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Nuclear Condensation: (threshold of cellular death

http://docs.abcam.com/ebooks/apoptosis-analysis-guide.pdf

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Surface exposure of phosphatidylserine during apoptosis of rat thymocytes precedes nuclear changes.

Eur J Cell Biol. 1998 May;76(1):77-83.

Stuart MC1, Damoiseaux JG, Frederik PM, Arends JW, Reutelingsperger CP.
Author information

1Department of Pathology, Maastricht University, The Netherlands.

Abstract

Cell surface exposure of phosphatidylserine (PS) during apoptosis serves recognition and removal of the dying cell by phagocytes. Loss of phospholipid asymmetry and PS exposure is investigated by immunocytochemistry and related to morphological changes. Loss of membrane asymmetry was determined on dexamethasone-treated rat thymocytes using the PS specific probe annexin V. Thymocytes incubated in the presence of dexamethasone were studied in time series during the execution of the apoptotic program. Thymocytes first start to expose PS at their cell surface. At this initial stage the barrier function of the plasma membrane remains intact. At a later stage the plasma membrane becomes leaky for compounds like propidium iodide and subsequently the cell disintegrates into apoptotic bodies. Microscopical evaluation of dexamethasone-treated thymocytes showed that the cells with an apoptotic morphology all bound annexin V. The cells with a normal viable morphology lacked annexin V binding except for those cells that started to shed small vesicles. These vesicles were positive for annexin V, indicating a local disturbance of the phospholipid asymmetry. The local exposure of PS is considered to be a very early event of apoptosis, preceding the full sequence of morphological changes at the ultrastructural level.

http://www.ncbi.nlm.nih.gov/pubmed/9650785

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Changes in intercellular junctions during apoptosis precede nuclear condensation or phosphatidylserine exposure on the cell surface

B M Corfe, C Dive and D R Garrod

Dear Editor

An essential feature of apoptosis in vivo is rapid removal of the dying cell before it ruptures to invoke an inflammatory response. Histology of epithelia reveals apoptotic cells to be detached from their neighbours and engulfed by phagocytes.1 This implies that following an apoptotic stimulus, detachment from neighbouring cells, substratum or extracellular matrix precedes or accompanies engulfment, and is likely to be a specific component of the apoptotic process.

Herein we report changes in cell-cell junctions during apoptosis in MDCK cells, a polarised epithelial line. We induced apoptosis by two different stimuli, a DNA damaging agent (etoposide) and a non-specific protein kinase inhibitor (staurosporine). Cells were cultured as confluent sheets and junctional changes examined in relation to characteristic apoptotic nuclear events and to phosphatidylserine exposure at the cell surface. The latter precedes nuclear condensation and is believed to be a signal for recognition of apoptotic cells by phagocytes. The concentrations of etoposide (50 muM) and staurosporine (400 nM) were chosen to allow sufficient time in which to examine changes in cell-cell interactions that might occur prior to the appearance of apoptotic morphology and cell detachment. Detached cells were shown to be apoptotic both by Hoechst 33528 staining for nuclear morphology and by DNA analysis for internucleosomal cleavage (DNA laddering). Apoptotic events induced by etoposide were also observed by time-lapse video microscopy of cell sheets.
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http://www.nature.com/cdd/journal/v7/n2/full/4400634a.html

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Three Distinct Stages of Apoptotic Nuclear Condensation Revealed by Time-Lapse Imaging, Biochemical and Electron Microscopy Analysis of Cell-Free Apoptosis

Shigenobu Toné,*,1 Kenji Sugimoto,# Kazue Tanda,* Taiji Suda,+ Kenzo Uehira,+ Hiroaki Kanouchi,* Kumiko Samejima,¶ Yohsuke Minatogawa,* and William C. Earnshaw¶,2

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 cell-free 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.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2705844/ TY BIOPHARM