The goal of this research is to test the hypothesis that the membrane skeleton protein adducin is critical to the assembly of the membrane skeleton during differentiation of erythrocytes and platelets. The membrane skeleton is crucial to the red cell, providing both support and flexibility as cells move rapidly through the circulation and traverse narrow capillaries. Defects in membrane skeleton proteins cause mild to severe hemolytic anemia and even hydrops fetalis. Inherited hemolytic anemia (spherocytosis or elliptocytosis) is one of the most common inherited diseases. The membrane skeleton is also crucial to normal platelet function. The following specific aims are designed to analyze adducin's role in both erythrocyte and platelet differentiation and function: I. Determine the role of alternatively spliced adducins in erythrocyte differentiation a. complete the analysis of adducin expression patterns during normal human and mouse erythroid differentiation. b. elucidate the role of adducin in erythroid differentiation and function in vivo using two approaches: adducin null """"""""knockout"""""""" mice and transgenic mice in which an erythroid specific promoter directs antisense mRNA production to block adducin expression. c. perform molecular dissection of the functions of the alternatively spliced isoforms in vivo by using an erythroid specific promoter to direct overexpression of tagged adducin isoforms (normal or mutant) to compete with normal adducin function in transgenic mice or to rescue knockout mice. II. Determine adducin's role in megakaryocyte differentiation and platelet function a. determine adducin expression patterns in mature platelets versus megakaryocytes b. determine the role of adducin in activation and aggregation of platelets c. elucidate the role of adducin in megakaryocyte differentiation and platelet function in vivo using two approaches: adducin null """"""""knockout"""""""" mice and transgenic mice in which a platelet specific promoter directs antisense mRNA production to block adducin expression. d. perform molecular dissection of the functions of the alternatively spliced isoforms in vivo by using a platelet specific promoter to direct overexpression of tagged adducin isoforms (normal or mutant) to compete with normal adducin function in transgenic mice or to rescue knockout mice.
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