We propose to extend the studies of cell junctions and cell membranes in the ocular lens of vertebrates. In order to maintain an avascular lens in a normal and transparent state, gap junctions are believed to play a crucial role in regulating ionic and metabolic communications between epithelial cells, between fiber cells, and between epithelial and fiber cells in the lens. Recent progress suggests that lens gap junctions consist of multiple connexins and structural types. The significance of the existence of multiple gap junction proteins and structures in the lens remains to be determined. We plan to investigate in greater detail the unique structural characteristics and functions of the controversial lens gap junctions. In addition, adherens junctions along with their cell adhesion molecules (cadherins) and actin bundles are thought to play an important role in governing lens development. We plan to study the differential expressions of cadherins during prenatal and postnatal lens development. Moreover, we will extend our research goals to gain some insights into the roles of cell membranes involved in receptor-mediated potocytosis. This newly-found mechanism may be involved in sequestering and transporting important small molecules necessary for normal lens growth. Finally, we will study the molecular motor, kinesin, and its association with microtubule-based organelle transport mechanisms required for the formation of a transparent lens. We will apply several new approaches and methodologies for these projects. These include scanning tunneling microscopy, rapid-freezing, freeze- substitution, deep freeze-etch electron microscopy, frozen-section immunofluorescence labeling, confocal laser scanning microscopy, immunocytochemistry using freeze-substitution preparations, thin-section electron microscopy using an improved fixative developed in our laboratory, tracer and cytochemical techniques, organ cultures, gel electrophoresis, and immunoblotting. The long-term objectives of this application are to gain a better understanding of the structure and functional role of cell junctions and cell membranes in the lens.
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