title: A functional knock-out of titin results in defective myofibril assembly.
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.
authors.affiliation: Department of Cell Biology, University of Potsdam, Lennéstr. 7a, D-14471 Potsdam, Germany. pvdven@rz.uni-potsdam.de
authors.forename: P F
authors.initials: PF
authors.lastname: van der Ven
authors.forename: J W
authors.initials: JW
authors.lastname: Bartsch
authors.forename: M
authors.initials: M
authors.lastname: Gautel
authors.forename: H
authors.initials: H
authors.lastname: Jockusch
authors.forename: D O
authors.initials: DO
authors.lastname: Fürst
mesh_terms: D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000502:physiology; D002460:Cell Line; D064211:Connectin; D005347:Fibroblasts / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D009124:Muscle Proteins / Q000502:physiology*; D018482:Muscle, Skeletal / Q000378:metabolism / Q000648:ultrastructure; D009210:Myofibrils / Q000502:physiology* / Q000648:ultrastructure*; D011494:Protein Kinases / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D017384:Sequence Deletion
chemical_list: D002148:Calmodulin-Binding Proteins; D064211:Connectin; D009124:Muscle Proteins; D012333:RNA, Messenger; C577285:TTN protein, human; D011494:Protein Kinases