BPS2026 – Dissecting sub-millisecond stepping dynamics of dynein with 1- and 2-color MINFLUX

Cytoplasmic dynein is a dimeric motor that drives minus-end-directed transport on microtubules (MTs). To couple ATP hydrolysis to a mechanical step, a dynein monomer must be released from the MT before undergoing a conformational change that generates a bias toward the minus end. How dynein couples ATP hydrolysis to a minus-end-directed step remains elusive. To address this question, we developed a cysteine-light mutant of yeast dynein, site-specifically labeled its MT-binding domain (MTBD), and characterized dynein stepping at physiological ATP using 1- and 2-color MINFLUX tracking at sub-millisecond resolution in vitro. We observed that dynein hydrolyzes one ATP per step and takes variable steps at multitudes of 8 nm that match the periodicity of tubulin subunits on the microtubule. As dynein’s inter-MTBD separation increases, the trailing monomer more often steps and has an increased stepping rate due to increased tension on the linker. Remarkably, steps are preceded by the transient movement of MTBD toward the plus end. A dynein mutant that slowly hydrolyzes ATP at the AAA1 site exhibits an increased proportion of steps preceded by these transient events. We propose that these backward “dips” correspond to the MT release of the active stepping monomer upon ATP binding and its subsequent diffusion around the tethered monomer. This is followed by the minus-end-directed movement of the stepping monomer upon ATP hydrolysis. Our results reveal the order and dynamics of sub-millisecond events that result in a productive step of dynein.