Key result
Expression of N-RAP deletion mutants in cultured chick cardiomyocytes demonstrated that the N-RAP super repeats are essential for organizing actin filaments during myofibril assembly.
N-RAP super repeats are essential for organizing actin filaments during myofibril assembly in cultured embryonic chick cardiomyocytes, supporting the N-RAP scaffolding model.
Supports N-RAP scaffolding model in chick cardiomyocytes; hypothesis-generating for in vivo myofibrillogenesis.
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.
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Carroll et al. (2003) studied Myofibril assembly. N-RAP deletion mutants (LIM-IB, IB-SR, LIM-SR) vs. Individual regions (LIM, IB, SR) was evaluated on alpha-actinin and actin organization into mature sarcomeres. Expression of N-RAP deletion mutants in cultured chick cardiomyocytes demonstrated that the N-RAP super repeats are essential for organizing actin filaments during myofibril assembly.
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