Biomimetic cytoskeleton and advanced microscopy to reveal intracellular DNA dynamics and distributions

仿生细胞骨架和先进显微镜揭示细胞内 DNA 动态和分布

• 批准号: 10203574
• 负责人: Ryan McGorty
• 金额: $ 39.51万
• 依托单位: UNIVERSITY OF SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10203574
• 关键词: 3-Dimensional; Actins; Algorithmic Analysis; Architecture; Automobile Driving; Benchmarking; Biomimetics; Cell membrane; Cells; Characteristics; Circular DNA; Complex; Contracts; Coupling; Crosslinker; Crowding; Custom; Cytoskeleton; DNA; Development; Diagnostic; Encapsulated; Engineering; Environment; Equilibrium; Exhibits; Filament; Gene Delivery; Goals; Health; Heterogeneity; Hour; Image; Image Analysis; In Vitro; Intracellular Space; Intracellular Transport; Kinesin; Label; Lead; Length; Light; Link; Lipid Bilayers; Lipids; Location; Macromolecular Complexes; Measures; Mechanics; Membrane; Membrane Lipids; Methods; Microscope; Microscopy; Microtubules; Modality; Molecular; Molecular Conformation; Molecular Motors; Motor; Motor Activity; Myosin Type II; Optics; Organelles; Positioning Attribute; Process; Property; Proteins; Research Personnel; Rheology; Role; Shapes; Spatial Distribution; Structure; System; Techniques; Time; Vesicle; Viral Genome; Work; base; biomacromolecule; crosslink; density; design; gene therapy; in vivo; insight; instrumentation; laser tweezer; macromolecule; millisecond; prevent; single molecule; spatiotemporal; submicron; success; tool; undergraduate student

Project Summary/Abstract The goal of this project is to understand how large biomacromolecules, like DNA, move through the crowded intracellular environment and what features of that environment and of DNA lead to the complex dynamics and spatial organization observed in cells. A highly tunable in vitro cytoskeleton system consisting of varying amounts of actin and microtubules, light-activated motor proteins, and crosslinkers will be developed and characterized with a suite of optical microscopy and rheology methods that span from the molecular-level to macroscopic scales. Advanced microscopy and analysis methods will allow for the dynamics of DNA molecules of controllable size and topologies, embedded in the biomimetic active cytoskeleton, to be precisely quantified across an unprecedented range of spatial and temporal scales. Further, the encapsulation of the active cytoskeleton and DNA molecules within lipid membranes will allow for elucidating the role of confinement and cytoskeleton-membrane interactions in determining cytoskeleton and DNA dynamics and structure. Studies of how macromolecules move through and spatially distribute within crowded intracellular spaces are often hampered either by the complexity of in vivo systems or the simplicity of in vitro environments. The proposed work finds a unique balance between complexity and tractability. The in vitro platform will be highly tunable and allow cell-like conditions to be recreated by independently allowing for control over: the relative concentrations of actin, microtubules, molecular motors and crosslinkers; the types of motors and crosslinkers; the location, timing, and strength of motor activity; and the properties of confining vesicles. Characteristics of this in vitro system will be linked to the dynamics and distributions of DNA molecules, which will be imaged with a custom-built light-sheet microscope and quantified with single-molecule conformational tracking and differential dynamic microscopy analysis methods. These methods allow for DNA dynamics to be captured across an unprecedented spatiotemporal range: from milliseconds to hours and from submicron to 100s of microns. The specific aims of this project are to: (1) design in vitro active cytoskeleton networks that exhibit tunable activity for probing non-equilibrium dynamics and rheological properties; (2) determine transport and conformational dynamics of linear and circular DNA within these active cytoskeleton networks; (3) link the time-varying spatial distributions of linear and circular DNA to cytoskeleton network properties and activity; and (4) incorporate active cytoskeleton networks and DNA into cell-mimicking lipid bilayer vesicles to determine the role of confinement and membrane interactions on results of Aims 1-3. Success in these Aims will allow for the complex macromolecular dynamics and distributions observed within cells to be recreated and understood. Further, this project will reveal how cells use their dynamic cytoskeleton to partition macromolecules and complexes and to aid or inhibit the transport of large molecules like viral genomes.

项目总结/摘要 这个项目的目标是了解大的生物大分子，如DNA，如何在拥挤的细胞内移动， 环境以及环境和DNA的哪些特征导致了复杂的动力学和空间组织 观察细胞。一种高度可调的体外细胞骨架系统，由不同量的肌动蛋白和微管组成， 光激活马达蛋白和交联剂将被开发，并用一套光学显微镜进行表征， 流变学方法，从分子水平到宏观尺度。先进的显微镜和分析方法 将允许可控大小和拓扑结构的DNA分子的动力学，嵌入仿生活性物质中， 细胞骨架，在一个前所未有的空间和时间尺度范围内精确量化。此夕h 脂质膜内活性细胞骨架和DNA分子的包封将允许阐明 限制和细胞骨架上的膜相互作用，以确定细胞骨架和DNA的动力学和结构。 大分子如何在拥挤的细胞内空间中移动和空间分布的研究通常是 受到体内系统的复杂性或体外环境的简单性的阻碍。拟议的工作发现， 复杂性和易处理性之间的独特平衡。该体外平台将是高度可调的，并允许细胞样的 通过独立地允许控制：肌动蛋白，微管， 分子马达和交联剂;马达和交联剂的类型;马达活动的位置、时间和强度; 以及限制囊泡的性质。该体外系统的特征将与动力学和 DNA分子的分布，这将是一个定制的光片显微镜成像和定量与 单分子构象追踪和微分动态显微镜分析方法。这些方法允许 DNA动态将在前所未有的时空范围内被捕获：从毫秒到小时， 亚微米到几百微米。本项目的具体目标是：（1）设计体外活性细胞骨架网络， 表现出可调的活性，用于探测非平衡动力学和流变学性质;（2）确定传输和 线性和环状DNA在这些活跃的细胞骨架网络中的构象动力学;（3）将随时间变化的 线性和环状DNA的空间分布对细胞骨架网络性质和活性的影响;以及（4）掺入活性 细胞骨架网络和DNA进入细胞模拟脂质双层囊泡，以确定限制和 膜相互作用对目标1-3结果的影响。这些目标的成功将允许复杂的大分子动力学 以及在细胞内观察到的分布，以便重新创建和理解。此外，该项目还将揭示细胞如何利用其 动态细胞骨架来分隔大分子和复合物，并帮助或抑制大分子的转运， 病毒基因组