In spite of strong evidence that the mammalian cell nucleus is a highly organized organelle, a
consensus on basic principles of global nuclear architecture has not so far been achieved.
The existence of major architectural features such as an organized interchromatin
compartment and higher order organization of chromatin postulated by some of the models is
questioned or even refused by the others. This study was set up to test predictions of the
various model views after manipulating nuclear architecture by applying the induced
formation of hypercondensed chromatin (HCC). This method leads to massive but
completely reversible conformational changes of chromatin arrangements in living cell nuclei,
but does not affect the cells survivability.
Nuclear functions like transcription, replication and cell cycling were immediately stalled
when HCC formation was induced, but were rapidly recovered upon recovery of normal
chromatin configurations. The emerging pattern of HCC revealed a 3D network of
interconnected chromosome territories. The surface of the emerging HCC bundles was the
site of preceding activity like RNA transcription or DNA replication, which confirmed the
existence of a distinct topological arrangement of functional processes with respect to the
architecture of chromatin. This arrangement could further be demonstrated by analyzing the
topography of defined chromatin modifications, showing that active chromatin is preferentially
located at the HCC bundle surfaces, whereas inactive chromatin regions are preferentially
found in the HCC bundle interior. The emerging patterns of HCC were further strikingly
similar in consecutively repeated cycles of HCC formation and recovery, demonstrating a
non-random but pre-existing and defined chromatin and interchromatin topography. All
results of this study were obtained using confocal laser scanning microscopy. A protocol for
deconvolution of confocal images was established to enhance confocal image quality to an
extent sufficient for subsequent image analysis.
In contribution to the present model views this study demonstrates: [1] That most chromatin
exists in the form of higher-order sub-compartments ('~1 Mb chromatin domains') above the
level of extended 30 nm fibers and [2] That an interchromatin compartment exists as a
dynamic, structurally distinct nuclear compartment, which is functionally linked with the
chromatin compartment.
An updated chromosome territory-interchromatin compartment model on the basis of the
gained results is presented at the end of this thesis together with an attempt to provide a
comprehensive view linking ultrastructural with light microscopic insights.