Tracking Down the Fast and Superprocessive KIF1A with Gold Scattering Microscopy

Abstract

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 μm/s and average run lengths > 5 μm, 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 labeled the C-terminus of a truncated KIF1A dimer with a 30-nm gold nanoparticle and used interferometric scattering microscopy (iSCAT) to observe the KIF1A stepping cycle at sub-millisecond time scales and nanometer-level precision. Our preliminary data reveal that KIF1A motility is characterized by periods of directed stepping interspersed by periods where the motor is statically bound to the microtubule, rather than diffusively tethered, as predicted by the K-loop model. An alternate explanation for superprocessivity, that could also account for the stepping speed, is that during each step the K-loop ensures the tethered head binds to the microtubule hundreds of times faster than the bound head detaches from the microtubule. Our ongoing investigations employ a KIF1A construct with a nanoparticle-functionalized motor domain to characterize the nature of these paused states, and the duration of the one-head bound state in the chemomechanical cycle.