Force-Dependent Detachment of Kinesin-2 Biases Track Switching at Cytoskeletal Filament Intersections

Abstract

Intracellular trafficking of organelles often involves cytoskeletal track switching. Organelles such as melanosomes are transported by multiple motors including kinesin-2, dynein, and myosin-V, which drive switching between microtubules and actin filaments during dispersion and aggregation. Here, we used optical trapping to determine the unitary and ensemble forces of kinesin-2, and to reconstitute cargo switching at cytoskeletal intersections in a minimal system with kinesin-2 and myosin-V motors bound to beads. Single kinesin-2 motors exerted forces up to ∼5 pN, similar to kinesin-1. However, kinesin-2 motors were more likely to detach at submaximal forces, and the duration of force maintenance was short as compared to kinesin-1. In multimotor assays, force increased with kinesin-2 density but was not affected by the presence of myosin-V. In crossed filament assays, switching frequencies of motor-bound beads were dependent on the starting track. At equal average forces, beads tended to switch from microtubules onto overlying actin filaments consistent with the relatively faster detachment of kinesin-2 at near-maximal forces. Thus, in addition to relative force, switching probability at filament intersections is determined by the dynamics of motor-filament interaction, such as the quick detachment of kinesin-2 under load. This may enable fine-tuning of filament switching in the cell.

Force measurements on cargoes in living cells reveal collective dynamics of microtubule motors

“…Consistent with this interpretation, in vitro singlemolecule studies of kinesin indicate that kinesin often dissociates before reaching the maximal stall force (27,34). Further, although kinesin-2 has a similar unitary stall force as kinesin-1, the detachment rate of kinesin-2 is very sensitive to force, increasing the frequency of detachments before stall (35). According to the magnitude of the forces generated, most anterograde events observed in living cells are driven by one kinesin, with infrequent events driven by multiple motors.…”

Delineating cooperative responses of processive motors in living cells

“…Multiple copies of kinesins, myosins, and other types of processive motors, including dynein, are often bound to the same cargo (2,15,16). The maximum force that a system of processive kinesin or myosinVa motors is capable of producing is predictive of which motor team will win a competition between these motors at filament intersections (30). The present responses suggest that the balance between kinesin and myosin can be tilted more sensitively via control over myosinVa number/activity level during these competitions.…”

Processivity of the Kinesin-2 KIF3A Results from Rear Head Gating and Not Front Head Gating

“…Structurally, the wild-type motor is a heterodimer with a 17-residue neck linker compared with the 14-residue kinesin-1 neck linker domain (28,54,55). Functionally, the wild-type motor is less processive than kinesin-1, and it dissociates more readily under load (18,28,29,56). From the biochemical perspective, however, little is known regarding differences between hydrolysis cycles of kinesin-2 and kinesin-1 that may explain their functional differences.…”