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 MINFLUX localization to elucidate the spatiotemporal changes underlying protein function in living cells.

中文翻译：

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MINFLUX 揭示了活神经元中的动力蛋白步进


动力蛋白是负责各种货物逆行细胞内运输的主要分子马达，在几毫秒内执行连续的纳米级步骤。由于已建立的分子追踪方法的时空精度有限，目前对动力蛋白步进的了解基本上仅限于体外的慢速测量。在这里，我们使用 MINFLUX 荧光团定位以纳米/毫秒的精度直接追踪活原代神经元中 CRISPR/Cas9 标记的内源性动力蛋白。我们表明内源性动力蛋白主要采取 8 nm 步骤，包括频繁的侧向步骤但很少向后步骤。引人注目的是，逆行和顺行运动之间的大多数方向反转发生在单步 （16 ms） 的时间尺度上，表明存在快速调节反转机制。在暂停或反转期间类似拔河的行为出乎意料地罕见。通过分析步骤之间的停留时间，我们得出结论，动力蛋白步进机制的基础是单个限速过程，可能是由于每个步骤中只需要一个腺苷 5′-三磷酸水解事件引起的。我们的研究强调了 MINFLUX 定位在阐明活细胞中蛋白质功能背后的时空变化的能力。