Myofibril assembly visualized by imaging N-RAP, alpha-actinin, and actin in living cardiomyocytes

Exp Cell Res. 2009 Jul 15;315(12):2126-39. doi: 10.1016/j.yexcr.2009.02.006. Epub 2009 Feb 20.


N-RAP is a striated muscle-specific scaffolding protein that organizes alpha-actinin and actin into symmetrical I-Z-I structures in developing myofibrils. Here we determined the order of events during myofibril assembly through time-lapse confocal microscopy of cultured embryonic chick cardiomyocytes coexpressing fluorescently tagged N-RAP and either alpha-actinin or actin. During de novo myofibril assembly, N-RAP assembled in fibrillar structures within the cell, with dots of alpha-actinin subsequently organizing along these structures. The initial fibrillar structures were reminiscent of actin fibrils, and coassembly of N-RAP and actin into newly formed fibrils supported this. The alpha-actinin dots subsequently broadened to Z-lines that were wider than the underlying N-RAP fibril, and N-RAP fluorescence intensity decreased. FRAP experiments showed that most of the alpha-actinin dynamically exchanged during all stages of myofibril assembly. In contrast, less than 20% of the N-RAP in premyofibrils was exchanged during 10-20 min after photobleaching, but this value increased to 70% during myofibril maturation. The results show that N-RAP assembles into an actin containing scaffold before alpha-actinin recruitment; that the N-RAP scaffold is much more stable than the assembling structural components; that N-RAP dynamics increase as assembly progresses; and that N-RAP leaves the structure after assembly is complete.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Actinin / metabolism*
  • Actins / metabolism*
  • Animals
  • Cells, Cultured
  • Chick Embryo
  • Humans
  • Muscle Proteins / metabolism*
  • Myocytes, Cardiac / physiology*
  • Myocytes, Cardiac / ultrastructure
  • Myofibrils / physiology*
  • Protein Binding


  • Actins
  • Muscle Proteins
  • Actinin