Fibronectin plays important roles in differentiation, adhesion and maintenance of cell morphology. The protein is heterogeneous in structure, and recent molecular studies indicate that the heterogeneity results from alternative splicing of three exons. Preliminary experiments show that the fibronectin RNA in embryonic chick cartilage differs from the fibronectin RNA in other tissues, and that the fibronectin RNA from chondrocytes in culture varies with the culture condition. The immediate goal of this project is to further define the structures of the fibronectin RNA and the resulting protein in intact cartilage and chondrocytes grown under various culture conditions. Nuclease protection assays will complete characterization of the fibronectin RNA from these tissues and cells. In addition, characterization of the fibronectin protein, both in intact cartilage and in cultured chondrocytes, will involve immunological studies with antibodies specific for each of the alternatively spliced regions of the fibronectin molecule. The long-term goal of this project is to understand the role of fibronectin in chondrocyte interactions with the cartilage extracellular matrix proteins and during chondrogenesis. This goal involves several types of experiments. First, investigation of the molecular interactions of the cartilage form of fibronectin with other cartilage extracellular matrix proteins will utilize direct binding assays and will determine the relative affinities of cartilage fibronectin for these matrix proteins. Second, investigation of the ability of cultured chondrocytes to interact with cartilage extracellular proteins, via cartilage fibronectin, will utilize standard cell adhesion and cell spreading assays. Finally, in situ hybridizations and indirect immunofluorescence with probes and antibodies specific for the alternatively spliced exons of fibronectin will characterize the forms of fibronectin present during all stages of chondrogenesis. Peptide competition and antibody interference assays will determine whether the alternatively spliced regions of the fibronectin molecule have any function in chondrocyte interactions with the cartilage extracellular matrix and during chondrogenesis. These studies will provide important information regarding the role of fibronectin in the cartilage matrix and during cartilage differentiation.
