Engineering Cytoskeletal Motors

工程细胞骨架马达

• 批准号: 10238890
• 负责人: Zev Bryant
• 金额: $ 31.98万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-10 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10238890
• 关键词: Actins; Adenylyl Imidodiphosphate; Biological Assay; Biology; Cell physiology; Cells; Chemicals; Collection; Complement; Complex; Cryoelectron Microscopy; Cues; Devices; Diagnostic; Dose; Drug Delivery Systems; Elements; Engineering; Exposure to; Filament; Gene Delivery; Generations; Genetic Recombination; Goals; Head; Image; In Vitro; Intracellular Transport; Ion Channel; Kinesin; Length; Light; Lighting; Measures; Mechanics; Messenger RNA; Microfilaments; Microtubules; Modeling; Molecular; Molecular Conformation; Molecular Motors; Molecular Structure; Motion; Motor; Myosin ATPase; Nanotechnology; Nature; Nuclear; Nucleotides; Optics; Organelles; Physiological; Pilot Projects; Positioning Attribute; Power stroke; Process; Property; Protein Engineering; Proteins; Research Design; Role; Running; Signal Transduction; Sorting - Cell Movement; Speed; Stroke; Structural Models; Structure; Structure-Activity Relationship; System; Technology; Tertiary Protein Structure; Testing; Transport Process; Variant; Walkers; Work; arm; base; cell motility; cellular pathology; design; experimental study; flexibility; fluorophore; improved; in silico; in vivo evaluation; insight; monomer; motor control; myosin VI; nanoGold; nanoparticle; nanoscale; novel; optical traps; optogenetics; programs; reconstruction; response; spatiotemporal; tool

SUMMARY Diverse cytoskeletal motors perform essential cellular functions including spindle assembly, nuclear positioning, and polarized transport of mRNA, proteins, and membranous cargos along microtubules and actin filaments. Engineering biomolecular motors with tunable and dynamically controllable properties can provide (1) rigorous tests of models relating molecular structures to mechanical functions, (2) novel tools for selective perturbation of mechanical processes inside living cells, and (3) optimized components for complex tasks such as molecular sorting and directed assembly in vitro. This project seeks to develop and characterize a comprehensive set of modified cytoskeletal motors with defined properties — including speed, direction, and force generation — than can be controlled using external cues such as light. A modular protein engineering approach will be applied to both actin-based and microtubule-based transport. During successive design cycles, chimeric motors will be constructed based on structural models, and then functionally characterized using gliding filament assays, single fluorophore imaging, gold nanoparticle tracking, and optical trapping. Complementary structural characterization using cryoelectron microscopy will be used to compare the experimental conformations of filament-bound motors to the original structural designs, and to yield new insights into class-specific structure-function relationships. Finally, pilot studies will be conducted to test the function of engineered motors inside living cells. The specific aims of this project are (1) to create diverse myosin motors that exploit dynamic changes in lever arm structure in order to shift gears — speed up, slow down, or change directions — when exposed to blue light; (2) to develop diverse microtubule-based motors with artificial lever arms, including light-activated gearshifts, by exploiting a mechanistic analogy between myosins and class-14 kinesins, and (3) to create processive multimeric assemblies of controllable engineered myosins and kinesins, and characterize their force-generating properties. If successful, this work will dramatically expand the potential applications of engineered molecular motors, and provide unprecedented control over nanoscale motion. Genetically encoded light- responsive motors will expand the optogenetics toolkit, complementing precise perturbations of ion channels and intracellular signaling with spatiotemporal control of cytoskeletal transport and contractility. Optogenetic control of bidirectional transport will enable dynamic relocalization of biomolecules and organelles; highly processive and controllable motors will have potential applications in gene and drug delivery; and controllable motors may be used to sort, shuttle, and concentrate analytes in microfabricated diagnostic devices.

概括 各种细胞骨架电动机执行必不可少的细胞功能，包括主轴组件，核 沿着微管和肌动蛋白的mRNA，蛋白质和膜碳的定位和极化转运 细丝。具有可调和动态控制特性的工程生物分子电动机可以提供 （1）将分子结构与机械函数相关的模型的严格测试，（2）选择性的新工具 活细胞内机械过程的扰动，以及（3）优化的组件，用于复杂任务 作为分子分类和定向组装体外。该项目旨在发展和表征 具有定义特性的全面修饰的细胞骨架电动机，包括速度，方向和 产生力 - 可以使用外部线索（例如光）来控制。模块化蛋白工程 方法将应用于基于肌动蛋白的基于肌动蛋白和基于微管的运输。在成功设计期间 循环，嵌合电动机将根据结构模型构建，然后在功能上表征 使用滑行丝测定，单荧光团成像，金纳米颗粒跟踪和光学诱捕。 使用冷冻光学显微镜的互补结构表征将用于比较 原始结构设计的细丝结合电动机的实验会议，并产生新的 洞悉特定类别的结构功能关系。最后，将进行试点研究以测试 活细胞内工程电动机的功能。 该项目的具体目的是（1）创建各种肌球蛋白电动机来利用动态变化 在杠杆臂结构中以移动齿轮 - 加快速度，放慢速度或更改方向 - 暴露于 蓝光； （2）开发具有人造杠杆臂的潜水员微管电动机，包括光激活 换档，通过利用肌球蛋白和14级驱动蛋白之间的机械类比，以及（3）创建 受控工程肌球蛋白和驱动蛋白的过程多媒体组件，并表征其 力生成特性。如果成功，这项工作将大大扩展 设计的分子电动机，并对纳米级运动提供前所未有的控制。遗传编码 轻响应电动机将扩大光遗传学工具包，完成离子的精确扰动 通道和细胞内信号传导，具有细胞骨骼转运和收缩力的时空控制。 双向转运的光遗传学控制将使生物分子和细胞器的动态重新定位； 高度加工和受控的电动机将在基因和药物输送中具有潜在的应用；和 控制器电动机可用于在微型诊断中进行分类，穿梭和浓缩分析物 设备。

项目成果

Optical control of fast and processive engineered myosins in vitro and in living cells.

• DOI: 10.1038/s41589-021-00740-7
• 发表时间: 2021-05
• 期刊: NATURE CHEMICAL BIOLOGY
• 影响因子: 14.8
• 作者: Ruijgrok, Paul V.;Ghosh, Rajarshi P.;Zemsky, Sasha;Nakamura, Muneaki;Gong, Rui;Ning, Lin;Chen, Robert;Vachharajani, Vipul T.;Chu, Alexander E.;Anand, Namrata;Eguchi, Raphael R.;Huang, Po-Ssu;Lin, Michael Z.;Alushin, Gregory M.;Liphardt, Jan T.;Bryant, Zev
• 通讯作者: Bryant, Zev

Exploitation of Engineered Light-Switchable Myosin XI for Nanotechnological Applications.

• DOI: 10.1021/acsnano.3c05137
• 发表时间: 2023-09-12
• 期刊: ACS NANO
• 影响因子: 17.1
• 作者: Salhotra, Aseem;Rahman, Mohammad A.;Ruijgrok, Paul, V;Meinecke, Christoph R.;Usaj, Marko;Zemsky, Sasha;Lindberg, Frida W.;Surendiran, Pradheebha;Lyttleton, Roman W.;Linke, Heiner;Korten, Till;Bryant, Zev;Mansson, Alf
• 通讯作者: Mansson, Alf

Introduction: Molecular Motors

• DOI: 10.1021/acs.chemrev.9b00819
• 发表时间: 2020-01-08
• 期刊: CHEMICAL REVIEWS
• 影响因子: 62.1
• 作者: Iino, Ryota;Kinbara, Kazushi;Bryant, Zev
• 通讯作者: Bryant, Zev

Motor processivity and speed determine structure and dynamics of microtubule-motor assemblies.

• DOI: 10.7554/elife.79402
• 发表时间: 2023-02-08
• 期刊: eLife
• 影响因子: 7.7
• 作者: Banks RA;Galstyan V;Lee HJ;Hirokawa S;Ierokomos A;Ross TD;Bryant Z;Thomson M;Phillips R
• 通讯作者: Phillips R