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, cofili n, ADF and profilin, are involved in the actin dynamics during myofibr illogenesis (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 b y means of quantitative immunoblotting and compared with that of G-act in pool. The sum of the amounts of cofilin, ADF and profilin was insuf ficient at early developmental stages but sufficient at later stages t o account for the pool of G-actin in muscle cells. We detected express ion of thymosin beta 4 at a considerable level in young embryonic but not in adult skeletal muscles. We, therefore, conclude that the G-acti n pool in young embryonic skeletal muscle is mainly due to cofilin, AD F, profilin and thymosin beta 4. Switching from a non-muscle-type (NM- ) cofilin to a muscle-type (M-) cofilin was observed during muscle dev elopment of mammals. In order to clarify cofilin-dependent regulation of actin assembly in muscle cells, cofilin tagged with fluorescence dy es was introduced into C2 myoblasts by a micro injection method. The e xogeneous cofilin, but not ADF, caused quick disassembly of actin fila ments 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 phosphoryl ation site, to Ala or Asp. When A3-cofilin and D3-cofilin were injecte d into living cells, the former was concentrated at the membrane ruffl es and cleavage furrow, while the latter showed only diffuse distribut ion in the cytoplasm. These results suggest that the subcellular distr ibution of cofilin as well as its interaction with actin in vivo is re gulated by its phosphorylation and dephosphorylation.