The nebulette repeat domain is necessary for proper maintenance of tropomyosin with the cardiac sarcomere.
Nebulette is a cardiac-specific isoform of the giant actin-binding protein nebulin. Nebulette, having a mass of approximately 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.
Bonzo
Jeremy R
JR
Department of Molecular and Cellular Biochemistry, University of Kentucky, 741 S. Limestone, Lexington, KY 40536, USA.
Norris
Andrea A
AA
Esham
Michael
M
Moncman
Carole L
CL
D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002478:Cells, Cultured; D002642:Chick Embryo; D002522:Chlorocebus aethiops; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D060588:LIM Domain Proteins; D032383:Myocytes, Cardiac / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012518:Sarcomeres / Q000378:metabolism*; D014335:Tropomyosin / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D018909:src Homology Domains
D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D060588:LIM Domain Proteins; C486890:NEBL protein, human; D014335:Tropomyosin