Research ArticleBIOPHYSICS

Force production of human cytoplasmic dynein is limited by its processivity

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Science Advances  08 Apr 2020:
Vol. 6, no. 15, eaaz4295
DOI: 10.1126/sciadv.aaz4295
  • Fig. 1 Optical trapping assay to probe dynein force generation.

    (A) Schematic of the optical tweezers assay (not to scale). A 0.9-μm-diameter carboxyl bead is nonspecifically bound to a purified single human dynein and is trapped by a near-infrared optical trapping beam focused via a high numerical aperture microscope objective lens. The trap holds the bead directly above an MT that is covalently linked to the glass surface of the coverslip. When the dynein binds to and moves along the MT in the presence of ATP, it pulls the attached bead with it. The trap resists this motion, exerting a force F = −k × ∆x on the bead-motor complex, where k is the trap stiffness and ∆x is the distance from the trap center to the center of the bead. (B) Example traces at 1 mM ATP and k = 0.01 pN/nm (see also fig. S2B). Stalling events (red horizontal bars) can be observed but are rare. Fast events, including large single forward-backward steps without any resolved intermediate steps (black star), are frequent. Events that are counted as force generation events are marked with black arrows. (C) Dilution curve counting beads as moving if forces equaled or exceeded 0.5 pN. Error bars were calculated assuming a binomial distribution. Twelve to 85 beads were tested for each dilution (Ntotal = 318). The curves are fits to equations assuming processive motors (Eq. 6, λ = 3.3 ± 0.1, solid line, R2 = 0.996) and nonprocessive motors (Eq. 7, λ = 7.9 ± 0.7, dashed line, R2 = 0.949).

  • Fig. 2 Force generation of human dynein increases with increasing trap stiffness.

    (A) Stall force histograms of StrepTrap-purified and MT-binding released human dynein counting stalling events ≥200 ms and excluding jump-like behavior measured at k = 0.01 pN/nm (black bars) and k = 0.03 pN/nm (gray bars). The Gaussian distributions (solid curves) are centered at 0.9 ± 0.3 pN (±SD; N = 77) and 1.3 ± 0.5 pN (±SD; N = 48). (B) All measured forces (“detachment forces”) acquired at k = 0.01 pN/nm (mean force: 0.64 pN; N = 572) and k = 0.03 pN/nm (mean force: 1.1 pN; N = 225). (C) Example record showing force generation events of a single dynein molecule bound to trapping bead measured at 0.01 pN/nm (left) and subsequently at 0.03 pN/nm (right), demonstrating an increase in force generation with increasing trap stiffness.

  • Fig. 3 Force production of human dynein is limited by its processivity.

    (A and B) CDFs of measured detachment forces (A) and corresponding displacements (B) acquired at various trap stiffnesses: black, k = 0.005 pN/nm; red, k = 0.01 pN/nm; blue, k = 0.02 pN/nm; green, k = 0.03 pN/nm; orange, k = 0.05 pN/nm; magenta, k = 0.06 pN/nm; brown, k = 0.08 pN/nm; cyan, 0.1 pN/nm. (C) Forces and distances as a function of trap stiffness [trap stiffnesses are displayed as mean ± 95% confidence intervals (CIs)]. The average distances (left y axis, black spheres) and average forces (right y axis, green spheres) (the error bars represent the 95% CI of the mean) were fit to Eqs. 5 and 4 (black curve, R2 = 0.983; green curve, R2 = 0.991), resulting in the force-free run length x0 = 91 ± 5 nm (±SEM) and the stall force Fs depicted by the dashed line (mean ± SEM). (D) Velocity versus force. Points are means, and error bars span 95% CIs of the mean. The solid line is a linear fit that intercepts the ordinate at 540 nm/s (±60 nm/s, ±SEM) and the abscissa at 1.9 pN (±0.2 pN, ±SEM; N = 83 to 84 at each force; 335 events total). (E) Stall force histogram compiling stall forces (≥200 ms) at k = 0.1 pN/nm (N = 115; mean ± SD from Gaussian fit).

  • Fig. 4 Force generation of yeast dynein is insensitive to changes within a practical trap stiffness range.

    (A) Example trace showing stalling events and premature detachments of full-length yeast dynein (k = 0.05 pN/nm). Yeast dynein was specifically attached to beads coated with anti-GFP antibodies. (B) Forces versus trap stiffness. Values of stall forces (≥10 s, black) and of all events (gray) do not depend on the trap stiffness in the measured trap stiffness range (the error bars represent the 95% CIs of the means of the measured forces and trap stiffnesses). (C) Histogram of stall forces measured at various trap stiffnesses (N = 486; mean ± SD).

  • Fig. 5 Kinesin force generation becomes sensitive to changes in trap stiffness with increasing ionic strength.

    (A) Example trace of K560 in BRB80 trapping buffer showing a stalling event (red horizontal bar) and events without stalling (k = 0.03 pN/nm). (B) Stall forces (stalling for ≥200 ms; black crosses) and detachment forces (gray crosses) measured in BRB80 (the error bars are the 95% CIs of the mean forces and trap stiffnesses). (C) Stall force histogram measured in BRB80 (k = 0.03 to 0.12 pN/nm, N = 409, Fstall = 5.7 ± 0.9 pN; mean ± SD). (D) Trap stiffness dependence of all events in Pipes-Hepes trapping buffer (blue, red), Pipes-Hepes trapping buffer +20 mM KAc (green, orange), + 30 mM KAc (dark cyan, purple), and + 40 mM KAc (light blue, magenta). Fitting Eq. 4 to the detachment forces (right y axis, circles, diamonds, hexagons, and crosses) and Eq. 5 to the distances (left y axis, triangles, squares, and plus signs) reveals the same hyperbolic dependence (solid curves) on the trap stiffness as human dynein (see Fig. 3C) (the error bars are the 95% CIs of the means of the measured forces, distances, and the trap stiffnesses). The dashed line depicts the average stall force in BRB80 [5.7 ± 0.9 pN; see (C) for histogram].

Supplementary Materials

  • Supplementary Materials

    Force production of human cytoplasmic dynein is limited by its processivity

    Sibylle Brenner, Florian Berger, Lu Rao, Matthew P. Nicholas, Arne Gennerich

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