From: C.Reutelingsperger@BIOCH.UNIMAAS.NL
Date: Tue Oct 22 1996 - 10:26:43 EST
Phosphatidylserine (PS) is a normal constituent of the plasma membrane (PM). The viable cell treats this phospholipid species in a specific manner by localizing PS predominantly in the membrane leaflet facing the cytosol. The outer leaflet of the PM facing the environment is almost devoid of PS. Aminophospholipid translocases are thought to be responsible for this asymmetric distribution of PS. The molecular identitie(s) of the translocases have not been resolved sofar (for a recent review see Diaz, C. and Schroit, A.J. J.Membrane Biol. 1996,151, 1-9). Translocase activity has been measured in all viable cell types tested sofar. During apoptosis the equilibrium distribution of PS changes by an increased appearance in the outer leaflet. Also in this case the machineries responsible for the transbilayer movement have not been identified. It is accepted though that specific intrinsic membrane proteins facilitate this movement. As was firstly shown by Valerie Fadok (Fadok, V. et al. J.Immunol 1992, 148, 2207-2216, J.Immunol. 1992, 149, 4029-4035) cell surface exposure of PS occurs during apoptosis of leukocytes. This exposed PS serves the function of recognition and phagocytosis by phagocytes. Hence, cell surface exposure of PS likely forms a functional and, consequently, integral part of the apoptotic program. From this perspective it is an attractive target for measuring apoptosis. The apoptosis associated loss of PM phospholipid asymmetry was firstly probed flow cytometrically using Merocyanine 540 (Fadok, V. et al. J.Immunol. 1992, 148.2207-2216). Valerie's papers triggered us to investigate Annexin V as a probe for cell surface exposed PS during apoptosis. Annexin V was subject of investigation in our laboratory for over a decade and was appreciated for its ability to bind to PS. Contrasting MC540, which intercalates in the bilayer, Annexin V binds to the PS exposing surface extrinsically in a calcium dependent manner. The first papers showed that Annexin V is powerful tool to measure apoptosis flow cytometrically (Koopman et al. Blood, 1994, 84, 1415-1420, Homburg et al. Blood, 1995, 85, 532-540, Vermes, et al. J.Immunol.Methods 1995, 184, 39-51). These papers also showed that an additional probe, like propidium iodide, should be included in order to discriminate between cells with intact and compromised PM integrity. These papers learned that PS exposure occurs during a phase of apoptosis where the PM integrity is still intact. The cell surface exposed PS likely originates from the existing pool of the cell. Whether PS overall content increases during apoptosis by synthesis is not known and has, to my knowledge, not been addressed sofar. It is a very interesting question though. Comprehensive studies of Seamus Martin learned that PS exposure precedes nuclear changes, occurs downstream of the Bcl-2 and Abl checkpoint and is a ubiquitous process of suspended cell types irrespective of the apoptosis initiating trigger (J.Exp.Med. 1995, 182, 1545-1557). Using Annexin V Van Engeland et al. (Cytometry, 1996, 24,131-139) showed that PS exposure also occurs during apoptosis of adherent cell types in culture. The state of the art is that cell surface exposure of PS is a ubiquitous and conserved part of apoptosis. This exposure can be simply assayed using Annexin V. It binds rapidly and with high affinity to the PS exposing cell. We have reached the point to consider seriously cell surface exposure of PS as the general Hallmark of apoptosis. I reckon that interesting and stimulating discussions on this subject are about to take place. Chris Reutelingsperger Department Biochemistry University Maastricht the Netherlands
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