Extracellular matices have important roles in the morphogenesis and development of multicellular animals. Many extracellular matrix components have been identified, but in most cases little is known about the details of their assembly and function. The long-term goals of this proposal are to understand how extracellular matrix proteins assemble into organized macromolecular complexes and how these complexes function in morphogenesis and development. The most abundant and ubiquitous extracellular matrix proteins are the collagens. Several human connective tissue diseases have been shown to result from mutations in collagens. The powerful genetic and molecular analyses possible in the nematode C. elegans, and its well characterized anatomy and development make it an excellent system for studying collagen structure and function. Mutations in several cuticle collagen genes in C. elegans have profound effects on the animals morphogenesis. Many of these mutations have been shown to define a region involved in proteolytic processing. Antibodies will be used to examine the fates of unprocessed mutant collagens. The gene encoding the proteolytic enzyme will be identified, using the two hybrid system of yeast, and characterized. A second group of mutations are cysteine replacements in the carboxyl-terminal domains that affect both disulfide and tyrosine- based covalent bonding of the collagens. The function of the carboxyl domains in collagen assembly will be studied by analyzing site-directed mutations in transgenic animals, both phenotypically and with collagen- specific antibodies. The two-hybrid system will also be used to examine protein-protein interactions between collagen carboxyl domains. C. elegans collagens have been shown to be crosslinked with di- and (iso)trityrosine residues. The site of one of these crosslinks has been determined. This domain will be used to identify the enzyme responsible for tyrosine oxidation and crosslink formation, using assays for dityrosine formation as well as the two-hybrid system. Mutations of the enzyme will be generated, by PCR selection for transposon insertion, in order to determine the effects of reducing or eliminating crosslinking. Tyrosine crosslinks make nematode cuticles extremely tough and insoluble. An understanding of the mechanism of crosslink formation may lead to novel approaches for controlling parasitic nematodes, as well as valuable technical tools for manipulating C. elegans. The persistence of collagen mutant phenotypes from one developmental stage to the next, when the collagen gene is not expressed, was proposed to result from maintenance of the phenotype by the epidermal cytoskeleton. This proposal will be tested by expressing mutant collagens using regulatable promoters in transgenic animals, and determining if phenotypic persistence is observed in the manner predicted. These studies may reveal developmental interplay between the extracellular matrix and the cytoskeleton. The mutations that provided these insights into collagen function were selected because they exhibit unusual genetic interactions. Additional interacting mutations have and will be generated and analyzed, as they may identify novel extracellular matrix components and their important functional domains.
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