Single-molecule imaging of stochastic interactions that drive dynein activation and cargo movement in cells

动力蛋白 动态素 微管 细胞生物学 运动蛋白 活体细胞成像 生物 细胞质 生物物理学 化学 细胞 生物化学
作者
Nireekshit Addanki Tirumala,Gregory Redpath,Sarah Viktoria Skerhut,Pritha Dolai,Natasha Kapoor‐Kaushik,Nicholas Ariotti,K. Vijay Kumar,Vaishnavi Ananthanarayanan
出处
期刊:Journal of Cell Biology [Rockefeller University Press]
卷期号:223 (3) 被引量:9
标识
DOI:10.1083/jcb.202210026
摘要

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.

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