Modulation of Kinesin’s Load-Bearing Capacity by Force Geometry and the Microtubule Track

Abstract

Kinesin motors and their associated microtubule tracks are essential for long-distance transport of cellular cargos. Intracellular activity and proper recruitment of kinesins is regulated by biochemical signaling, cargo adaptors, microtubule-associated proteins, and mechanical forces. In this study, we found that the effect of opposing forces on the kinesin-microtubule attachment duration depends strongly on experimental assay geometry. Using optical tweezers and the conventional singlebead assay, we show that detachment of kinesin from the microtubule is likely accelerated by forces vertical to the long axis of the microtubule due to contact of the single bead with the underlying microtubule. We used the three-bead assay to minimize the vertical force component and found that when the opposing forces are mainly parallel to the microtubule, the median value of attachment durations between kinesin and microtubules can be up to 10-fold longer than observed using the single-bead assay. Using the three-bead assay, we also found that not all microtubule protofilaments are equivalent interacting substrates for kinesin and that the median value of attachment durations of kinesin varies by more than 10-fold, depending on the relative angular position of the forces along the circumference of the microtubule. Thus, depending on the geometry of forces across the microtubule, kinesin can switch from a fast detaching motor (median attachment duration <0.2 s) to a persistent motor that sustains attachment (median attachment duration >3 s) at high forces (5 pN). Our data show that the load-bearing capacity of the kinesin motor is highly variable and can be dramatically affected by off-axis forces and forces across the microtubule lattice, which has implications for a range of cellular activities, including cell division and organelle transport. SIGNIFICANCE Kinesins are cytoskeletal motors that transport cargos along microtubules. Single-bead, optical-trapping assays have been used to show that the speed and run length of kinesin are affected by mechanical load. We found that this widely used assay introduces a vertical force on the motor not accounted for in previous experiments, and this force decreases kinesin's attachment duration. Using an assay geometry that minimizes vertical forces, we found that a 180 change in the azimuthal position between a pair of net opposing forces applied between kinesin and microtubules can lead up to a 10-fold increase in the kinesin attachment duration. These results reveal a previously unknown versatility of kinesin's load-bearing capacity, which has implications for the physiological roles of kinesin.

Processivity of molecular motors under vectorial loads

“…This is explained by the catch-bond behavior that allows kinesin to stay attached to MT under high loads F x . This behaviour has been observed in recent experiments where the effects of horizontal and vertical loads on the attachment duration of a single kinesin were investigated using a three-bead assay [38]. When kinesin was subject to horizontal forces, threefold increase in the attachment duration was observed.…”

“…However, multiple kinesins can produce forces much larger than the single-motor force when the vertical load component is small, e.g., in gliding assays [33,34] and bead assays [35][36][37], due to catch-bond behaviour. This later prediction has been recently confirmed in a three-bead assay where lower detachment rates were observed for a single kinesin with low kinesin-MT angle [38]. [14].…”

Highly-parallel microfluidics-based force spectroscopy on single cytoskeletal motors

“…Such a prolonged spacer could, in principle, be implemented in conventional optical tweezer experiments. A reduction of vertical force component in optical tweezers can also be achieved in a three-bead assay with suspended microtubules, as recently demonstrated 37 . To explore the influence of loading geometry on the measured motility parameters in our assay, experiments with varying linker length or varying vertical magnetic force could be performed.…”

“…showing a three-fold decrease at 2 pN resisting load 34 . This discrepancy can be attributed to the high vertical forces in conventional optical tweezers experiments that cause pre-mature detachment of motors compared to forces applied more horizontally 35–37 . The mean run-length value of 0.62 μm observed at low loads, corresponds well to the previously observed value of 0.67 μm for unloaded motors 33 .…”

“…Although the force is applied horizontally onto the bead in a conventional optical trap experiment, the molecule under investigation is experiencing a vertical load which, in fact, can surpass the applied horizontal force in magnitude 43 . Such substantial vertical load is pulling the motor away from the filament and can influence its detachment rate 35–37,43,46 . In our approach, the introduction of a spacer between the motor and the bead reduces the vertical force component to less than 15% of the applied force (see Figure 4e and Supplementary Table 1), thus applying forces more stringently in the direction of motor movement.…”