Myofibril assembly visualized by imaging N-RAP, alpha-actinin, and actin in living cardiomyocytes.
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.
Manisastry
Shyam M
SM
National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA.
Zaal
Kristien J M
KJ
Horowits
Robert
R
D000185:Actinin / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D006801:Humans; D009124:Muscle Proteins / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000502:physiology* / Q000648:ultrastructure; D009210:Myofibrils / Q000502:physiology*; D011485:Protein Binding
D000199:Actins; D009124:Muscle Proteins; D000185:Actinin