Force generation of KIF1C is impaired by pathogenic mutations

Abstract

Intracellular transport is essential for neuronal function and survival. The fastest neuronal transporter is the kinesin-3 KIF1C. Mutations in KIF1C cause hereditary spastic paraplegia and cerebellar dysfunction in human patients. However, neither the force generation of the KIF1C motor protein, nor the biophysical and functional properties of pathogenic mutant proteins have been studied thus far. Here we show that full length KIF1C is a processive motor that can generate forces up to 5.7 pN. We find that KIF1C single molecule processivity relies on its ability to slip and re-engage under load and that its slightly reduced stall force compared to kinesin-1 relates to its enhanced probability to backslip. Two pathogenic mutations P176L and R169W that cause hereditary spastic paraplegia in humans 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 establish a baseline for the single molecule mechanics of Kif1C 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.