Three distinct stages of apoptotic nuclear condensation revealed by time-lapse imaging, biochemical and electron microscopy analysis of cell-free apoptosis

Abstract

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. We have previously used a cell-free system to examine the roles of caspases or other proteases in apoptotic chromatin condensation and nuclear disassembly. But so far, the role of DNase activity or ATP hydrolysis in this system has not yet been elucidated. Here, in order to better define the stages of nuclear disassembly in apoptosis, we have characterized the apoptotic condensation using a cellfree system and time-lapse imaging. 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. 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. Electron microscopy revealed that neither chromatin nor detectible subnuclear structures were present inside the stage 1 ring-condensed structures. DNase activity was not essential for stage 1 ring condensation, which could occur in apoptotic extracts depleted of all detectible DNase activity. However, DNase(s) were required for stage 2 necklace condensation. Finally, we demonstrated that hydrolysable ATP is required for stage 3 nuclear collapse/disassembly. This requirement for ATP hydrolysis further distinguished stage 2 from stage 3. Together, these experiments provide the first steps towards a systematic biochemical characterization of chromatin condensation during apoptosis.

Observation of DNA and protein distributions in mammalian cell nuclei using STXM

“…3(j), (k) and (l). These morphological changes of the nuclei in apoptosis seem similar to the observation by the fluorescence microscopy [8]. At all of the four stages, the distributions of DNA and protein are similar but that of the constant component differs from them obviously.…”

“…Stage 0) normal nucleus, stage 1) the chromatin condenses like a ring shape, Stage 2) the chromatin transforms into necklace shape and stage 3) the nucleus collapses [8]. These stages of apoptosis were investigated by the method described in the previous section.…”

Caspase-activated DNase Is Necessary and Sufficient for Oligonucleosomal DNA Breakdown, but Not for Chromatin Disassembly during Caspase-dependent Apoptosis of LN-18 Glioblastoma Cells

“…When they were incubated with preactivated cytoplasms from SH-SY5Y cells, the chromatin adopted a necklace shape, compatible with an intermediate stage II apoptotic nuclear morphology (35). However, when incubated with cyto- plasms from staurosporine-treated LN-18 cells, SH-SY5Y nuclei collapsed, showing a chromatin condensation similar to that observed in LN-18 cells in vivo (Fig.…”

Negative short-term salt effects on the soybean–Bradyrhizobium japonicum interaction and partial reversion by calcium addition

“…In this work we evaluated in detail the root hair death induced by saline treatments and in saline treatments supplemented with calcium. The progression of chromatin condensation can be classified into three stages during animal cell apoptosis: stage I, or ring condensation, stage II, or necklace condensation, and stage III, or nuclear collapse/disassembly (Toné et al 2007). These stages are less clearly defined in plant cells, but the process of chromatin condensation progresses essentially in the same way and culminates in the formation of discrete domains of condensed and finally fragmented chromatin (Domínguez and Cejudo 2006;Yamada et al 2006).…”