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

项目摘要/摘要 这个项目的目标是了解像DNA这样的大生物大分子是如何在拥挤的细胞内移动的 环境以及环境和DNA的什么特征导致了复杂的动力学和空间组织 在细胞中观察到。一种高度可调的体外细胞骨架系统，由不同数量的肌动蛋白和微管组成， 将开发光激活的马达蛋白和交联剂，并用一套光学显微镜和 从分子水平到宏观尺度的流变学方法。先进的显微镜和分析方法 将允许动态的DNA分子的大小和拓扑结构可控，嵌入仿生活性 细胞骨架，将在前所未有的空间和时间尺度上被精确量化。此外， 将活性细胞骨架和DNA分子包裹在脂膜中将有助于阐明 限制和细胞骨架-膜相互作用在确定细胞骨架和DNA动力学和结构中的作用。 对大分子如何在拥挤的细胞内空间内移动和分布的研究通常是 受制于体内系统的复杂性或体外环境的简单性。拟议的工作发现 在复杂性和易操纵性之间取得独特的平衡。体外平台将是高度可调且允许细胞样细胞 通过独立地允许控制以下各项来重新创造条件：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。这个体外系统的特性将与动力学和 DNA分子的分布，将用定制的光片显微镜成像，并用 单分子构象跟踪和差示动态显微镜分析方法。这些方法允许 将在前所未有的时空范围内捕获DNA动力学：从毫秒到几小时，从 亚微米到100微米。该项目的具体目标是：(1)设计体外活性细胞骨架网络， 表现出可调节的活性，用于探测非平衡动力学和流变性；(2)确定传输和 线状和环状DNA在这些活跃的细胞骨架网络中的构象动力学；(3)连接时变的 线状和环状DNA对细胞骨架网络特性和活性的空间分布；以及(4)结合活性 细胞骨架网络和DNA进入类细胞脂质双层囊泡，以确定限制和 膜相互作用对AIMS结果的影响。这些目标的成功将使复杂的大分子动力学成为可能 以及在细胞内观察到的分布有待重建和理解。此外，这个项目将揭示细胞是如何使用它们的 动态细胞骨架，用于分割大分子和复合体，并帮助或抑制大分子的运输，如 病毒基因组。

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.