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马达如何为真核细胞内各种大分子的运输提供动力，使它们能够有效地组织其内容物，移动，分裂和响应信号。这一建议的重点是细胞质动力蛋白，最大的，最复杂的，和了解最少的细胞骨架马达。具体目标是确定单个动力蛋白二聚体是如何进行运动的，马达是如何有效地移动货物的，以及细胞中进行运动的作用。理解这些重要的运动特征的一个重要障碍是缺乏精确控制体外马达-马达和马达-货物相互作用的工具。利用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碱基配对产生稳定的、功能性的动力蛋白异二聚体。其次，利用三维（3D） DNA纳米技术，我们构建了合成货物，DNA连接的动力蛋白或动力蛋白马达可以以确定的数量和间距附着在货物上。为了确定动力蛋白如何沿着微管采取连续的步骤，单分子技术，包括高精度，多色荧光显微镜和单分子福斯特共振能量转移（smFRET），将被应用于跟踪单个运动动力蛋白分子的行为。这些实验的结果将用于构建动力蛋白如何在微管上运动的模型。为了确定动力蛋白马达之间或动力蛋白与动力蛋白马达之间的协调如何影响货物的运动性，不同数量的动力蛋白或动力蛋白与动力蛋白混合将被附着在3D合成DNA货物上。通过在单分子运动分析中分析货物和单个货物附着电机的行为，将确定多电机运输的生物物理特性。长途运输被认为需要过程运动性。然而，我们最近发现动力蛋白是次最大化进程的。采用体内和体外方法，我们将检验亚最大加工能力对细胞质动力蛋白特别重要的假设。由于细胞质动力蛋白在所有真核生物中仅由一个基因编码，但执行广泛的任务，亚最大的处理能力可能允许它被调整以执行各种细胞功能。这项研究将提供基本的，机械的见解如何无处不在的和必要的动力马达工作。此外，这里产生的DNA纳米技术工具将作为研究其他蛋白质寡聚化状态的一般工程原理，或以更生理相关的方式研究任何分子马达阵列。