LENS CELL COUPLING--ASSEMBLY AND ROLE OF GAP JUNCTIONS

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Mature lens fiber cells lack biosynthetic organelles and are therefore uniquely dependent on intercellular communication with the metabolically active anterior lens epithelial cells for the nutrients and ions required to maintain lens homeostasis and prevent cataract formation. Three closely related types of gap junction proteins (connexins) participate in joining lens cells into a syncytium with respect to molecules smaller than approximately 1 kD: connexin43 in epithelial cells, and connexins 46 and 50 (or their chick homologues connexins 56 and 45.6) which are found predominantly in fiber cells. I have developed biochemical assays for the major steps in gap junction assembly (connexin oligomerization, transport to the plasma membrane, phosphorylation, and clustering into gap junctional plaques) and have previously used these techniques to study gap junction formation in tissue culture fibroblasts. The first goal of the proposed studies is to apply and extend the knowledge obtained from this model system to determine how gap junctions are established and regulated in the vertebrate lens, processes that are likely to be essential for lens clarity. These experiments will be performed using intact embryonic chick lenses and primary cultures derived from them, systems in which I have previously characterized connexin43 expression on a molecular, morphological, and biochemical level. Specific issues to be addressed include: l) do the different lens connexins ever coassemble in the same cell, potentially creating new channel phenotypes? 2) what are the proteins (chaperones) and intracellular conditions responsible for the novel, post-endoplasmic reticulum oligomerization pathway by which newly synthesized connexins are assembled into half-channels (connexins)? 3) what are the roles of Ca++-dependent and -independent cell-cell adhesion molecules (CAMs) in the establishment and maintenance of gap junctions in the lens, and is there evidence for signal transduction between connexins and CAMs? The second objective of this proposal is to design a mutant form of a lens fiber connexin (cx46) that will coassemble with, and inhibit the channel-forming function of, wild-type lens connexins. Such a dominant-negative approach has been successfully used to perturb gap junction-mediated intercellular communication in early Xenopus embryos (Paul et al., 1993). Ultimately, this cx46 mutant would be expressed in transgenic mice under the control of the lens-selective alphaA crystallin promoter to provide the first molecular evidence defining the in vivo role of gap junctions in lens development, physiology, and cataract pathology.