Tracking Down the Fast and Superprocessive KIF1A with Gold Scattering Microscopy

The kinesin-3 family member KIF1A is a neuronal kinesin that performs long-distance anterograde vesicle transport in axons and dendrites. Single molecule studies observe KIF1A velocities > 1 mm/s and average run lengths > 5 mm, making KIF1A one of the fastest and most processive members of the kinesin superfamily; however, the mechanistic basis of these high speeds and long run lengths is unknown. One prevailing model for superprocessivity holds that the positively-charged “K-loop” in the KIF1A motor domain diffusively tethers the motor to the negatively-charged microtubule, which prevents complete dissociation of the motor and effectively links together short runs. However, this model does not account for how KIF1A reaches such high speeds, or what role the K-loop plays in the ATP-driven stepping mechanism. To address these questions, we used biochemical assays in conjunction with direct observations of stepping of wild type KIF1A and k-loop mutants. We captured the transient states of the stepping cycle by tracking a 30-nm gold nanoparticle-functionalized motor domain via interferometric scattering microscopy (iSCAT), which enables fast acquisition of gold particles < 40 nm and simultaneous visualization of the microtubule tracks. We find that the chemomechanical cycle of KIF1A is distinct from other neuronal transport kinesins.