Disease-associated mutations in TPM2 alter regulation of actin filament stability and cofilin-dependent dynamics

ABSTRACT Missense mutations in the TPM2 gene encoding skeletal muscle tropomyosin Tpm2.2 cause congenital myopathies associated with hyper- and hypocontractile phenotypes. Mutation-dependent defects in thin filament stability and length maintenance may contribute to sarcomere dysfunction. To address this possibility, four disease-associated substitutions in Tpm2.2 were analyzed: hypercontractile D20H and E181K, and hypocontractile E41K and N202K. Recombinant proteins were examined in vitro for their effects on actin filament polymerization, stability, and cofilin-2-dependent filament length regulation in the absence and presence of troponin (+Ca2+). Wild-type Tpm2.2 inhibited spontaneous actin polymerization and reduced polymerization cooperativity in the presence of cofilin-2. Hypercontractile substitutions D20H and E181K further decreased the polymerization rate, whereas hypocontractile variants had little effect. Under ATP-driven actomyosin interactions, E41K and N202K stabilized filaments, resulting in increased filament length, but this effect was abolished by troponin. All variants slightly decreased cofilin-2 affinity for F-actin without affecting cooperativity. Troponin prevented displacement of Tpm2.2 from the filament at increasing cofilin-2 occupancy, indicating concomitant binding of all proteins to the thin filament, consistent with a structural model based on high-resolution F-actin–Tpm–Tn and cofilactin structures.Tpm2.2-N202K inhibited cofilin-2-dependent depolymerization, whereas Tpm2.2-E181K increased susceptibility to depolymerization. Although cofilin-2 induced filament severing in all cases, the Tpm2.2–Tn complex protected filaments from disassembly. These findings support a model in which the Tpm2.2–Tn complex forms a cooperative regulatory strand that constrains filament dynamics and transmits structural perturbations along the filament. Disease-causing substitutions differentially alter filament length and stability, potentially contributing to the pathogenesis of myopathies. Competing Interest Statement The authors have declared no competing interest. Abbreviations - Tpm - tropomyosin - Tpm2.2 - skeletal muscle tropomyosin isoform 2.2 - TPM2 - gene encoding Tpm2.2 - Tn - troponin complex - TnI - troponin I - TnT - troponin T - TnC - troponin C - HMM - heavy meromyosin - S1 - myosin subfragment 1 - G-actin - monomeric actin - F-actin - filamentous actin - ADF - actin-depolymerizing factor - DTT - 1,4-dithiothreitol - Hepes - 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid - MOPS - (3-(N-morpholino)propanesulfonic acid) - ATP - adenosine triphosphate - MW - molecular weight - TLCK - Tosyl-L-lysyl-chloromethane hydrochloride - SDS - sodium dodecyl sulfate - EGTA - ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid - BSA - bovine serum albumin - GST - Glutathione S-transferase - PIA - N-(1-pyrene)iodoacetamide - LS - Light scattering - TRC - tetramethyl-rhodamine cadaverine