The long-term goal is to understand the mechanism by which myofibrils assemble from their components, and how these precise structures are maintained in the face of repeated muscle activity. This goal has relevance to many types of human muscle disease, including those of skeletal and cardiac muscle. This proposal outlines studies of three genes in C. elegans, unc-98, unc-96 and unc-97 which are required for proper myofibril assembly and/or maintenance. UNC-98 is a novel 310 residue polypeptide consisting of 4 C2H2 Zn fingers and predicted NLS and NES sequences. By use of UNC-98 antibodies and UNC-98:GFP fusions, UNC-98 resides at M-lines, dense bodies (Z line analogs) and muscle cell nuclei. UNC-98 interacts with UNC-97 (PINCH in mammals), a LIM domain protein required for muscle focal adhesion assembly. Like UNC-98, UNC-97:GFP localizes to dense bodies, M-lines and nuclei. It is hypothesized that UNC-98 and UNC-97 function in muscle focal adhesion homeostasis, in which these proteins when localized to the adhesion sites monitor myofibril structure or activity, and travel to the nucleus to affect gene expression, unc-96 has a similar mutant phenotype to unc-98, interacts with unc-98 genetically, and may protect the sarcomere from breakdown resulting from normal muscle usage. We have determined that UNC-96 is a novel 418 aa protein and by 2 hybrid interacts with two conserved LIM domain proteins that also interact with UNC-97. Goals include: (1) for UNC-98, prove that its nuclear localization is important for its function, show that it can move from myofibrils into the nucleus, determine whether it can influence the expression of other genes, and whether it interacts with paramyosin in thick filaments; (2) for UNC-96, find out where it is localized in the sarcomere, provide further evidence that it interacts with paramyosin, UNC-98 and two LIM domain proteins; (3) for UNC-97 study its dynamics, whether it influences gene expression, whether its nuclear localization depends on UNC-98, and whether it indeed interacts with the two LIM domain proteins.
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