Modulation of kinesin’s load-bearing capacity by force geometry and the microtubule track

AbstractKinesin 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 single-bead 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 surface. 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 attachment duration 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 attachment duration (median-Δt) 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-Δt < 0.2 s) to a persistent motor that sustains attachment (median-Δt > 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 StatementKinesins are cytoskeletal motors responsible for the transport of cargoes along microtubules. It is well known that opposing forces decrease kinesin’s speed and run length. In this study, we found that when the pair of opposing forces applied on the kinesin-microtubule complex are parallel to the microtubule, the ability of kinesin to remain attached to the microtubule can vary by more than an order of magnitude depending on the relative azimuthal position of the pair of forces along the periphery of the microtubule. These results reveal a previously unknown versatility of kinesin’s load bearing capacity and as such have implications for the potential physiological roles of kinesin in a wide range of cell activities, including organelle transport and cell division.