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.

项目概要 双向运输对于细胞内的货物运输至关重要，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。 基于微管运动的运输与许多神经退行性疾病有关，包括阿尔茨海默病、 帕金森病、脊髓性肌萎缩症、肌萎缩侧索硬化症和亨廷顿病； 了解双向传输的机制对于理解传输至关重要 尽管在理解方面取得了重要进展，但疾病状态和开发潜在治疗方法仍存在缺陷。 尽管单个电机的机械化学特性，但关于电机如何工作仍存在许多问题 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模型。 运输是一种“拔河”模型，其中动力蛋白和驱动蛋白团队向相反的方向拉动，获胜者 团队决定运输方向但是，该模型无法解释运动协调和运动协调。 先前的建模工作将负载相关的脱离率确定为 决定驱动蛋白或动力蛋白在电机拔河比赛中获胜的关键参数，以及最近的实验 理论工作表明，广泛使用的单珠光镊几何结构固有的垂直力 与此一致的是，当驱动蛋白和动力蛋白连接时。 通过 DNA 连接，使得力仅平行于微管，这两个运动复合体仍然存在 附着时间比光镊实验中的时间长得多该项目的第一个目标是 建立一种使用 ssDNA 作为 pN 级弹簧的新技术，以准确确定电机步进 在没有垂直力的情况下，模仿生理条件的特性将测试 Aim1 的能力。 运输驱动蛋白和动力蛋白-动力蛋白-BicD2 复合物，以维持对抗阻碍负载的步伐 最初，将通过 TIRF 显微镜用荧光探针跟踪电机， 随后，金纳米粒子将用于以高分辨率跟踪驱动蛋白中负载依赖性的转变 目标 2 将使用 DNA 折纸支架来配对金纳米粒子标记的驱动蛋白和动力蛋白。 一起并通过干涉散射 (iSCAT) 显微镜跟踪它们。 双向传输轨迹将使用驱动蛋白-动力蛋白传输的计算模型来解释。 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的方式 几何形状影响驱动蛋白和动力蛋白团队之间的竞争，揭示潜在的运动动力学。 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输到神经元和其他细胞中的适当位置。