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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR052133-05
Application #
7674655
Study Section
Skeletal Muscle and Exercise Physiology Study Section (SMEP)
Program Officer
Boyce, Amanda T
Project Start
2005-09-01
Project End
2011-08-31
Budget Start
2009-09-01
Budget End
2011-08-31
Support Year
5
Fiscal Year
2009
Total Cost
$234,226
Indirect Cost
Name
Emory University
Department
Pathology
Type
Schools of Medicine
DUNS #
066469933
City
Atlanta
State
GA
Country
United States
Zip Code
30322
Lecroisey, Claire; Brouilly, Nicolas; Qadota, Hiroshi et al. (2013) ZYX-1, the unique zyxin protein of Caenorhabditis elegans, is involved in dystrophin-dependent muscle degeneration. Mol Biol Cell 24:1232-49
Warner, Adam; Xiong, Ge; Qadota, Hiroshi et al. (2013) CPNA-1, a copine domain protein, is located at integrin adhesion sites and is required for myofilament stability in Caenorhabditis elegans. Mol Biol Cell 24:601-16
Wilson, Kristy J; Qadota, Hiroshi; Benian, Guy M (2012) Immunofluorescent localization of proteins in Caenorhabditis elegans muscle. Methods Mol Biol 798:171-81
Qadota, Hiroshi; Moerman, Donald G; Benian, Guy M (2012) A molecular mechanism for the requirement of PAT-4 (integrin-linked kinase (ILK)) for the localization of UNC-112 (Kindlin) to integrin adhesion sites. J Biol Chem 287:28537-51
Warner, Adam; Qadota, Hiroshi; Benian, Guy M et al. (2011) The Caenorhabditis elegans paxillin orthologue, PXL-1, is required for pharyngeal muscle contraction and for viability. Mol Biol Cell 22:2551-63
Qadota, Hiroshi; Benian, Guy M (2010) Molecular structure of sarcomere-to-membrane attachment at M-Lines in C. elegans muscle. J Biomed Biotechnol 2010:864749
Moulder, Gary L; Cremona, Gina H; Duerr, Janet et al. (2010) ?-actinin is required for the proper assembly of Z-disk/focal-adhesion-like structures and for efficient locomotion in Caenorhabditis elegans. J Mol Biol 403:516-28
Miller, Rachel K; Qadota, Hiroshi; Stark, Thomas J et al. (2009) CSN-5, a component of the COP9 signalosome complex, regulates the levels of UNC-96 and UNC-98, two components of M-lines in Caenorhabditis elegans muscle. Mol Biol Cell 20:3608-16
Miller, Rachel K; Qadota, Hiroshi; Mercer, Kristina B et al. (2008) UNC-98 and UNC-96 interact with paramyosin to promote its incorporation into thick filaments of Caenorhabditis elegans. Mol Biol Cell 19:1529-39
Stevenson, Tesheka O; Mercer, Kristina B; Cox, Elisabeth A et al. (2007) unc-94 encodes a tropomodulin in Caenorhabditis elegans. J Mol Biol 374:936-50

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