Molecular motor dynamics underlying bidirectional cargo transport in cells

细胞内双向货物运输的分子运动动力学

• 批准号: 10679824
• 负责人: Crystal Renea Noell
• 金额: $ 6.91万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10679824
• 关键词: Acceleration; Affect; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Binding; Binding Sites; Cell division; Cells; Characteristics; Complex; Computer Models; DNA; Defect; Diffusion; Disease; Dynein ATPase; Ensure; Fluorescence Microscopy; Fluorescent Probes; Geometry; Goals; Gold; Growth; Head; Huntington Disease; Individual; Intracellular Transport; Investigation; Kinesin; Kinetics; Label; Link; Linker DNA; Location; Microscopy; Microtubules; Modeling; Molecular; Molecular Motors; Motor; Neurodegenerative Disorders; Neurons; Parkinson Disease; Physiological; Property; Resolution; Single-Stranded DNA; Spinal Muscular Atrophy; System; Techniques; Testing; Total Internal Reflection Fluorescent; War; Work; dimer; dynactin; experimental study; in vivo; insight; laser tweezer; nanoGold; novel; optic tweezer; particle; scaffold; simulation; single molecule; tool; trafficking

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.

项目摘要 双向运输对于细胞中的货物运输是必不可少的，并且是正常生长和细胞分裂所必需的。 驱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。缺陷 基于微管马达的运输与许多神经退行性疾病有关， 帕金森氏症、脊髓性肌萎缩症、肌萎缩侧索硬化症和亨廷顿氏病;因此， 了解双向运输的基本机制对于理解运输至关重要 疾病状态和开发潜在的治疗方法的缺陷。尽管在理解上取得了重要进展 尽管单个马达的机械化学性质，但关于马达如何工作的许多问题仍然存在， 团队，以及驱动蛋白和动力蛋白如何相互协调。一种广泛支持的双向 运输是一种“拔河”模式，动力蛋白和驱动蛋白向相反的方向拉扯， 团队决定运输的方向。然而，该模型不能解释运动协调， 其他监管因素。先前的建模工作将载荷依赖的脱离率确定为 决定驱动蛋白或动力蛋白在运动拔河中获胜的关键参数，最近的实验 理论工作表明，广泛使用的单珠光镊几何形状所固有的垂直力 显著加速了运动神经分离率与此相一致的是，当驱动蛋白和动力蛋白连接在一起时， 通过DNA连接，使力只与微管平行，这两个马达复合体仍然存在， 附着的时间比光镊实验中看到的要长得多。该项目的第一个目标是 建立了一种新的技术，使用ssDNA作为pN刻度弹簧，以准确地确定电机步进 在没有垂直力的情况下的特性，模仿生理条件。AIM 1将测试 运输驱动蛋白和动力蛋白-动力蛋白-BicD 2复合物，以维持对阻碍性负荷定向的步进 与微管平行。最初，将通过TIRF显微镜用荧光探针跟踪电机， 稍后，金纳米颗粒将用于高分辨率地跟踪驱动蛋白中的负载依赖性跃迁 步进循环目标2将使用DNA折纸支架将金纳米颗粒标记的驱动蛋白和动力蛋白配对 并通过干涉散射（iSCAT）显微镜跟踪它们。运动动力学的基础是 将使用驱动蛋白-动力蛋白运输的计算模型来解释双向运输轨迹。在 目标3，将跟踪电机组，以测试多电机固有的辅助和阻碍负载 几何形状影响驱动蛋白和动力蛋白团队之间的竞争。揭示运动动力学的基础 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。