Dynactin p150 promotes processive motility of DDB complexes by minimizing diffusional behavior of dynein
Dynactin p150 promotes processive motility of DDB complexes by minimizing diffusional behavior of dynein
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DOI：
10.1091/mbc.e19-09-0495
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发表时间：
2020-04-01
影响因子：
3.3
通讯作者：
Hancock, William O.
中科院分区：
文献类型：
--
作者：
Feng, Qingzhou;Gicking, Allison M.;Hancock, William O.
胞质动力蛋白通过与动力蛋白和适应蛋白BicD2形成复合物而被激活。我们使用干涉散射（iSCAT）显微镜跟踪体外dynein-dynactin-BicD2 （DDB）复合物，并开发了一种基于回归的算法来分类在进程、扩散和粘滞运动状态之间的切换。我们发现，DDB 65%的时间经历了渐进步进，4%的时间经历了一维扩散，剩下的时间短暂地粘附在微管上。尽管p150亚基先前被证明能够使dynactin沿着微管扩散，但阻断p150增加了DDB扩散的时间比例，并减少了DDB进程行走的时间。因此，DDB的扩散行为很可能是由于动力蛋白切换到非活性（扩散）状态，而不是p150将复合物束缚在微管上。使用DNA适配器形成的DDB-kinesin-1复合物缓慢而持久地移动，阻断p150导致复合物平均速度的70 nm/s +端移动，这与分离的DDB向扩散状态的移动在定量上一致。这些数据表明，在DDB激活模型中，dynactin p150不仅通过充当维持微管关联的扩散系索来增强动力蛋白的加工能力，而且还作为一种变构激活剂来促进动力蛋白的优化构象。在由kinesin和dynein- dyactin - bicd2的相反活动驱动的双向货物运输中，dynactin p150亚基促进逆行运输，可以作为运输调节的靶标。
Cytoplasmic dynein is activated by forming a complex with dynactin and the adaptor protein BicD2. We used interferometric scattering (iSCAT) microscopy to track dynein-dynactin-BicD2 (DDB) complexes in vitro and developed a regression-based algorithm to classify switching between processive, diffusive, and stuck motility states. We find that DDB spends 65% of its time undergoing processive stepping, 4% undergoing 1D diffusion, and the remaining time transiently stuck to the microtubule. Although the p150 subunit was previously shown to enable dynactin diffusion along microtubules, blocking p150 enhanced the proportion of time DDB diffused and reduced the time DDB processively walked. Thus, DDB diffusive behavior most likely results from dynein switching into an inactive (diffusive) state, rather than p150 tethering the complex to the microtubule. DDB-kinesin-1 complexes, formed using a DNA adapter, moved slowly and persistently, and blocking p150 led to a 70 nm/s plus-end shift in the average velocity of the complexes, in quantitative agreement with the shift of isolated DDB into the diffusive state. The data suggest a DDB activation model in which dynactin p150 enhances dynein processivity not solely by acting as a diffusive tether that maintains microtubule association, but rather by acting as an allosteric activator that promotes a conformation of dynein optimal for processive stepping. In bidirectional cargo transport driven by the opposing activities of kinesin and dynein-dynactin-BicD2, the dynactin p150 subunit promotes retrograde transport and could serve as a target for regulators of transport.