title: Alteration of tropomyosin-binding properties of tropomodulin-1 affects its capping ability and localization in skeletal myocytes.
abstract: Tropomodulin (Tmod) is an actin-capping protein that binds to the two tropomyosins (TM) at the pointed end of the actin filament to prevent further actin polymerization and depolymerization. Therefore, understanding the role of Tmod is very important when studying actin filament dependent processes such as muscle contraction and intracellular transport. The capping ability of Tmod is highly influenced by TM and is 1000-fold greater in the presence of TM. There are four Tmod isoforms (Tmod1-4), three of which, Tmod1, Tmod3, and Tmod4, are expressed in skeletal muscles. The affinity of Tmod1 to skeletal striated TM (stTM) is higher than that of Tmod3 and Tmod4 to stTM. In this study, we tested mutations in the TM-binding sites of Tmod1, using circular dichroism (CD) and prediction analysis (PONDR). The mutations R11K, D12N, and Q144K were chosen because they decreased the affinity of Tmod1 to stTM, making it similar to that of affinity of Tmod3 and Tmod4 to stTM. Significant reduction of inhibition of actin pointed-end polymerization in the presence of stTM was shown for Tmod1 (R11K/D12N/Q144K) as compared with WT Tmod1. When GFP-Tmod1 and mutants were expressed in primary chicken skeletal myocytes, decreased assembly of Tmod1 mutants was revealed. This indicates a direct correlation between TM-binding and the actin-capping abilities of Tmod. Our data confirmed the hypothesis that assembly of Tmod at the pointed-end of the actin filament depends on its TM-binding affinity.
authors.affiliation: Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, Washington 99164, USA.
authors.forename: Natalia A
authors.initials: NA
authors.lastname: Moroz
authors.forename: Stefanie M
authors.initials: SM
authors.lastname: Novak
authors.forename: Ricardo
authors.initials: R
authors.lastname: Azevedo
authors.forename: Mert
authors.initials: M
authors.lastname: Colpan
authors.forename: Vladimir N
authors.initials: VN
authors.lastname: Uversky
authors.forename: Carol C
authors.initials: CC
authors.lastname: Gregorio
authors.forename: Alla S
authors.initials: AS
authors.lastname: Kostyukova
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry; D000199:Actins / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D002942:Circular Dichroism; D005786:Gene Expression Regulation; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D008969:Molecular Sequence Data; D032342:Muscle Cells / Q000166:cytology*; D018482:Muscle, Skeletal / Q000166:cytology*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D020033:Protein Isoforms; D017386:Sequence Homology, Amino Acid; D051348:Tropomodulin / Q000737:chemistry* / Q000235:genetics*; D014335:Tropomyosin / Q000737:chemistry*
chemical_list: D000199:Actins; D020033:Protein Isoforms; D051348:Tropomodulin; D014335:Tropomyosin