Underlying the plasma membrane of the fiber cells of the vertebrate eye lens is a highly cross-linked cytoskeletal network of spectrin, actin filaments and associated proteins that is referred to as the membrane skeleton. The long term objectives of this research are (1) to characterize the molecular organization of the lens fiber cell membrane skeleton, (2) to establish the mechanism of membrane skeleton assembly and (3) to determine the role of the membrane skeleton in generating and maintaining specialized plasma membrane domains in lens fiber cells. In this proposal, we will focus on the expression, localization and molecular associations of lens tropomodulin, a novel tropomyosin-binding and actin filament pointed end capping protein that is likely to play a critical role in regulating the assembly and organization of the actin filaments in the lens fiber cell membrane skeleton.
The specific aims are as follows: (1) The expression and assembly of tropomodulin, tropomyosin and spectrin will be examined in adult bovine lenses as a function of lens fiber cell differentiation and maturation, using immunofluorescence staining of cryosections together with microdissection, subcellular fractionation and western blotting. In addition, the association of membrane skeleton components with specialized plasma membrane subdomains in the lens fiber cells (e.g., adherens junctions, ball-and-socket interdigitations, gap junctions) will be examined by immunolocalization at the ultrastructural level. (2) To determine whether soluble unassembled tropomodulin is associated with other membrane skeleton components in a supramolecular complex, tropomodulin from bovine lens cytosol will be subjected to sucrose gradient sedimentation, co-immunoprecipitation and ultrastructural analysis. The possibility that this complex could represent an unassembled """"""""subunit"""""""" of the lens fiber cell membrane skeleton will also be examined. (3) To test the hypothesis that soluble tropomodulin (and other membrane skeleton components) are precursors destined for assembly into the membrane skeleton, 35S-methionine, pulse-chase labelling of primary culture cells of embryonic chick lens epithelial cells followed by extraction with Triton X-100 and immunoprecipitation will be performed. (4) In addition, cDNA clones for chick lens isoforms of tropomodulin and tropomyosin will be isolated and sequenced and (5) the expression and subcellular distribution of tropomodulin and tropomyosin proteins and these messages will be examined. The results of our studies will have broad implications for the role of the membrane skeleton in generating plasma membrane domains in other nonerythroid cells and will also provide the essential groundwork for future studies on the role of the membrane skeleton in visual accommodation and in the biogenesis of lens fiber cell pathologies such as cataracts.
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