Studies on co-operative properties of tropomyosin-actin and tropomyosin-troponin-actin complexes by the use of N-ethylmaleimide-treated and untreated species of myosin subfragment 1

Nagashima, Hajime; Asakura, Shô

doi:10.1016/0022-2836(82)90479-x

Cited by 91 publications

(57 citation statements)

References 30 publications

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“…5). This reversal of inhibition required less than 0.4 NEM-S-1 per actin, thus confirming that it was acting cooperatively upon tropomyosin in the same way as it does with wild type striated muscle actin-tropomyosin (28,34). E93K actin-tropomyosin filaments were also reactivated by adding troponin in the presence of Ca 2ϩ (Fig.…”

Section: Glu 93mentioning

confidence: 53%

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Tropomyosin and Troponin Regulation of Wild Type and E93K Mutant Actin Filaments from Drosophila Flight Muscle

Bing¹,

Razzaq²,

Sparrow³

et al. 1998

Journal of Biological Chemistry

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In the Drosophila flight muscle actin mutant E93K there is a charge reversal on the surface of actin close to the proposed position of tropomyosin when it is in the off state. Using a quantitative in vitro motility assay we have found that the wild type Drosophila ACT88F actin behaved like rabbit skeletal muscle actin when tropomyosin and troponin were added at pCa5 and pCa9. In contrast the effect of tropomyosin upon the E93K mutant actin filament movement was completely different from wild type and resembled the response of wild type with tropomyosin؉troponin at pCa9 (i.e. the filaments were switched off). Velocity of E93K actin did not increase, and the fraction of filaments motile was reduced to less than 15% by adding up to 30 nM tropomyosin. When myosin subfragment-1 modified by N-ethylmaleimide was mixed with mutant E93K actin-tropomyosin filaments we observed that it restored motility of the filaments to the level observed with E93K actin alone. We conclude that electrostatic charge on the surface of domain 2 of actin plays a critical role in determining the state of actin-tropomyosin that is a central feature of the steric blocking mechanism of actin filament regulation.

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Section: Glu 93mentioning

confidence: 53%

“…NEM-S-1 forms strong bonds to actin, even in the presence of ATP, thus it switches actin-tropomyosin to the on state independently of regulatory proteins (29,34). We have shown previously that adding NEM-S-1 to actin-tropomyosin resulted in an increase in filament motility (28).…”

Section: Glu 93mentioning

confidence: 99%

Tropomyosin and Troponin Regulation of Wild Type and E93K Mutant Actin Filaments from Drosophila Flight Muscle

Bing¹,

Razzaq²,

Sparrow³

et al. 1998

Journal of Biological Chemistry

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“…Because the velocity of A-Tm filaments was inhibited >40% for most Tm constructs compared with actin alone, we carried out motility assays in the presence of NEM-S1, a mimic of the rigor complex between actin and myosin S1, known to activate regulated thin filaments (26)(27)(28). NEM-S1 binds tightly and activates thin filaments in the presence of MgATP.…”

Section: Resultsmentioning

confidence: 99%

Regulation of actin-myosin interaction by conserved periodic sites of tropomyosin

Barua

Winkelmann

White

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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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.actin filament structure | cell motility | cytoskeleton | muscle contraction | motility assay A ctin and myosin are found in almost all eukaryotic cells and are required for diverse cellular functions, including muscle contraction, cell motility, cell adhesion, cytokinesis, and organelle transport (1). The actin-myosin interaction produces two types of movements: force generation between actin filaments leading to contractions, such as in muscle contraction, cell motility, and cytokinesis; and transport of subcellular organelles and macromolecular complexes by myosin motors along actin filaments. The actomyosin contractile apparatus is best characterized in striated muscle contraction, which is regulated in a Ca 2+ -dependent manner by the proteins, tropomyosin (Tm) and troponin (Tn).Tm is a universal regulator of the actin cytoskeleton. It is a twochained, α-helical coiled-coil actin binding protein that associates end-to-end to form a continuous strand along both sides of actin filaments and regulates the functions and stability of actin filaments (2, 3). Tm regulates the interaction of actin filaments with actin binding proteins, including myosin, Tn, ADF-cofilin, Arp2/3, formin, and tropomodulin. Tm can activate or inhibit actomyosin, depending on the myosin and Tm isoforms (4-7). The Tm-dependent regulation is widespread, having been reported in fission yeast and mammalian muscle and nonmuscle systems. The regulation of actomyosin by Tm may be viewed as universal, although there is little understanding of the mechanism or structural basis, the focus of the present work.In striated muscle, regulation of myosin binding to the thin filament (actin-Tm-Tn) is described by a three-state model, where the three kinetic states are thought to correspond to three positions of Tm on the actin filament (8-1...

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“…NEM-S1 as a Thin Filament Activator-The position of Tm on the filament was controlled through NEM-S1, which has a binding constant of 1.4 ϫ 10 6 M Ϫ1 with bare actin and 6.3 ϫ 10 6 M Ϫ1 with Tm-actin (34) and should result in 11% of the binding sites on the Tm-actin filament being occupied at 20 nM NEM-S1. This value corresponds to about 1 regulatory unit per bound NEM-S1.…”

Section: Discussionmentioning

confidence: 99%

Force Spectroscopy Reveals Multiple “Closed States” of the Muscle Thin Filament

Rao

Clobes

Guilford

2011

Journal of Biological Chemistry

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Tropomyosin (Tm) plays a critical role in regulating the contraction of striated muscle. The three-state model of activation posits that Tm exists in three positions on the thin filament: "blocked" in the absence of calcium when myosin cannot bind, "closed" when calcium binds troponin and Tm partially covers the myosin binding site, and "open" after myosin binding forces Tm completely off neighboring sites. However, we recently showed that actin filaments decorated with phosphorylated Tm are driven by myosin with greater force than bare actin filaments. This result cannot be explained by simple steric hindrance and suggests that Tm may have additional effects on actin-myosin interactions. We therefore tested the hypothesis that Tm and its phosphorylation state affect the rate at which single actin-myosin bonds form and rupture. Using a laser trap, we measured the time necessary for the first bond to form between actin and rigor heavy meromyosin and the load-dependent durations of those bonds. Measurements were repeated in the presence of subsaturating myosin-S1 to force Tm from the closed to the open state. Maximum bond lifetimes increased in the open state, but only when Tm was phosphorylated. While the frequency with which bonds formed was extremely low in the closed state, when a bond did form it took significantly less time to do so than with bare actin. These data suggest there are at least two closed states of the thin filament, and that Tm provides additional points of contact for myosin.The actin-binding protein tropomyosin (Tm) 2 is thought to operate in opposing roles as an inhibitor or an activator in the cross-bridge cycle by sterically hindering myosin binding to actin in the absence of calcium and, conversely, promoting the cooperative relief of inhibition over the span of one regulatory unit when an initial myosin binds (1-3). Data suggest that the transition of Tm from an inhibitory to a permissive state proceeds through at least one intermediate step. The three-state model of cooperative activation suggests that Tm exists in three positions on the thin filament: 1) blocked, in the absence of calcium when myosin cannot bind; 2) closed, when calcium binds troponin and Tm still partially covers the myosin binding site; and 3) open, after initial myosin binding forces Tm completely off neighboring myosin binding sites (4, 5).These state changes are thought to be propagated to neighboring regulatory units on the thin filament. Tm polymerizes in a head-to-tail manner with neighboring Tm dimers (6), and evidence suggests that Tm can transmit conformational changes over long distances via end-to-end interactions (7-9). In the ␣ isoform of Tm found in striated muscle, there is a phosphorylation site near the carboxyl terminus at . This phosphorylation site is in the head-to-tail overlap between neighboring Tm dimers, and we showed previously that Tm phosphorylation is necessary to extend cooperative activation beyond one regulatory unit (11). We also found that actin filaments decorated with phosphoryl...

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Studies on co-operative properties of tropomyosin-actin and tropomyosin-troponin-actin complexes by the use of N-ethylmaleimide-treated and untreated species of myosin subfragment 1

Cited by 91 publications

References 30 publications

Tropomyosin and Troponin Regulation of Wild Type and E93K Mutant Actin Filaments from Drosophila Flight Muscle

Tropomyosin and Troponin Regulation of Wild Type and E93K Mutant Actin Filaments from Drosophila Flight Muscle

Regulation of actin-myosin interaction by conserved periodic sites of tropomyosin

Force Spectroscopy Reveals Multiple “Closed States” of the Muscle Thin Filament

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