Single-molecule imaging reveals the concerted release of myosin from regulated thin filaments.
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ABSTRACT:
SUBMITTER: Smith QM
PROVIDER: S-EPMC8476120 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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.
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Project description:Myosin binding protein-C (MyBP-C) is a key regulatory protein in heart muscle, and mutations in the MYBPC3 gene are frequently associated with cardiomyopathy. However, the mechanism of action of MyBP-C remains poorly understood, and both activating and inhibitory effects of MyBP-C on contractility have been reported. To clarify the function of the regulatory N-terminal domains of MyBP-C, we determined their effects on the structure of thick (myosin-containing) and thin (actin-containing) filaments in intact sarcomeres of heart muscle. We used fluorescent probes on troponin C in the thin filaments and on myosin regulatory light chain in the thick filaments to monitor structural changes associated with activation of demembranated trabeculae from rat ventricle by the C1mC2 region of rat MyBP-C. C1mC2 induced larger structural changes in thin filaments than calcium activation, and these were still present when active force was blocked with blebbistatin, showing that C1mC2 directly activates the thin filaments. In contrast, structural changes in thick filaments induced by C1mC2 were smaller than those associated with calcium activation and were abolished or reversed by blebbistatin. Low concentrations of C1mC2 did not affect resting force but increased calcium sensitivity and reduced cooperativity of force and structural changes in both thin and thick filaments. These results show that the N-terminal region of MyBP-C stabilizes the ON state of thin filaments and the OFF state of thick filaments and lead to a novel hypothesis for the physiological role of MyBP-C in the regulation of cardiac contractility.
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Project description:Myosin-5B is one of three members of the myosin-5 family of actin-based molecular motors fundamental in recycling endosome trafficking and collective actin network dynamics. Through single-molecule motility assays, we recently demonstrated that myosin-5B can proceed in 36-nm steps along actin filaments as single motor. By analyzing trajectories of single myosin-5B along actin filaments we showed that its velocity is dependent on ATP concentration, while its run length is independent on ATP concentration, as a landmark of processivity. Here, we share image stacks acquired under total internal reflection fluorescence (TIRF) microscopy and representative trajectories of single myosin-5B molecules labelled with Quantum Dots (QD-myo-5B) moving along actin filaments at different ATP concentrations (0.3-1000 μM). Localization of QD-myo-5B was performed with the PROOF software, which is freely available [1]. The data can be valuable for researchers interested in molecular motors motility, both from an experimental and modeling point of view, as well as to researchers developing single particle tracking algorithms. The data is related to the research article "Dissecting myosin-5B mechanosensitivity and calcium regulation at the single molecule level" Gardini et al., 2015.
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Project description:Myosin is a large family of actin-based molecular motors, which includes efficient intracellular transporters that move cargoes and material essential for cell's life. Here, we describe protocols for labelling single myosin motors with quantum dots, tracking them in an in vitro reconstituted single-molecule motility assay, acquiring image stacks and analyzing them. We describe the required steps to obtain trajectories of single myosin motors from which fundamental biophysical parameters such as the motor velocity, run length and step size can be derived. We also describe protocols for an ensemble actin gliding assay, which is valuable to test the motor viability and its ensemble properties. The protocols allow probing the effect of changes in nucleotides, ions, and buffer composition on the motor properties and are easily generalizable to track the movements of different motor proteins.
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Project description:How mechanical stress applied to the actin network modifies actin turnover has attracted considerable attention. Actomyosin exerts the major force on the actin network, which has been implicated in actin stability regulation. However, direct monitoring of immediate changes in F-actin stability on alteration of actomyosin contraction has not been achieved. Here we reexamine myosin regulation of actin stability by using single-molecule speckle analysis of actin. To avoid possible errors attributable to actin-binding probes, we employed DyLight-labeled actin that distributes identical to F-actin in lamellipodia. We performed time-resolved analysis of the effect of blebbistatin on actin turnover. Blebbistatin enhanced actin disassembly in lamellipodia of fish keratocytes and lamellar of Xenopus XTC cells at an early stage of the inhibition, indicating that actomyosin contraction stabilizes cellular F-actin. In addition, our data show a previously unrecognized relationship between the actin network-driving force and the actin turnover rates in lamellipodia. These findings point to the power of direct viewing of molecular behavior in elucidating force regulation of actin filament turnover.
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Project description:Cardiac muscle contraction is triggered by calcium binding to troponin. The consequent movement of tropomyosin permits myosin binding to actin, generating force. Cardiac myosin-binding protein C (cMyBP-C) plays a modulatory role in this activation process. One potential mechanism for the N-terminal domains of cMyBP-C to achieve this is by binding directly to the actin-thin filament at low calcium levels to enhance the movement of tropomyosin. To determine the molecular mechanisms by which cMyBP-C enhances myosin recruitment to the actin-thin filament, we directly visualized fluorescently labeled cMyBP-C N-terminal fragments and GFP-labeled myosin molecules binding to suspended actin-thin filaments in a fluorescence-based single-molecule microscopy assay. Binding of the C0C3 N-terminal cMyBP-C fragment to the thin filament enhanced myosin association at low calcium levels. However, at high calcium levels, C0C3 bound in clusters, blocking myosin binding. Dynamic imaging of thin filament-bound Cy3-C0C3 molecules demonstrated that these fragments diffuse along the thin filament before statically binding, suggesting a mechanism that involves a weak-binding mode to search for access to the thin filament and a tight-binding mode to sensitize the thin filament to calcium, thus enhancing myosin binding. Although shorter N-terminal fragments (Cy3-C0C1 and Cy3-C0C1f) bound to the thin filaments and displayed modes of motion on the thin filament similar to that of the Cy3-C0C3 fragment, the shorter fragments were unable to sensitize the thin filament. Therefore, the longer N-terminal fragment (C0C3) must possess the requisite domains needed to bind specifically to the thin filament in order for the cMyBP-C N terminus to modulate cardiac contractility.
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Project description:Mutations in titin are responsible for many cardiac and muscle diseases, yet the underlying mechanisms remain largely unexplained. Numerous studies have established roles for titin in muscle function, and Ca2+-dependent interactions between titin and actin have been suggested to play a role in muscle contraction. The present study used co-sedimentation assays, dynamic force spectroscopy (DFS), and in vitro motility (IVM) assays to determine whether the N2A region of titin, overlooked in previous studies, interacts with actin in the presence of Ca2+. Co-sedimentation demonstrated that N2A - F-actin binding increases with increasing protein and Ca2+ concentration, DFS demonstrated increased rupture forces and decreased koff in the presence of Ca2+, and IVM demonstrated a Ca2+-dependent reduction in motility of F-actin and reconstituted thin filaments in the presence of N2A. These results indicate that Ca2+ increases the strength and stability of N2A - actin interactions, supporting the hypothesis that titin plays a regulatory role in muscle contraction. The results further support a model in which N2A - actin binding in active muscle increases titin stiffness, and that impairment of this mechanism contributes to the phenotype in muscular dystrophy with myositis. Future studies are required to determine whether titin - actin binding occurs in skeletal muscle sarcomeres in vivo.
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