We propose to extend the studies of junctional complexes and cell membranes in the ocular lens of vertebrates. The long-term objectives of this application are to gain a better understanding in structural characteristics, distributions and functional roles of various types of cell junctions in the lens. Cell membranes are the major component of al cell junctions. It is believed that cell junctions and cell membranes play important roles in maintaining normal architecture physiology, and transparency of the lens. We extend our research goals to gain some insights into the roles of cell membranes involved in endocytosis and vesicular transport of useful macromolecules into the lens.
The specific aims of the research plan are as follows: (1) Direct structural analysis and comparison of native gap-junction connexons in lens epithelium and fiber cells, using rapid-freezing techniques; (2) Study association of gap junctions with actin bundles and actin-associated components, using cytochalasin-D treatments, and an improved fixation containing glutaraldehyde, lysine and tannic acid; (3) Immunocytochemical localization of gap junction proteins (MP70 and/or MP26), actin and actin- bundling proteins; (4) Ultrastructural and immunocytochemical analyses of formation of adherens junctions during chick lens morphogenesis; (5) Morphological and immunocytochemical studies of the occurrence, structure and nature of desmosomes and associated cytoskeletal components in lens epithelium of various species; (6) Ultrastructural and immunocytochemical investigations of unique membrane domains (clathrin-like) associated with formation of ball-and-socket junctions in fiber cells; (7) Visualization of structure and distribution of coated vesicles and non-coated vesicles in the lens of various species, using improved fixation and protein tracer methods; (8) Study the effects of growth factors and other factors on fluid-phase and receptor-mediated endocytosis in the lens; (9) Immunocytochemical determination of possible involvements of some growth factors in endocytosis using their specific antibodies as probes. Methodology used incudes (1) transmission electron microscopy using the improved fixation method developed in this laboratory; (2) rapid-freezing in liquid helium temperature without prior chemical fixation, and in conjunction with free-substitution, freeze-fracture and deep-etching techniques; (3) conventional freeze-fracture TEM; (4) immunofluorescence and immunogold labeling techniques, and (5) protein tracer and cytochemical methods.
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