Abstract: Intracellular transport is essential for neuronal function and survival. The most effective plus end-directed neuronal transporter is the kinesin-3 KIF1C, which transports large secretory vesicles and endosomes in both axons and dendrites. Cytoplasmic dynein is the major minus-end directed transporter. Thus both motors transport cargo with opposite polarity. KIF1C depletion reduces both microtubule plus and minus end-directed vesicle transport in cells, suggesting that KIF1C facilitates dynein-mediated transport by an unknown mechanism. Such a co-dependence of opposite polarity motors for bidirectional cargo transport has also been observed for kinesin-1 and dynein. However, when both motors were linked together artificially in vitro, they undertook a tug-of-war with little net motility. Here we reconstituted complexes of dynein and KIF1C in the presence of dynactin and cargo adapters from purified recombinant human proteins and show that both motors can bind simultaneously to cargo adapters Hook3, BICD2 and BICDR1 to form co-motile complexes. Quaternary complexes of dynein, dynactin, hook3 and KIF1C (DDHK) form most efficiently and show directional, processive motility towards the plus and the minus end of microtubules in single molecule assays. KIF1C increases the initiation, duration and distance of minus end-directed runs, both by acting as a processivity tether and because KIF1C facilitates the recruitment of a second dynein to dynactin and hook3. Based on intensity measurements of motile complexes we propose a stoichiometry of two dynein dimers, two KIF1C dimers, two hook3 dimers and one dynactin in DDHK complexes. Directional switching of DDHK complexes was relatively rare, suggesting that the adapter-mediated coupling of opposite polarity motors primarily supports processive unidirectional transport, prevents tug-of-war and enables the super-processive KIF1C to extend dynein-driven runs.

Mutations in KIF1C cause hereditary spastic paraplegia and cerebellar dysfunction in human patients. Two pathogenic mutations (P176L and R169W) maintain fast, processive single molecule motility in vitro, but with decreased run length and slightly increased unloaded velocity compared to the wildtype motor. Under load in an optical trap, force generation by these mutants is severely reduced. In cells, the same mutants are impaired in producing sufficient force to efficiently relocate organelles. Our results show how its mechanics supports KIF1C’s role as an intracellular transporter and explain how pathogenic mutations at the microtubule-binding interface of KIF1C impair the cellular function of these long-distance transporters and result in neuronal disease.