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Author Notes:

Address correspondence to: Guy M. Benian (Email: pathgb@emory.edu)

We are grateful to Megan Landsverk and Henry Epstein for patiently teaching us how to purify nematode thick filaments.

We thank Phil Anderson for providing us with r291 mutant worms, and Pam Hoppe for suggesting that we stain unc-96 mutants with anti-paramyosin antibodies.

We thank Vardges Ter-Hovhannisyan and Mark Borodovsky for calculating the amino acid composition of the average C. elegans protein, Robert Santoianni for the EM images, Kim Gernert for help in analyzing the UNC-96 protein sequence, CamTu Thi Nguyen for cosmid preps, Denise Flaherty and Paula Checchi for some of the immunofluorescent staining of unc-96 mutants, and Tesheka Stevenson for comments on the manuscript.

We also thank Andy Fire for the promoterless GFP vector, Alan Coulson for cosmid clones, Kozo Kaibuchi for plasmid pMAL-KK1, Krishna Bhat for use of a confocal microscope, and Dan Kalman for use of a deconvolution microscopy system.

We thank David Miller for providing us monoclonal antibodies 5-6 (to MHC A) and 5-8 (to MHC B), Pam Hoppe for providing us monoclonal antibodies 5-23 (to paramyosin), MH42 (to UNC-89), and MH35 (to α-actinin), and Hiroaki Kagawa for mAb Mab 5B5 (to paramyosin).

In later stages of this work, monoclonal antibodies 5-6 and 5-23, developed by David Miller and Henry Epstein, were purchased from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by Department of Biological Sciences, The University of Iowa, (Iowa City, IA).

Some strains used in this work were provided by the Caenhorhabditis Genetics Center, which is supported by the National Center for Research Resources of the National Institutes of Health.

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Research Funding:

Funds for these studies were derived from Grant 0255157B (to G.M.B.) and predoctoral fellowship 0415274B (to R.K.M.) from the American Heart Association Southeast Affiliate and Grants AR-051466 and AR-052133 from National Institutes of Health (to G.M.B).

Caenorhabditis elegans UNC-96 Is a New Component of M-Lines That Interacts with UNC-98 and Paramyosin and Is Required in Adult Muscle for Assembly and/or Maintenance of Thick Filaments

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Journal Title:

Molecular Biology of the Cell

Volume:

Volume 17, Number 9

Publisher:

, Pages 3832-3847

Type of Work:

Article | Final Publisher PDF

Abstract:

To gain further insight into the molecular architecture, assembly, and maintenance of the sarcomere, we have carried out a molecular analysis of the UNC-96 protein in the muscle of Caenorhabditis elegans. By polarized light microscopy of body wall muscle, unc-96 mutants display reduced myofibrillar organization and characteristic birefringent “needles.” By immunofluorescent staining of known myofibril components, unc-96 mutants show major defects in the organization of M-lines and in the localization of a major thick filament component, paramyosin. In unc-96 mutants, the birefringent needles, which contain both UNC-98 and paramyosin, can be suppressed by starvation or by exposure to reduced temperature. UNC-96 is a novel ∼47-kDa polypeptide that has no recognizable domains. Antibodies generated to UNC-96 localize the protein to the M-line, a region of the sarcomere in which thick filaments are cross-linked. By genetic and biochemical criteria, UNC-96 interacts with UNC-98, a previously described component of M-lines, and paramyosin. Additionally, UNC-96 copurifies with native thick filaments. A model is presented in which UNC-96 is required in adult muscle to promote thick filament assembly and/or maintenance.

Copyright information:

© 2006 by The American Society for Cell Biology

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