Journal
JOURNAL OF CELL SCIENCE
Volume 119, Issue 20, Pages 4322-4331Publisher
COMPANY BIOLOGISTS LTD
DOI: 10.1242/jcs.03198
Keywords
titin; muscle; myofibrillogenesis; gene targeting
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Funding
- BBSRC [BB/D524875/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/D524875/1] Funding Source: Medline
- Medical Research Council [G0200496] Funding Source: Medline
- Wellcome Trust Funding Source: Medline
- Biotechnology and Biological Sciences Research Council [BB/D524875/1] Funding Source: researchfish
- Medical Research Council [G0300058B, G0200496] Funding Source: researchfish
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Titin, a multifunctional protein that stretches from the Z-disk to the M-band in heart and skeletal muscle, contains a kinase domain, phosphorylation sites and multiple binding sites for structural and signalling proteins in the M-band. To determine whether this region is crucial for normal sarcomere development, we created mouse embryonic stem cell (ES) lines in which either one or both alleles contained a targeted deletion of the entire M-band-coding region, leaving Z-disk-binding and myosin-filament-binding sites intact. ES cells were differentiated into cardiomyocytes, and myofibrillogenesis investigated by immunofluorescence microscopy. Surprisingly, deletion of one allele did not markedly affect differentiation into cardiomyocytes, suggesting that a single intact copy of the titin gene is sufficient for normal myofibrillogenesis. By contrast, deletion of both alleles resulted in a failure of differentiation beyond an early stage of myofibrillogenesis. Sarcomeric myosin remained in non-striated structures, Z-disk proteins, such as alpha-actinin, were mainly found in primitive dot-like structures on actin stress fibres, M-band-associated proteins (myomesin, obscurin, Nbr1, p62 and MURF2) remained punctate. These results show that integration of the M-band region of titin is required for myosin filament assembly, M-band formation and maturation of the Z-disk.
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