Figure 4.

Establishment of higher order nuclear compartments after mitosis and the underlying chromosomal structure. HeLa S6 cells synchronized in early S phase were labeled for 30 min with IdU and 9.5 h later for 30 min with CldU (see labeling scheme at the top). Therefore, initially labeled cells simultaneously displayed a type I pattern labeled by IdU and a pattern typical for the second half of S phase labeled by CldU. Daughter cells of the initially labeled cells were fixed 14 (a–c) or 69 h (d–f), respectively, after the CldU pulse and analyzed by confocal microscopy. Identical nuclear planes were imaged regarding TRITC (IdU detection, depicted in red) and FITC (CldU detection, depicted in green) fluorescence. The corresponding merged TRITC and FITC signals (colocalizing signals appear yellow) are shown in a–d. e and f display only the TRITC (e) or FITC (f) signals of the merged image in d (midnuclear plane). The two cells depicted in a–c display the typical morphology of early G1 cells shortly after mitosis. Both G1 cells display an IdU (red) type I pattern and a CldU (green) type III pattern present in their mother cell. Perinucleolar labeling is indicated by an arrowhead in c. After 69 h (d–f), initially labeled cells went through at least two mitoses as indicated by the presence of single-labeled chromosome territories (double-labeled patches within the nucleus depicted in d). Double-labeled chromosome territories reveal that the nuclear type I (IdU, red) and type III patterns are due to a reproducibly distinct distribution of IdU- and CldU-labeled DNA within single chromosome territories. CldU-labeled DNA is concentrated at subchromosomal positions near the nuclear and nucleolar peripheries, whereas IdU-labeled DNA is located at subchromosomal positions between these compartments.