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 双向转运对于货物贩运细胞至关重要，对于适当的生长和细胞分裂是必需的。 动力素和动力蛋白是负责双向货物运输细胞的微管电机。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，脊柱肌肉萎缩，肌萎缩性侧面硬化症和亨廷顿氏病；因此， 了解双向运输的基础机制对于理解运输至关重要 疾病状态的缺陷和发展潜在治疗。尽管有重要的理解进展 单个电动机的机械化学特性，关于电动机的工作方式仍然存在许多问题 团队，以及动机和动力蛋白如何彼此协调。一个广泛支持的双向模型 运输是“拔河”模型，在其中，动力恩和动机团队朝着相反的方向和获胜 团队确定运输方向。但是，该模型无法解释电动机协调性和 涉及的其他监管因素。以前的建模工作确定了载荷依赖的脱离率 确定动力蛋白或动力蛋白是否在电动机拔河中获胜的关键参数以及最近的实验 理论工作表明，垂直力继承为广泛使用的单孔光学镊子几何形状 明显加速运动脱离率。与此相一致，当动机和动力蛋白连接时 通过DNA链接，使力仅与微管平行，这些两运动复合物保持 连接的时间比光学镊子实验中的时间长得多。该项目的第一个目标是 建立一种新型技术，该技术将ssDNA用作PN尺度弹簧，以准确确定电动机垫脚 在没有垂直力的情况下的特征，模仿生理条件。 AIM1将测试 运输驱动蛋白和Dynein-dynactin-BICD2复合物可维持面向阻碍负载的阶梯 仅平行于微管。最初，将通过TIRF显微镜使用荧光探针跟踪电动机， 后来，将使用金纳米颗粒来跟踪高分辨率的载荷依赖性跃迁 步进周期。 AIM 2将使用DNA折纸支架配对金纳米颗粒标记的驱动蛋白和动力蛋白 一起通过干涉散射（ISCAT）显微镜跟踪它们。电动机动力学 双向传输轨迹将使用驱动蛋白 - 二氧蛋白传输的计算模型来解释。在 AIM 3，将跟踪电动机团队，以测试如何协助和阻碍继承的多次计量 几何影响会影响动力蛋白和Dynein团队之间的竞争。揭开电动机动力学的基础 这些复杂的多项运动系统对于理解细胞内双向运输如何确保 该特定的货物可靠地运输到其在神经元和其他细胞中的适当位置。