Schleske, Jonas M.Jonas M.SchleskeHubrich, JasmineJasmineHubrichWirth, Jan OttoJan OttoWirthD’Este, ElisaElisaD’EsteEngelhardt, JohannJohannEngelhardtHell, Stefan W.Stefan W.Hell2024-10-042024-10-042024https://resolver.sub.uni-goettingen.de/purl?gro-2/145761Dynein 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, likely arising from just one adenosine 5′-triphosphate hydrolysis event being required during each step. Our study underscores the power of MINFLUX localization to elucidate the spatiotemporal changes underlying protein function in living cells.enhttps://creativecommons.org/licenses/by/4.0/journal_article10.1073/pnas.2412241121