Cy3-labeled DNA foci in HeLa and SH-EP N14 cells were shown to undergo constrained random diffusion with rare examples of directional motion correlating with changes in cell shape . Later, a more sophisticated analysis of temporal dynamics was performed using a,10 Mbp artificial repeat that was able to bind lacI-GFP . However, largely because of technical limitations, we have only limited understanding of dynamic changes that occur when DNA foci engage DNA or RNA polymerases to function as a synthetic template . As PI-103 PI3K inhibitor structural transitions related to chromatin function must increase the probability of DNA mixing within the inter-chromatin domain, we next wanted to evaluate if live cell imaging could be used to define the structural stability of DNA foci. Because DNA foci within euchromatin and heterochromatin have well-characterized Masitinib nuclear organization and dynamic properties , it is possible to use foci labeled at different times of S phase as metastable landmarks to map the relative movement of individual foci. A typical example of this approach is shown in Figure 5. Replication foci were labeled with AF488-dUTP during early S phase and Cy3- dUTP 5 hours later . Using this labeling program, the relative spatial stability of heterochromatic foci labeled during mid- S phase can be used as anchor points to align CTs at different times during the imaging series and so increase the confidence with which the location of individual foci can be assigned. In the example shown, the overall shape of the CTs and local architecture of individual foci is maintained throughout the imaging time-course even though it is not unusual to see local transformations in the shape of individual CTs; the example shown here is seen to rotate around its vertical axis . Even though the quality of foci is limited by the imaging set-up used during live cell imaging, the type of data shown in Figure 5 allows unambiguous identification of discrete foci using image processing software . In this typical example, individual foci are most obvious along the periphery of CTs . In such areas of the sample, the ability to track and assign co-ordinates for individual foci during time-lapse imaging allows the location and movement of individual foci to be monitored with confidence. As noted before , we found euchromatic foci to be locally dynamic, typically moving,0.5 mm over periods of 15 minutes . However, dramatic directional movements were never sustained for long periods. Instead, foci appeared to oscillate within CTs so that individual territories maintained their relative position and general shape for many hours. Relative to euchromatic foci, heterochromatic foci were frequently clustered and showed significantly reduced mobility . This correlates with heterochromatic foci being preserved as temporally stable clusters of structurally inert chromatin. The architecture of mid/late replicating DNA foci correlates with the structural polarization of CTs and corresponding programme of DNA synthesis in mammalian cells .