Cytoplasmic dynein 1 (dynein) is the primary minus end–directed motor protein in most eukaryotic cells. Dynein remains in an inactive conformation until the formation of a tripartite complex comprising dynein, its regulator dynactin, and a cargo adaptor. How this process of dynein activation occurs is unclear since it entails the formation of a three-protein complex inside the crowded environs of a cell. Here, we employed live-cell, single-molecule imaging to visualize and track fluorescently tagged dynein. First, we observed that only ∼30% of dynein molecules that bound to the microtubule (MT) engaged in minus end–directed movement, and that too for a short duration of ∼0.6 s. Next, using high-resolution imaging in live and fixed cells and using correlative light and electron microscopy, we discovered that dynactin and endosomal cargo remained in proximity to each other and to MTs. We then employed two-color imaging to visualize cargo movement effected by single motor binding. Finally, we performed long-term imaging to show that short movements are sufficient to drive cargo to the perinuclear region of the cell. Taken together, we discovered a search mechanism that is facilitated by dynein’s frequent MT binding–unbinding kinetics: (i) in a futile event when dynein does not encounter cargo anchored in proximity to the MT, dynein dissociates and diffuses into the cytoplasm, (ii) when dynein encounters cargo and dynactin upon MT binding, it moves cargo in a short run. Several of these short runs are undertaken in succession for long-range directed movement. In conclusion, we demonstrate that dynein activation and cargo capture are coupled in a step that relies on the reduction of dimensionality to enable minus end–directed transport in cellulo and that complex cargo behavior emerges from stochastic motor–cargo interactions.

中文翻译：

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驱动细胞内动力蛋白激活和货物运动的随机相互作用的单分子成像


细胞质动力蛋白 1（动力蛋白）是大多数真核细胞中主要的负端定向运动蛋白。动力蛋白保持非活性构象，直到形成由动力蛋白、其调节剂动力蛋白和货物接头组成的三联复合物。动力蛋白激活过程如何发生尚不清楚，因为它需要在细胞拥挤的环境内形成三蛋白复合物。在这里，我们采用活细胞、单分子成像来可视化和跟踪荧光标记的动力蛋白。首先，我们观察到只有 ~30% 与微管 (MT) 结合的动力蛋白分子参与负端定向运动，而且持续时间也很短 ~0.6 秒。接下来，通过对活细胞和固定细胞进行高分辨率成像，并使用相关光学和电子显微镜，我们发现动力蛋白和内体货物仍然彼此接近并与 MT 接近。然后，我们采用双色成像来可视化单电机绑定所影响的货物运动。最后，我们进行了长期成像，以表明短距离运动足以将货物驱动到细胞的核周区域。综上所述，我们发现了一种由动力蛋白频繁的 MT 结合-解除结合动力学促进的搜索机制：（i）在无效事件中，当动力蛋白没有遇到锚定在 MT 附近的货物时，动力蛋白解离并扩散到细胞质中，（ii） ）当动力蛋白在 MT 结合时遇到货物和动力蛋白时，它会在短期内移动货物。这些短跑中的几次连续进行以进行长距离定向运动。 总之，我们证明了动力蛋白激活和货物捕获在一个步骤中耦合，该步骤依赖于维数的降低，以实现纤维中的负端定向运输，并且复杂的货物行为是由随机的马达-货物相互作用产生的。