F) PcGFP.

View larger version (33K): [in a new window]   Fig. 8. FRAP on bound PcG proteins in the salivary gland nuclei. Individual bands in the salivary gland nuclei were photobleached and redistribution of fluorescence was measured over time. Recovery times (t0, the time required for the fluorescence intensity to reach 63% of the final height of the recovery curve) were plotted as a histogram in A (blue, PhGFP; red, PcGFP). Redistribution of the fluorescence in both cases occurs in less than 6 minutes. (B) Lack of correlation between the concentration of binding sites and recovery time t0. Individual nuclei where several loci were analyzed are color coded (solid colored symbols for PhGFP and open colored symbols for PcGFP). Data from simulations show a linear dependence of the t0 on the concentration of binding sites (connected points for three different dissociation rate constants).

View larger version (21K): [in a new window]   Fig. 9. Dissociation rate constants for PhGFP and PcGFP and reassociation rate constants for PhGFP. (A) Dissociation rate constants for PhGFP (blue) and PcGFP (red) plotted against the normalized concentration of binding sites for individual loci. (B) Pseudo-reassociation rate constants for PhGFP (blue). See text for details.

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