Electron Microscope Center, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192 (Japan).

Laboratory of Applied Molecular Biology, Division of Bioscience and Informatics, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531 (Japan).

Department of Biochemistry, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192 (Japan).

Together, these experiments provide the first steps towards a systematic biochemical characterization of chromatin condensation during apoptosis. This requirement for ATP hydrolysis further distinguished stage 2 from stage 3. Finally, we demonstrated more » that hydrolyzable ATP is required for stage 3 nuclear collapse/disassembly.

However, DNase(s) were required for stage 2 necklace condensation. DNase activity was not essential for stage 1 ring condensation, which could occur in apoptotic extracts depleted of all detectable DNase activity. Electron microscopy revealed that neither chromatin nor detectable subnuclear structures were present inside the stage 1 ring-condensed structures. This enabled us to define three stages of apoptotic chromatin condensation: stage 1 ring condensation stage 2 necklace condensation and stage 3 nuclear collapse/disassembly.

We demonstrated that the population of nuclei undergoing apoptosis in vitro appears to follow a reproducible program of nuclear condensation, suggesting the existence of an ordered biochemical pathway. Here, in order to better define the stages of nuclear disassembly in apoptosis, we have characterized the apoptotic condensation using a cell-free system and time-lapse imaging. But so far, the role of DNase activity or ATP hydrolysis in this system has not yet been elucidated. We have previously used a cell-free system to examine the roles of caspases or other proteases in apoptotic chromatin condensation and nuclear disassembly. During apoptotic execution, chromatin undergoes a phase change from a heterogeneous, genetically active network to an inert highly condensed form that is fragmented and packaged into apoptotic bodies.

Nuclear time lapse