9728762 Benian Actin is one of the most abundant proteins in eukaryotic cells It can polymerize into microfilaments that provide structure to eukaryotic cells, either as individual dispersed filaments or bundled together by actin binding proteins into cables. Together with its associated motor proteins, the myosins, actin microfilaments form the basis for a significant amount of intracellular movement, including muscle contraction. Although actin is one of the most intensively studied eukaryotic proteins, there is much about its function and regulation that remains poorly understood. This project promises significant insight into the understanding of how certain proteins regulate the assembly of the actin cytoskeleton. The model system to be used is the nematode worm, C. elegans, a model organism in which many mutations affecting muscle proteins have been catalogued. The unc-60 ("uncoordinated") gene encodes two proteins, both of which are members of a known family of actin-binding proteins (the ADF/cofilin family) which have been found in all eukaryotic cells examined to date. Biochemical studies of these proteins in vertebrates have shown that they are involved primarily in depolymerizing actin filaments. Unc-60 mutants provide a unique opportunity in that they are the only available mutants in this type of muscle protein in any multicellular organism. Unc-60 mutant worms are paralyzed, and their muscles have abnormally large accumulations of actin filaments. The working hypothesis for this study is that in normal worms, depolymerization by unc-60 serves to regulate the amount of actin in polymerized form, and lack of such regulation results in the abnormal accumulation of filaments. The Benian laboratory has successfully expressed both UNC-60 isoforms in bacteria and has purified and initially characterized the proteins. Microinjection of synthetic RNA into the germline of C. elegans often results in a mutant phenotype of the corresponding gene. Using this method, the Be nian lab has shown that injection of unc-60A RNA but not unc-60B RNA results in embryonic lethality. This suggests that the normal function of UNC-60A is different from UNC-60B and that UNC-60A has an essential role in early development. The goals of the project are: 1, to determine the sequence alterations of 12 unc-60 alleles and interpret these with respect to phenotypic severity and position of the mutation on the UNC-60 protein structure; 2, to determine the spatial and temporal patterns of expression of the two proteins encoded by unc-60, to evaluate the hypothesis that one of them is muscle-specific and the other is embryonic or non-muscle; 3, to study the effect of bacterially expressed unc-60A and B on actin polymerization in vitro and look for possible regulation by phosphoinositides and serine phosphorylation; 3a, to attempt to crystallize the proteins for structural analysis; 4, to analyze an unc-60-specific suppressor gene, sup-12, to see if SUP-12 interacts with, or modifies either UNC-60 or actin. The nature of the SUP-12 proteins is likely to provide insight into the regulation of thin filament number, presently not easily obtainable through biochemical means. These studies will provide important insights into how cells regulate the polymerization of actin microfilaments.
