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.

总结 不同的细胞骨架马达执行基本的细胞功能，包括纺锤体组装，核 mRNA、蛋白质和膜质货物沿着微管和肌动蛋白的定位和极化转运，沿着微管和肌动蛋白 细丝。具有可调和动态可控特性的工程生物分子马达可以提供 (1)严格的测试模型相关的分子结构的机械功能，（2）新的工具，选择性 活细胞内机械过程的扰动，以及（3）用于复杂任务的优化组件， 如体外分子分选和定向组装。该项目旨在开发和表征一种 一套全面的修改细胞骨架电机与定义的属性-包括速度，方向， 力的产生-可以使用外部线索如光来控制。一种模块化蛋白质工程 方法将适用于肌动蛋白为基础的和微管为基础的运输。在后续设计中 周期，嵌合电机将构建基于结构模型，然后功能上的特点 使用滑动细丝测定、单荧光团成像、金纳米颗粒跟踪和光学捕获。 使用冷冻电子显微镜进行的补充结构表征将用于比较 实验构象的顺从绑定电机的原始结构设计，并产生新的 深入了解特定类别的结构-功能关系。最后，将进行试点研究，以测试 活细胞内工程马达的功能。 本计画的具体目标为：（1）利用动态变化，创造出多样化的肌球蛋白马达 在杠杆臂结构中，当暴露于 （2）开发各种具有人工杠杆臂的微管马达，包括光激活的 换档，通过利用肌球蛋白和14类驱动蛋白之间的机械类比，和（3）创建 可控工程化肌球蛋白和驱动蛋白的进行性多聚体组装，并表征其 力生成特性。如果成功的话，这项工作将极大地扩展 工程分子马达，并提供前所未有的控制纳米运动。遗传编码 光响应马达将扩展光遗传学工具包，补充离子的精确扰动。 通道和细胞内信号传导与时空控制的细胞骨架运输和收缩性。 双向运输的光遗传学控制将使生物分子和细胞器的动态再定位成为可能; 高度加工性和可控的马达将在基因和药物输送方面具有潜在的应用; 可控马达可用于在微制造诊断中分选、穿梭和浓缩分析物 装置.

项目成果

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

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