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A functional knock-out of titin results in defective myofibril assembly
- Source :
- Journal of Cell Science; April 2000, Vol. 113 Issue: 8 p1405-1414, 10p
- Publication Year :
- 2000
-
Abstract
- Titin, also called connectin, is a giant muscle protein that spans the distance from the sarcomeric Z-disc to the M-band. Titin is thought to direct the assembly of sarcomeres and to maintain sarcomeric integrity by interacting with numerous sarcomeric proteins and providing a mechanical linkage. Since severe defects of such an important molecule are likely to result in embryonic lethality, a cell culture model should offer the best practicable tool to probe the cellular functions of titin. The myofibroblast cell line BHK-21/C13 was described to assemble myofibrils in culture. We have now characterized the sub-line BHK-21-Bi, which bears a small deletion within the titin gene. RNA analysis revealed that in this mutant cell line only a small internal portion of the titin mRNA is deleted. However, western blots, immunofluorescence microscopy and immunoprecipitation experiments showed that only the N-terminal, approx. 100 kDa central Z-disc portion of the 3 MDa titin protein is expressed, due to the homozygous deletion in the gene. Most importantly, in BHK-21-Bi cells the formation of thick myosin filaments and the assembly of myofibrils are impaired, although sarcomeric proteins are expressed. Lack of thick filament formation and of ordered actin-myosin arrays was confirmed by electron microscopy. Myogenisation induced by transfection with MyoD yielded myofibrils only in myotubes formed from wild type and not from mutant cells, ruling out that a principal failure in myogenic commitment of the BHK-21-Bi cells might cause the observed effects. These experiments provide the first direct evidence for the crucial role of titin in both thick filament formation as a molecular ruler and in the coordination of myofibrillogenesis.
Details
- Language :
- English
- ISSN :
- 00219533 and 14779137
- Volume :
- 113
- Issue :
- 8
- Database :
- Supplemental Index
- Journal :
- Journal of Cell Science
- Publication Type :
- Periodical
- Accession number :
- ejs58996808
- Full Text :
- https://doi.org/10.1242/jcs.113.8.1405