MINFLUX reveals dynein stepping in live neurons

• 成果类型: Article
• 署名作者: Schleske, Jonas M.; Hubrich, Jasmine; Wirth, Jan Otto; D'Este, Elisa; Engelhardt, Johann; Hell, Stefan W.
• 署名单位: Max Planck Society
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-14881
• DOI: 10.1073/pnas.2412241121
• 发表日期: 2024-09-17
• 关键词: tug-of-war; bidirectional cargo transport; cytoplasmic dynein; axonal-transport; dynactin recruits; living cells; in-vitro; kinesin; microtubules; mechanism

摘要

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, likely arising from just one adenosine 5 '- triphosphate hydrolysis event being required during each step. Our study underscores the power of function in living cells.