G146V Mutation at the Hinge Region of Actin Reveals a Myosin Class-specific Requirement of Actin Conformations for Motility

Noguchi, Tarow; Komori, Tomotaka; Umeki, Nobuhisa; Demizu, Noriyuki; Ito, Kohji; Iwane, Atsuko H.; Tokuraku, Kiyotaka; Yanagida, Toshio; Uyeda, Taro Q.P.

doi:10.1074/jbc.m111.321752

Cited by 18 publications

(21 citation statements)

References 41 publications

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“…It is noteworthy that Rng2CHD did not inhibit actin filament movement on myosin V HMM, which is well established to move actin filaments processively with a lever arm swing (Forkey et al, 2003, Kodera et al, 2010. In line with this finding, two actin mutations, M47A (Kubota et al, 2009) and G146V (Noguchi et al, 2012), both of which likely perturb conformational changes of actin filaments, inhibit actin movements on myosin II, but not on myosin V. We suggest that the movement by myosin II, which is a fast and non-processive motor, requires some unidentified structural property of actin filaments which is dispensable for the slow and processive myosin V involving the lever arm swing. Further studies are needed to understand the mechanism by which actin filaments structurally altered by Rng2CHD fails to move on myosin II.…”

Section: The Mechanism By Which Structural Changes Of Actin Filamentssupporting

confidence: 66%

Actin binding domain of Rng2 sparsely bound on F-actin strongly inhibits actin movement on myosin II

Hayakawa

Takaine

Imai

et al. 2020

Preprint

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The contraction of contractile rings (CRs) depends on sliding between actin filaments and myosin II filaments. The rate of contraction in the fission yeast Schizosaccharomyces pombe is less than 1/120 of the velocity of acto-myosin II movement in vitro, but the mechanism of inhibition has not been described. Here, we found that the calponin-homology actin binding domain of fission yeast IQGAP Rng2 (Rng2CHD) strongly inhibits the motility of actin filaments on skeletal muscle myosin II fragments in vitro, even at a low ratio of bound Rng2CHD to actin protomers, reducing the sliding velocity to half when the binding ratio was 1/75. Rng2CHD also induced structural changes of actin filaments and reduced the affinity between actin filaments and subfragment 1 (S1) of muscle myosin II carrying ADP. Intriguingly, actin-activated ATPase of S1 was only mildly inhibited, even by high concentrations of Rng2CHD. Moreover, the motility of actin filaments by myosin V was not inhibited by Rng2CHD. We propose a new regulatory mechanism for acto-myosin II movement that involves Rng2CHD-induced structural changes of actin filaments.

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Section: The Mechanism By Which Structural Changes Of Actin Filamentssupporting

confidence: 66%

Actin binding domain of Rng2 sparsely bound on F-actin strongly inhibits actin movement on myosin II

Hayakawa

Takaine

Imai

et al. 2020

Preprint

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“…In this scenario, the on state has a higher FRET efficiency than the off state, and the G146V mutant actin does not easily take the on state, either kinetically or thermodynamically. Consistent with this hypothesis, our earlier biochemical study suggests that the motility defect of G146V actin filaments with myosin II derives from two defects; i.e., slower transition from the weakly bound state to the strongly bound state and the lower affinity during the strongly bound state [ 29 ].…”

Section: Resultsmentioning

confidence: 65%

“…The buffer contained 20 mM HEPES (pH 7.8), 5 mM MgCl 2 , 25 mM KCl, 1 mM EGTA, 0.5 mM DTT, 0.5 mM ATP, and 1.2-fold molar excess of phalloidin (Sigma). Phalloidin has no effect on the motility defect of sk MII HMM with G146V mutation [ 29 ].…”

Section: Methodsmentioning

confidence: 99%

“…The absolutely conserved Gly146 (in the Dictyostelium Act15 sequence without the removed first Met) of actin sits at the hinge between the two domains. We previously reported that the G146V mutation of actin dominantly inhibits the growth of yeast cells [ 28 ] and impairs the in vitro gliding motility by skeletal myosin II but not of myosin V [ 29 ]. We speculated that the G146V mutation somehow affects the propeller rotation between the two major domains, which in turn impairs the motility of myosin II but not that of myosin V. In the present study, we performed filament depolymerization assays and single-molecule intramolecular FRET measurements to test this hypothesis.…”

Section: Introductionmentioning

confidence: 99%

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The Role of Structural Dynamics of Actin in Class-Specific Myosin Motility

et al. 2015

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The structural dynamics of actin, including the tilting motion between the small and large domains, are essential for proper interactions with actin-binding proteins. Gly146 is situated at the hinge between the two domains, and we previously showed that a G146V mutation leads to severe motility defects in skeletal myosin but has no effect on motility of myosin V. The present study tested the hypothesis that G146V mutation impaired rotation between the two domains, leading to such functional defects. First, our study showed that depolymerization of G146V filaments was slower than that of wild-type filaments. This result is consistent with the distinction of structural states of G146V filaments from those of the wild type, considering the recent report that stabilization of actin filaments involves rotation of the two domains. Next, we measured intramolecular FRET efficiencies between two fluorophores in the two domains with or without skeletal muscle heavy meromyosin or the heavy meromyosin equivalent of myosin V in the presence of ATP. Single-molecule FRET measurements showed that the conformations of actin subunits of control and G146V actin filaments were different in the presence of skeletal muscle heavy meromyosin. This altered conformation of G146V subunits may lead to motility defects in myosin II. In contrast, distributions of FRET efficiencies of control and G146V subunits were similar in the presence of myosin V, consistent with the lack of motility defects in G146V actin with myosin V. The distribution of FRET efficiencies in the presence of myosin V was different from that in the presence of skeletal muscle heavy meromyosin, implying that the roles of actin conformation in myosin motility depend on the type of myosin.

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“…Finally, Uyeda presented effects of point mutations in actin expected to perturb conformational dynamics within or between actin molecules. When tested with in vitro motility assays these mutations inhibit the sliding velocity of actin filaments by myosin II immobilized on glass, but not by immobilized myosin V (Kubota, Mikhailenko, Okabe, Taguchi, & Ishiwata, ; Noguchi et al, ). These results indicate that myosin II requires conformational dynamics of actin protomers or the propagation of conformational changes along an actin filament to work as an assembled system, but the processive myosin V motor does not.…”

Section: Bodymentioning

confidence: 99%

Keeping the focus on biophysics and actin filaments in Nagoya: A report of the 2016 “now in actin” symposium

Fujiwara

Narita

2017

Cytoskeleton

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Regulatory systems in living cells are highly organized, enabling cells to response to various changes in their environments. Actin polymerization and depolymerization are crucial to establish cytoskeletal networks to maintain muscle contraction, cell motility, cell division, adhesion, organism development and more. To share and promote the biophysical understanding of such mechanisms in living creatures, the "Now in Actin Study: -Motor protein research reaching a new stage-" symposium was organized at Nagoya University, Japan on 12 and 13, December 2016.The organizers invited emeritus professor of Nagoya and Osaka Universities Fumio Oosawa and leading scientists worldwide as keynote speakers, in addition to poster presentations on cell motility studies by many researchers. Studies employing various biophysical, biochemical, cell and molecular biological and mathematical approaches provided the latest understanding of mechanisms of cell motility functions driven by actin, microtubules, actin-binding proteins, and other motor proteins.actin, actin binding proteins, cell cytoskeleton and cellular motilities, meeting summary, Nagoya

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G146V Mutation at the Hinge Region of Actin Reveals a Myosin Class-specific Requirement of Actin Conformations for Motility

Cited by 18 publications

References 41 publications

Actin binding domain of Rng2 sparsely bound on F-actin strongly inhibits actin movement on myosin II

Actin binding domain of Rng2 sparsely bound on F-actin strongly inhibits actin movement on myosin II

The Role of Structural Dynamics of Actin in Class-Specific Myosin Motility

Keeping the focus on biophysics and actin filaments in Nagoya: A report of the 2016 “now in actin” symposium

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