The long term goal of this study is to understand the composition and function of the spectrin-based membrane skeleton in non-erythroid cells. The membrane skeleton was discovered in erythrocytes and is now very well defined in that cell type. Isoforms of many of the erythroid membrane skeleton components exists in many if not most non- erythroid cell types. Although it has been postulated that regional variation in the membrane skeleton may participate in the process of generating cell polarity in non-erythroid tissues, the specific roles of the various spectrin isoforms with distinct tissue and subcellular localization's in non-erythroid cells is still unclear. We have chosen Drosophila as a model system since it provides a unique and powerful combination of genetic, molecular and cellular tools with which to achieve our goals. Specifically, this proposal will study beta/heavy-spectrin (betaH), an isoform of beta-spectrin in this organism. We have created mutations in the betaH gene, karst, and find that these are lethal. This indicates that betaH is crucial for the development of the fly, and provides a unique opportunity to study the role of a specific beta-spectrin isoform in vivo. Preliminary analysis of the karst phenotype has revealed defects in tissues of epithelial origin. This application focuses on one of these defects: failure of the receptor tyrosine kinase/ras-based sevenless signaling pathway that normally determines a specific cell fate in the developing eye imaginal disc. Based on our preliminary data, we hypothesize that betaH is involved either in the apical restriction of sevenless signaling components, in cell adhesion between the communicating cells or in building microvillar structures containing signaling components. We will take a number of complementary approaches to identify the most likely of these hypotheses.
Our specific aims are: i) to make a rescue construct suitable for directed mutagenesis of the betaH sequence, ii) to characterize defects in epithelial cell architecture in karst mutant eyes by staining with a battery of antibodies to key proteins, iii) to identify specific domains of betaH that play a role in sevenless signaling, iv) to identify interacting proteins by characterizing complexes containing betaH that we will identify by immunoprecipitation. The proposed experiments will make a fundamental contribution to our understanding of epithelial structure: one the first lines of defense against most human pathogens. They will also illuminate the relationship between ras signaling and the cytoskeleton, a relationship that is important for oncogenic and metastatic processes.
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