Dissecting dynein motor function using DNA nanotechnology

使用 DNA 纳米技术剖析动力蛋白运动功能

• 批准号: 8436011
• 负责人: SAMARA L RECK-PETERSON
• 金额: $ 31.4万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-01 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8436011
• 关键词: ATPase Domain; Address; Affect; Aspergillus nidulans; Base Pairing; Behavior; Binding; Biological Assay; Cell physiology; Cells; Color; Complementary DNA; Complex; Coupled; DNA; Data; Defect; Disease; Dynein ATPase; Endosomes; Energy Transfer; Engineering; Eukaryota; Eukaryotic Cell; Fluorescence Microscopy; Genes; Goals; In Vitro; Individual; Kinesin; Label; Lead; Libraries; Life; Link; Maps; Methods; Microtubules; Mitotic spindle; Modeling; Molecular; Molecular Motors; Motor; Movement; Mutation; Nanotechnology; Neurodegenerative Disorders; Neurons; Positioning Attribute; Property; Proteins; Protomer; Relative (related person); Research; Resolution; Role; Signal Transduction; Sorting - Cell Movement; Structure; System; Techniques; Testing; Work; Yeasts; base; cell motility; design; dimer; fluorophore; in vivo; insight; macromolecule; motor control; novel; public health relevance; research study; single molecule; single-molecule FRET; spatial relationship; synthetic construct; tool

DESCRIPTION (provided by applicant): The long term research goal of this project is to understand how cytoskeletal motors power the transport of diverse macromolecules within eukaryotic cells, enabling them to effectively organize their contents, move, divide, and respond to signals. This proposal focuses on cytoplasmic dynein, the largest, most complex, and least understood of the cytoskeletal motors. The specific objectives are to determine how single dynein dimers move processively, how ensembles of motors efficiently move cargo, and the role of processive movement in cells. A significant obstacle to understanding these important features of motility is a lack of tools to precisely control motor-motor and motor-cargo interactions in vitro. Using DNA nanotechnology, we have developed methods to achieve this. First, we generate stable, functional dynein heterodimers through DNA base pairing. Second, using three-dimensional (3D) DNA nanotechnology, we build synthetic cargo to which DNA-linked dynein or kinesin motors can be attached with defined numbers and spacing. To determine how dynein takes consecutive steps along microtubules, single-molecule techniques, including high-precision, multi-color fluorescence microscopy and single-molecule Forster resonance energy transfer (smFRET), will be applied to track the behavior of individual moving dynein molecules. The results of these experiments will be used to construct a model for how dynein moves processively on microtubules. To determine how coordination among dynein motors or between dynein and kinesin motors affects cargo motility, varying numbers of dynein or dynein mixed with kinesin will be attached to a 3D, synthetic DNA cargo. By analyzing the behavior of both the cargo and individual, cargo-attached motors in single-molecule motility assays, the biophysical properties of multi-motor-based transport will be determined. Long distance transport is thought to require processive motility. However, we recently discovered that dynein is sub-maximally processive. Using in vivo and in vitro approaches, we will test the hypothesis that sub-maximal processivity is especially critical for cytoplasmic dynein. Because cytoplasmic dynein is encoded by only a single gene in all eukaryotes but carries out a wide range of tasks, sub-maximal processivity may allow it to be tuned to perform a variety of cellular functions. This research will provide fundamental, mechanistic insights into how the ubiquitous and essential dynein motor works. In addition, the DNA nanotechnology tools generated here will serve as general engineering principles for studying the oligomerization state of other proteins or for studying arrays of any molecular motor in a more physiologically relevant manner.

描述（由申请人提供）：该项目的长期研究目标是了解细胞骨架电动机如何为真核细胞内的各种大分子的运输提供动力，从而使它们能够有效地组织其内容，移动，分裂和对信号的反应。该提议的重点是细胞质动力蛋白，这是细胞骨架电动机中最大，最复杂，最不了解的。具体目标是确定单个动力蛋白二聚体如何进行过程移动，电动机的合奏如何有效移动货物以及过程中的过程中的作用。理解运动的这些重要特征的一个重要障碍是缺乏在体外精确控制电动机运动和电动货车相互作用的工具。使用DNA纳米技术，我们开发了实现这一目标的方法。首先，我们通过DNA碱基配对生成稳定的功能性动力蛋白异二聚体。其次，使用三维（3D）DNA纳米技术，我们建造合成货物，可以将DNA连接的动力蛋白或驱动蛋白电动机与定义的数字和间距连接。为了确定Dynein如何沿着微管连续步骤，将应用单分子技术，包括高精度，多色荧光显微镜和单分子Forster forster resonance Enermance Energy Tranfcer（SMFRET），以跟踪单个移动Dynein Molecules的行为。这些实验的结果将用于构建一个模型，以在微管上进行过程移动。为了确定动力蛋白电动机之间或动力蛋白和驱动蛋白电动机之间的协调如何影响货物运动，将变化的动力蛋白或动力蛋白与驱动蛋白混合在一起，将附加到3D合成的DNA货物上。通过分析货物和个体，货物连接的电动机在单分子运动分析中的行为，将确定基于多运动的运输的生物物理特性。长距离运输被认为需要过程上的运动。但是，我们最近发现动力蛋白是亚最大的过程。使用体内和体外方法，我们将测试以下假设：亚最大加工性对于细胞质动力蛋白尤其重要。由于细胞质动力蛋白在所有真核生物中仅由单个基因编码，但执行了多种任务，因此亚最大加工性可以使其调整以执行多种细胞功能。这项研究将为无处不在和必不可少的动力蛋白运动的工作原理提供基本的机械见解。此外，此处生成的DNA纳米技术工具将作为研究其他蛋白质的寡聚状态或以更相关的方式研究任何分子运动阵列的通用工程原理。