The long range goal of this project is an understanding of the diverse functions of a membrane skeletal protein, adducin. Initially identified in erythrocytes, adducin is expressed in nearly all cells. This proposal will focus on adducin function in two cell types: 1) cultured epithelial cells, where adducin is localized to cell-cell borders, and 2) cultured monocytic cell lines, where adducin is localized to phagosomes. Membrane skeletal proteins are essential for maintenance of epithelial integrity and disruption of cytoskeletal organization is a feature of carcinomas. Membrane skeletal proteins are also important for host defense; chemotaxis, adherence, and phagocytosis by leukocytes are dependent upon membrane skeletal protein function. The cloning of erythrocytes cDNAs encoding both alpha and beta subunits of adducin (see preliminary data) will allow a combined approach using molecular and cell biology techniques to study adducin function in epithelia and monocytes.
The specific aims i nclude: I.) Analysis of physiological functions of adducin, a.) Epithelial cell lines: 1. Interrupt endogenous adducin function by transfection of epithelial cell lines with expression vectors encoding truncated forms of alpha and beta adducin or antisense mRNA. 2. Analyze effects of transfection on junction formation using immunofluorescence, electron microscopy and in vivo phosphorylation. 3. Determine whether adducin phosphorylation is cell-cycle dependent. b.) Monocytic cell lines: 1. Characterize adducin's role in phagocytosis by microinjection of cultured macrophages with anti-adducin antibodies or fluorescently tagged domains of adducin. 2. Microinjection (or transfection) of monocytes with adducin expression vectors. II.) Characterization of tissue-specific adducin isoforms, a.) cDNA cloning of additional beta adducin mRNAs: 1. Amplify the alternative 3' end of erythrocyte beta adducin using PCR primers based upon the cloned rat spleen cDNA. 2. Isolate clones from a human brain cDNA library using erythrocyte beta adducin cDNA probes. b.) Correlation of adducin isoform expression with cellular functions 1. Analyze tissue-specific protein expression using isoform-specific antibodies for immunoblotting and immunofluorescence microscopy. 2. Expand current knowledge of adducin localization to include nervous tissue and cardiac muscle. III.) Identification of adducin homologues in lower eukaryotes a. Determine whether adducin homologues are expressed in Drosophila, C. elegans, Dictyostelium, or Paramecium by immunoblotting whole cells or embryos. b. Isolate cDNA clones encoding adducin homologues. c. Create strains with adducin mutations in order to further analyze functions of adducin.
|Gilligan, D M; Lozovatsky, L; Gwynn, B et al. (1999) Targeted disruption of the beta adducin gene (Add2) causes red blood cell spherocytosis in mice. Proc Natl Acad Sci U S A 96:10717-22|
|Gilligan, D M; Lozovatsky, L; Silberfein, A (1997) Organization of the human beta-adducin gene (ADD2). Genomics 43:141-8|