Dissecting dynein motor function using DNA nanotechnology

描述(申请人提供)：这个项目的长期研究目标是了解细胞骨架马达如何驱动真核细胞内不同大分子的运输，使它们能够有效地组织其内容物、移动、分裂和对信号做出反应。这项建议集中在细胞质动力蛋白，最大的，最复杂的，也是最不了解的细胞骨架马达。具体的目标是确定单个动力蛋白二聚体如何进行过程运动，马达集合如何有效地移动货物，以及过程运动在细胞中的作用。理解运动性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机-电机和电机-货物相互作用的工具。利用DNA纳米技术，我们已经开发出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、功能强大的动力蛋白异源二聚体。其次，使用三维(3D)DNA纳米技术，我们制造了合成货物，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连接到这些货物上。为了确定动力蛋白如何沿着微管连续前进，将使用单分子技术，包括高精度的多色荧光显微镜和单分子福斯特共振能量转移(SmFRET)，来跟踪单个动力蛋白分子的运动行为。这些实验的结果将被用来构建动力蛋白如何在微管上连续运动的模型。为了确定动力蛋白发动机之间或动力蛋白和动力蛋白发动机之间的协调如何影响货物的运动性，将不同数量的动力蛋白或动力蛋白与动力蛋白混合连接到3D合成DNA货物上。通过分析货物和单个货物附属马达在单分子运动性分析中的行为，将确定基于多马达的运输的生物物理性质。长途运输被认为需要前进的动力。然而，我们最近发现，动力蛋白是亚最大进程的。使用体内和体外方法，我们将检验这一假说，即亚最大过程对细胞质动力蛋白特别关键。由于细胞质动力蛋白在所有真核生物中只由一个基因编码，但执行广泛的任务，亚最大处理能力可能允许它被调节以执行各种细胞功能。这项研究将为无处不在的、必不可少的动力蛋白马达如何工作提供基本的、机械性的见解。此外，这里产生的DNA纳米技术工具将作为一般工程原理，用于研究其他蛋白质的寡聚化状态，或以更具生理学意义的方式研究任何分子马达的阵列。

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.