N-RAP scaffolds I-Z-I assembly during myofibrillogenesis in cultured chick cardiomyocytes.
N-RAP is a muscle-specific protein with an N-terminal LIM domain (LIM), C-terminal actin-binding super repeats homologous to nebulin (SR) and nebulin-related simple repeats (IB) in between the two. Based on biochemical data, immunofluorescence analysis of cultured embryonic chick cardiomyocytes and the targeting and phenotypic effects of these individual GFP-tagged regions of N-RAP, we proposed a novel model for the initiation of myofibril assembly in which N-RAP organizes alpha-actinin and actin into the premyofibril I-Z-I complexes. We tested the proposed model by expressing deletion mutants of N-RAP (i.e. constructs containing two of the three regions of N-RAP) in chick cardiomyocytes and observing the effects on alpha-actinin and actin organization into mature sarcomeres. Although individually expressing either the LIM, IB, or SR regions of N-RAP inhibited alpha-actinin assembly into Z-lines, expression of either the LIM-IB fusion or the IB-SR fusion permitted normal alpha-actinin organization. In contrast, the LIM-SR fusion (LIM-SR) inhibited alpha-actinin organization into Z-lines, indicating that the IB region is critical for Z-line assembly. While permitting normal Z-line assembly, LIM-IB and IB-SR decreased sarcomeric actin staining intensity; however, the effects of LIM-IB on actin assembly were significantly more severe, as estimated both by morphological assessment and by quantitative measurement of actin staining intensity. In addition, LIM-IB was consistently retained in mature Z-lines, while mature Z-lines without significant IB-SR incorporation were often observed. We conclude that the N-RAP super repeats are essential for organizing actin filaments during myofibril assembly in cultured embryonic chick cardiomyocytes, and that they also play an important role in removal of the N-RAP scaffold from the completed myofibrillar structure. This work strongly supports the N-RAP scaffolding model of premyofibril assembly.
Carroll
Stefanie
S
Laboratory of Muscle Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health/DHHS, Bethesda, MD 20892, USA.
Lu
Shajia
S
Herrera
Amy H
AH
Horowits
Robert
R
D000199:Actins / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D024510:Muscle Development / Q000502:physiology*; D009124:Muscle Proteins / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000502:physiology; D009154:Mutation; D032383:Myocytes, Cardiac / Q000166:cytology / Q000502:physiology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D000199:Actins; D008164:Luminescent Proteins; D009124:Muscle Proteins; C035620:nebulin; D049452:Green Fluorescent Proteins