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Biochemistry Jun 2014The actin cytoskeleton carries out cellular functions, including division, migration, adhesion, and intracellular transport, that require a variety of actin binding...
The actin cytoskeleton carries out cellular functions, including division, migration, adhesion, and intracellular transport, that require a variety of actin binding proteins, including myosins. Our focus here is on class II nonmuscle myosin isoforms, NMIIA, NMIIB, and NMIIC, and their regulation by the actin binding protein, tropomyosin. NMII myosins are localized to different populations of stress fibers and the contractile ring, structures involved in force generation required for cell migration, adhesion, and cytokinesis. The stress fibers and contractile ring that contain NMII myosins also contain tropomyosin. Four mammalian genes encode more than 40 tropomyosins. Tropomyosins inhibit or activate actomyosin MgATPase and motility depending on the myosin and tropomyosin isoform. In vivo, tropomyosins play a role in cell migration, adhesion, cytokinesis, and NMII isoform localization in an isoform-specific manner. We postulate that the isoform-specific tropomyosin localization and effect on NMII isoform localization reflect modulation of NMII actomyosin kinetics and motile function. In this study, we compare the ability of different tropomyosin isoforms to support actin filament motility with NMIIA, NMIIB, and NMIIC as well as skeletal muscle myosin. Tropomyosins activated, inhibited, or had no effect on motility depending on the myosin, indicating that the myosin isoform is the primary determinant of the isoform-specific effect of tropomyosin on actomyosin regulation. Activation of motility of nonmuscle tropomyosin-actin filaments by NMII myosin correlates with an increased Vmax of the myosin MgATPase, implying a direct effect on the myosin MgATPase, in contrast to the skeletal tropomyosin-actin filament that has no effect on the Vmax or maximal filament velocity.
Topics: Actins; Adenosine Triphosphatases; Animals; Cell Movement; Humans; Myosin Subfragments; Myosin Type II; Rats; Tropomyosin
PubMed: 24873380
DOI: 10.1021/bi500162z -
International Journal of Molecular... May 2021Tropomyosin (Tpm) is one of the major protein partners of actin. Tpm molecules are -helical coiled-coil protein dimers forming a continuous head-to-tail polymer along...
Tropomyosin (Tpm) is one of the major protein partners of actin. Tpm molecules are -helical coiled-coil protein dimers forming a continuous head-to-tail polymer along the actin filament. Human cells produce a large number of Tpm isoforms that are thought to play a significant role in determining actin cytoskeletal functions. Even though the role of these Tpm isoforms in different non-muscle cells is more or less studied in many laboratories, little is known about their structural and functional properties. In the present work, we have applied various methods to investigate the properties of five cytoplasmic Tpm isoforms (Tpm1.5, Tpm 1.6, Tpm1.7, Tpm1.12, and Tpm 4.2), which are the products of two different genes, and , and also significantly differ by alternatively spliced exons: N-terminal exons 1a2b or 1b, internal exons 6a or 6b, and C-terminal exons 9a, 9c or 9d. Our results demonstrate that structural and functional properties of these Tpm isoforms are quite different depending on sequence variations in alternatively spliced regions of their molecules. The revealed differences can be important in further studies to explain why various Tpm isoforms interact uniquely with actin filaments, thus playing an important role in the organization and dynamics of the cytoskeleton.
Topics: Actin Cytoskeleton; Alternative Splicing; Cytoplasm; Exons; Humans; Protein Isoforms; Tropomyosin
PubMed: 34067970
DOI: 10.3390/ijms22105141 -
Cell Jul 2012Regulation of myosin and filamentous actin interaction by tropomyosin is a central feature of contractile events in muscle and nonmuscle cells. However, little is known...
Regulation of myosin and filamentous actin interaction by tropomyosin is a central feature of contractile events in muscle and nonmuscle cells. However, little is known about molecular interactions within the complex and the trajectory of tropomyosin movement between its "open" and "closed" positions on the actin filament. Here, we report the 8 Å resolution structure of the rigor (nucleotide-free) actin-tropomyosin-myosin complex determined by cryo-electron microscopy. The pseudoatomic model of the complex, obtained from fitting crystal structures into the map, defines the large interface involving two adjacent actin monomers and one tropomyosin pseudorepeat per myosin contact. Severe forms of hereditary myopathies are linked to mutations that critically perturb this interface. Myosin binding results in a 23 Å shift of tropomyosin along actin. Complex domain motions occur in myosin, but not in actin. Based on our results, we propose a structural model for the tropomyosin-dependent modulation of myosin binding to actin.
Topics: Actins; Animals; Cryoelectron Microscopy; Humans; Models, Molecular; Multiprotein Complexes; Muscle, Skeletal; Muscular Diseases; Myosins; Rabbits; Tropomyosin
PubMed: 22817895
DOI: 10.1016/j.cell.2012.05.037 -
Fimbrin and tropomyosin competition regulates endocytosis and cytokinesis kinetics in fission yeast.Current Biology : CB Aug 2010Tropomyosin is an important actin filament-stabilizing protein that controls the access of other essential proteins to filaments, including myosin motors, Arp2/3...
BACKGROUND
Tropomyosin is an important actin filament-stabilizing protein that controls the access of other essential proteins to filaments, including myosin motors, Arp2/3 complex, formin, and cofilin. It is therefore critical to establish mechanisms for regulating the actin filament binding of tropomyosin. We examined how the actin filament crosslinking protein fimbrin Fim1p and tropomyosin Cdc8p affect each other's ability to bind filaments, localize to particular cellular structures, and regulate filament severing by cofilin Adf1p in fission yeast Schizosaccharomyces pombe.
RESULTS
We discovered a novel mechanism for regulating actin filament dynamics in fission yeast. Fim1p inhibits Cdc8p binding to actin filaments in vitro, which permits Adf1p-mediated severing in the presence of Cdc8p. In cells, the balance between Fim1p and Cdc8p is important for both endocytic actin patch kinetics and contractile ring assembly during cytokinesis. High Fim1p concentrations prevent Cdc8p from associating with actin patches, allowing rapid patch turnover and motility. In the absence of Fim1p, ectopic localization of Cdc8p to actin patches increases patch lifetime while decreasing patch motility. Fim1p and Cdc8p also play antagonistic roles during cytokinesis, in which the deletion of Fim1p rescues the contractile ring assembly defects caused by mutation of Cdc8p.
CONCLUSION
Fimbrin Fim1p dissociates tropomyosin Cdc8p from actin filaments, permitting cofilin Adf1p-mediated severing. Therefore, we propose that in addition to actin filament crosslinking, Fim1p has a novel role as a positive actin-binding "selector" protein that promotes the access of other proteins to actin filaments by inhibiting Cdc8p.
Topics: Actin Cytoskeleton; Actin Depolymerizing Factors; Cell Cycle Proteins; Cytokinesis; Endocytosis; Membrane Glycoproteins; Microfilament Proteins; Models, Biological; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Tropomyosin
PubMed: 20705466
DOI: 10.1016/j.cub.2010.06.020 -
Biochemistry Dec 2010Tropomyosin is a ubiquitous actin-binding protein with an extended coiled-coil structure. Tropomyosin-actin interactions are weak and loosely specific, but they potently...
Tropomyosin is a ubiquitous actin-binding protein with an extended coiled-coil structure. Tropomyosin-actin interactions are weak and loosely specific, but they potently influence myosin. One such influence is inhibitory and is due to tropomyosin's statistically preferred positions on actin that sterically interfere with actin's strong attachment site for myosin. Contrastingly, tropomyosin's other influence is activating. It increases myosin's overall actin affinity ∼4-fold. Stoichiometric considerations cause this activating effect to equate to an ∼4(7)-fold effect of myosin on the actin affinity of tropomyosin. These positive, mutual, myosin-tropomyosin effects are absent if Saccharomyces cerevisiae tropomyosin replaces mammalian tropomyosin. To investigate these phenomena, chimeric tropomyosins were generated in which 38-residue muscle tropomyosin segments replaced a natural duplication within S. cerevisiae tropomyosin TPM1. Two such chimeric tropomyosins were sufficiently folded coiled coils to allow functional study. The two chimeras differed from TPM1 but in opposite ways. Consistent with steric interference, myosin greatly decreased the actin affinity of chimera 7, which contained muscle tropomyosin residues 228-265. On the other hand, myosin S1 increased by an order of magnitude the actin affinity of chimera 3, which contained muscle tropomyosin residues 74-111. Similarly, myosin S1-ADP binding to actin was strengthened 2-fold by substitution of chimera 3 tropomyosin for wild-type TPM1. Thus, a yeast tropomyosin was induced to mimic the activating behavior of mammalian tropomyosin by inserting a mammalian tropomyosin sequence. The data were not consistent with direct tropomyosin-myosin binding. Rather, they suggest an allosteric mechanism, in which myosin and tropomyosin share an effect on the actin filament.
Topics: Actins; Amino Acid Sequence; Animals; Cattle; Muscle Proteins; Mutant Chimeric Proteins; Myosin Subfragments; Myosins; Protein Binding; Protein Folding; Protein Stability; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Tropomyosin
PubMed: 21114337
DOI: 10.1021/bi101632f -
Genes Dec 2022Tropomyosin (TPM) is a contractile protein responsible for muscle contraction through its actin-binding activity. The complete sequence of in () was 2160 bp, encoding...
Tropomyosin (TPM) is a contractile protein responsible for muscle contraction through its actin-binding activity. The complete sequence of in () was 2160 bp, encoding 284 amino acids, and contained a TPM signature motif and a TPM domain. Gene ontology (GO) analysis based on the amino acid sequence predicted Hdh-TPM to have an actin-binding function in the cytoskeleton. The 3D analysis predicted the Hdh-TPM to have a coiled-coil α-helical structure. Phylogenetically, Hdh-TPM formed a cluster with other TPM/TPM1 proteins during analysis. The tissue-specific mRNA expression analysis found the higher expression of in the heart and muscles; however, during embryonic and larval development (ELD), the higher expression was found in the trochophore larvae and veliger larvae. expression was upregulated in fast-growing abalone. Increasing thermal stress over a long period decreased expression. Long-term starvation (>1 week) reduced the mRNA expression of in muscle; however, the mRNA expression of was significantly higher in the mantle, which may indicate overexpression. This study is the first comprehensive study to characterize the gene in Pacific abalone and to report the expression of in different organs, and during ELD, different growth patterns, thermal stress, seasonal changes, and starvation.
Topics: Animals; Tropomyosin; Actins; Gastropoda; Muscle Contraction; RNA, Messenger
PubMed: 36672743
DOI: 10.3390/genes14010002 -
Biophysical Journal May 2003Tropomyosin binds end to end along the actin filament. Tropomyosin ends, and the complex they form, are required for actin binding, cooperative regulation of actin... (Comparative Study)
Comparative Study
Tropomyosin binds end to end along the actin filament. Tropomyosin ends, and the complex they form, are required for actin binding, cooperative regulation of actin filaments by myosin, and binding to the regulatory protein, troponin T. The aim of the work was to understand the isoform and structural specificity of the end-to-end association of tropomyosin. The ability of N-terminal and C-terminal model peptides with sequences of alternate alpha-tropomyosin isoforms, and a troponin T fragment that binds to the tropomyosin overlap, to form complexes was analyzed using circular dichroism spectroscopy. Analysis of N-terminal extensions (N-acetylation, Gly, AlaSer) showed that to form an overlap complex between the N-terminus and the C-terminus requires that the N-terminus be able to form a coiled coil. Formation of a ternary complex with the troponin T fragment, however, effectively takes place only when the overlap complex sequences are those found in striated muscle tropomyosins. Striated muscle tropomyosins with N-terminal modifications formed ternary complexes with troponin T that varied in affinity in the order: N-acetylated > Gly > AlaSer > unacetylated. The circular dichroism results were corroborated by native gel electrophoresis, and the ability of the troponin T fragment to promote binding of full-length tropomyosins to filamentous actin.
Topics: Amino Acid Sequence; Animals; Binding Sites; Chickens; Macromolecular Substances; Molecular Motor Proteins; Molecular Sequence Data; Muscle, Skeletal; Protein Binding; Protein Conformation; Protein Isoforms; Sensitivity and Specificity; Structure-Activity Relationship; Temperature; Tropomyosin; Troponin T
PubMed: 12719247
DOI: 10.1016/S0006-3495(03)70042-3 -
Mutations and expressions of the tropomyosin gene and the troponin C gene of Caenorhabditis elegans.Cell Structure and Function Feb 1997How does muscle gene mutation affect the muscle structure and function of an animal? Mutant animals of the tropomyosin and troponin C genes of Caenorhabditis elegans...
How does muscle gene mutation affect the muscle structure and function of an animal? Mutant animals of the tropomyosin and troponin C genes of Caenorhabditis elegans show Pat (paralyzed, arrested elongation at twofold) phenotypes together with abnormal muscle filament assembly. We present evidence that the mutation sites of lev-11 gene was in the tropomyosin gene, tmy-1 and that of pat-10 was in the troponin C gene, tnc-1, of the worm, respectively. The lev-11 (st557) mutation occurred at the splice donor site of exon 1 and results in translation termination. Although the gene product from heterozygous (+/st557) animal was not detected, our result could be the reason for the Pat phenotype of this mutation. The lev-11(x12) mutation, isolated as an allele of levamisole resistance, occurred in exon 7 and results in amino acid substitution at 234 from Glu to Lys. This substitution give a charge change from - to + at this point which is common in three isoforms. There may be functional importance of this region for molecular interaction of the tropomyosin. Mutation site of pat-10(st575) was Asp64 to Asn and Trp153 to termination in the troponin C. The first mutation site was in the second calcium binding site and the second mutation raised the deletion of H helix in the troponin C. Both might affect the calcium binding or the retaining of the conformation for its function. Results presented here will be useful to understand the interaction site between the tropomyosin and troponin complex.
Topics: Animals; Caenorhabditis elegans; Chromosome Mapping; Gene Expression Regulation; Models, Molecular; Muscle Development; Mutation; Protein Conformation; Tropomyosin; Troponin C
PubMed: 9113409
DOI: 10.1247/csf.22.213 -
Pflugers Archiv : European Journal of... Aug 2009The effects of tropomyosin on muscle mechanics and kinetics were examined in skeletal myofibrils using a novel method to remove tropomyosin (Tm) and troponin (Tn) and...
The effects of tropomyosin on muscle mechanics and kinetics were examined in skeletal myofibrils using a novel method to remove tropomyosin (Tm) and troponin (Tn) and then replace these proteins with altered versions. Extraction employed a low ionic strength rigor solution, followed by sequential reconstitution at physiological ionic strength with Tm then Tn. SDS-PAGE analysis was consistent with full reconstitution, and fluorescence imaging after reconstitution using Oregon-green-labeled Tm indicated the expected localization. Myofibrils remained mechanically viable: maximum isometric forces of myofibrils after sTm/sTn reconstitution (control) were comparable (~84%) to the forces generated by non-reconstituted preparations, and the reconstitution minimally affected the rate of isometric activation (k (act)), calcium sensitivity (pCa(50)), and cooperativity (n (H)). Reconstitutions using various combinations of cardiac and skeletal Tm and Tn indicated that isoforms of both Tm and Tn influence calcium sensitivity of force development in opposite directions, but the isoforms do not otherwise alter cross-bridge kinetics. Myofibrils reconstituted with Delta23Tm, a deletion mutant lacking the second and third of Tm's seven quasi-repeats, exhibited greatly depressed maximal force, moderately slower k (act) rates and reduced n (H). Delta23Tm similarly decreased the cooperativity of calcium binding to the troponin regulatory sites of isolated thin filaments in solution. The mechanisms behind these effects of Delta23Tm also were investigated using P ( i ) and ADP jumps. P ( i ) and ADP kinetics were indistinguishable in Delta23Tm myofibrils compared to controls. The results suggest that the deleted region of tropomyosin is important for cooperative thin filament activation by calcium.
Topics: Animals; Cells, Cultured; Female; Kinetics; Mechanics; Muscle Contraction; Myofibrils; Rabbits; Stress, Mechanical; Tropomyosin
PubMed: 19255776
DOI: 10.1007/s00424-009-0653-3 -
The FEBS Journal Sep 2013We have investigated the interactions between the actin-binding proteins gelsolin and tropomyosin, with special respect to any effects on the functional properties of...
We have investigated the interactions between the actin-binding proteins gelsolin and tropomyosin, with special respect to any effects on the functional properties of gelsolin. Limited proteolysis indicated that the loop connecting the gelsolin domains G3 and G4 is involved in tropomyosin binding. Under nonpolymerizing conditions, binding of tropomyosin neither prevented the formation of a 2: 1actin-gelsolin complex, nor did it affect the nucleating activity of gelsolin in actin polymerization, likely as a result of competitive displacement of tropomyosin from gelsolin. To evaluate the effect of tropomyosin on the actin filament severing activity of gelsolin, we measured both filamentous actin (F-actin) viscosity and the relative number concentrations of filaments after fragmentation, either by gelsolin alone or by gelsolin-tropomyosin complexes. The interaction of gelsolin with tropomyosin caused a reduction in F-actin severing activity of up to 80% compared to gelsolin alone. Thus, being bound to gelsolin, tropomyosin prevented gelsolin from severing actin filaments. By contrast, the severing activity of gelsolin for F-actin/tropomyosin was similar to that for F-actin alone even at a tropomyosin : actin saturation ratio of 1: 7. Thus, when bound to actin filaments, tropomyosin did not significantly inhibit the severing of filaments by gelsolin. The interaction between gelsolin and tropomyosin was largely independent of the muscle actin and tropomyosin isoforms investigated. The results obtained in the present study suggest that tropomyosin is involved in the modulation of actin dynamics not via the protection of filaments against severing, but rather by binding gelsolin in solution to prevent it from severing and to promote the formation of new actin filaments.
Topics: Actin Cytoskeleton; Actins; Animals; Binding Sites; Electrophoresis, Polyacrylamide Gel; Gelsolin; Humans; Kinetics; Muscle, Smooth; Protein Binding; Protein Isoforms; Rabbits; Solutions; Swine; Tropomyosin; Viscosity
PubMed: 23844991
DOI: 10.1111/febs.12431