Quantitative motion analysis of subchromosomal foci in living cells using four-dimensional microscopy

The motion of subchromosomal foci and of whole chromosome territories in li ve human cell nuclei was investigated in four-dimensional space-time images . Visualization of subchromosomal fool was achieved by incorporating Cy3-dU TP into the nuclear DNA of two different cell types after microinjection, A subsequent segregation of the labeled cell nuclei led to the presence of o nly a few labeled chromosome territories on a background of nonlabeled chro matin (Zink et al., 1998. Hum. Genet. 102.241-251). This procedure yielded many distinct signals in a given cell nucleus. Motion analysis in four-dime nsional space-time images was performed using single-particle tracking and a statistical approach to the detection of a possible directional motion of foci relative to the center of mass of a chromosome territory. The accurac y of the analysis was tested using simulated data sets that closely mirrore d the experimental setup and using microparticles of known size. Applicatio n of the analysis tools to experimental data showed that mutual diffusion-l ike movements between foci located on different chromosomes were more prono unced than inside the territories. In the time range observed, movements of individual foci could best be described by a random diffusion process. The statistical test for joint directed motion of several foci inside chromoso me territories revealed that foci occasionally switched from random to dire ctional motion inside the territories.