Equipment supplement - Refeyn TwoMP iSCAT microscope

设备补充 - Refeyn TwoMP iSCAT 显微镜

• 批准号: 10784112
• 负责人: David M Warshaw
• 金额: $ 22.36万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10784112
• 关键词: 3-Dimensional; Actins; Adaptor Signaling Protein; Address; Binding Proteins; Biological Models; Cell membrane; Cell physiology; Cells; Complex; Cytoskeleton; Data; Destinations; Electrostatics; Environment; Equipment; Event; Foundations; Human; In Vitro; Intracellular Transport; Kinesin; Life; Link; Lipid Binding; Liposomes; MYO5A gene; Mechanics; Membrane; Microfilaments; Microscope; Microtubules; Modeling; Molecular; Molecular Motors; Mothers; Motor; Myosin ATPase; Nature; Physically Challenged; Physiological; Property; Research Personnel; Sorting; Surface; Synaptic Vesicles; System; Transport Process; Tropomyosin; War; biophysical techniques; design; experimental study; in silico; in vitro Model; in vivo; innovation; insulin granule; knowledgebase; presynaptic; receptor; single molecule; tau Proteins; temporal measurement; vesicle transport

Project Summary/Abstract Intracellular cargo transport, such as lipid-bound insulin granules and presynaptic vesicles that are destined for secretion at the plasma membrane, rely on the concerted effort of kinesin-1 (kin1) and myosin Va (myoVa) molecular motors. These double-headed molecular motors carry their common cargo by stepping processively for considerable distances along their respective cytoskeletal tracks, i.e. microtubules (MTs) for kin1 and actin filaments for myoVa. To successfully deliver cargo, teams of kin1 and myoVa motors on the cargo surface, must overcome the physical challenges presented by the 3-dimensional (3D) complex network of MTs and actin filaments that comprise the cell's cytoskeleton, which also serves as these motors' highways. To determine how efficient intracellular cargo transport and delivery are accomplished despite the physical challenges presented by the cell's complex cytoskeletal highway, we have developed a near-physiological in vitro model system of kin1 and myoVa transport that is composed of a complex, but well-defined, 3- dimensional (3D), MT and actin filament network. Physiologically-relevant lipid-bound liposomes will be formed having Rab receptor proteins embedded in the liposome membranes so that molecular motors can be linked to the Rab receptors through their respective adapter proteins as in vivo. Once formed, motor-coated liposomes are introduced into the 3D networks and their transport trajectories defined using state-of-the-art single molecule biophysical techniques with high spatial and temporal resolution. Track-binding proteins to MTs (MAP7, Tau) and actin filaments (tropomyosins) will be added to dictate the direction of liposome transport. Key questions to be addressed using this well-defined model system are: 1) How are motors loaded onto the cargo surface? 2) Are motors on the cargo surface that are not engaged in transport, passive hitchhikers or are they cargo tethers that electrostatically interact with the heterologous track to enhance cargo transport by the actively engaged motors? 3) Is cargo hand-off from MT- to actin-based transport a coordinated event or the result of a tug of war? 4) Do track-binding proteins help to sort cargo by enhancing or inhibiting transport on specific MT and actin filaments? To interpret the results of these experiments, we will develop a mechanistic in silico transport model that incorporates the mechanical interactions between motor teams on the liposome surface. We propose that a functional interplay exists between the properties of the cytoskeletal tracks, motors, and cargos, which determines how teams of molecular motors meet the cellular demands placed on them by 3D cytoskeletal highways. The data obtained will provide a rich, mechano-spatial knowledgebase for the field and serve as a foundation for understanding molecular motor transport in the complex cytoarchitectural environment of the cell and how efficient motor transport systems are designed for delivery and retention of cargo at its destination.

项目概要/摘要 细胞内货物运输，例如脂质结合胰岛素颗粒和突触前囊泡 质膜分泌，依靠驱动蛋白-1（kin1）和肌球蛋白Va（myoVa）的协同作用 分子马达。这些双头分子马达通过逐步步进来携带它们的共同货物 沿着各自的细胞骨架轨道（即 kin1 和肌动蛋白的微管 (MT)）有相当长的距离 myoVa 的细丝。为了成功运送货物，kin1和myoVa电机团队在货面上， 必须克服 3 维 (3D) 复杂 MT 网络带来的物理挑战， 构成细胞细胞骨架的肌动蛋白丝，也充当这些马达的高速公路。到 确定如何有效地完成细胞内货物运输和交付，尽管物理 为了应对细胞复杂的细胞骨架高速公路带来的挑战，我们开发了一种近乎生理学的方法 kin1 和 myoVa 运输的体外模型系统由复杂但明确的 3- 三维 (3D)、MT 和肌动蛋白丝网络。将形成生理相关的脂质结合脂质体 将 Rab 受体蛋白嵌入脂质体膜中，以便分子马达可以连接 Rab 受体通过其各自的接头蛋白与体内一样。一旦形成，电机包被的脂质体 被引入到 3D 网络中，并使用最先进的单通道定义其传输轨迹 具有高空间和时间分辨率的分子生物物理技术。 MT 的轨道结合蛋白 （MAP7、Tau）和肌动蛋白丝（原肌球蛋白）将被添加来决定脂质体运输的方向。 使用这个定义明确的模型系统要解决的关键问题是：1）电机如何加载到 货面？ 2) 货物表面上的发动机是否不参与运输、被动搭便车或 它们的货物系绳与异源轨道发生静电相互作用，以增强货物运输 积极参与电机？ 3) 货物从 MT 到基于肌动蛋白的运输的交接是一个协调事件还是 拉锯战的结果？ 4) 轨道结合蛋白是否通过增强或抑制运输来帮助分类货物 特定的 MT 和肌动蛋白丝？为了解释这些实验的结果，我们将开发一种机制 计算机模拟运输模型，结合了脂质体上运动团队之间的机械相互作用 表面。我们认为细胞骨架轨道、马达、 和货物，这决定了分子马达团队如何满足细胞对它们的需求 3D 细胞骨架高速公路。获得的数据将为该领域提供丰富的力空间知识库 并作为理解复杂细胞结构中分子运动运输的基础 细胞环境以及如何设计高效的汽车运输系统来输送和保留 货物到达目的地。

项目成果

Modeling myosin Va liposome transport through actin filament networks reveals a percolation threshold that modulates transport properties.

对肌球蛋白 Va 脂质体通过肌动蛋白丝网络的运输进行建模揭示了调节运输特性的渗滤阈值。

• 发表时间: 2022-02-01
• 影响因子: 3.3
• 作者: Walcott, S;Warshaw, D M
• 通讯作者: Warshaw, D M

MyosinA is a druggable target in the widespread protozoan parasite Toxoplasma gondii.

肌球蛋白 A 是广泛存在的原生动物寄生虫弓形虫中的药物靶点。

• 发表时间: 2023-05
• 影响因子: 9.8
• 作者: Kelsen, Anne;Kent, Robyn S;Snyder, Anne K;Wehri, Eddie;Bishop, Stephen J;Stadler, Rachel V;Powell, Cameron;Martorelli di Genova, Bruno;Rompikuntal, Pramod K;Boulanger, Martin J;Warshaw, David M;Westwood, Nicholas J;Schaletzky, Julia;Ward, Gar
• 通讯作者: Ward, Gar