The manner in which extracellular matrix components are assembled into a tissue specific structure with a unique function will be investigated during development and repair of the chick embryo tendon. Synthesis and deposition of collagen involves multiple steps beginning in the cell and continuing into extracellular compartments. The passage of matrix components from intracellular compartments, where synthesis and early aggregation occur, to extracellular compartments, where further biochemical processing and deposition take place, is a spatial, temporal and functional continuum. The structural study of these compartments, in situ, requires a three dimensional appreciation of their topography. The functions of these various compartments are defined by their content and assembly related activity, and their study, in situ, will require autoradiography and immunocytochemistry. The structure of the extracellular compartments formed by the tendon cell surface during collagen fibril, bundle and macro-aggregate formation will be studied by conventional (60 kv) and intermediate voltage (120 kv) transmission electron microscopy (IVEM), stereoscopic examinations of 0.5 Mum sections, and computer assisted three dimensional reconstruction of 0.5 Mum serial sections. Extracellular compartments control focal regions of the extracellular space for such biochemical steps as procollagen processing, covalent cross-linking, and also serve to define the actual sites of matrix deposition. The temporal and spatial organization of collagen fibrils, bundles and larger aggregates in the extracellular space will be studied using pulse-chase autoradiography to define the pathway of type I collagen through the extracellular compartments. The unique, in situ, biochemical character of each of the compartments will be studied. Specific antibodies and immunoelectron microscopy will be used to identify structures and/or enzymes specific for the processing and assembly of type I collagen, viz. amino and carboxy extension peptides of procollagen; the carboxy terminal processing enzyme; lysyl oxidase, the cross-linking enzyme; and cell surface collagen binding proteins. These studies characterizing the structural and biochemical compartmentalization of the extracellular space will contribute to our understanding of the mechanisms by which the precise collagen architecture associated with tissue integrity are controlled during development, growth, injury and repair.
Showing the most recent 10 out of 11 publications
