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 immuno-cytochemistry 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.
Highlights
Apoptosis entails gene-regulated processes of cell suicide, silent death and removal without inflammatory response
In this study we investigated the loss of membrane phospholipid asymmetry in relation to morphological alterations of apoptotic thymocytes, using immunoelectron microscopy and flow cytometry
Annexin V single positive cells, annexin V propidium iodide double positive cells with normal amount of DNA and annexin V propidium iodide double positive cells with a reduced amount of DNA (Fig. 1A).The double negative population consists of normal viable thymocytes because there is no indication of cell damage and loss of membrane asymmetry and integrity as probed by the fluorescent markers
Summary
Apoptosis entails gene-regulated processes of cell suicide, silent death and removal without inflammatory response. These apoptotic processes are characterized by a sequence of morphological alterations, which are clearly distinguishable from necrosis [13]. Rapid blebbing of the cell membrane [22] and chromatin condensation [26] can be observed during the first stage of apoptosis These changes are often but not always accompanied by nuclear convolution, followed by nuclear pycnosis and cell shrinkage. The main biochemical feature of apoptosis is DNA cleavage at the internucleosomal sites, by calcium-activated endonucleases [2, 11,32] This results in the typical DNA laddering on agarose gels, which is considered the biochemical hallmark of apoptosis. Contrary to the apoptotic cell the viable cell retains PS exclusively in the inner leaflet by energy-dependent processes [8, 35]
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