The aim of this proposal is to determine the structure of fibronectin fibrils and the role of collagen on the assembly of fibronectin fibrils. The project will use immunofluorescence microscopy, high voltage immunoelectron microscopy (HVEM), biochemical and molecular biology techniques in an integrated approach to study fibronectin fibril formation. To determine the structure of fibronectin fibrils, homotypic plasma fibronectin fibrils formed in cycloheximide treated subconfluent cultures will be studied. Homotypic fibrils formed in these cultures are well- suited for immunoelectron microscopy studies, because these fibrils contain only one isotype of fibronectin and the fibril can easily be followed along its entire length. The arrangement of fibronectin in fibrils will be determined by using monoclonal antibodies to various domains of fibronectin to map the distances between domains. Regions of fibronectin involved in linear and lateral growth of fibrils will be identified by using monoclonal antibodies, fibronectin fragments and recombinant fibronectin fragments missing sequences to inhibit linear and lateral growth of fibrils. Sequences involved in disulfide exchange between fibronectin dimers will be identified by two dimensional gel electrophoresis and amino acid analysis. To determine the role of the fibronectin binding site in collagen on the assembly of fibronectin fibrils, cycloheximide treated subconfluent cultures of fibroblasts will be incubated with exogenous plasma fibronectin and either normal type I collagen or type I collagen containing mutations in its fibronectin binding site. Collagen monomers with mutations in their fibronectin binding site will be purified from the media of cells transfected with full length cDNAs for alpha1(I) collagen chains that contain mutations in the fibronectin binding site. Immunoelectron microscopy will be used to determine if fibronectin and collagen can be co- assembled into fibrils and whether the periodic arrangement of fibronectin in collagen fibrils is altered in the presence of mutated collagen chains. Metabolic labeling studies, northern blots and immunofluorescence microscopy will be used to determine if cells transfected with the mutated collagen chains are unable to assemble fibronectin because the mutated alpha1(I) chains have altered the secretion and turnover of collagen or inhibited the expression of fibronectin receptors necessary for fibril formation.
