Troponin I and troponin T interact with troponin C to produce different Ca2+-dependent effects on actin-tropomyosin filament motility.
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ABSTRACT:
SUBMITTER: Bing W
PROVIDER: S-EPMC1218798 | biostudies-other | 1997 Oct
REPOSITORIES: biostudies-other
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Project description:Tropomyosin binds to actin filaments and is implicated in stabilization of actin cytoskeleton. We examined biochemical and cell biological properties of Caenorhabditis elegans tropomyosin (CeTM) and obtained evidence that CeTM is antagonistic to ADF/cofilin-dependent actin filament dynamics. We purified CeTM, actin, and UNC-60B (a muscle-specific ADF/cofilin isoform), all of which are derived from C. elegans, and showed that CeTM and UNC-60B bound to F-actin in a mutually exclusive manner. CeTM inhibited UNC-60B-induced actin depolymerization and enhancement of actin polymerization. Within isolated native thin filaments, actin and CeTM were detected as major components, whereas UNC-60B was present at a trace amount. Purified UNC-60B was unable to interact with the native thin filaments unless CeTM and other associated proteins were removed by high-salt extraction. Purified CeTM was sufficient to restore the resistance of the salt-extracted filaments from UNC-60B. In muscle cells, CeTM and UNC-60B were localized in different patterns. Suppression of CeTM by RNA interference resulted in disorganized actin filaments and paralyzed worms in wild-type background. However, in an ADF/cofilin mutant background, suppression of CeTM did not worsen actin organization and worm motility. These results suggest that tropomyosin is a physiological inhibitor of ADF/cofilin-dependent actin dynamics.
| S-EPMC2173459 | biostudies-literature
Project description:Elongated tropomyosin, associated with actin-subunits along the surface of thin filaments, makes electrostatic interactions with clusters of conserved residues, K326, K328, and R147, on actin. The association is weak, permitting low-energy cost regulatory movement of tropomyosin across the filament during muscle activation. Interestingly, acidic D292 on actin, also evolutionarily conserved, lies adjacent to the three-residue cluster of basic amino acids and thus may moderate the combined local positive charge, diminishing tropomyosin-actin interaction and facilitating regulatory-switching. Indeed, charge neutralization of D292 is connected to muscle hypotonia in individuals with D292V actin mutations and linked to congenital fiber-type disproportion. Here, the D292V mutation may predispose tropomyosin-actin positioning to a myosin-blocking state, aberrantly favoring muscle relaxation, thus mimicking the low-Ca2+ effect of troponin even in activated muscles. To test this hypothesis, interaction energetics and in vitro function of wild-type and D292V filaments were measured. Energy landscapes based on F-actin-tropomyosin models show the mutation localizes tropomyosin in a blocked-state position on actin defined by a deeper energy minimum, consistent with augmented steric-interference of actin-myosin binding. In addition, whereas myosin-dependent motility of troponin/tropomyosin-free D292V F-actin is normal, motility is dramatically inhibited after addition of tropomyosin to the mutant actin. Thus, D292V-induced blocked-state stabilization appears to disrupt the delicately poised energy balance governing thin filament regulation. Our results validate the premise that stereospecific but necessarily weak binding of tropomyosin to F-actin is required for effective thin filament function.
| S-EPMC5768522 | biostudies-literature
Project description:RATIONALE:Ca(2+) control of troponin-tropomyosin position on actin regulates cardiac muscle contraction. The inhibitory subunit of troponin, cardiac troponin (cTn)I is primarily responsible for maintaining a tropomyosin conformation that prevents crossbridge cycling. Despite extensive characterization of cTnI, the precise role of its C-terminal domain (residues 193 to 210) is unclear. Mutations within this region are associated with restrictive cardiomyopathy, and C-terminal deletion of cTnI, in some species, has been associated with myocardial stunning. OBJECTIVE:We sought to investigate the effect of a cTnI deletion-removal of 17 amino acids from the C terminus- on the structure of troponin-regulated tropomyosin bound to actin. METHODS AND RESULTS:A truncated form of human cTnI (cTnI(1-192)) was expressed and reconstituted with troponin C and troponin T to form a mutant troponin. Using electron microscopy and 3D image reconstruction, we show that the mutant troponin perturbs the positional equilibrium dynamics of tropomyosin in the presence of Ca(2+). Specifically, it biases tropomyosin position toward an "enhanced C-state" that exposes more of the myosin-binding site on actin than found with wild-type troponin. CONCLUSIONS:In addition to its well-established role of promoting the so-called "blocked-state" or "B-state," cTnI participates in proper stabilization of tropomyosin in the "Ca(2+)-activated state" or "C-state." The last 17 amino acids perform this stabilizing role. The data are consistent with a "fly-casting" model in which the mobile C terminus of cTnI ensures proper conformational switching of troponin-tropomyosin. Loss of actin-sensing function within this domain, by pathological proteolysis or cardiomyopathic mutation, may be sufficient to perturb tropomyosin conformation.
| S-EPMC2834238 | biostudies-literature
Project description:Cellular actin networks exhibit diverse filamentous architectures and turnover dynamics, but how these differences are specified remains poorly understood. Here, we used multicolor total internal reflection fluorescence microscopy to ask how decoration of actin filaments by five biologically prominent Tropomyosin (TPM) isoforms influences disassembly induced by Cofilin alone, or by the collaborative effects of Cofilin, Coronin, and AIP1 (CCA). TPM decoration restricted Cofilin binding to pointed ends, while not interfering with Coronin binding to filament sides. Different isoforms of TPM provided variable levels of protection against disassembly, with the strongest protection by Tpm3.1 and the weakest by Tpm1.6. In biomimetic assays in which filaments were simultaneously assembled by formins and disassembled by CCA, these TPM isoform-specific effects persisted, giving rise to filaments with different lengths and treadmilling behavior. Together, our data reveal that TPM isoforms have quantitatively distinct abilities to tune actin filament length and turnover.
| S-EPMC6589703 | biostudies-literature
Project description:In an effort to elucidate the mechanism of calmodulin regulation of muscle contraction, we investigated the interaction between calmodulin and troponin components in the presence of Ca2+ or Sr2+ by the use of ultracentrifugation methods and polyacrylamide-gel electrophoresis. Skeletal-muscle troponin C bound to troponin I and dissociated it from the tropomyosin-actin complex in the presence of Ca2+ or Sr2+. When troponin T was absent, calmodulin bound to troponin I and dissociated it from the tropomyosin-actin complex in the presence of Ca2+ or Sr2+. When troponin T was present, calmodulin hardly bound to troponin I even in the presence of bivalent cations. Trifluoperazine, a calmodulin antagonist, inhibited the bivalent-cation-dependent interaction between calmodulin and troponin I. Calmodulin migrated more slowly in the presence of Sr2+ than it did in the presence of EGTA but faster than it did in the presence of Ca2+ on polyacrylamide-gel electrophoresis under non-denaturing conditions. It is concluded that troponin T is not required in the calmodulin regulation of muscle contraction because troponin T inhibits the bivalent-cation-dependent interaction between calmodulin and troponin I and because calmodulin binds to troponin I and dissociates it from the tropomyosin-actin complex in a bivalent-cation-dependent manner. Sr2+-induced exposure of the hydrophobic region enables calmodulin to bind to troponin I, as is the case with Ca2+.
| S-EPMC1147646 | biostudies-other
Project description:Actin filaments assemble into a variety of networks to provide force for diverse cellular processes [1]. Tropomyosins are coiled-coil dimers that form head-to-tail polymers along actin filaments and regulate interactions of other proteins, including actin-depolymerizing factor (ADF)/cofilins and myosins, with actin [2-5]. In mammals, >40 tropomyosin isoforms can be generated through alternative splicing from four tropomyosin genes. Different isoforms display non-redundant functions and partially non-overlapping localization patterns, for example within the stress fiber network [6, 7]. Based on cell biological studies, it was thus proposed that tropomyosin isoforms may specify the functional properties of different actin filament populations [2]. To test this hypothesis, we analyzed the properties of actin filaments decorated by stress-fiber-associated tropomyosins (Tpm1.6, Tpm1.7, Tpm2.1, Tpm3.1, Tpm3.2, and Tpm4.2). These proteins bound F-actin with high affinity and competed with α-actinin for actin filament binding. Importantly, total internal reflection fluorescence (TIRF) microscopy of fluorescently tagged proteins revealed that most tropomyosin isoforms cannot co-polymerize with each other on actin filaments. These isoforms also bind actin with different dynamics, which correlate with their effects on actin-binding proteins. The long isoforms Tpm1.6 and Tpm1.7 displayed stable interactions with actin filaments and protected filaments from ADF/cofilin-mediated disassembly, but did not activate non-muscle myosin IIa (NMIIa). In contrast, the short isoforms Tpm3.1, Tpm3.2, and Tpm4.2 displayed rapid dynamics on actin filaments and stimulated the ATPase activity of NMIIa, but did not efficiently protect filaments from ADF/cofilin. Together, these data provide experimental evidence that tropomyosin isoforms segregate to different actin filaments and specify functional properties of distinct actin filament populations.
| S-EPMC5344678 | biostudies-literature
Project description:The actin cytoskeleton is a dynamic network of filaments that is involved in virtually every cellular process. Most actin filaments in metazoa exist as a co-polymer of actin and tropomyosin (Tpm) and the function of an actin filament is primarily defined by the specific Tpm isoform associated with it. However, there is little information on the interdependence of these co-polymers during filament assembly and disassembly. We addressed this by investigating the recovery kinetics of fluorescently tagged isoform Tpm3.1 into actin filament bundles using FRAP analysis in cell culture and in vivo in rats using intracellular intravital microscopy, in the presence or absence of the actin-targeting drug jasplakinolide. The mobile fraction of Tpm3.1 is between 50% and 70% depending on whether the tag is at the C- or N-terminus and whether the analysis is in vivo or in cultured cells. We find that the continuous dynamic exchange of Tpm3.1 is not significantly impacted by jasplakinolide, unlike tagged actin. We conclude that tagged Tpm3.1 may be able to undergo exchange in actin filament bundles largely independent of the assembly and turnover of actin.
| S-EPMC5158027 | biostudies-literature
Project description:Striated muscle contraction is governed by the thin filament regulatory proteins troponin and tropomyosin. Here, we investigate the molecular mechanisms by which troponin-tropomyosin inhibits myosin's interactions with the thin filament in the absence of calcium by using a laser trap. The displacement events for a single-myosin molecule interacting with a reconstituted thin filament were shorter (step size = 5 nm) and prolonged (69 ms) compared with actin alone (11 nm and 26 ms, respectively). However, these changes alone do not account for the degree of inhibition of thin filament movement observed in an ensemble assay. Our investigations of single- and multiple-myosin molecules with regulated thin filaments suggest the primary basis for this inhibition derives from an approximately 100-fold decrease in the probability of myosin attaching to actin. At higher myosin concentrations, short bursts of motility are observed in a laser trap consistent with the strong binding of a single-myosin crossbridge, resulting in cooperative binding of other cycling crossbridges. We confirmed this cooperativity in the in vitro motility assay by observing thin filament translocation in the absence of calcium but at low [ATP], consistent with rigor activation. We have developed a simple mechanistic model that reproduces and provides insight into both the observed single-myosin molecule and ensemble data in the absence of Ca(2+). These data support the hypothesis that thin filament inhibition in the absence of Ca(2+) is largely achieved by modulating the rate of attachment and/or transition from the weakly to strongly bound state.
| S-EPMC1287988 | biostudies-literature
Project description:Tropomyosin and cofilin are involved in the regulation of actin filament dynamic polymerization and depolymerization. Binding of cofilin changes actin filaments structure, leading to their severing and depolymerization. Non-muscle tropomyosin isoforms were shown before to differentially regulate the activity of cofilin 1; products of TPM1 gene stabilized actin filaments, but products of TPM3 gene promoted cofilin-dependent severing and depolymerization. Here, conformational changes at the longitudinal and lateral interface between actin subunits resulting from tropomyosin and cofilin 1 binding were studied using skeletal actin and yeast wild type and mutant Q41C and S265C actins. Cross-linking of F-actin and fluorescence changes in F-actin labeled with acrylodan at Cys41 (in D-loop) or Cys265 (in H-loop) showed that tropomyosin isoforms differentially regulated cofilin-induced conformational rearrangements at longitudinal and lateral filament interfaces. Tryptic digestion of F-Mg-actin confirmed the differences between tropomyosin isoforms in their regulation of cofilin-dependent changes at actin-actin interfaces. Changes in the fluorescence of AEDANS attached to C-terminal Cys of actin, as well as FRET between Trp residues in actin subdomain 1 and AEDANS, did not show differences in the conformation of the C-terminal segment of F-actin in the presence of different tropomyosins ± cofilin 1. Therefore, actin's D- and H-loop are the sites involved in regulation of cofilin activity by tropomyosin isoforms.
| S-EPMC7291848 | biostudies-literature
Project description:BackgroundTropomyosin is a prototypical coiled coil along its length with subtle variations in structure that allow interactions with actin and other proteins. Actin binding globally stabilizes tropomyosin. Tropomyosin-actin interaction occurs periodically along the length of tropomyosin. However, it is not well understood how tropomyosin binds actin.Principal findingsTropomyosin's periodic binding sites make differential contributions to two components of actin binding, cooperativity and affinity, and can be classified as primary or secondary sites. We show through mutagenesis and analysis of recombinant striated muscle alpha-tropomyosins that primary actin binding sites have a destabilizing coiled-coil interface, typically alanine-rich, embedded within a non-interface recognition sequence. Introduction of an Ala cluster in place of the native, more stable interface in period 2 and/or period 3 sites (of seven) increased the affinity or cooperativity of actin binding, analysed by cosedimentation and differential scanning calorimetry. Replacement of period 3 with period 5 sequence, an unstable region of known importance for cooperative actin binding, increased the cooperativity of binding. Introduction of the fluorescent probe, pyrene, near the mutation sites in periods 2 and 3 reported local instability, stabilization by actin binding, and local unfolding before or coincident with dissociation from actin (measured using light scattering), and chain dissociation (analyzed using circular dichroism).ConclusionsThis, and previous work, suggests that regions of tropomyosin involved in binding actin have non-interface residues specific for interaction with actin and an unstable interface that is locally stabilized upon binding. The destabilized interface allows residues on the coiled-coil surface to obtain an optimal conformation for interaction with actin by increasing the number of local substates that the side chains can sample. We suggest that local disorder is a property typical of coiled coil binding sites and proteins that have multiple binding partners, of which tropomyosin is one type.
| S-EPMC2710508 | biostudies-literature