This application proposes to identify the protein components of the lens fiber intercellular junctions. The strategy for this identification will be to prepare monoclonal antibodies and polyclonal antisers using isolated lens fiber membranes as antigen. These antibodies will be screened by selecting for reagents which produce a macular staining pattern on immunofluorescently-labeled frozen sections of whole lens, which recognize specific polypeptides on immune replicas, and which interfere with lens fiber intercellular communication as assayed by intracellular microinjection of the fluorescent dye, Lucifer Yellow CH. Antigens will be visualized by immunoelectron microscopy to verify their location on lens fiber junctions. Successfully screened antisera will be used to demonstrate the molecular components of lens epithelium to lens fiber junctions, and epithelial cell-to-epithelial cell junctions. These antisera will be used to study the in vitro synthesis of junctional proteins in cell-free systems, and to define the junctional interactions between differentiated and undifferentiated cells in culture. The antisera will also be used to attempt cloning the lens fiber junction gene, using a lens cDNA library cloned into the Gamma gtll expression vector. If successful, the derived amino acid sequence from this gene will permit raising additional antisera to defined amino acid sequences within the junctional molecule, and topological mapping of the protein structure within the junctional membranes. If the antisera demonstrate native cell junctions between the cultured cells, direct effects of cataractogenic conditions on intercellular communication will be attempted.

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National Eye Institute (NEI)
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Visual Sciences A Study Section (VISA)
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Harvard University
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Chai, Zhifang; Goodenough, Daniel A; Paul, David L (2011) Cx50 requires an intact PDZ-binding motif and ZO-1 for the formation of functional intercellular channels. Mol Biol Cell 22:4503-12
Magnotti, Laura M; Goodenough, Daniel A; Paul, David L (2011) Deletion of oligodendrocyte Cx32 and astrocyte Cx43 causes white matter vacuolation, astrocyte loss and early mortality. Glia 59:1064-74
Magnotti, Laura M; Goodenough, Daniel A; Paul, David L (2011) Functional heterotypic interactions between astrocyte and oligodendrocyte connexins. Glia 59:26-34
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