Characterizing dynamics of productive dynein stepping by MINFLUX.

Cytoplasmic dynein drives minus-end-directed motility along microtubules by converting ATP hydrolysis into coordinated structural changes but how this process produces directional stepping remains unresolved. The dynamics of dynein stepping have previously been characterized by tracking flexible regions of the motor with limited resolution. Here we site-specifically labeled the microtubule-binding domain of yeast dynein using a cysteine-light mutant and tracked stepping with submillisecond, nanometer precision at physiological ATP using minimal fluorescence photon flux (MINFLUX). We show that dynein hydrolyzes one ATP per step and moves in multiples of 8 nm. Steps are preceded by a transient plus-end-directed displacement ('dip'), corresponding to microtubule release upon ATP binding and diffusion of the stepping head around its partner. A slow ATP-hydrolyzing mutant shows more frequent dips, supporting a model in which ATP hydrolysis produces net forward movement. These results clarify the sequence of mechanochemical events underlying productive dynein stepping.