Noell Crystal R, Ma Tzu-Chen, Jiang Rui, McKinley Scott A, Hancock William O
Department of Biomedical Engineering, Pennsylvania State University, University Park, Pennsylvania, USA.
Department of Mathematics, Tulane University, New Orleans, Louisiana, USA.
bioRxiv. 2025 Mar 25:2024.12.03.626575. doi: 10.1101/2024.12.03.626575.
Bidirectional cargo transport by kinesin and dynein is essential for cell viability and defects are linked to neurodegenerative diseases. Computational modeling suggests that the load-dependent off-rate is the strongest determinant of which motor 'wins' a kinesin-dynein tug-of-war, and optical tweezer experiments find that the load-dependent detachment sensitivity of transport kinesins is kinesin-3 > kinesin-2 > kinesin-1. However, in reconstituted kinesin-dynein pairs vitro, all three kinesin families compete nearly equally well against dynein. Modeling and experiments have confirmed that vertical forces inherent to the large trapping beads enhance kinesin-1 dissociation rates. In vivo, vertical forces are expected to range from negligible to dominant, depending on cargo and microtubule geometries. To investigate the detachment and reattachment kinetics of kinesin-1, 2 and 3 motors against loads oriented parallel to the microtubule, we created a DNA tensiometer comprising a DNA entropic spring attached to the microtubule on one end and a motor on the other. Kinesin dissociation rates at stall were slower than detachment rates during unloaded runs, and the complex reattachment kinetics were consistent with a weakly-bound 'slip' state preceding detachment. Kinesin-3 behaviors under load suggested that long KIF1A run lengths result from the concatenation of multiple short runs connected by diffusive episodes. Stochastic simulations were able to recapitulate the load-dependent detachment and reattachment kinetics for all three motors and provide direct comparison of key transition rates between families. These results provide insight into how kinesin-1, -2 and -3 families transport cargo in complex cellular geometries and compete against dynein during bidirectional transport.
驱动蛋白和动力蛋白的双向货物运输对细胞活力至关重要，其缺陷与神经退行性疾病有关。计算模型表明，负载依赖的解离速率是驱动蛋白 - 动力蛋白拔河比赛中哪个马达“获胜”的最强决定因素，光镊实验发现运输驱动蛋白的负载依赖解离敏感性为驱动蛋白 - 3 > 驱动蛋白 - 2 > 驱动蛋白 - 1。然而，在体外重组的驱动蛋白 - 动力蛋白对中，所有三个驱动蛋白家族与动力蛋白的竞争能力几乎相同。建模和实验已经证实，大捕获珠固有的垂直力会提高驱动蛋白 - 1的解离速率。在体内，垂直力预计从可忽略不计到占主导地位不等，这取决于货物和微管的几何形状。为了研究驱动蛋白 - 1、2和3马达相对于平行于微管定向的负载的分离和重新附着动力学，我们创建了一种DNA张力计，其一端是连接到微管的DNA熵弹簧，另一端是马达。驱动蛋白在失速时的解离速率比空载运行时的分离速率慢，复杂的重新附着动力学与分离前的弱结合“滑动”状态一致。负载下驱动蛋白 - 3的行为表明，长的KIF1A运行长度是由通过扩散事件连接的多个短运行串联而成的。随机模拟能够概括所有三个马达的负载依赖分离和重新附着动力学，并提供家族之间关键转变速率的直接比较。这些结果为驱动蛋白 - 1、 - 2和 - 3家族如何在复杂的细胞几何形状中运输货物以及在双向运输过程中与动力蛋白竞争提供了见解。
