Kinesin-1 Autoinhibition Tunes Cargo Transport by Motor Ensembles

Intracellular vesicular transport by kinesin-1 motors through numerous 3-dimensional (3D) microtubule (MT) intersections must be regulated to support proper vesicle delivery. Knowing kinesin-1 can be regulated via autoinhibition, does kinesin-1 exhibit autoinhibition on cargo, and could this regulate vesicular transport through 3D MT intersections in vitro? To answer this question, we compared liposome transport by ~10 nearly full-length kinesin-1 motors with KLC bound (KinΔC) versus constitutively active control (K543). In 3D MT intersections, KinΔC-liposomes terminate (48%), go straight (43%), but rarely turn (9%), starkly contrasting K543-liposomes which go straight (57%), turn (31%), but rarely terminate (12%). On single MTs, KinΔC-liposomes have reduced run lengths and detachment forces versus K543-liposomes, suggesting autoinhibition reduces MT engagement, as supported by 3-fold lower KinΔC MT landing rates versus K543, and mechanistic in silico modeling. Furthermore, kinesore, a small molecule that overcomes kinesin-1 autoinhibition, restores KinΔC's MT engagement. Thus, we propose that partial kinesin-1 autoinhibition while cargo-bound may fine-tune cargo delivery to support physiological demands.