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 has been hypothesized that a molecular template works with actin filament capping proteins to regulate thin filament lengths. Nebulin is a giant protein ( approximately 800 kDa) in skeletal muscle that has been proposed to act as a molecular ruler to specify the thin filament lengths characteristic of different muscles. Tropomodulin (Tmod), a pointed end thin filament capping protein, has been shown to maintain the final length of the thin filaments. Immunofluorescence microscopy revealed that the N-terminal end of nebulin colocalizes with Tmod at the pointed ends of thin filaments. The three extreme N-terminal modules (M1-M2-M3) of nebulin bind specifically to Tmod as demonstrated by blot overlay, bead binding, and solid phase binding assays. These data demonstrate that the N terminus of the nebulin molecule extends to the extreme end of the thin filament and also establish a novel biochemical function for this end. Two Tmod isoforms, erythrocyte Tmod (E-Tmod), expressed in embryonic and slow skeletal muscle, and skeletal Tmod (Sk-Tmod), expressed late in fast skeletal muscle differentiation, bind on overlapping sites to recombinant N-terminal nebulin fragments. Sk-Tmod binds nebulin with higher affinity than E-Tmod does, suggesting that the Tmod/nebulin interaction exhibits isoform specificity. These data provide evidence that Tmod and nebulin may work together as a linked mechanism to control thin filament lengths in skeletal muscle.
McElhinny
A S
AS
Department of Cell Biology and Anatomy, University of Arizona, Tucson, Arizona 85724, USA.
Kolmerer
B
B
Fowler
V M
VM
Labeit
S
S
Gregorio
C C
CC
D008841:Actin Cytoskeleton / Q000737:chemistry* / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D019921:Biotinylation; D002352:Carrier Proteins / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry* / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012996:Solutions; D013379:Substrate Specificity; D051348:Tropomodulin; D014335:Tropomyosin / Q000378:metabolism
D000199:Actins; D002352:Carrier Proteins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012996:Solutions; C495829:TMOD1 protein, human; C495832:Tmod1 protein, rat; D051348:Tropomodulin; D014335:Tropomyosin; C035620:nebulin