Fig. 3. DNA shows only local rearrangements whereas the replication machinery appears at neighbouring sites. The nucleus was labelled according to the labelling scheme depicted in Fig. 1a. (a) Time series (minutes after microinjection) of a perinucleolar region. The nucleolus appears dark and is labelled N. Projections representing four consecutive light optical sections (z=300 nm) are depicted for each time point. The merged images of GFP (green) and Cy3 fluorescence (red) are shown with colocalization appearing yellow. The upper three panels represent the whole area whereas enlargements of two regions of nascent DNA synthesis labelled 1 and 2 are displayed in the lower six images. A large arrowhead indicates the first site of synthesis at region 1. A small arrowhead points to a GFP-labelled focus appearing in a closely adjacent region first visible at 58 minutes. Nascent DNA extruded away from the nucleolar border at 58 minutes at site 2 is labelled with a large arrow (the GFP-labelled focus at site 2 remains associated with the nucleolar periphery). At 82 minutes all nascent DNA is again closely associated with the nuclear periphery. In regions at the nucleolar periphery so far devoid of nascent DNA, new GFP-PCNA foci appeared. A small arrow indicates a GFP-labelled focus that appeared at 82 minutes in such a region devoid of nascent DNA. (b) Fluorescence intensity scans along the lines indicated on the images in a (arrowheads and arrows point to the same regions as in a), with 0 µm corresponding to the left end of the line in a. (c) Consecutive light optical sections (confocal raw data, z=300 nm) from the enlarged region at 32 minutes and at 58 minutes (arrowheads as in a and b). The new GFP-labelled focus appearing at the left of site 1 at 58 minutes did not relocalize to this site from another nuclear plane. (d) Projections of three consecutive light optical sections from the same perinucleolar area at 348 minutes after microinjection. The nucleus progressed normally into G2. During G2, GFP-PCNA is uniformly distributed over the extranucleolar nuclear regions. The nucleolus appears dark and the Cy3-labelled DNA is still associated with the nucleolar periphery. Bars, 1 µm.