Low molecular-weight G-actin binding proteins involved in the regulation of actin assembly during myofibrillogenesis.

We previously demonstrated that small G-actin binding proteins, cofilin, ADF and profilin, are involved in the actin dynamics during myofibrillogenesis (OBINATA, T. (1993). Int. Rev. Cytol., 143: 153-189.). To better understand how they are responsible for the regulation of actin assembly, the amounts of the actin-binding proteins were quantified by means of quantitative immunoblotting and compared with that of G-actin pool. The sum of the amounts of cofilin, ADF and profilin was insufficient at early developmental stages but sufficient at later stages to account for the pool of G-actin in muscle cells. We detected expression of thymosin beta 4 at a considerable level in young embryonic but not in adult skeletal muscles. We, therefore, conclude that the G-actin pool in young embryonic skeletal muscle is mainly due to cofilin, ADF, profilin and thymosin beta 4. Switching from a non-muscle-type (NM-) cofilin to a muscle-type (M-) cofilin was observed during muscle development of mammals. In order to clarify cofilin-dependent regulation of actin assembly in muscle cells, cofilin tagged with fluorescence dyes was introduced into C2 myoblasts by a micro injection method. The exogeneous cofilin, but not ADF, caused quick disassembly of actin filaments and accumulated in furrow region of dividing cells. The analogs of the unphosphorylated form (A3-cofilin) and the phosphorylated form (D3-cofilin) were prepared by converting Ser3, a regulatory phosphorylation site, to Ala or Asp. When A3-cofilin and D3-cofilin were injected into living cells, the former was concentrated at the membrane ruffles and cleavage furrow, while the latter showed only diffuse distribution in the cytoplasm. These results suggest that the subcellular distribution of cofilin as well as its interaction with actin in vivo is regulated by its phosphorylation and dephosphorylation.