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A novel actin binding site of myosin required for effective muscle contraction

Abstract

F-actin serves as a track for myosin's motor functions and activates its ATPase activity by several orders of magnitude, enabling actomyosin to produce effective force against load. Although actin activation is a ubiquitous property of all myosin isoforms, the molecular mechanism and physiological role of this activation are unclear. Here we describe a conserved actin-binding region of myosin named the 'activation loop', which interacts with the N-terminal segment of actin. We demonstrate by biochemical, biophysical and in vivo approaches using transgenic Caenorhabditis elegans strains that the interaction between the activation loop and actin accelerates the movement of the relay, stimulating myosin's ATPase activity. This interaction results in efficient force generation, but it is not essential for the unloaded motility. We conclude that the binding of actin to myosin's activation loop specifically increases the ratio of mechanically productive to futile myosin heads, leading to efficient muscle contraction.

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Figure 1: Myosin and its structural changes in its cyclic interactions with actin.
Figure 2: Interactions between the activation loop and the N-terminal segment of actin in three states of actomyosin obtained from molecular dynamics simulations.
Figure 3: Actin-binding properties of wild-type and mutant D. discoideum (Dd) myosin motor domain (MD).
Figure 4: Mutations in the activation loop of myosin suppress actin activation, whereas in vitro motility function is unaffected.
Figure 5: C. elegans bearing a loss-of-function mutation in the activation loop show reduced mechanical efficiency and a reduced force-generating capability of myosin.

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Acknowledgements

We thank A.G. Szent-Györgyi, C.R. Bagshaw and M. Kovács for insightful discussions and helpful comments. We thank E. Málnási-Csizmadia for the processing of videos. This work was supported by the European Research Council (European Community's Seventh Framework Programme (FP7/2007-2013)/European Research Council grant agreement no. 208319), the European Union in collaboration with the European Social Fund (grant agreement no. TAMOP-4.2.1/B-09/1/KMR), the National Office for Research and Technology and the European Union (European Regional Development Fund), under the sponsorship of the National Technology Programme (NTP TECH_08_A1/2-2008-0106). T.V. is a grantee of the János Bolyai scholarship.

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Authors and Affiliations

Authors

Contributions

B.H.V. and A.M.-C. designed, conducted and analyzed all experiments and wrote the paper. Z.Y. conducted the in silico experiments. B.K. designed experiments and contributed to the writing of the paper. T.V. and P.E. contributed to the C. elegans experiments. I.B.-N. contributed to the in vitro motility experiments. A.L.K. carried out electron microscopy. M.K. designed and conducted the AFM experiment. P.H. designed the analysis of the movement of C. elegans.

Corresponding author

Correspondence to András Málnási-Csizmadia.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2, Supplementary Table 1, Supplementary Discussion and Supplementary Methods (PDF 388 kb)

Supplementary Movie 1

Fluorescent videos of transgenic animals. Fluorescent video recordings of transgenic animals from strains Δunc-54;unc-54::gfp and Δunc-54;unc-54K525E::gfp expressing UNC-54::GFP or UNC-54K525E::GFP, respectively. Recordings were taken with a Zeiss Lumar.V12 microscope combined with Zeiss AxioCam using AxioVision 4.8 software. GFP was excited with a UV lamp and a combination of 470nm-510nm and 525nm-575nm transmission filters were used on the excitation and emission side, respectively. (MOV 1043 kb)

Supplementary Movie 1a. Fluorescent video recording of a Δunc-54;unc-54K525E::gfp animal.

Supplementary Movie 1b. Fluorescent video recording of a Δunc-54;unc-54::gfp animal.

Supplementary Movie 2

Videos of the four C. elegans strains during AFM experiments. Video recordings of the four C. elegans strains (Δunc-54, Δunc-54;unc-54::gfp, Δunc-54;unc-54K525E::gfp and wild type) during the force measurement experiments with AFM. One typical behaviour from each strain is presented here. In the field of view the animal, the base of the cantilever and the cantilever can be seen from above. Recordings were taken from the moment of catching the animal with the tip of the cantilever until ~40 seconds or until their escape. During this period, several push-and-pull phases took place on one animal. The animal's mechanical activity was measured between a push and a pull phase. Animals from different strains were of similar size. The spring constant of the cantilever was kept around 50pN/μm2 within all experiments. (MOV 10386 kb)

Supplementary Movie 2a. Video recording of a Δunc-54 animal.

Supplementary Movie 2b. Video recording of a Δunc-54;unc-54K525E::gfp animal.

Supplementary Movie 2c. Video recording of a Δunc-54;unc-54::gfp animal.

Supplementary Movie 2d. Video recording of a wild type animal.

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Várkuti, B., Yang, Z., Kintses, B. et al. A novel actin binding site of myosin required for effective muscle contraction. Nat Struct Mol Biol 19, 299–306 (2012). https://doi.org/10.1038/nsmb.2216

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