Effects of cardiomyopathy-linked mutations K15N and R21H in tropomyosin on thin-filament regulation and pointed-end dynamics

Missense mutations K15N and R21H in striated muscle tropomyosin are linked to dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM), respectively. Tropomyosin, together with the troponin complex, regulates muscle contraction and, along with tropomodulin and leiomodin, controls the uniform thin-filament lengths crucial for normal sarcomere structure and function. We used Förster resonance energy transfer to study effects of the tropomyosin mutations on the structure and kinetics of the cardiac troponin core domain associated with the Ca2+-dependent regulation of cardiac thin filaments. We found that the K15N mutation desensitizes thin filaments to Ca2+ and slows the kinetics of structural changes in troponin induced by Ca2+ dissociation from troponin, while the R21H mutation has almost no effect on these parameters. Expression of the K15N mutant in cardiomyocytes decreases leiomodin’s thin-filament pointed-end assembly but does not affect tropomodulin’s assembly at the pointed end. Our in vitro assays show that the R21H mutation causes a twofold decrease in tropomyosin’s affinity for F-actin and affects leiomodin’s function. We suggest that the K15N mutation causes DCM by altering Ca2+-dependent thin-filament regulation and that one of the possible HCM-causing mechanisms by the R21H mutation is through alteration of leiomodin’s function

Tropomodulin’s Actin-Binding Abilities Are Required to Modulate Dendrite Development

There are many unanswered questions about the roles of the actin pointed end capping and actin nucleation by tropomodulins (Tmod) in regulating neural morphology. Previous studies indicate that Tmod1 and Tmod2 regulate morphology of the dendritic arbor and spines. Tmod3, which is expressed in the brain, had only a minor influence on morphology. Although these studies established a defined role of Tmod in regulating dendritic and synaptic morphology, the mechanisms by which Tmods exert these effects are unknown. Here, we overexpressed a series of mutated forms of Tmod1 and Tmod2 with disrupted actin-binding sites in hippocampal neurons and found that Tmod1 and Tmod2 require both of their actin-binding sites to regulate dendritic morphology and dendritic spine shape. Proximity ligation assays (PLAs) indicate that these mutations impact the interaction of Tmod1 and Tmod2 with tropomyosins Tpm3.1 and Tpm3.2. This impact on Tmod/Tpm interaction may contribute to the morphological changes observed. Finally, we use molecular dynamics simulations (MDS) to characterize the structural changes, caused by mutations in the C-terminal helix of the leucine-rich repeat (LRR) domain of Tmod1 and Tmod2 alone and when bound onto actin monomers. Our results expand our understanding of how neurons utilize the different Tmod isoforms in development

MOLECULAR MECHANISMS OF ISOFORM-DEPENDENT INTERACTIONS OF PROTEINS FROM THE TROPOMODULIN FAMILY WITH THEIR BINDING PARTNERS

Actin filaments are major components of the cytoskeleton in eukaryotic cells and are involved in vital cellular functions such as cell motility and muscle contraction. Polymerization of actin filament is regulated by fast-growing (barbed) end and slow-growing (pointed) end-binding proteins. Proteins of the tropomodulin (Tmod) family bind to the pointed end of actin filaments in a tropomyosin (TM)-dependent fashion. Tmod isoforms cap the pointed end to inhibit actin polymerization or depolymerization, whereas leiomodin (Lmod) isoforms allow actin filaments to elongate from this end.This dissertation describes the molecular basis of isoform-dependent interactions of Tmod protein family members. The actin-binding abilities of three Tmod isoforms, Tmod1, Tmod2 and Tmod3, were compared in the presence of Tpm3.1, a TM isoform expressed in the brain. Tmod3’s cooperative binding to Tpm3.1 was found to be the driving force for its preferential binding to actin filaments in the presence of other isoforms. These findings describe the long-standing phenomenon of Tmod3’s preferential association with Tpm3.1-coated actin filaments and demonstrate a TM-mediated competition mechanism between Tmod isoforms for binding actin filaments.In addition, we localized the Lmod-binding site on striated muscle -tropomyosin (Tpm1.1) to 21 N-terminal residues of Tpm1.1. We studied the isoform-dependent effect of the dilated cardiomyopathy (DCM) associated-mutation K15N in Tpm1.1, and found that it disrupted the coiled-coil structure of Tpm1.1 and decreased its binding to Tmod and Lmod isoforms and actin. We showed that the mutation decreased Tmod and Lmod binding at the pointed end of actin filaments and altered the dynamics of actin polymerization. Our findings suggest a molecular explanation for DCM development caused by the K15N mutation in Tpm1.1 for the first time. A fundamental understanding of the isoform-dependent interactions of the proteins of the Tmod family with TM and actin revealed the molecular basis of disease development in cardiac muscle

Tropomodulins and tropomyosins: working as a team

Actin filaments are major components of the cytoskeleton in eukaryotic cells and are involved in vital cellular functions such as cell motility and muscle contraction. Tmod and TM are crucial constituents of the actin filament network, making their presence indispensable in living cells. Tropomyosin (TM) is an alpha-helical, coiled coil protein that covers the grooves of actin filaments and stabilizes them. Actin filament length is optimized by tropomodulin (Tmod), which caps the slow growing (pointed end) of thin filaments to inhibit polymerization or depolymerization. Tmod consists of two structurally distinct regions: the N-terminal and the C-terminal domains. The N-terminal domain contains two TM-binding sites and one TM-dependent actin-binding site, whereas the C-terminal domain contains a TM-independent actin-binding site. Tmod binds to two TM molecules and at least one actin molecule during capping. The interaction of Tmod with TM is a key regulatory factor for actin filament organization. The binding efficacy of Tmod to TM is isoform-dependent. The affinities of Tmod/TM binding influence the proper localization and capping efficiency of Tmod at the pointed end of actin filaments in cells. Here we describe how a small difference in the sequence of the TM-binding sites of Tmod may result in dramatic change in localization of Tmod in muscle cells or morphology of non-muscle cells. We also suggest most promising directions to study and elucidate the role of Tmod–TM interaction in formation and maintenance of sarcomeric and cytoskeletal structure

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The precise assembly of actin-based thin filaments is crucial for muscle contraction. Dysregulation of actin dynamics at thin filament pointed ends results in skeletal and cardiac myopathies. Here, we discovered adenylyl cyclase-associated protein 2 (CAP2) as a unique component of thin filament pointed ends in cardiac muscle. CAP2 has critical functions in cardiomyocytes as it depolymerizes and inhibits actin incorporation into thin filaments. Strikingly distinct from other pointed-end proteins, CAP2’s function is not enhanced but inhibited by tropomyosin and it does not directly control thin filament lengths. Furthermore, CAP2 plays an essential role in cardiomyocyte maturation by modulating pre-sarcomeric actin assembly and regulating α-actin composition in mature thin filaments. Identification of CAP2’s multifunctional roles provides missing links in our understanding of how thin filament architecture is regulated in striated muscle and it reveals there are additional factors, beyond Tmod1 and Lmod2, that modulate actin dynamics at thin filament pointed ends.</p