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First published online December 1, 2003
doi: 10.1242/10.1242/jcs.00847

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N-RAP scaffolds I-Z-I assembly during myofibrillogenesis in cultured chick cardiomyocytes

Laboratory of Muscle Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA

^* Author for correspondence (e-mail: horowits{at}helix.nih.gov)

Accepted 27 August 2003

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 -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 -actinin and actin organization into mature sarcomeres. Although individually expressing either the LIM, IB, or SR regions of N-RAP inhibited -actinin assembly into Z-lines, expression of either the LIM-IB fusion or the IB-SR fusion permitted normal -actinin organization. In contrast, the LIM-SR fusion (LIM-SR) inhibited -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.

Key words: Myofibrillogenesis, Cardiomyocytes, N-RAP, Actin

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