Dynamics of actin assembly during myofibrillogenesis and troponin

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1) Further characterization of actin-binding proteins which regulate actin assembly in developing muscle cells Previously, we reported that cofilin, ADF and profilin is involved in the regulation of actin assembly in developing muscle. In this study, two cofilin variants, muscle-type and non-muscle-type, were distinguished in mammals, and the transition from the non-muscle-type to the muscle-type was observed during muscle development. The cDNA for the muscle-type cofilin isoform was cloned by a PCR method and the cDNA sequence was determined. Northern blot analysis showed that expression of the muscle cofilin is induced as muscle differentiates. In addition, cDNA of profilin was cloned from the cDNA library of embryonic chicken skeletal muscle and the sequence was determined. 2) Functional analysis of cofilin in cultured muscle cells Cofilin amount in myogenic cells was increased either by 1) microinjection of cofilin or 2) transfection of cofilin cDNA, and change in cytoplasmic actin … More filaments was examined. When cofilin produced in E. Coli was introduced into cultured muscle cells, actin microfilaments were disassembled quickly and marked actin/cofilin rods were generated in the cytoplasm. However, the rod structures disappeared and microfilaments were recovered within 24 hrs, suggesting that activity of cofilin was weakened gradually. On the other hand, in the muscle cells with transfection of cofilin cDNA, actin filaments were remained unaffected regardless of over-expression of cofilin. Heat-shock or DMSO-stimulation caused drastic disassembly of actin filaments in these cells. These results suggest that cofilin is modulated in the cytoplasm to alter the functional activity. Evidence was obtained which suggests that phosphorylation is one of the way of modulation. 3) Increased expression of cofilin in degenerating muscle cells Marked increase in the expression of cofilin was detected in denervated or dystrophic degenerating chicken skeletal muscle. This fact suggests that cofilin may be involved in dynamic redistribution or turnover of actin molecules during muscle degeneration. Less