Stepping dynamics of dynein characterized by MINFLUX

Abstract

Cytoplasmic dynein is the principal motor for minus-end-directed motility and force generation functions along microtubules (MTs)1. Dynein converts the chemical energy of ATP hydrolysis into coordinated structural changes to step processively along MTs, but how dynein couples ATP hydrolysis to a minus-end-directed step remains controversial2, 3. The dynamics of dynein stepping have previously been characterized by tracking flexible regions of the motor with limited resolution4-6. Here, we site-specifically labeled yeast dynein at its MT-binding domain by developing a cysteine-light mutant and tracked its stepping at sub-millisecond and nanometer resolution at physiological ATP concentrations using MINFLUX7. We show that dynein hydrolyzes one ATP per step and takes multiples of 8 nm steps. Steps are preceded by a transient movement towards the plus end. These backward "dips" correspond to MT release upon ATP binding and subsequent diffusion of the stepping monomer around its MT-bound partner. Functional assays showed that dips terminate with a minus-end-directed movement upon ATP hydrolysis. These results provide critical insights into the order of mechanochemical events that result in a productive step of dynein.