Transport by populations of fast and slow kinesins uncovers novel family-dependent motor characteristics important for in vivo function.

Intracellular cargo transport frequently involves multiple motor types, either having opposite directionality or having the same directionality but different speeds. Although significant progress has been made in characterizing kinesin motors at the single-molecule level, predicting their ensemble behavior is challenging and requires tight coupling between experiments and modeling to uncover the underlying motor behavior. To understand how diverse kinesins attached to the same cargo coordinate their movement, we carried out microtubule gliding assays using pairwise mixtures of motors from the kinesin-1, -2, -3, -5, and -7 families engineered to have identical run lengths and surface attachments. Uniform motor densities were used and microtubule gliding speeds were measured for varying proportions of fast and slow motors. A coarse-grained computational model of gliding assays was developed and found to recapitulate the experiments. Simulations incorporated published force-dependent velocities and run lengths, along with mechanical interactions between motors bound to the same microtubule. The simulations show that the force-dependence of detachment is the key parameter that determines gliding speed in multimotor assays, while motor compliance, surface density, and stall force all play minimal roles. Simulations also provide estimates for force-dependent dissociation rates, suggesting that kinesin-1 and the mitotic motors kinesin-5 and -7 maintain microtubule association against loads, whereas kinesin-2 and -3 readily detach. This work uncovers unexpected motor behavior in multimotor ensembles and clarifies functional differences between kinesins that carry out distinct mechanical tasks in cells.