The nebulette repeat domain is necessary for proper maintenance of tropomyosin with the cardiac sarcomere

• Published in: Experimental cell research 2008-11, Vol.314 (19), p.3519-3530
• Main Authors: Bonzo, Jeremy R., Norris, Andrea A., Esham, Michael, Moncman, Carole L.
• Format: Article
• Language: English
• Subjects: Actin-binding proteins; Animals; Binding sites; Carrier Proteins - chemistry; Carrier Proteins - metabolism; Cells, Cultured; Cellular biology; Cercopithecus aethiops; Chick Embryo; COS Cells; Cytoskeletal Proteins - chemistry; Cytoskeletal Proteins - metabolism; Deoxyribonucleic acid; DNA; DNA, Complementary - metabolism; Fluorescent Antibody Technique; Humans; LIM Domain Proteins; Myocytes, Cardiac - metabolism; Myofibrillogenesis; Nebulin; Poultry; Protein Structure, Tertiary; Proteins; Sarcomeres - metabolism; src Homology Domains; Titin; Tropomyosin - metabolism; Two-Hybrid System Techniques
• ISSN: 0014-4827; 1090-2422
• DOI: 10.1016/j.yexcr.2008.09.001

Nebulette is a cardiac-specific isoform of the giant actin-binding protein nebulin. Nebulette, having a mass of ∼ 100 kDa, is only predicted to extend 150 nm from the edge of the Z-lines. Overexpression of the nebulette C-terminal linker and/or SH3 domains in chicken cardiomyocytes results in a loss of endogenous nebulette with a concomitant loss of tropomyosin (TPM) and troponin, as well as a shortening of the thin filaments. These data suggest that nebulette's position in the sarcomere is important for the maintenance of TPM, troponin and thin filament length. To evaluate this hypothesis, N-terminal nested truncations tagged with GFP were expressed in chicken cardiomyocytes and the cells were analyzed for the distribution of myofilament proteins. Minimal effects on the myofilaments were observed with N-terminal deletions of up to 10 modules; however, deletion of 15 modules replicated the phenotype observed with expression of the C-terminal fragments. Expression of internal deletions of nebulette verifies that a site between module 10 and 15 is important for TPM maintenance within the sarcomeric lattice. We have additionally isolated TPM cDNAs from a yeast two hybrid (Y2H) analysis. These data indicate the importance of the nebulette–TPM interactions in the maintenance and stability of the thin filaments.