The N-terminal end of nebulin interacts with tropomodulin at the pointed ends of the thin filaments

Strict regulation of actin thin filament length is critical for the proper functioning of sarcomeres, the basic contractile units of myofibrils. It ha s been hypothesized that a molecular template works with actin filament cap ping proteins to regulate thin filament lengths. Nebulin is a giant protein (similar to 800 kDa) in skeletal muscle that has been proposed to act as a molecular ruler to specify the thin filament lengths characteristic of dif ferent muscles. Tropomodulin (Tmod), a pointed end thin filament capping pr otein, has been shown to maintain the final length of the thin filaments. I mmunofluorescence microscopy revealed that the N-terminal end of nebulin co localizes with Tmod at the pointed ends of thin filaments. The three extrem e N-terminal modules (M1-M2-M3) of nebulin bind specifically to Tmod as dem onstrated by blot overlay, bead binding, and solid phase binding assays. Th ese data demonstrate that the N terminus of the nebulin molecule extends to the extreme end of the thin filament and also establish a novel biochemica l function for this end. Two Tmod isoforms, erythrocyte Tmod (E-Tmod), expr essed in embryonic and slow skeletal muscle, and skeletal Tmod (Sk-Tmod), e xpressed late in fast skeletal muscle differentiation, bind on overlapping sites to recombinant N-terminal nebulin fragments. Sk-Tmod binds nebulin wi th higher affinity than E-Tmod does, suggesting that the Tmod/ nebulin inte raction exhibits isoform specificity.These data provide evidence that Tmod and nebulin may work together as a linked mechanism to control thin filamen t lengths in skeletal muscle.