Molecular basis of axonal transport described by high-resolution 3D imaging

描述(申请人提供)：细胞器、膜小泡和蛋白质沿着轴突内的微管(MT)细胞骨架进行远距离的主动运输对我们神经系统的功能至关重要。这种以三磷酸腺苷为燃料的重要细胞质成分的穿梭是由两种类型的马达完成的--动蛋白和动力蛋白，前者向轴突末端运送货物，后者以逆行方向移动。虽然基于动蛋白的转运的分子基础已经很好地描述了，但我们对更复杂的动力蛋白马达的了解严重不足。货物与动力蛋白的结合是由一种名为动力蛋白的多蛋白复合体介导的，该复合体与动力蛋白尾部和MTS相互作用，显著提高动力蛋白的处理能力。动力蛋白沿MTS的运动还受到一系列MT相关蛋白(MAP)的严重影响，这些蛋白在稳定神经元中的MT高速公路方面也发挥着关键作用。与以下方面的缺陷有关的神经系统疾病清单 动力蛋白/动力蛋白依赖的运输包括亨廷顿、帕金森、阿尔茨海默氏症、小头畸形、无脑畸形、佩里综合征和脊柱-小脑性共济失调等，强调了这一系统的生物医学相关性。利用低温电子显微镜，我计划阐明导致细胞内货物装载和运输的结构-功能关系 由动力蛋白-动力蛋白复合体沿着MAP稳定的MTS。我们已经获得了动力蛋白的低分辨率三维结构，并开发了分析动力蛋白柔性组分的算法。在接下来的几年里，我们将以近原子的精度解决动力蛋白募集到动力蛋白的分子细节，概述影响货物系留的特定相互作用，并促进沿MTS的动力蛋白加工。同时，我们将探索参与MAP诱导MT稳定的结构基序。由此产生的货物运输系统的机械框架将为导致上述疾病的已知突变提供结构背景，突出正常神经功能所需的关键相互作用或构象开关。然后，这些特定的dynein和dynactin组分将通过使用重组表达系统来进一步探索，以确定功能丧失的确切原因，为未来旨在恢复突变亚基的自然行为的工作提供基础。通过这些研究，我们还将开发和优化一种用于EM定位蛋白质的新的分子标记技术，这将在该领域具有深远的意义。

DESCRIPTION (provided by applicant): The active transport of organelles, membrane vesicles, and proteins across great distances along the microtubule (MT) cytoskeleton within axons is critical to the function of our nervous system. This ATP-fueled shuttling of vital cytoplasmic components is accomplished by two types of motors - kinesins, which transport cargo towards the axon tips, and dyneins, which move in the retrograde direction. While the molecular basis of kinesin-based transport is well- characterized, our understanding of the more sophisticated dynein motor is severely lacking. Cargo-binding to dynein is mediated by a multiprotein complex named dynactin, which interacts with the dynein tail as well as MTs, and significantly increases dynein processivity. Dynein movement along MTs is also heavily influenced by a wide array of MT-associated proteins (MAPs), which also play a critical role in stabilizing the MT highways in neurons. The list of neurological diseases linked to deficiencies in dynein/dynactin-dependent transport include Huntington's, Parkinson's, Alzheimer's, microcephaly, lissencephaly, Perry syndrome, and spino-cerebellar ataxia to name a few, underscoring the biomedical relevance of this system. Using cryoEM, I plan to elucidate the structure-function relationships that give rise to the loading and transport of intracellular cargo by the dynein-dynactin complex along MAP- stabilized MTs. We have already obtained a low-resolution 3D structure of dynactin, and developed algorithms for analyzing the flexible components of dynein. Over the next few years, we will resolve the molecular details of dynactin recruitment to dynein with near-atomic precision, outlining the specific interactions that influenc cargo tethering and promote dynein processivity along MTs. Concurrently, we will explore the structural motifs involved in MAP-induced MT stabilization. The resulting mechanistic framework for this cargo transport system will provide a structural context for the known mutations leading to the aforementioned diseases, highlighting key interactions or conformational switches that are required for proper neurological function. These specific dynein and dynactin components will then be further probed by cryoEM using recombinant expression systems in order to identify the precise causes for abrogation of function, providing the basis for future work aimed at restoring native behavior to mutated subunits. Through the course of these studies, we will additionally develop and optimize a novel molecular labeling technique for protein localization by EM, which will have far-reaching implications in the field.