This project is concerned with the process(es) involved in the assembly of the extracellular matrix in connective tissues. Specifically, the project is designed to determine 1) the interactions among fibronectin, procollagen and the cell surface during matrix formation and 2) the orientation of fibronectin in fibronectin-containing filaments and collagen-containing filaments. To study these processes, fluorescently-labeled procollagen (precursor molecule to collagen), plasma fibronectin, a fibronectin binding fragment of collagen (CB.7) and fibronectin fragments specific for cell adhesion, collagen binding and matrix formation will be added to freshly seeded cultures of human skin fibroblasts. The movement of fibronectin and procollagen into the matrix and the location of the respective fragments will be monitored by fluorescent microscopy and time lapse video-enhanced microcinematography. Ultrastructural analyses of fibronectin orientation in filaments and inter-actions among fibronectin, procollagen and the cell surface will be undertaken with the high voltage electron microscope using colloidal gold attached to antibodies specific for procollagen, fibronectin, or the fluorescent markers (Fluorescein, dansylcadaverine, N-Iodoacetyl dansyl or 5-iodoacetamido fluorescein). The last three markers can be precisely incorporated into the amino terminal or free sulfhydryls of fibronectin, thereby revealing the periodic arrangement of fibronectin in fibrils after labeling with anti-fluorescein or anti-dansyl gold antibodies.