A functional knock-out of titin results in defective myofibril assembly

Titin, also called connectin, is a giant muscle protein that spans the dist ance from the sarcomeric Z-disc to the M-band. Titin is thought to direct t he assembly of sarcomeres and to maintain sarcomeric integrity by interacti ng with numerous sarcomeric proteins and providing a mechanical linkage. Si nce severe defects of such an important molecule are likely to result in em bryonic lethality, a cell culture model should offer the best practicable t ool to probe the cellular functions of titin. The myofibroblast cell line B HK-21/C13 was described to assemble myofibrils in culture. We have now char acterized the sub-line BHK-21-Bi, which bears a small deletion within the t itin gene. RNA analysis revealed that in this mutant cell line only a small internal portion of the titin mRNA is deleted. However, western blots, imm unofluorescence microscopy and immunoprecipitation experiments showed that only the N-terminal, approx. 100 kDa central Z-disc portion of the 3 MDa ti tin 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 arr ays was confirmed by electron microscopy. Myogenisation induced by transfec tion with MyoD yielded myofibrils only in myotubes formed from wild type an d not from mutant cells, ruling out that a principal failure in myogenic co mmitment of the BHK-21-Bi cells might cause the observed effects. These exp eriments 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.