Myosin-5 varies its steps along the irregular F-actin track

Adam Fineberg; Yasuharu Takagi; Kavitha Thirumurugan; Joanna Andrecka; Neil Billington; Gavin Young; Daniel Cole; Stan A Burgess; Alistair P Curd; John A Hammer; James R Sellers; Philipp Kukura; Peter J Knight

doi:10.1101/2023.07.16.549178

Myosin-5 varies its steps along the irregular F-actin track

bioRxiv [Preprint]. 2023 Jul 16:2023.07.16.549178. doi: 10.1101/2023.07.16.549178.

Authors

Adam Fineberg^{1

2}, Yasuharu Takagi³, Kavitha Thirumurugan^{4

5}, Joanna Andrecka^{1

6}, Neil Billington^{3

7}, Gavin Young^{1

8}, Daniel Cole^{1

8}, Stan A Burgess⁴, Alistair P Curd⁴, John A Hammer⁹, James R Sellers³, Philipp Kukura^{1

10}, Peter J Knight⁴

Affiliations

¹ Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3QZ, U.K.
² Laboratory of Single Molecule Biophysics, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, U.S.A.
³ Laboratory of Molecular Physiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, U.S.A.
⁴ Astbury Centre for Structural Molecular Biology, and Institute of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, U.K.
⁵ Present address: Structural Biology Lab, Pearl Research Park, SBST, Vellore Institute of Technology, Vellore-632 014, India.
⁶ Present address: Human Technopole, Viale Rita Levi-Montalcini 1, 20157, Milan, Italy.
⁷ Present address: Department of Biochemistry and Molecular Medicine, West Virginia University, Morgantown, WV, U.S.A.
⁸ Present address: Refeyn Ltd., Unit 9, Trade City, Sandy Ln W, Littlemore, Oxford OX4 6FF, U.K.
⁹ Cell and Developmental Biology Center, NHLBI, National Institutes of Health, Bethesda, MD 20892, U.S.A.
¹⁰ The Kavli Institute for Nanoscience Discovery, Dorothy Crowfoot Hodgkin Building, University of Oxford, South Parks Rd, Oxford OX1 3QU, U.K.

Abstract

Molecular motors employ chemical energy to generate unidirectional mechanical output against a track. By contrast to the majority of macroscopic machines, they need to navigate a chaotic cellular environment, potential disorder in the track and Brownian motion. Nevertheless, decades of nanometer-precise optical studies suggest that myosin-5a, one of the prototypical molecular motors, takes uniform steps spanning 13 subunits (36 nm) along its F-actin track. Here, we use high-resolution interferometric scattering (iSCAT) microscopy to reveal that myosin takes strides spanning 22 to 34 actin subunits, despite walking straight along the helical actin filament. We show that cumulative angular disorder in F-actin accounts for the observed proportion of each stride length, akin to crossing a river on variably-spaced stepping stones. Electron microscopy revealed the structure of the stepping molecule. Our results indicate that both motor and track are soft materials that can adapt to function in complex cellular conditions.

Keywords: Actin Filament; Cryogenic Electron Microscopy (cryoEM); Cumulative Angular Disorder (CAD); Interferometric Scattering (iSCAT) Microscopy; Myosin-5; Negative-stain Electron Microscopy (nsEM); Single Molecule Biophysics.

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