Anti-actin antibodies. An immunological approach to the myosin-actin and the tropomyosin-actin interfaces.
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ABSTRACT:
SUBMITTER: Mejean C
PROVIDER: S-EPMC1148034 | biostudies-other | 1987 Jun
REPOSITORIES: biostudies-other
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Project description: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.
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Project description:Tropomyosin (Tpm) isoforms decorate actin with distinct spatial and temporal localization patterns in cells and thus may function to sort actomyosin processes by modifying the actin track affinity for specific myosin isoforms. We examined the effect of three Tpm isoforms on the ability of myosin Va (myoVa) to engage with actin in vitro in the absence or presence of the cargo adapter melanophilin (Mlph), which links myoVa to Rab27a-melanosomes for in vivo transport. We show that both the myosin motor domain and the cargo adapter Mlph, which has an actin-binding domain that acts as a tether, are sensitive to the Tpm isoform. Actin-Tpm3.1 and actin-Tpm1.8 were equal or better tracks compared to bare actin for myoVa-HMM based on event frequency, run length, and speed. The full-length myoVa-Mlph complex showed high-frequency engagement with actin-Tpm3.1 but not with actin-Tpm1.8. Actin-Tpm4.2 excluded both myoVa-HMM and full-length myoVa-Mlph from productive interactions. Of importance, Tpm3.1 is enriched in the dendritic protrusions and cortical actin of melanocytes, where myoVa-Mlph engages in melanosome transport. These results support the hypothesis that Tpm isoforms constitute an "actin-Tpm code" that allows for spatial and temporal sorting of actomyosin function in the cell.
| S-EPMC5042576 | biostudies-literature
Project description:Actin/myosin interactions in vertebrate striated muscles are believed to be regulated by the "steric blocking" mechanism whereby the binding of calcium to the troponin complex allows tropomyosin (TM) to change position on actin, acting as a molecular switch that blocks or allows myosin heads to interact with actin. Movement of TM during activation is initiated by interaction of Ca(2+) with troponin, then completed by further displacement by strong binding cross-bridges. We report x-ray evidence that TM in insect flight muscle (IFM) moves in a manner consistent with the steric blocking mechanism. We find that both isometric contraction, at high [Ca(2+)], and stretch activation, at lower [Ca(2+)], develop similarly high x-ray intensities on the IFM fourth actin layer line because of TM movement, coinciding with x-ray signals of strong-binding cross-bridge attachment to helically favored "actin target zones." Vanadate (Vi), a phosphate analog that inhibits active cross-bridge cycling, abolishes all active force in IFM, allowing high [Ca(2+)] to elicit initial TM movement without cross-bridge attachment or other changes from relaxed structure. However, when stretched in high [Ca(2+)], Vi-"paralyzed" fibers produce force substantially above passive response at pCa approximately 9, concurrent with full conversion from resting to active x-ray pattern, including x-ray signals of cross-bridge strong-binding and TM movement. This argues that myosin heads can be recruited as strong-binding "brakes" by backward-sliding, calcium-activated thin filaments, and are as effective in moving TM as actively force-producing cross-bridges. Such recruitment of myosin as brakes may be the major mechanism resisting extension during lengthening contractions.
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Project description:The conformational elasticity of the actin cytoskeleton is essential for its versatile biological functions. Increasing evidence supports that the interplay between the structural and functional properties of actin filaments is finely regulated by actin-binding proteins; however, the underlying mechanisms and biological consequences are not completely understood. Previous studies showed that the binding of formins to the barbed end induces conformational transitions in actin filaments by making them more flexible through long range allosteric interactions. These conformational changes are accompanied by altered functional properties of the filaments. To get insight into the conformational regulation of formin-nucleated actin structures, in the present work we investigated in detail how binding partners of formin-generated actin structures, myosin and tropomyosin, affect the conformation of the formin-nucleated actin filaments using fluorescence spectroscopic approaches. Time-dependent fluorescence anisotropy and temperature-dependent Förster-type resonance energy transfer measurements revealed that heavy meromyosin, similarly to tropomyosin, restores the formin-induced effects and stabilizes the conformation of actin filaments. The stabilizing effect of heavy meromyosin is cooperative. The kinetic analysis revealed that despite the qualitatively similar effects of heavy meromyosin and tropomyosin on the conformational dynamics of actin filaments the mechanisms of the conformational transition are different for the two proteins. Heavy meromyosin stabilizes the formin-nucleated actin filaments in an apparently single step reaction upon binding, whereas the stabilization by tropomyosin occurs after complex formation. These observations support the idea that actin-binding proteins are key elements of the molecular mechanisms that regulate the conformational and functional diversity of actin filaments in living cells.
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Project description:Cooperative activation of actin-myosin interaction by tropomyosin (Tm) is central to regulation of contraction in muscle cells and cellular and intracellular movements in nonmuscle cells. The steric blocking model of muscle regulation proposed 40 y ago has been substantiated at both the kinetic and structural levels. Even with atomic resolution structures of the major players, how Tm binds and is designed for regulatory function has remained a mystery. Here we show that a set of periodically distributed evolutionarily conserved surface residues of Tm is required for cooperative regulation of actomyosin. Based on our results, we propose a model of Tm on a structure of actin-Tm-myosin in the "open" (on) state showing potential electrostatic interactions of the residues with both actin and myosin. The sites alternate with a second set of conserved surface residues that are important for actin binding in the inhibitory state in the absence of myosin. The transition from the closed to open states requires the sites identified here, even when troponin + Ca(2+) is present. The evolutionarily conserved residues are important for actomyosin regulation, a universal function of Tm that has a common structural basis and mechanism.
| S-EPMC3494946 | 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.
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Project description:Myo51, a class V myosin in fission yeast, localizes to and assists in the assembly of the contractile ring, a conserved eukaryotic actomyosin structure that facilitates cytokinesis. Rng8 and Rng9 are binding partners that dictate the cellular localization and function of Myo51. Myo51 was expressed in insect cells in the presence or absence of Rng8/9. Surprisingly, electron microscopy of negatively stained images and hydrodynamic measurements showed that Myo51 is single headed, unlike most class V myosins. When Myo51-Rng8/9 was bound to actin-tropomyosin, two attachment sites were observed: the typical ATP-dependent motor domain attachment and a novel ATP-independent binding of the tail mediated by Rng8/9. A modified motility assay showed that this additional binding site anchors Myo51-Rng8/9 so that it can cross-link and slide actin-tropomyosin filaments relative to one another, functions that may explain the role of this motor in contractile ring assembly.
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Project description:A hallmark of class-V myosins is their processivity--the ability to take multiple steps along actin filaments without dissociating. Our previous work suggested, however, that the fission yeast myosin-V (Myo52p) is a nonprocessive motor whose activity is enhanced by tropomyosin (Cdc8p). Here we investigate the molecular mechanism and physiological relevance of tropomyosin-mediated regulation of Myo52p transport, using a combination of in vitro and in vivo approaches. Single molecules of Myo52p, visualized by total internal reflection fluorescence microscopy, moved processively only when Cdc8p was present on actin filaments. Small ensembles of Myo52p bound to a quantum dot, mimicking the number of motors bound to physiological cargo, also required Cdc8p for continuous motion. Although a truncated form of Myo52p that lacked a cargo-binding domain failed to support function in vivo, it still underwent actin-dependent movement to polarized growth sites. This result suggests that truncated Myo52p lacking cargo, or single molecules of wild-type Myo52p with small cargoes, can undergo processive movement along actin-Cdc8p cables in vivo. Our findings outline a mechanism by which tropomyosin facilitates sorting of transport to specific actin tracks within the cell by switching on myosin processivity.
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Project description:We show that the mutations D137L and G126R, which stabilize the central part of the tropomyosin (Tm) molecule, increase both the maximal sliding velocity of the regulated actin filaments in the in vitro motility assay at high ??(2+) concentrations and the ??(2+)-sensitivity of the actin-myosin interaction underlying this sliding. Based on an analysis of the recently published data on the structure of the actin-Tm-myosin complex, we suppose that the physiological effects of these mutations in Tm can be accounted for by their influence on the interactions between the central part of Tm and certain sites of the myosin head.
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Project description:The motor protein myosin drives muscle and nonmuscle motility by binding to and moving along actin of thin filaments. Myosin binding to actin also modulates interactions of the regulatory protein, tropomyosin, on thin filaments, and conversely tropomyosin affects myosin binding to actin. Insight into this reciprocity will facilitate a molecular level elucidation of tropomyosin regulation of myosin interaction with actin in muscle contraction, and in turn, promote better understanding of nonmuscle cell motility. Indeed, experimental approaches such as fiber diffraction, cryoelectron microscopy, and three-dimensional reconstruction have long been used to define regulatory interaction of tropomyosin and myosin on actin at a structural level. However, their limited resolution has not proven sufficient to determine tropomyosin and myosin contacts at an atomic-level and thus to fully substantiate possible functional contributions. To overcome this deficiency, we have followed a hybrid approach by performing new cryogenic electron microscopy reconstruction of myosin-S1-decorated F-actin-tropomyosin together with atomic scale protein-protein docking of tropomyosin to the EM models. Here, cryo-EM data were derived from filaments reconstituted with α1-actin, cardiac αα-tropomyosin, and masseter muscle β-myosin complexes; masseter myosin, which shares sequence identity with β-cardiac myosin-heavy chain, was used because of its stability in vitro. The data were used to build an atomic model of the tropomyosin cable that fits onto the actin filament between the tip of the myosin head and a cleft on the innermost edge of actin subunits. The docking and atomic scale fitting showed multiple discrete interactions of myosin loop 4 and acidic residues on successive 39-42 residue-long tropomyosin pseudorepeats. The contacts between S1 and tropomyosin on actin appear to compete with and displace ones normally found between actin and tropomyosin on myosin-free thin filaments in relaxed muscle, thus restructuring the filament during myosin-induced activation.
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