MINFLUX Reveals Dynein Stepping in Live Neurons

Dynein is the primary molecular motor responsible for retrograde intracellular transport of a variety of cargoes, performing successive nanometer-sized steps within milliseconds. Due to the limited spatiotemporal precision of established methods for molecular tracking, current knowledge of dynein stepping is essentially limited to slowed-down measurements in vitro. Here, we use MINFLUX fluorophore localization to directly track CRISPR/Cas9-tagged endogenous dynein with nanometer/millisecond precision in living primary neurons. We show that endogenous dynein primarily takes 8 nm steps, including frequent sideways steps but few backward steps. Strikingly, the majority of direction reversals between retrograde and anterograde movement occurred on the time scale of single steps (16 ms), suggesting a rapid regulatory reversal mechanism. Tug-of-war-like behavior during pauses or reversals was unexpectedly rare. By analyzing the dwell time between steps, we concluded that a single rate-limiting process underlies the dynein stepping mechanism whereby dynein consumes one adenosine 5’-triphosphate (ATP) per step. Our study underscores the power of MINFLUX localization to elucidate the spatiotemporal changes underlying protein function in living cells. ### Competing Interest Statement S.W.H. has revenues through patents on MINFLUX owned by the Max Planck Society, and through shares of AI. J.H. also advises AI in technical preparation of samples. All other authors declare no competing interests.