Processive kinesins require loose mechanical coupling for efficient collective motility

Abstract

Processive motor proteins are stochastic steppers that perform actual mechanical steps for only a minor fraction of the time they are bound to the filament track. Motors usually work in teams and therefore the question arises whether the stochasticity of stepping can cause mutual interference when motors are mechanically coupled. We used biocompatible surfaces to immobilize processive kinesin-1 motors at controlled surface densities in a mechanically well-defined way. This helped us to study quantitatively how mechanical coupling between motors affects the efficiency of collective microtubule transport. We found that kinesin-1 constructs that lack most of the non-motor sequence slow each other down when collectively transporting a microtubule, depending on the number of interacting motors. This negative interference observed for a motor ensemble can be explained quantitatively by a mathematical model using the known physical properties of individual molecules of kinesin-1. The non-motor extension of kinesin-1 reduces this mutual interference, indicating that loose mechanical coupling between motors is required for efficient transport by ensembles of processive motors.

Figure 1

Properties of single kinesins measured by single-molecule imaging. (A) Domain architecture of kinesin 1 constructs. (B) Scheme showing an immobilized microtubule on biotin-PEG functionalized glass, enabling single-molecule imaging by total internal reflection fluorescence microscopy. (C) Examples of space–time plots (kymographs) for each construct as indicated. Monomeric Kin₃₄₀GFP is not processive. Scale bar, 2 μm (horizontal), 2 s (vertical). (D) Histogram of the mean velocity (left) and dwell time (right) with model fits for the long construct Kin₆₁₂GFP in low ionic strength buffer (B12). (E) Motility data of Kin₆₁₂GFP and Kin₄₀₁GFP in low ionic strength buffer. Numbers in parentheses represent the 95% confidence interval. CC1, coiled coil 1; CC2, coiled coil 2; H, hinge; M, motor domain; N, neck; PEG, polyethylene glycol; S, swivel; T, tail.

Figure 2

Mutual inhibition of collective microtubule transport by kinesin constructs. (A) Chemical structure of Ni-Tris-NTA-PEG coupled to glass (left) and schematic illustration of histidine (His)-tagged motors immobilized on a Ni-Tris-NTA-PEG surface (right). (B) Domain architecture of the two kinesin 1 constructs used. Symbols are as in Fig 1A. (C) Scheme of several immobilized kinesins transporting a microtubule. (D) Sequence of three filtered images showing the transport of fluorescently labelled microtubules by Kin₆₁₂His immobilized on a Ni-Tris-NTA-PEG surface. The fourth image is a processed image, in which the microtubule at t=0 s is shown in white and the segments of alternating colour show the trajectories of microtubule movement, each segment representing the distance covered during 6 s. Scale bar, 10 μm. (E) Molecular surface densities of immobilized Kin₆₁₂His (blue) and Kin₄₀₁His (red) as calculated from the motor mass on the surface as determined by RIfS (inset), plotted as a function of the concentration used to incubate Ni-Tris-NTA-PEG surfaces for 10 min to allow functional immobilization. (F) Dependence of the average microtubule-gliding velocity v on the measured density of Kin₆₁₂His (blue) and Kin₄₀₁His (red) in medium ionic strength buffer (B80). (G) Dependence of the average microtubule-gliding velocity v on the measured density of Kin₄₀₁His in medium (B80, dark red) and low (B12, light red) ionic strength buffer. Inset: variation of the microtubule transport velocity at a density of 11,000 Kin₄₀₁His molecules per square micrometre in response to a sequential buffer change, as indicated, in the same experiment. Error bars of measured velocities are standard deviations. Ni-Tris-NTA-PEG, nickel-Tris-nitrilotriacetic acid polyethylene glycol; RIfS, reflectometric interference spectroscopy.

Figure 3

Global fit of the kinetic model for mutual interference of mechanically coupled motors to the experimental data. (A) Schematic illustration of the stepping behaviour of an individual motor. (B) Schematic illustration of the spatial dimensions of immobilized Kin₄₀₁His (left) and Kin₆₁₂His (right) transporting a microtubule. Polyethylene glycol (PEG; red), coiled coil segments of kinesin (yellow), motor domains (blue) and the microtubule (grey) are drawn roughly to scale. (C) Global least-squares fit of the kinetic model of mutual interference (lines) to four sets of experimental data (circles) as indicated. The normalized microtubule transport velocity is plotted as a function of the motor density shown on a logarithmic scale. Error bars of the velocities represent standard deviations. (D) Global fit and data as in (C), shown on a linear density scale. (E) Normalized microtubule transport velocities and global fit (same data as in (C,D)) replotted as a function of the number of motors bound per microtubule as predicted by the kinetic model. (F) Increase in the dissociation constant as predicted by the model as a function of the number of motors bound per microtubule. Colour code in (C–F) as in Fig 2F,G.