Dr. Shohet hypothesizes that the distance between the spectrin-based membrane skeleton and the bilayer increases in certain types of hereditary spherocytosis due to defects or deficiencies in membrane proteins. This physical uncoupling is thought to increase the motion of spectrin, weaken distance-dependent interactions between the skeleton and the bilayer, and destabilize membrane lipids. To test this hypothesis, Dr. Shohet will pursue the following specific aims: 1.Measure the distances from the two major anchorage sites of the membrane skeleton to the lipid bilayer in intact red cell membranes; i.e., the distance from the spectrin:ankyrin binding region to the bilayer and the distance from the spectrin: band 4.1 binding region to the bilayer. These measurements will be determined by fluorescence energy transfer (FET) and single-photon radioluminescence. Novel methodologic approaches include: alkylation of thiophosphorylated phosphorylation sites to label ankyrin and band 3; a fluoresceinated recombinant 52-amino acid peptide of band 4.1 containing the spectrin binding domain to label spectrin at its docking site on the junctional complex; and a non-fluorescent """"""""dark"""""""" acceptor to increase FET distance range. 2. Measure the distance from the more mobile spectrin span region of the membrane skeleton to the lipid bilayer and define the dynamic behavior of this region. First, the average distance from spectrin to the bilayer will be measured utilizing uniformly fluorescently labeled spectrin and the novel technique of total internal reflectance (TIR) developed by the UCSF group to detect fluorophores at distances 100-2000 beneath the membrane surface. Second, the distance from the beta-chain carboxy terminus in the middle of the spectrin oligomer to the bilayer will be measured using a fusion of B-spectrin with the green fluorescent protein from jellyfish. A similar distance estimate will be generated using a fluorescent Fab against the 10th repeat of the a-spectrin subunit. Third, the extent of motion of the central region of the spectrin tetramer will be measured using fluorescence recovery after photo-bleaching combined with total internal reflectance. 3. Test the hypothesis that bilayer coupling is defective in certain spherocytic hemolytic anemias by measuring bilayer:skeletal distances and spectrin motion in pathologic cells. The above distance and dynamics measurements will be made on spherocytic erythrocytes with mutations or deficiencies in ankyrin or band 4. Similar measurements will be conducted on Southeast Asian Ovalocytes which are more rigid than normal cells and which may exhibit increased cytoskeletal-bilayer coupling with reduced spectrin motion. In aggregate, the role of bilayer-skeletal interactions in red cell membrane structure and stability will be more clearly defined.
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