The proteins of red cell membrane skeleton have been partially characterized in terms of ultrastructure, function and their associations in the membrane. In contrast, we have only a limited understanding of altered assembly of these proteins in abnormal cells and their functional consequences. Our proposal is focused to the following: (1) Cytoskeletal alterations in hereditary elliptocytosis (HE) and pyropoikilocytosis (HPP). These studies test the hypothesis that these disorders are related to an abnormal (presumably mutant) spectrin or other skeletal protein and aim to define (1) functional properties of this protein such as dimer-tetramer equilibria, association with other skeletal proteins and binding to the membrane, (2) the contribution of the presumptive skeletal defects to altered membrane stability and abnormal cell shape. In those HE and HPP patients in whom we have recently observed altered association of spectrin dimers to tetramers, we will further attempt to purify the putative mutants and define their physical properties (such as circular dichroism and sedimentation coefficient). Ultimately, we will isolate the tryptic peptides involved in dimer-dimer contact and subject them to amino acid sequencing studies. (2) Dynamics of spectrin equilibrium in the membrane. We plan to characterize the dynamics of the equilibria among spectrin dimers, tetramers and the spectrin-actin-4.1 complexes in the membrane in situ and their modulation by calcium, magnesium, ATP and spectrin phosphorylation. Our major methodological approaches include extraction of membrane skeletal proteins, studies of equilibria among spectrin dimers, tetramers and the spectrin-actin-4.1 complexes both in solution and in the membrane, spectrin binding to inside out membrane vesicles, reconstitution studies of purified skeletal components, studies of ultrastructure and stability of membrane skeletons and peptide analysis of spectrin dimer-dimer binding site.
Showing the most recent 10 out of 55 publications
