Dissecting myosin-5B mechanosensitivity and calcium regulation at the single molecule level

Figures

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  Fig. 1. Processive movement of myosin-5B on F-actin in the single-molecule in vitro motility assay. a Schematic of the myosin-5B single molecule in vitro motility assay. A single biotinilated myosin-5B conjugated with a streptavidinated quantum dot (strep-QD) interacts with a single-actin filament. Actin filaments are anchored to the coverslip surface through N-ethylmaleimide modified myosin II (NEM). Schematic not drawn to scale. b Three consecutive frames (5 s interval) of a processive run of QD-labeled myosin-5B (red) on an actin filament (green). The yellow arrow indicates the QD position. Scale bar is 2 µm

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  Fig. 2. Signatures of single myosin-5B molecule motility under unloaded condition. a The velocity of myosin-5B movement (n = 127) on F-actin increases with increasing ATP following Michaelis–Menten kinetics (red curve), with vmax = 691 ± 25 nm s−1 and Kapp = 22 ± 2 µM. Error bars, s.d. b The run length (n = 127) is ATP-independent within error bars. Average run length is 780 ± 2 nm (red line). Error bars, s.d. c Representative time trace of myosin-5B stepping forward in discrete 36 nm increments on the actin filament. Average step 36 ± 4 nm (n = 42, s.e.m., [ATP] = 0.3 µM)

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  Fig. 3. Load dependence of myosin-5B stepping signatures investigated with high-speed optical tweezers. a Schematic of ultrafast force-clamp spectroscopy applied to a processive myosin-5B motor. A single myosin-5B molecule is attached to a glass bead pedestal through a streptavidin-biotin link. A single actin filament is trapped by suspending it between alpha-actinin coated beads. Black arrows represent the force clamped on the right (F1) and left bead (F2), red arrow represents the net force (F) on the dumbbell. F is alternated back and forth to maintain the dumbbell within a limited oscillation range when myosin is not bound to actin. b Left: cartoon showing the position of the right bead when: (1) myosin is unbound and the dumbbell oscillates; (2) myosin attaches to actin; and (3) myosin moves towards the actin barbed end. Right: example trace showing displacement and force during the corresponding phases of dumbbell oscillation, myosin-5B attachment, and processive runs under assistive and resistive loads. c Position record showing myosin-5B processive runs and the step and run detection algorithm. Detected beginning and end of each run are indicated by green and cyan vertical lines,…

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  Fig. 4. Myosin-5B velocity and run length and detachment rate from actin are force sensitive. Force dependence of myosin-5B average velocity (a), run length (b), and detachment rate (c). Detachment rate is calculated as k = 1/<τ>, where <τ> is the average run duration. In all panels, assistive forces are negative, resistive forces are positive. Red squares are the unloaded velocity (a), run length (b), and detachment rate (c) measured with the single-molecule motility assay (n = 30). Cyan curves in b and c are fits of the exponential model equations \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$L = L_0^ \pm {\mathrm{exp}}\left( { - \frac{{F \cdot d_L^ \pm }}{{{{k}}_{\mathrm{B}} \cdot {{T}}}}} \right)$$\end{document}L=L0±exp-F⋅dL±kB⋅T and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$k = k_0^ \pm {\mathrm{exp}}\left( {\frac{{F…

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  Fig. 5. The ratio of forward to backward steps determines myosin-5B run length and velocity under resistive load. a Force dependence of the average step size. Box plot in Supplementary Fig. 1c. b Step size distributions at ±1.2 pN (left panel) and ±2.4 pN (right panel). For resistive forces close to the stall force (+2.4 pN), the distributions of forward (>0) and backward (<0) steps become nearly symmetric. c Force dependence of the average forward (black squares) and backward (cyan squares) step size. d Force dependence of the ratio of forward to backward steps. The dotted cyan line is the fit of the exponential model equation \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$R = R_0^ + {\mathrm{exp}}\left( { - \frac{{F \cdot d_{\rm{fwd/bwd}}^ + }}{{{{k}}_{\mathrm{B}} \cdot {{T}}}}} \right)$$\end{document}R=R0+exp-F⋅dfwd∕bwd+kB⋅T to data for positive forces below the stall force, which gives R0+ = 54 ± 8 and dfwd/bwd+ = 8.2 ± 0.6 nm. e Force dependence of forward (black squares) and backward (cyan squares)…

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  Fig. 6. Effect of Ca2+ and CaM on single-molecule processivity and ensemble kinetics of myosin-5B. Dependence of myosin-5B velocity (a) and number of processive runs (b) on Ca2+ and CaM in the single-molecule motility assay. In this particular set of measurements, we observed 0.0125 runs s−1 with an average velocity of 559 ± 64 nm s−1 in the absence of Ca2+ and CaM. In the presence of 0.1 mM Ca2+, no significant movement (NSM) was observed. In the presence of 0.1 mM Ca2+ and 2 μM CaM, myosin processivity and velocity were partially rescued to 0.0055 runs s−1 and 383 ± 93 nm s−1 at [ATP] = 1000 μM (n = 12, p value = 0.02). Error bars, s.d. No error bars are reported in b because data are from single experiments. c Dependence of myosin-5B velocity on Ca2+ and CaM in the gliding assay. Myosin-5B translocated actin filaments with a speed of 413 ± 128 nm s−1 (n = 417) in the in vitro gliding assay. Filaments movement stalled (NSM) in the presence of 0.2 mM Ca2+ but was partially restored (264 ± 92 nm s−1, n = 439, p value = 4E−17) in the presence of 5 µM CaM. Error bars, s.d. Student’s t test applied on a and c, *P < 0.1, ****P < 0.0001. d Actin-activated steady-state ATPase activity of…

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  Fig. 7. Proposed model of myosin-5B regulation by force and Ca2+. a In the presence of assistive forces, myosin-5B moves in the direction of the force at full speed by consecutive steps towards the plus actin end. However, assistive forces drastically reduce myosin run length as a consequence of accelerated detachment of the motor from actin. On the other hand, resistive forces slow down the motor and reduce its run length as a consequence of increased back stepping. b (1) In the absence of Ca2+, myosin-5B folds in an enzymatically and mechanically inactive conformation where head-tail interaction hinders binding to actin. (2) As Ca2+ concentration increases, myosin-5B switches to an open conformation that can bind to actin, thus becoming enzymatically active. However, Ca2+-induced detachment of 25% of CaM molecules results in loss of myosin processivity. (3) In the presence of excess CaM (2–5 μM), new CaM molecules are available for occupying the vacant binding sites on the light chains, thus restoring myosin capability to move along actin