Phosphatidylserine on the cell surface

Reviews of the role of aminophospholipid translocase and scramblase (Schlegel et al, 2000) and the consequences of the appearance of phosphatidylserine on the cell surface (Williamson et al, 2001) in apoptosis of thymocytes have been published. The precise relationship between membrane phospholipid asymmetry and apoptosis is currently a topic of considerable interest. 

The control of the processes of phosphohpid translocation and scrambling by cytoplasmic calcium concentration is known to be one factor in regulating the appearance of phosphatidylserine on the cell surface. There is now evidence that other factors are involved in modulating membrane phospholipid distribution and for preserving phospholipid homeostasis. Current research is directed to clarify the role of these agents and to establish any connections with the initiation of apoptosis. 

Apoptosis Apoptosis is an ordered, active process that brings about the death of a cell as an important part of the maintenance of organismal homeostasis. Apoptosis can be assayed, in flow cytometry, by, for example, looking at the expression of phosphatidylserine on the cell surface, by looking for nuclei with less-than-normal (sub-GO/Gl) amounts of DNA, and by looking for an increase in DNA fragment termini. 

The appearance of anionic phospholipids, particularly phosphatidylserine, on the cell surface activates prothrombinase complex culminating in the formation of thrombin (Bevers et al., 1982 Connor et al., 1989). The assay can be performed with pure coagulation proteins and specific chromogenic substrates to produce a very sensitive test to detect the appearance of phosphatidylserine on cell surfaces. Nevertheless, it has been shown that changes in the disposition of phosphatidylethanolamine and sphingomyelin may interfere with the ability of phosphatidylserine-containing membranes to activate prothrombinase (Smeets et al., 1996). 

Vermes, I., Haanen, C., and Reutelingsperger, C. (1995) A novel assay for apoptosis based upon flow cytometric detection of phosphatidylserine on the cell surface with use of F1TC-labelled annexin V. Clin. Chem. 41, 91. 

One of the early events of the apoptotic process involves the translocation of phosphatidylserine on the surface of cell membranes annexin V binding and propidium iodide uptake reveals various cellular states. After treatment with organotin(IV) compounds the cells could be categorized into populations vital cells (annexin V /P ), early apoptotic cells (annexin V /P ), late apoptotic cells (annexin V /P ), and necrotic cells (annexin V /P" ). Cells are observed with a fluorescence microscope and it is possible to observe translocation of phosphatidylserine (PS) from the inner side of the plasma membrane to the outer one and to see a green stain for annexin V FLUOS bound to PS, and a red stain for propidium iodide. 

Methods used to demonstrate the existence of membrane phospholipid asymmetry, such as chemical labelling and susceptibility to hydrolysis or modification by phospholipases and other enzymes, are rmsuitable for dynamic studies because the rates of chemical and biochemical reactions are of a different order compared to the transmembrane translocahon of the phospholipids. Indirect methods have therefore been developed to measure the translocation rate which are consequent on the loss of membrane phospholipid asymmetry. Thus time scales appropriate to rates of lipid scrambling under resting conditions or when the forces preserving the asymmetric phospholipid distribution are disturbed can be monitored. Generally the methods rely on detecting the appearance of phosphatidylserine on the surface of cells. Methods of demonstrating Upid translocation in mammalian cells has been the subject of a recent review (Bevers etal., 1999). 

Shiratsuchi, A., Umeda, M., Ohba, Y., and Nakanishi, Y., 1997, Recognition of phosphatidylserine on the surface of apoptotic spermatogenic cells and subsequent phagocytosis by Sertoh cells of the rat. J. Biol. Chem. 272 2354-2358. 

Plasma membrane lipids are asymmetrically distributed between the two monolayers of the bilayer, although the asymmetry, unlike that of membrane proteins, is not absolute. In the plasma membrane of the erythrocyte, for example, choline-containing lipids (phosphatidylcholine and sphingomyelin) are typically found in the outer (extracellular or exoplasmic) leaflet (Fig. 11-5), whereas phosphatidylserine, phosphatidyl-ethanolamine, and the phosphatidylinositols are much more common in the inner (cytoplasmic) leaflet. Changes in the distribution of lipids between plasma membrane leaflets have biological consequences. For example, only when the phosphatidylserine in the plasma membrane moves into the outer leaflet is a platelet able to play its role in formation of a blood clot. For many other cells types, phosphatidylserine exposure on the outer surface marks a cell for destruction by programmed cell death. 

Annexin V Annexin V is a molecule that binds to phosphatidylserine and, therefore, if conjugated to a fluorochrome, will identify apoptotic cells (which express phosphatidylserine on their surface). In the assay for apoptosis, annexin V must be used in conjunction with propidium iodide in order to exclude dead cells (which express phosphatidylserine on the internal side of their cytoplasmic membranes). 

The negatively charged phospholipid phosphatidylserine is asymmetrically distributed in mammalian cell membranes, primarily on the inner leaflet. Upon exposure to collagen or thrombin, the distribution of phospholipids changes with increasing phosphatidylserine in the external membrane leaf (I). The increased expression of phosphatidylserine on the outer leaflet of the membrane creates a procoagulant surface on which several steps of the coagulation cascade take place. 

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