In Vitro Reconstitution of Dynein Force Exertion in a Bulk Viscous Medium
In Vitro Reconstitution of Dynein Force Exertion in a Bulk Viscous Medium
复制标题
散装粘性介质中动力蛋白作用力的体外重构
DOI：
10.1016/j.cub.2020.08.078
复制
发表时间：
2020
期刊：
影响因子：
9.2
通讯作者：
Minc Nicolas
中科院分区：
文献类型：
--
作者：
Palenzuela Heliciane;Lacroix Benjamin;Salle Jeremy;Minami Katsuhiko;Shima Tomohiro;Jegou Antoine;Romet-Lemonne Guillaume;Minc Nicolas
微管(MT)及其相关马达产生的力协调了从囊泡运输到中心体定位的基本细胞过程[1，2]。到目前为止，大多数研究都集中在固定在静态表面的电机施加的MT力，如体外活着的玻璃表面的细胞皮质[2-4]。然而，马达也运输大型货物和内膜网络，它们与粘性细胞质的流体动力相互作用应该会产生相当大的散装作用力。这样的力量可能有助于MT星体的集中、组织和定向[5-14]，但尚未在最小的重组系统中得到证明和研究。通过发展一种基于稳定的MTS和动力蛋白包裹的珠子自由漂浮在远离任何表面的粘性介质中的整体运动性分析，我们证明了货物的运动对MT施加拉力并推动它向相反的方向移动。对不同马达、不同马达速度、不同货物大小和中等粘度的MT移动量的量化结果表明，这种机制的效率主要由货物大小和MT长度决定。货物所施加的力是相加的，使我们能够概括拔河的情况或最小星座的二维运动。这些数据还揭示了粘性人群的性质以及货物和MT之间的流体动力相互作用的未被意识到的影响，这可能与理解这种在活细胞中施加的力模式有关。这项研究强化了膜内转运可以对MTS施加显著力量的概念。
The forces generated by microtubules (MTs) and their associated motors orchestrate essential cellular processes ranging from vesicular trafficking to centrosome positioning [1, 2]. To date, most studies have focused on MT force exertion by motors anchored to a static surface, such as the cell cortexin vivoor glass surfacesin vitro[2–4]. However, motors also transport large cargos and endomembrane networks, whose hydrodynamic interactions with the viscous cytoplasm should generate sizable forces in bulk. Such forces may contribute to MT aster centration, organization, and orientation [5–14] but have yet to be evidenced and studied in a minimal reconstituted system. By developing a bulk motility assay, based on stabilized MTs and dynein-coated beads freely floating in a viscous medium away from any surface, we demonstrate that the motion of a cargo exerts a pulling force on the MT and propels it in opposite direction. Quantification of resulting MT movements for different motors, motor velocities, over a range of cargo sizes and medium viscosities shows that the efficiency of this mechanism is primarily determined by cargo size and MT length. Forces exerted by cargos are additive, allowing us to recapitulate tug-of-war situations or bi-dimensional motions of minimal asters. These data also reveal unappreciated effects of the nature of viscous crowders and hydrodynamic interactions between cargos and MTs, likely relevant to understand this mode of force exertion in living cells. This study reinforces the notion that endomembrane transport can exert significant forces on MTs.