Dual-color MINFLUX: Kinesin-1 takes Chasse-Inchworm steps

Despite tremendous efforts using various techniques, many central questions regarding the walking mechanism of the ATP-driven motor protein kinesin-1 remained contradictory and puzzling. Still, it is widely believed that kinesin-1 walks hand-over-hand, meaning that the two motor domains (heads) sequentially overtake each other with a 16 nm step so that, after each step, the two heads are 8 nm apart. Here we developed dual-color fluorophore tracking by MINFLUX, which enabled us to follow the individual steps of the two heads simultaneously, up to physiological ATP concentrations. We found that, besides hand-over-hand, kinesin-1 frequently walks in a previously undescribed chasse-inchworm mode, whereby one of the heads advances by 16 nm, whereas the other one follows suit, binding in close proximity to the first. In this mode, the two heads are either 16 nm or [~]0 nm apart. The transition between the two walking mechanisms is initiated by one of the heads making a large (>20 nm) passing step. MINFLUX also revealed that load-free kinesin-1 does not show significant limping and that the stalk folds back, pointing leftwards. The finding of the chasse-inchworm mechanism reconciles many of the contradictory results gained with other techniques, highlighting the power of multicolor MINFLUX tracking to reveal protein operation.