Targeting and functional role of N-RAP, a nebulin-related LIM protein, during myofibril assembly in cultured chick cardiomyocytes.
Targeting and functional effects of N-RAP domains were studied by expression as GFP-tagged fusion proteins in cultured embryonic chick cardiomyocytes. GFP-tagged N-RAP was targeted to myofibril precursors, myofibril ends and cell contacts, expression patterns that are similar to endogenous N-RAP. The GFP-tagged N-RAP LIM domain (GFP-N-RAP-LIM) was targeted to the membrane in cells with myofibril precursors and cell-cell contacts. The GFP-tagged super repeats (N-RAP-SR) and the GFP-tagged domain normally found in between the super repeats and the LIM domain (N-RAP-IB) were each observed at sites of myofibril assembly, incorporating into myofibril precursors in a manner similar to full length N-RAP. However, unlike full-length N-RAP, N-RAP-SR and N-RAP-IB were also found in mature myofibrils, associating with the sarcomeric actin filaments and the Z-lines, respectively. N-RAP-IB was also colocalized with alpha-actinin at cell contacts. Each of the N-RAP constructs could inhibit the formation of mature myofibrils in cultured cardiomyocytes, with the effects of N-RAP-SR and N-RAP-IB depending on the time of transfection. The results show that each region of N-RAP is crucial for myofibril assembly. Combining the targeting and functional effects of N-RAP domains with information in the literature, we propose a new model for initiation of myofibrillogenesis.
Carroll
S L
SL
Laboratory of Muscle Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Herrera
A H
AH
Horowits
R
R
D000185:Actinin / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D060588:LIM Domain Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D024510:Muscle Development / Q000502:physiology*; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D009210:Myofibrils / Q000378:metabolism / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D013997:Time Factors
D060588:LIM Domain Proteins; D008164:Luminescent Proteins; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; C090114:cysteine and glycine-rich protein 3; C035620:nebulin; D000185:Actinin; D049452:Green Fluorescent Proteins