UNIVERSITY OF ILLINOIS
With the support from the ARRA 2009, we will devote the next two years to test the deleterious effects of dematin deficiency on erythrocyte membrane stability with direct implications in improving our understanding of the molecular etiology of hemolytic anemia in humans. Our previous studies have demonstrated that the deletion of the headpiece domain of mouse dematin results in compensated anemia, microcytosis, and spherocytosis. A similar phenotype has also been reported for the beta adducin null mice. Importantly, when the two mutations were combined in our laboratory, the double knockout mice displayed a severe phenotype with highly fragile erythrocytes, suggesting an essential role of dematin and adducin in the regulation of erythrocyte membrane stability and shape. To elucidate the mechanism of weakened spectrin-actin junctions, we plan to perform a detailed characterization of the erythrocyte membrane in the double knockout mice. These studies will include biochemical evaluation of the actin protofilaments, rescue of the membrane instability defect by reconstitution of dematin and adducin domains, and the effect of double mutation on the horizontal and vertical interactions in the plasma membrane. The possibility of direct physical interaction between dematin and adducin will be investigated with a view that dematin?s interactions with other proteins are likely to be regulated by its headpiece, phosphorylation, and oligomerization domains. Finally, using the double knockout mice that completely lack dematin and beta adducin, we will investigate whether the residual core domain of dematin exerts a dominant negative on the stability of the erythrocyte membrane. Together, these studies will test the model that protein interactions mediated by dematin and adducin provide a critical node for the regulation of erythrocyte membrane stability and cell shape.