Divisions of Advanced Materials, Instituto Potosino de Investigación Científica y Tecnológica, Camino a la Presa San José 2055, 78216 San Luis Potosí, Mexico; Molecular Biology, Instituto Potosino de Investigación Científica y Tecnológica, Camino a la Presa San José 2055, 78216 San Luis Potosí, Mexico.
Molecular Biology, Instituto Potosino de Investigación Científica y Tecnológica, Camino a la Presa San José 2055, 78216 San Luis Potosí, Mexico.
Biophys Chem. 2018 Nov;242:28-33. doi: 10.1016/j.bpc.2018.08.007. Epub 2018 Sep 3.
The in vitro transport of cargo by motor proteins constitutes a model system to understand mechanisms of vesicle trafficking inside cells. Here we apply the classic bead assay with a short, stiff kinesin protein to test the effect of multiple motors on essential transport parameters: distance, force and microtubule binding rate. Measurements of unloaded run length show that the transition from single- to multiple-motor behavior can be characterized by the appearance of extended runs, in accordance with a recently proposed model that quantifies the probability of multiple-motor engagement. In this transition, application of mechanical load using optical tweezers allows us to register maximum force values above single kinesin levels (8 pN). Yet, averages of run length and maximum force undergo little change as the probability of multiple-motor participation increases. In contrast, the measured rate of bead binding to microtubules scales linearly with the average number of motors per bead. These observations suggest that multiple motors bound randomly to the same cargo mainly increase the probability of attachment of these cargoes to the cytoskeletal filament network.
马达蛋白介导的货物体外运输构成了理解细胞内囊泡运输机制的模型体系。在这里，我们应用带有短而硬的驱动蛋白的经典珠子检测法，以测试多个马达对基本运输参数（距离、力和微管结合速率）的影响。空载运行长度的测量表明，从单马达行为到多马达行为的转变可以通过延伸运行的出现来表征，这符合最近提出的一种量化多马达结合概率的模型。在这种转变中，使用光学镊子施加机械负载使我们能够记录超过单个驱动蛋白水平（8 pN）的最大力值。然而，随着多马达参与概率的增加，运行长度和最大力的平均值变化很小。相比之下，与微管结合的珠子的测量结合速率与每个珠子上的平均马达数量呈线性关系。这些观察结果表明，随机结合到同一货物上的多个马达主要增加了这些货物与细胞骨架丝状网络附着的概率。
