The long-term objectives of the proposal are to understand both the physiological and molecular basis of the specificity which the fluorescent dye merocyanine 540 (MC540) displays for leukemia cells. The physiological basis of staining appears to involve the pathological retention by leukemia cells of an immature or activated phenotype of normal cells. The molecular basis of staining appears to reside in the lipid bilayer of the plasma membrane. This proposal is designed to investigate the mechanism controlling whether the bilayer is stained or not. MC540 preferentially intercalates into membranes whose lipids are loosely-packed. In erythrocytes, lipid packing is dependent on the transbilayer distribution of phospholipids, which in turn appears to be dependent on lipid-cytoskeletal interactions. Ghosts prepared from normal erythrocytes will be used as models to examine the role of protein modification in controlling these interactions. A photoactivable lipid analogue will be developed to determine whether these interactions are controlled by regulating the physical proximity of the spectrin cytoskeletal network to the membrane bilayer. The importance of plasma membrane lipid transport in altering transbilayer lipid distribution will be assessed. The results from these studies will be compared with results from similar experiments carried out with erythrocytes from patients with chronic myelogenous leukemia in order to determine the molecular basis for the staining of these simple leukemia cells. Finally, the role of these mechanisms in controlling the staining of activated and non-activated normal leukocytes will be evaluated as a first step in establishing the lesion in leukemic leukocytes responsible for their staining.
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