Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Ted W. Huiatt
Titin is a giant muscle protein that spans the distance from the Z line to the M line in sarcomeres of striated muscle myofibrils. One of the functions proposed for titin is that it could act as a molecular scaffold to direct sarcomere assembly. To investigate this role of titin, titin expression was transiently altered in the cultured mouse skeletal muscle cell line C2C12 by either a dominant-negative approach or an antisense mRNA approach. The time course of myofibril assembly was monitored by immunofluorescence labeling with antibodies to actin, alpha-actinin, titin, and myosin from 1 to 5 d after myoblast differentiation was induced. The dominant-negative approach was implemented by transiently transfecting cells with a fusion construct consisting of a cDNA for GFP linked to cDNA encoding a portion of the A band region of titin. The protein encoded by this titin cDNA was expressed in bacteria and shown to interact with myosin in vitro by solid phase binding assays. Transfected myotubes expressing high levels of this fusion protein demonstrated delayed myofibrillar A band assembly compared to non-transfected myotubes and myotubes transfected with GFP vector alone. Assembly of the Z line and thin filament regions of the sarcomere were not affected by overexpression of the A band titin. The antisense approach was implemented by transiently transfecting cells with a fusion construct consisting of a 0.5 kb cDNA for the N-terminal (Z line) region of titin in antisense orientation linked to GFP cDNA. This partial Z line titin cDNA contains the 5' untranslated region and the start codon of the titin message. Expression of this anti-sense titin RNA decreased titin expression and delayed titin organization. Delayed myofibrillar A band assembly was also observed in myotubes transfected with this construct in comparison with non-transfected myotubes and myotubes transfected with the GFP vector alone, while the assembly of alpha-actinin and actin filaments were not affected. Overall, the results from both approaches indicate that titin indeed plays an important role in myofibril assembly. Appropriate titin-myosin interaction seems to be crucial for organizing the myofibrillar A band in embryonic skeletal muscle.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Dong, Qunfeng, "Effects of altering titin expression on myofibril assembly in a skeletal muscle cell line " (2000). Retrospective Theses and Dissertations. 12680.