Charles Philippe Leblond, Ph.D. - US grants

The collagens are elaborated within cells as procollagens along a complex biosynthetic pathway, which has been partly identified in the case of type I and IV collagens but is little known in the case of type III and V collagens. The proposal is to identify the pathway in the latter two and to measure the rates of passage of the precursors through various cell compartments in all four. In addition, the incidental findings of new structures in basement membrane had led to a study of their tridimensional architecture and immunocharacterization. For the intracellular biogenesis of the collagens, two main methods will be utilized: a) immunostaining of Lowicryl embedded tissues by protein A-gold; and counting gold particles per unit area over the structures under study; b) high resolution radioautography at various times after a 3H-proline injection. The results obtained by the two approaches will be combined to prepare specific activity time curves for each organelle in the producer cells and thus work out steps and rates of migration along the biogenetic pathway of the various collagens. In practice, the antibodies capable of reacting with the intracellular precursor of collagen will be used for two purposes: firstly, to identify the cells producing the collagen and secondly, to examine the intracellular steps in the biogenesis of this collagen. The cells selected to study biogenesis are cells which are clearly immunostained for only one type of collagen: odontoblasts for type I collagen, periodontal fibroblasts for type III collagen, and endodermal cells associated with a multilayered basement membrane of rat embryos (Reichert's membrane) for type IV collagen. As for type V collagen, exploratory work on rat placenta and other tissues will be needed to detect producer cells and use them for the study of biogenesis. Finally, incidental results on the structure of Reichert's membrane offer hope of clarifying the organization of type IV collagen within basement membranes. In Reichert's membrane, type IV collagen is present as an axial filament in cords arranged into a network. These obervations have to be extended to the basement membranes of tooth and other tissues. Moreover, the finding of rigid hollow rods called basotubules and dots called double-pegs has to be confirmed in common basement membranes. It is thus hoped to extend our knowledge of collagen biogenesis in general and basement membranes in particular.