MECHANISMS OF THIN FILAMENT ASSEMBLY IN EMBRYONIC CHICK CARDIAC MYOCYTES - TROPOMODULIN REQUIRES TROPOMYOSIN FOR ASSEMBLY

Tropomodulin is a pointed end capping protein for tropomyosin-coated a ctin filaments that is hypothesized to play a role in regulating the p recise lengths of striated muscle thin filaments (Fowler, V. M., M. A. Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cel l Biol. 120:411-420; Weber, A., C. C. Pennise, G. G. Babcock, and V. M . Fowler. 1994. J. Cell Biol. 127:1627-1635). To gain insight into the mechanisms of thin filament assembly and the role of tropomodulin the rein, we have characterized the temporal appearance, biosynthesis and mechanisms of assembly of tropomodulin onto the pointed ends of thin f ilaments during the formation of striated myofibrils in primary embryo nic chick cardiomyocyte cultures. Our results demonstrate that tropomo dulin is not assembled coordinately with other thin filament proteins. Double immunofluorescence staining and ultrastructural immunolocaliza tion demonstrate that tropomodulin is incorporated in its characterist ic sarcomeric location at the pointed ends of the thin filaments after the thin filaments have become organized into periodic I bands. In fa ct, tropomodulin assembles later than all other well characterized myo fibrillar proteins studied including: actin, tropomyosin, alpha-actini n, titin, myosin and C-protein. Nevertheless, at steady state, a signi ficant proportion (similar to 39%) of tropomodulin is present in a sol uble pool throughout myofibril assembly. Thus, the absence of tropomod ulin in some striated myofibrils is not due to limiting quantities of the protein. In addition, kinetic data obtained from [S-35]methionine pulse-chase experiments indicate that tropomodulin assembles more slow ly into myofibrils than does tropomyosin. This observation, together w ith results obtained using a novel permeabilized cell model for thin f ilament assembly, indicate that tropomodulin assembly is dependent on the prior association of tropomyosin with actin filaments. We conclude that tropomodulin is a late marker for the assembly of striated myofi brils in cardiomyocytes; its assembly appears to be linked to their ma turity. We propose that tropomodulin is involved in maintaining and st abilizing the final lengths of thin filaments after they are assembled .