CAP2 is a regulator of actin pointed end dynamics and myofibrillogenesis in cardiac muscle.
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ABSTRACT:
SUBMITTER: Colpan M
PROVIDER: S-EPMC7979805 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The actin filament has clear polarity where one end, the pointed end, has a much slower polymerization and depolymerization rate than the other end, the barbed end. This intrinsic difference of the ends significantly affects all actin dynamics in the cell, which has central roles in a wide spectrum of cellular functions. The detailed mechanism underlying this difference has remained elusive, because high-resolution structures of the filament ends have not been available. Here, we present the structure of the actin filament pointed end obtained using a single particle analysis of cryo-electron micrographs. We determined that the terminal pointed end subunit is tilted towards the penultimate subunit, allowing specific and extra loop-to-loop inter-strand contacts between the two end subunits, which is not possible in other parts of the filament. These specific contacts prevent the end subunit from dissociating. For elongation, the loop-to-loop contacts also inhibit the incorporation of another actin monomer at the pointed end. These observations are likely to account for the less dynamic pointed end.
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Project description:Correct assembly of thin filaments composed of actin and actin-binding proteins is of crucial importance for properly functioning muscle cells. Tropomyosin (Tpm) mediates the binding of tropomodulin (Tmod) and leiomodin (Lmod) at the slow-growing, or pointed, ends of the thin filaments. Together these proteins regulate thin filament lengths and actin dynamics in cardiac muscle. The K15N mutation in the TPM1 gene is associated with familial dilated cardiomyopathy (DCM) but the effect of this mutation on Tpm's function is unknown. In this study, we introduced the K15N mutation in striated muscle ?-Tpm (Tpm1.1) and investigated its interaction with actin, Tmod and Lmod. The mutation caused a ?3-fold decrease in the affinity of Tpm1.1 for actin. The binding of Lmod and Tmod to Tpm1.1-covered actin filaments also decreased in the presence of the K15N mutation. Furthermore, the K15N mutation in Tpm1.1 disrupted the inhibition of actin polymerization and affected the competition between Tmod1 and Lmod2 for binding at the pointed ends. Our data demonstrate that the K15N mutation alters pointed end dynamics by affecting molecular interactions between Tpm1.1, Lmod2 and Tmod1.
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Project description:Proteins that cap the ends of the actin filament are essential regulators of cytoskeleton dynamics. Whereas several proteins cap the rapidly growing barbed end, tropomodulin (Tmod) is the only protein known to cap the slowly growing pointed end. The lack of structural information severely limits our understanding of Tmod's capping mechanism. We describe crystal structures of actin complexes with the unstructured amino-terminal and the leucine-rich repeat carboxy-terminal domains of Tmod. The structures and biochemical analysis of structure-inspired mutants showed that one Tmod molecule interacts with three actin subunits at the pointed end, while also contacting two tropomyosin molecules on each side of the filament. We found that Tmod achieves high-affinity binding through several discrete low-affinity interactions, which suggests a mechanism for controlled subunit exchange at the pointed end.
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Project description:The premyofibril model proposes a three-stage process for the de novo assembly of myofibrils in cardiac and skeletal muscles: premyofibrils to nascent myofibrils to mature myofibrils. FRAP experiments and jasplakinolide, a drug that stabilizes F-actin, permitted us to determine how decreasing the dynamics of actin filaments affected the dynamics of tropomyosin, troponin-T, troponin-C, and two Z-Band proteins (alpha-actinin, FATZ) in premyofibrils versus mature myofibrils. Jasplakinolide reduced markedly the dynamics of actin in premyofibrils and in mature myofibrils in skeletal muscles. Two isoforms of tropomyosin-1 (TPM1α, TPM1κ) are more dynamic in premyofibrils than in mature myofibrils in control skeletal muscles. Jasplakinolide reduced the exchange rates of tropomyosins in premyofibrils but not in mature myofibrils. The reduced tropomyosin recoveries did not match the YFP-actin recoveries in premyofibrils in jasplakinolide. There were no significant differences in the effects of jasplakinolide on the dynamics of troponins in the thin filaments or of two Z-band proteins in premyofibrils or skeletal mature myofibrils. Cardiac control mature myofibrils lack nebulin, and small decreases in actin (∼5%) and two tropomyosin isoforms (∼10-15%) dynamics are detected in premyofibril to mature myofibril transformations compared with skeletal muscle. In contrast to skeletal muscle, jasplakinolide lowered the dynamics of actin and tropomyosin isoforms in the cardiac mature myofibrils. These results suggest that the dynamics of tropomyosins in control muscle cells are related to actin exchange. These results also suggest a stabilizing role for nebulin, an actin and tropomyosin-binding protein, present in mature myofibrils but not in premyofibrils of skeletal muscles.
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Project description:Cytoskeletal structures characterized by actin filaments with uniform lengths, including the thin filaments of striated muscles and the spectrin-based membrane skeleton, use barbed and pointed-end capping proteins to control subunit addition/dissociation at filament ends. While several proteins cap the barbed end, tropomodulins (Tmods), a family of four closely related isoforms in vertebrates, are the only proteins known to specifically cap the pointed end. Tmods are ∼350 amino acids in length, and comprise alternating tropomyosin- and actin-binding sites (TMBS1, ABS1, TMBS2, and ABS2). Leiomodins (Lmods) are related in sequence to Tmods, but display important differences, including most notably the lack of TMBS2 and the presence of a C-terminal extension featuring a proline-rich domain and an actin-binding WASP-Homology 2 domain. The Lmod subfamily comprises three somewhat divergent isoforms expressed predominantly in muscle cells. Biochemically, Lmods differ from Tmods, acting as powerful nucleators of actin polymerization, not capping proteins. Structurally, Lmods and Tmods display crucial differences that correlate well with their different biochemical activities. Physiologically, loss of Lmods in striated muscle results in cardiomyopathy or nemaline myopathy, whereas complete loss of Tmods leads to failure of myofibril assembly and developmental defects. Yet, interpretation of some of the in vivo data has led to the idea that Tmods and Lmods are interchangeable or, at best, different variants of two subfamilies of pointed-end capping proteins. Here, we review and contrast the existing literature on Tmods and Lmods, and propose a model of Lmod function that attempts to reconcile the in vitro and in vivo data, whereby Lmods nucleate actin filaments that are subsequently capped by Tmods during sarcomere assembly, turnover, and repair.
| S-EPMC5425412 | biostudies-literature
Project description:Many actin-binding proteins have been shown to possess multiple activities to regulate filament dynamics. Tropomodulins (Tmod1-4) are a conserved family of actin filament pointed end-capping proteins. Our previous work has demonstrated that Tmod3 binds to monomeric actin in addition to capping pointed ends. Here, we show a novel actin-nucleating activity in mammalian Tmods. Comparison of Tmod isoforms revealed that Tmod1-3 but not Tmod4 nucleate actin filament assembly. All Tmods bind to monomeric actin, and Tmod3 forms a 1:1 complex with actin. By truncation and mutagenesis studies, we demonstrated that the second α-helix in the N-terminal domain of Tmod3 is essential for actin monomer binding. Chemical cross-linking and LC-MS/MS further indicated that residues in this second α-helix interact with actin subdomain 2, whereas Tmod3 N-terminal domain peptides distal to this α-helix interact with actin subdomain 1. Mutagenesis of Leu-73 to Asp, which disrupts the second α-helix of Tmod3, decreases both its actin monomer-binding and -nucleating activities. On the other hand, point mutations of residues in the C-terminal leucine-rich repeat domain of Tmod3 (Lys-317 in the fifth leucine-rich repeat β-sheet and Lys-344 or Arg-345/Arg-346 in the C-terminal α6-helix) significantly reduced pointed end-capping and nucleation without altering actin monomer binding. Taken together, our data indicate that Tmod3 binds actin monomers over an extended interface and that nucleating activity depends on actin monomer binding and pointed end-capping activities, contributed by N- and C-terminal domains of Tmod3, respectively. Tmod3 nucleation of actin assembly may regulate the cytoskeleton in dynamic cellular contexts.
| S-EPMC2963411 | biostudies-literature
Project description:VopL and VopF (VopL/F) are tandem WH2-domain actin assembly factors used by infectious Vibrio species to induce actin assembly in host cells. There is disagreement about the filament assembly mechanism of VopL/F, including whether they associate with the filament barbed or pointed end. Here, we used multicolor total internal reflection fluorescence microscopy to directly observe actin assembly with fluorescently labeled VopL/F. In actin monomer assembly reactions, VopL/F exclusively nucleate actin filament assemblies, remaining only briefly associated with the pointed end. VopL/F do not associate with the ends of preassembled filaments. In assembly reactions with saturating profilin, ∼85% of VopL/F molecules also promote nucleation from the pointed end, whereas a smaller fraction (<15%) associate for ∼25 s with the barbed end of preassembled filaments, inhibiting their elongation. We conclude that VopL/F function primarily as actin nucleation factors that remain briefly (∼100 s) associated with the pointed end.
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Project description:Improper lengths of actin-thin filaments are associated with altered contractile activity and lethal myopathies. Leiomodin, a member of the tropomodulin family of proteins, is critical in thin filament assembly and maintenance; however, its role is under dispute. Using nuclear magnetic resonance data and molecular dynamics simulations, we generated the first atomic structural model of the binding interface between the tropomyosin-binding site of cardiac leiomodin and the N-terminus of striated muscle tropomyosin. Our structural data indicate that the leiomodin/tropomyosin complex only forms at the pointed end of thin filaments, where the tropomyosin N-terminus is not blocked by an adjacent tropomyosin protomer. This discovery provides evidence supporting the debated mechanism where leiomodin and tropomodulin regulate thin filament lengths by competing for thin filament binding. Data from experiments performed in cardiomyocytes provide additional support for the competition model; specifically, expression of a leiomodin mutant that is unable to interact with tropomyosin fails to displace tropomodulin at thin filament pointed ends and fails to elongate thin filaments. Together with previous structural and biochemical data, we now propose a molecular mechanism of actin polymerization at the pointed end in the presence of bound leiomodin. In the proposed model, the N-terminal actin-binding site of leiomodin can act as a "swinging gate" allowing limited actin polymerization, thus making leiomodin a leaky pointed-end cap. Results presented in this work answer long-standing questions about the role of leiomodin in thin filament length regulation and maintenance.
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Project description:Regulation of actin filament assembly is essential for efficient contractile activity in striated muscle. Leiomodin is an actin-binding protein and homolog of the pointed-end capping protein, tropomodulin. These proteins are structurally similar, sharing a common domain organization that includes two actin-binding sites. Leiomodin also contains a unique C-terminal extension that has a third actin-binding WH2 domain. Recently, the striated-muscle-specific isoform of leiomodin (Lmod2) was reported to be an actin nucleator in cardiomyocytes. Here, we have identified a function of Lmod2 in the regulation of thin filament lengths. We show that Lmod2 localizes to the pointed ends of thin filaments, where it competes for binding with tropomodulin-1 (Tmod1). Overexpression of Lmod2 results in loss of Tmod1 assembly and elongation of the thin filaments from their pointed ends. The Lmod2 WH2 domain is required for lengthening because its removal results in a molecule that caps the pointed ends similarly to Tmod1. Furthermore, Lmod2 transcripts are first detected in the heart after it has begun to beat, suggesting that the primary function of Lmod2 is to maintain thin filament lengths in the mature heart. Thus, Lmod2 antagonizes the function of Tmod1, and together, these molecules might fine-tune thin filament lengths.
| S-EPMC2931607 | biostudies-literature
Project description:Actin remodeling is crucial for dendritic spine development, morphology and density. CAP2 is a regulator of actin dynamics through sequestering G-actin and severing F-actin. In a mouse model, ablation of CAP2 leads to cardiovascular defects and delayed wound healing. This report investigates the role of CAP2 in the brain using Cap2(gt/gt) mice. Dendritic complexity, the number and morphology of dendritic spines were altered in Cap2(gt/gt) with increased number of excitatory synapses. This was accompanied by increased F-actin content and F-actin accumulation in cultured Cap2(gt/gt) neurons. Moreover, reduced surface GluA1 was observed in mutant neurons under basal condition and after induction of chemical LTP. Additionally, we show an interaction between CAP2 and n-cofilin, presumably mediated through the C-terminal domain of CAP2 and dependent on cofilin Ser3 phosphorylation. In vivo, the consequences of this interaction were altered phosphorylated cofilin levels and formation of cofilin aggregates in the neurons. Thus, our studies identify a novel role of CAP2 in neuronal development and neuronal actin dynamics.
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