This is a renewal proposal for five years of support to continue biophysical studies of the behavior of normal and pathological human red cells under mechanical stress. The long term goals of this research are to correlate protein interactions among structural components of erythrocyte membranes with precisely defined mechanical methods including extensional rigidity (length of membrane extension into a micropipette as a function of pressure) bending rigidity (pressure required to buckle or wrinkle the membrane in a micropipette), membrane viscosity (rate of recovery of a red cell distorted by stretching induced with opposed pipettes), membrane plastic flow (extent of irreversible deformation following aspiration of membrane into a micropipette for various times). In addition, yield strength of membranes will be measured from behavior in the presence of shear stress in an ektcytometer. The lateral mobility of integral membrane proteins will be measured by fluorescence recovery following photobleaching. Individual cell-cell interactions will be examined by micromechanical methods such that cells attached to micropipettes can be opposed and the force required to separate them can be measured. This array of measurements will be applied to answer the following questions: 1) What are the contributions of skeletal proteins including spectrin, ankyrin, protein 4.1, protein 4.2, and adducin to membrane mechanical properties. The experimental approach will be to evaluate the consequences of incorporation of protein fragments and antibodies against defined functional domains of these proteins into red cells. In addition abnormal erythrocytes with defined defects will be examined, and in some cases the abnormal cells will be rescued by replacement of missing proteins. 2) What are the roles of cytoplasmic domains of integral membrance proteins such as band 3, glycophorin A, and glycophorin C. Again, the approach will be to reconstitute normal red cells with potential inhibitors of protein interacts (protein fragments, peptides, antibodies against functional domains), and to utilize abnormal or mutant red cells with defects in integral proteins. Examples of such mutatants include Glycophorin C-deficient cells and Melanesian red cells with altered cytoplasmic domain of band 3. Another issue to be addressed with integral membrane proteins is whether association with external ligands alters coupling to the membrane skeleton as determined by measurement of lateral mobility. 3) What are the consequences of association of abnormal hemoglobins with red cell membranes. Red cells will be examined from patients with thalassemias, hemoglobin C, and hemoglobin E as well as oxidized normal red cells. 4) What are effects of membrane protein oxidation, immunoglobulin binding, and abnormal hemoglobins on cell-cell interactions among red cells and of red cells with macrophages/monocytes.
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