Regulation of KIF1A motility via polyglutamylation of tubulin C-terminal tails

Axonal transport is a highly regulated cellular process responsible for site-specific neuronal cargo delivery. This process is mediated in part by KIF1A, a member of the kinesin-3 family of molecular motors. It is imperative that KIF1A9s highly efficient, superprocessive motility along microtubules is tightly regulated as misregulation of KIF1A cargo delivery is observed in many neurodegenerative diseases. However, the regulatory mechanisms responsible for KIF1A9s motility, and subsequent proper spatiotemporal cargo delivery, are largely unknown. One potential regulatory mechanism of KIF1A motility is through the posttranslational modifications (PTMs) of axonal microtubules. These PTMs, often occurring on the C-terminal tails of the microtubule tracks, act as molecular "traffic signals" helping to direct kinesin motor cargo delivery. Occurring on neuronal microtubules, C-terminal tail polygutamylation is known to be important for KIF1A cargo transport. KIF1A9s initial interaction with microtubule C-terminal tails is facilitated by the K-loop, a positively charged surface loop of the KIF1A motor domain. However, the K-loop9s role in KIF1A motility and response to perturbations in C-terminal tail polyglutamylation is underexplored. Using single-molecule imaging, we present evidence of KIF1A9s previously unreported pausing behavior on multiple microtubule structures. Further analysis revealed that these pauses link multiple processive segments together, contributing to KIF1A9s characteristic superprocessive run length. We further demonstrate that KIF1A pausing is mediated by a K-loop/polyglutamylated C-terminal tail interaction and is a regulatory mechanism of KIF1A motility. In summary, we introduce a new mechanism of KIF1A motility regulation, providing further insight into KIF1A9s role in axonal transport.