Requirement of pointed-end capping by tropomodulin to maintain actin filament length in embryonic chick cardiac myocytes

Abstract

Control of actin filament length and dynamics is important for cell motility and architecture and is regulated in part by capping proteins that block elongation and depolymerization at both the fast-growing (barbed) and slow-growing (pointed) ends. Tropomodulin is a capping protein for the pointed end of the actin filament; it is associated with the free, pointed ends of the thin filaments in striated muscle, where it is thought to bind to both tropomyosin and actin. In embryonic chick cardiac myocytes, tropomodulin assembles after the thin, as well as the thick, filaments have become organized into periodic I and A bands, suggesting that tropomodulin might be involved in maintaining actin filament length. Here we show that microinjection of an antibody that inhibits tropomodulin's pointed-end-capping activity in vitro results in a marked elongation of actin filaments from their pointed ends and a > 80% reduction in the percentage of beating cells. This demonstrates that pointed-end capping by tropomodulin is required to maintain actin filament length in vivo and that this is essential for contractile function in embryonic chick cardiac myocytes.

Cytoplasmic Nuclear Transfer of the Actin-capping Protein Tropomodulin

“…continued expression of Tmod transgenes. Overexpression of Tmod in proliferating C2 cells does not appear to be as cytotoxic as in well differentiated muscle cells as previously described (10,11,31). The delay of myogenic markers expressed in cells transduced with NES mutants does not appear to be caused by a loss of transgene-expressing cells (Figs.…”

“…This isoform, initially isolated from human erythrocyte membranes and hence dubbed E-Tmod, is highly expressed in the cytoplasm of striated muscles and at lower levels in a number of nonmuscle tissues (3,6,11,(13)(14)(15). Unexpectedly we find that E-Tmod can traffic through the nucleus.…”

“…Tmod is well established as a major regulatory component for the architecture of the sarcomere in muscle cells and of the membrane skeleton in non-muscle cells (6). It has also been shown that overexpression of Tmod or depletion of Tmod by introduction of anti-Tmod monoclonal antibody in well differentiated cardiac myocytes can disrupt the sarcomeric structures of myofibrils (10,11). In a transgenic model, overexpression of Tmod in the heart leads to a dilated cardiomyopathic phenotype (10).…”

Identification of a Novel Tropomodulin Isoform, Skeletal Tropomodulin, That Caps Actin Filament Pointed Ends in Fast Skeletal Muscle

“…When embryonic chick cardiac myocytes are microinjected with an antibody that blocks tropomodulin's ability to cap actin filament pointed ends in vitro, actin elongates from the thin filament pointed ends (10). Furthermore, the cells are no longer able to beat, demonstrating that maintenance of thin filament length by tropomodulin is critical for normal contraction (10).…”

Effect of the Structure of the N Terminus of Tropomyosin on Tropomodulin Function

“…Erythrocyte tropomodulin (Tmod1) 1 binds weakly to actin alone (K d ϳ0.3-0.4 mM) but much stronger in the presence of tropomyosin (K d ϳ50 pM) (27). Analysis of tropomodulin fragments and antibodies to specific domains has identified the actin and tropomyosin binding domains to be the C-and N-terminal parts of the molecule, respectively (28,29). The atomic structure of the C-terminal domain is a right-handed super-helix composed of alternating ␣-helices and ␤-strands (30).…”