9319104 Williamson, Patrick Schlegel, Robert Apoptosis, or programmed cell death, is a developmental process whose result is the death and engulfment of cells without the pathological consequences which attend cell death due to injury (necrosis). The presence of phosphatidylserine (PS) on the cell surface has been implicated as one of the signals for recognition and phagocytosis of apoptotic lymphocytes. The exposure of PS requires alteration of the normal distribution of plasma membrane phospholipids, and thus regulation of the processes which control this distribution must be part of the program of apoptotic cell death. Studies of erythrocytes have demonstrated the existence of two distinct pathways governing transbilayer lipid distribution. The first is an ATP-dependent transport of the aminophospholipids, and particularly PS, to the inner leaflet; the second is an energy independent randomization of all phospholipids between the two leaflets, a process which brings PS to the cell surface. In this project, flow cytometric techniques will be used in combination with specific fluorescent phospholipid probes to analyze transbilayer lipid movements in apoptotic cells. The normal, ATP- dependent, NEM-sensitive movement of PS to the inside of lymphocytes will be characterized in cells before and after induction of apoptosis, to determine whether and when this pathway is inactivated. NEM-insensitive movement of phosphatidylcholine (PC) from the outer to the inner leaflet (a marker for the second pathway) will be characterized before and after induction of apoptosis to determine whether and when lipid randomization occurs during apoptosis. The generality of these events will be tested by comparing the results in cells induced to undergo apoptosis by steroid hormone treatment and by inactivation-induced suicide, and by extension of the results from tissue culture cells to thymocytes in the mouse. The role of elevation of cytoplasmic calcium as a regulatory me diator of alterations in transbilayer lipid movements will be assessed, and the role of several specific calcium-related signalling pathways in this regulation will be investigated. %%% Fatty, organic molecules called lipids form the basis of the structure of the membrane that separates a cell interior from its external environment. Despite their importance, however, little is known about the mechanisms by which lipids are organized in membranes and play their roles in cell membrane structure and function. Recent evidence indicates that one of the molecular species of phospholipid in the cell membrane (phosphatidylserine or "PS") serves as a signal for recognition and removal of cells undergoing programmed cell death. Programmed cell death is an orderly process activated during tissue remodeling in development, allowing replacement of one cell type by another, or, in the adult organism, to remove cells with a limited life span. Removal of dying cells by specialized scavenger cells prior to their disintegration is important to this orderly process. The goal of this research is to understand the molecular mechanisms which regulate the exposure of PS on the surfaces of cells at the time they begin programmed cell death so that they are targeted for removal.***
