The human red blood cell membrane is a dynamic structure which must respond to changes in its environment in order to assure continued cell viability. The submenbranous erythrocyte cytoskeleton plays a crucial role in defining red cell stability, deformability, and shape, properties which, in turn, influence interactions between the red cell and cells of the reticuloendothelial system. This proposal focuses on a set of dynamic membrane phenomena which are controlled by the cytoskeleton: the distribution and lateral mobility of the major transmembrane proteins of the human red blood cell -- band 3 and glycophorin. These phenomena will be examined on membranes which have been labeled with covalent fluorescent reagents specific for band 3 and/or glycophorin, using the techniques of fluorescence microscopy, fluorescence phtobleaching recovery, and fluorescence energy transfer. The intermolecular interactions among band 3, glycophorin, and cytoskeletal components will be probed, differentiating among various dynamic control mechanisms such as steric hindrance, specific binding, and complex formation. The mechanism of immobilization of glycophorin in the membrane by elevated intracellular calcium, and the possible role of calmodulin therein, will also be investigated. Detailed study of transmembrane protein dynamics in normal and abnormal erythrocyte membranes -- such as those from patients with hereditary spherocytosis, hereditary elliptocytosis, hereditary pyropoikilocytosis and sickle cell anemia -- is expected to provide new information about the structure and function of the red cell membrane in health and disease. Alterations in transmembrane protein dynamics may, for example, adversely affect interactions between red cells and cells of the reticuloendothelial system, leading to abnormal adherence, sequenstration, or hemolysis. The goal of this research is the understanding, in molecular terms, of the cytoskeletal forces controlllng the distribution and lateral mobility of transmembrane proteins in the human erythrocyte membrane, and of the relationship between derangements in these control mechanisms and the pathophysiology of disease in various abnormal red cell membrane states.
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