title: Myosin-10 produces its power-stroke in two phases and moves processively along a single actin filament under low load.
abstract: Myosin-10 is an actin-based molecular motor that participates in essential intracellular processes such as filopodia formation/extension, phagocytosis, cell migration, and mitotic spindle maintenance. To study this motor protein's mechano-chemical properties, we used a recombinant, truncated form of myosin-10 consisting of the first 936 amino acids, followed by a GCN4 leucine zipper motif, to force dimerization. Negative-stain electron microscopy reveals that the majority of molecules are dimeric with a head-to-head contour distance of ∼50 nm. In vitro motility assays show that myosin-10 moves actin filaments smoothly with a velocity of ∼310 nm/s. Steady-state and transient kinetic analysis of the ATPase cycle shows that the ADP release rate (∼13 s(-1)) is similar to the maximum ATPase activity (∼12-14 s(-1)) and therefore contributes to rate limitation of the enzymatic cycle. Single molecule optical tweezers experiments show that under intermediate load (∼0.5 pN), myosin-10 interacts intermittently with actin and produces a power stroke of ∼17 nm, composed of an initial 15-nm and subsequent 2-nm movement. At low optical trap loads, we observed staircase-like processive movements of myosin-10 interacting with the actin filament, consisting of up to six ∼35-nm steps per binding interaction. We discuss the implications of this load-dependent processivity of myosin-10 as a filopodial transport motor.
authors.affiliation: Laboratory of Molecular Physiology, National Heart, Lung and Blood, Institute, National Institutes of Health, Bethesda, MD 20892.
authors.forename: Yasuharu
authors.initials: Y
authors.lastname: Takagi
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Farrow
authors.forename: Neil
authors.initials: N
authors.lastname: Billington
authors.forename: Attila
authors.initials: A
authors.lastname: Nagy
authors.forename: Christopher
authors.initials: C
authors.lastname: Batters
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
authors.forename: James R
authors.initials: JR
authors.lastname: Sellers
authors.forename: Justin E
authors.initials: JE
authors.lastname: Molloy
mesh_terms: D000199:Actins / Q000737:chemistry / Q000502:physiology*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001696:Biomechanical Phenomena; D002417:Cattle; D066298:In Vitro Techniques; D007700:Kinetics; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018995:Myosin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D015879:Myosin Subfragments / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D052898:Optical Tweezers; D054730:Protein Interaction Domains and Motifs; D020836:Protein Structure, Quaternary; D011554:Pseudopodia / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D000199:Actins; D015879:Myosin Subfragments; D011993:Recombinant Fusion Proteins; D000244:Adenosine Diphosphate; D018995:Myosin Heavy Chains
keywords: actomyosin; myosin X; myosin-5a; optical trapping; stable single alpha-helix
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