Molecular motor dynamics underlying bidirectional cargo transport in cells

项目摘要 双向运输对细胞内的货物贩运至关重要，对细胞的正常生长和分裂也是必要的。 动蛋白和动力蛋白是负责细胞内双向货物运输的微管马达。中的缺陷 基于微管运动的运输与许多神经退行性疾病有关，包括阿尔茨海默氏症， 帕金森氏症、脊髓性肌萎缩症、肌萎缩侧索硬化症和亨廷顿病；因此， 了解双向运输的机制对于理解运输是至关重要的 疾病状态的缺陷和开发潜在的治疗方法。尽管在认识上取得了重大进展 关于单个马达的机械力化学性质，关于马达如何作为 团队，以及动蛋白和动力蛋白如何相互协调。一种广泛支持的双向模型 交通是一种‘拔河’模式，在这种模式中，动力蛋白和动蛋白组成的队伍向相反的方向拉动，获胜的是 团队决定了运输的方向。然而，该模型不能考虑运动的协调性和 其他监管因素也牵涉其中。以前的建模工作将与载荷相关的剥离率确定为 决定激动素或动力蛋白在汽车拔河比赛中获胜的关键参数，以及最近的实验 而理论工作表明，广泛使用的单珠光钳几何结构所固有的垂直力 显著加快了运动脱落率。与此一致的是，当动蛋白和动力蛋白联系在一起时 通过DNA连接，作用力只平行于微管，这些双马达复合体保留 附着的时间比在光镊子实验中看到的要长得多。这个项目的第一个目标是 建立一种使用单链DNA作为PN标尺弹簧的新技术，以准确地确定电机的步进 在没有垂直力的情况下，模仿生理条件。Aim1将测试 运输动蛋白和dynein-dynactin-BicD2复合体以维持对抗阻碍负荷导向的步态 完全平行于微管。最初，马达将通过TIRF显微镜用荧光探针进行跟踪， 随后，金纳米颗粒将被用来高分辨率地跟踪运动蛋白中依赖于负载的转变 步进循环。Aim 2将使用DNA折纸支架将金纳米颗粒标记的动蛋白和动力蛋白配对 并通过干涉散射(ISCAT)显微镜对其进行跟踪。其背后的马达动力学 双向传输轨迹将使用动蛋白-动力蛋白传输的计算模型来解释。在……里面 目标3，将跟踪马达团队，以测试多马达固有的辅助和阻碍负载 几何形状会影响动蛋白和动力蛋白团队之间的竞争。揭示背后的马达动力学 这些复杂的多运动系统对于理解细胞内双向运输如何确保 特定的货物被可靠地运送到神经元和其他细胞中的适当位置。

Project Summary Bidirectional transport is essential for cargo trafficking in cells and is required for proper growth and cell division. Kinesin and dynein are microtubule motors responsible for bidirectional cargo transport in cells. Defects in microtubule motor-based transport are linked to many neurodegenerative diseases including Alzheimer’s, Parkinson’s, spinal muscular atrophy, amyotrophic lateral sclerosis, and Huntington’s disease; thus, understanding the mechanisms underlying bidirectional transport is crucial to understanding transport deficiencies in disease states and developing potential treatments. Despite important advances in understanding the mechanochemical properties of individual motors, many questions remain regarding how motors work as teams, and how kinesins and dyneins coordinate with one another. A widely supported model for bidirectional transport is the ‘tug-of-war’ model in which teams of dynein and kinesin pull in opposite directions and the winning team determines the direction of transport. However, this model cannot account for the motor coordination and other regulatory factors involved. Previous modeling work identified the load-dependent detachment rate as the key parameter that determines whether kinesin or dynein wins in a motor tug-of-war, and recent experimental and theoretical work showed that vertical force inherent to widely used single-bead optical tweezer geometry significantly accelerates motor detachment rates. Consistent with this, when kinesin and dynein were connected through DNA linkages such that forces are only parallel to the microtubule, these two-motor complexes remained attached for much longer times than seen in optical tweezer experiments. The first goal of this project is to establish a novel technique that uses ssDNA as a pN-scale spring, to accurately determine motor stepping characteristics in the absence of vertical forces, mimicking physiological conditions. Aim1 will test the ability of transport kinesins and the dynein-dynactin-BicD2 complex to maintain stepping against a hindering load oriented solely parallel to the microtubule. Initially, motors will be tracked with a fluorescent probe via TIRF microscopy, and later a gold nanoparticle will be used to track in high resolution the load-dependent transitions in the kinesin stepping cycle. Aim 2 will use a DNA origami scaffold to pair gold nanoparticle-labeled kinesin and dynein together and track them via Interferometric Scattering (iSCAT) microscopy. The motor dynamics underlying the bidirectional transport trajectories will be interpreted using a computational model of kinesin-dynein transport. In Aim 3, teams of motors will be tracked to test how assisting and hindering loads inherent to multimotor geometries affect the competition between kinesin and dynein teams. Uncovering the motor dynamics underlying these complex multimotor systems is essential for understanding how intracellular bidirectional transport ensures that specific cargoes are reliably transported to their proper locations in neurons and other cells.