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

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

INTERACTION OF ANNEXINS WITH MEMBRANES - THE N-TERMINUS AS A GOVERNING PARAMETER AS REVEALED WITH A CHIMERIC ANNEXIN

The modulating effect of the variable N-terminus of annexins on the properties of these Ca2+-binding proteins was investigated. To this end, the interaction of annexin V and a mutant annexin, I(N)V(C), consisting of the N-terminus of annexin I (amino acids 1-45) and the core of annexin V (19-320), with large unilamellar phosphatidylserine (PS) vesicles was examined. In contrast to annexin V, the mutant annexin mediated Ca2+-dependent aggregation of the lipid vesicles at neutral pH. However, annexin V induces Ca2+-dependent aggregation at mild acidic pH. Moreover, both proteins can engage in hydrophobic interactions with PS vesicles, which results in release of the vesicle contents. These membrane-perturbing properties are expressed by both annexins in the absence of Ca2+ and occur at neutral and mild acidic pH. Interestingly, addition of Ca2+ inhibits annexin V-induced release, but sustains the release induced by the mutant annexin I(N)V(C). The Ca2+-dependent effects on the release of vesicle contents are reversed upon EDTA addition. Conformational changes revealed by binding of the hydrophobic probe, 4,4'-bis(1-anilino-8-naphthalensulfonate), underly the observed Ca2+-modulated effects on leakage. However, low-pH-mediated aggregation by annexin V does not seem to be related to macroscopic conformational changes. Annexin I(N)V(C) also affects Ca2+-induced fusion of PS vesicles, displaying synergistic properties in conjunction with Ca2+ at neutral pH. By contrast, annexin V does not display similar properties at mild acidic pH, in spite of its ability to aggregate vesicles under such conditions. Since the cores of annexins V and I(N)V(C) are identical, the present results emphasize the role of the N-terminus in governing annexin-membrane interaction properties. It is furthermore of interest that, in addition, the properties of annexins might be regulated by pH, which would extend their physiological range of operation.</p

The Annexin A5-1C/T polymorphism in ischemic stroke: a case-control study.

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