Fig. 1. Overall nuclear dynamics during S-phase progression in living HeLa cells. (a) Labelling scheme. (b-d) Time series (time points indicated in minutes after microinjection) of a double-labelled nucleus (green, GFP-PCNA; red, Cy3). Colocalizing Cy3 and GFP fluorescence appears yellow. Each panel displays a projection of four focal planes (z=600 nm). The GFP-PCNA pattern proceeds from a mid (45 minutes) to a late (330 minutes) S-phase pattern. After initial colocalization of nascent DNA with GFP-PCNA foci (45 minutes), GFP-PCNA foci appear at sites adjacent to nascent DNA at 145 minutes and at increasingly distant sites during S-phase progression (see enlargements of the framed regions in the insets). The large arrowhead indicates a region where GFP-PCNA foci disappeared after DNA synthesis. Two replication sites (small arrows) at the nucleolar (N) periphery are shown enlarged in Fig. 2. Small arrowheads indicate Cy3-labelled cytoplasmic vesicles. Bar, 5 µm; inset bar, 1 µm. (e,f) The distance between the two foci of a given pair of Cy3-labelled DNA foci was measured at two different time points (t=25 minutes). The bars indicate the percentages of pairs of foci showing changes in their distances within a given interval (0-100 nm, 101-200 nm etc.) after 25 minutes. (e) To determine DNA dynamics during replication, it was ensured that at both time points both DNA foci of a given pair colocalized with GFP-PCNA. (f) DNA dynamics after replication of the foci were measured in the same nuclei at later time points, when GFP-PCNA foci occupied different nuclear regions. n, number of pairs of foci evaluated.