The long-term objective of this grant is to determine the functional and structural relationships between the basement membrane, corneal epithelial cytoskeleton and organelle distribution (RER, Golgi). It has been established that cultured cells form focal adhesions in response to extracellar matrix proteins that contain cell adhesion molecules, actin associated proteins, kinases and calcium binding proteins. In addition, cell adhesion molecules act as signaling receptors by causing tyrosine phosphorylation, Ca2+ influx, an increase in cytoplasmic pH and phospholipid turnover. We will expand this previous work to determine if these proteins and mechanisms are also necessary for reorganizing actin in the embryonic corneal epithelial model. The sequence of events that occur during actin cortical mat reorganization in response to extracellular matrix molecules will be dissected to determine the relationships between the ECM, cell adhesion molecules, cytoskeleton and organelle distribution. We will study the following specific areas and questions: I. Cell-cell and cell-matrix interactions during corneal epithelial development. We will test the hypothesis that the reorganization of the actin cortical depends upon a cascade of events including receptor binding, actin associated protein rearrangements, and signal transduction. What are the specific link proteins between cell-cell junctions or extracellular matrix receptors and actin? How is the non-actin cytoskeleton organized in embryonic corneal epithelial cells? Is the reorganization of the actin cortical mat dependent on signal transduction? II. Factors controlling mRNA distribution during corneal differentiation. We have shown that beta- actin mRNA has a polarized distribution in corneal epithelia similar to F- actin protein. Specific collagen mRNAs also appear to have a distinct distribution in chondrocytes and corneal epithelia. We will test the hypothesis that actin and collagen mRNA are organized in the corneal epithelium by a cytoskeletal dependent mechanism and that reorganization of the cytoskeleton is a factor in up-regulating matrix synthesis. How does the cell organize actin and collagen mRNA? Will disruption of the cytoskeleton also disrupt collagen mRNA distribution? III. Factors controlling organelle distribution during actin cortical mat reorganization. We have previously shown that disrupting actin with cytochalasin D caused the RER in the basal compartment to decrease in epithelia isolated with the basal lamina. We will test the hypothesis that cytoskeletal elements organize protein secretion organelles and translation cofactors to regulate expression of secreted proteins. Are translational cofactors or endoplasmic reticulum stabilized by attachment to the cytoskeleton? What mechanisms are responsible for this attachment? In summary, a cell biological model for corneal epithelial interactions between the extracellular environment and cellular organization has been developed.