Kinesin-1 autoinhibition tunes cargo transport by motor ensembles.

Intracellular vesicular transport by kinesin-1 motors through numerous three-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 these questions, we compared liposome transport by ∼10 nearly full-length kinesin-1 motors with kinesin light chains bound (KinΔC) versus a constitutively active control (K543). In 3D MT intersections, KinΔC-liposomes terminate (48%) or go straight (43%) but rarely turn (9%), starkly contrasting K543-liposomes, which go straight (57%) or 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 threefold 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.