Gillies John P, Little Saffron R, Hancock William O, DeSantis Morgan E
University of Michigan, Department of Molecular, Cellular, and Developmental Biology, Ann Arbor, MI 48109.
Pennsylvania State University, Departments of Biomedical Engineering and Chemistry, University Park, PA 16802.
bioRxiv. 2024 Oct 12:2024.10.09.617440. doi: 10.1101/2024.10.09.617440.
Cytoplasmic dynein-1 (dynein), the primary retrograde motor in most eukaryotes, supports the movement of hundreds of distinct cargos, each with specific trafficking requirements. To achieve this functional diversity, dynein must bind to the multi-subunit complex dynactin and one of a family of cargo adaptors to be converted into an active, processive motor complex. Very little is known about the dynamic processes that promote the formation of this complex. To delineate the kinetic steps that lead to dynein activation, we developed a single-molecule fluorescence assay to visualize the real-time formation of dynein-dynactin-adaptor complexes in vitro. We found that dynactin and adaptors bind dynein independently rather than cooperatively. We also found that different dynein adaptors promote dynein-dynactin-adaptor assembly with dramatically different kinetics, which results in complex formation occurring via different assembly pathways. Despite differences in association rates or mechanism of assembly, all adaptors tested can generate a population of tripartite complexes that are very stable. Our work provides a model for how modulating the kinetics of dynein-dynactin-adaptor binding can be harnessed to promote differential dynein activation and reveals a new facet of the functional diversity of the dynein motor.
细胞质动力蛋白-1（动力蛋白）是大多数真核生物中的主要逆行马达蛋白，它支持数百种不同货物的运输，每种货物都有特定的运输需求。为实现这种功能多样性，动力蛋白必须与多亚基复合物动力肌动蛋白以及一类货物衔接蛋白中的一种结合，才能转化为有活性的、持续运动的马达蛋白复合物。对于促进该复合物形成的动态过程，我们了解甚少。为了描绘导致动力蛋白激活的动力学步骤，我们开发了一种单分子荧光测定法，以在体外可视化动力蛋白-动力肌动蛋白-衔接蛋白复合物的实时形成。我们发现动力肌动蛋白和衔接蛋白独立而非协同地结合动力蛋白。我们还发现，不同的动力蛋白衔接蛋白以截然不同的动力学促进动力蛋白-动力肌动蛋白-衔接蛋白组装，这导致复合物通过不同的组装途径形成。尽管结合速率或组装机制存在差异，但所有测试的衔接蛋白都能产生一群非常稳定的三方复合物。我们的工作提供了一个模型，说明如何利用调节动力蛋白-动力肌动蛋白-衔接蛋白结合的动力学来促进动力蛋白的差异激活，并揭示了动力蛋白马达功能多样性的一个新方面。
