Phenotypic analysis of transgenic worms of troponin C mutations in Caenorhabditis elegans

Tomohide Takaya, Hiromi Terami, Hiroaki Kagawa.

Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan.

East Asia C. elegans Meeting (Awaji, Japan), 2004/06/28 (Talk).

Abstract

There is two muscle tissues in Caenorhabditis elegans: pharynx for feeding and body wall for locomotion. The TnC gene, pat-10, encodes 161 amino acid residues of CeTnC-1 and is expressed in body wall muscles. TnC-1 shows 45% and 34% identities with TnCs of Drosophila and cardiac of vertebrate, respectively. The mutations in pat-10 lead to animals Pat phenotype (paralyzed arrest at embryonic two-fold stage). The pat-10(st568) mutation has occurred at two sites in TnC-1; D64 to N and W153 to stop. Mutated proteins produced in bacteria show that the D64 site is necessary for Ca²⁺-dependent conformation change and the C-terminal H-helix is essential for troponin I- and Ca²⁺-bindings. (J.Cell Biol. (1999) 146: 193-202). We constructed TnC-1 genomic vectors having the mutation of m1: D64N or m2: W153stop and injected each vector to the animal RW3613 (pat-10(st568)/dpy-5(e61), unc-29(e1072)). Segregation rates of F2 phenotypes suggest that the m1/pat-10 (st568) animal is similar to the wild type, but the m2/pat-10(st568) animal still has Pat phenotype. This means that calcium binding to site II is not important for TnC function but calcium binding to site IV and the H-helices are essential for animal development. For studying more details about relations between calcium- and TnI-bindings and phenotypes of transgenic animals we constructed deletion- and substitution- mutations in the H helices. TnI-bindigs of mutant TnCs in the H-helix were assayed in the presence and absence of Ca²⁺ in vitro by Western analysis and phenotypes of the transgenic animals of these mutations were observed by microscope. Substitution of negative charge for calcium binding at site IV did not affect TnI binding. Length and direction of H-helix were important for TnI binding. These results confirm that Ca2+ binding at site IV and TnI binding of H-helix of TnC-1 are essential for filament assembly and animal development.