Engineering myosins for long-range transport on actin filaments

Abstract

Cytoskeletal motors act as cargo transporters in cells and may be harnessed for directed transport applications in molecular detection and diagnostic devices. High processivity, the ability to take many steps along a track before dissociating, is often a desirable characteristic because it allows nanoscale motors to transport cargoes over distances on the scale of micrometres, in vivo and in vitro. Natural processive myosins are dimeric and use internal tension to coordinate the detachment cycles of the two heads. Here, we show that processivity can be enhanced in engineered myosins using two non-natural strategies designed to optimize the effectiveness of random, uncoordinated stepping: (1) the formation of three-headed and four-headed myosins and (2) the introduction of flexible elements between heads. We quantify improvements using systematic single-molecule characterization of a panel of engineered motors. To test the modularity of our approach, we design a controllably bidirectional myosin that is robustly processive in both forward and backward directions, and also produce the fastest processive cytoskeletal motor measured so far, reaching a speed of 10 µm s(-1).

Figure 1

Multimerization effects on engineered myosin VI processivity and stepping behaviour. (a) Single molecule fluorescence trajectories for M6DI₈₁₆2R dimers (red), trimers (green), and tetramers (blue) recorded at 2mM ATPtogether with corresponding cartoons and block diagrams, showing differences due to oligomerization state. (b) Mean run lengths as a function of [ATP]. M6DI₈₁₆2R~DIM is fit to a 3-state model10, yielding k_rebind=290 ± 30 s⁻¹, k^ADP_off=5.8 ± 0.1 s⁻¹, kATP_on=0.015 ± 0.001 s⁻¹μM⁻¹, and the defect parameter d=63 ± 6 steps. (c) Motor velocities. M6DI₈₁₆2R~DIM is fit to the 3-state model, and M6DI₈₁₆2R~TRI and M6DI₈₁₆2R~TET are fit to Michaelis Menten kinetics. Trimer: K_M=740 ± 40 μM and V_max=120 ± 2 nm/s; tetramer: K_M=630 ± 50 μM and V_max=99 ± 3 nm s⁻¹. Data are displayed as mean ± s.e.m. (d-e) Gold nanoparticle tracking motor stepping traces. (d) Raw trajectories (colored traces, collected at 500 Hz) are shown along with fits (black) generated by an automatic stepfinding procedure (see Methods). (e) Step size distributions. Histograms of displacements generated by stepfinding (N > 1000 steps for each construct) are shown together with fits (black) to sums of multiple Gaussian distributions. Peak locations for dimer: −12 ± 0.1 nm and 20 ± 0.3 nm; trimer: −9 ± 0.1 nm, 9 ± 0.1 nm, and 21 ± 0.3 nm; tetramer: −10 ± 0.1 nm, 10 ± 0.1 nm, and 23 ± 0.3 nm. Mean displacements per step for dimer: 17.5 ± 0.4 nm; trimer: 14.4 ± 0.4 nm; tetramer: 13.2 ± 0.5 nm.

Figure 2

Effects of increased flexibility on myosin VI constructs with short lever arms. Single molecule fluorescence trajectories for dimers (red), trimers (green), and tetramers (blue) were recorded at 2mM ATP or (*) 50 μM, showing differences due to addition of a slack element (~1R~) or insertion of a flexible hinge (~).

Figure 3

Characterization of engineered myosin XI motors. (a) Cartoons and block diagrams for constructs using the Nicotiana tabacum (light brown) and Chara corallina myosin XI (dark brown) catalytic domains. (b) Run lengths and (c) velocities are shown (mean ± s.e.m.) as a function of [ATP].

Figure 4

Characterization of design targets for combining processivity with other desirable characteristics. (a) MCaR-2IQ~1R~TET schematic. (b) MCaR-2IQ~1R~TET is processive at 2mM ATP in both low (EGTA) and high (pCa 4) calcium conditions. Randomly selected motor trajectories illustrate predominantly minus-end directed movement in EGTA (99.4% minus (dark blue), 0.6% plus, N=165) and predominantly plus-end directed movement in pCa 4 (83.3% plus (light blue), 2.1% mixed (gray), and 14.6% minus (dark blue), N=96). Minus-end directed run lengths in EGTA and plus-end directed run lengths in pCa 4 are displayed as mean ± s.e.m. Filament orientations were determined using M6DI₈₁₆2R~DIM as a minus-end directed control18. Average velocities were <V>=8 ± 1 nm/s for minus-end directed runs in EGTA and <V>=1 ± 0.1 nm/s for plus-end directed runs in pCa4 (mean ± s.e.m.). (c) Dynamic switching of individual motors was accomplished using in situ buffer exchange. Duration of buffer exchange is approximate. (d) CM11CD₇₄₆2R~1R~TET schematic. (e) CM11CD₇₄₆2R~1R~TET is processive over a range of [ATP], processive velocities up to <V>=10.0 ± 0.3 μm s⁻¹. Velocity and run length data are shown as mean ± s.e.m.