Spectrin function will be analyzed in a model system, Drosophila melanogaster. Spectrin is a membrane-associated cytoskeletal protein that plays a key role in the determination of erythrocyte cell shape. spectrin also plays unknown, but essential, roles in non-erythroid cells. As a result of our recent work that has identified alpha-spectrin mutants, we are in the singular position of being able to directly assess the functions of spectrin in non-erythroid cells. Using a combination of biochemical, molecular genetic, and classical genetic analyses, we will test various hypotheses for spectrin function in non-erythroid tissue at the cellular, developmental and organismal level. Beta-spectrin mutants will be produced and these, together with recently identified alpha-spectrin mutants, will be utilized to investigate spectrin function in vivo. Phenotype analysis will determine the effects of the complete absence of functional spectrin; mosaic analysis will determine the effects of spectrin absence at selected developmental stages. This phenotype information will be used to design experiments that rescue null mutants with spectrin-DNA constructs that will test the cellular or developmental role of a specific aspect of spectrin organization, such as membrane binding, or spectrin chain- length. Although spectrin isoforms are present in nearly all human cells, the neurological and general health consequences of defective spectrin expression in non-erythroid cells are only beginning to be explored. The proposed experiments will provide an in-depth understanding of spectrin's role in non-erythroid cells.
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