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

描述（由申请人提供）：细胞器、膜囊泡和蛋白质沿轴突内微管（MT）细胞骨架沿着长距离主动转运对我们的神经系统功能至关重要。这种以ATP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-驱动蛋白，其将货物运输到轴突尖端，以及动力蛋白，其在逆行方向上移动。虽然基于驱动蛋白的转运的分子基础被很好地表征，但我们对更复杂的动力蛋白马达的理解严重缺乏。与动力蛋白的货物结合是由一种名为动力蛋白的多蛋白复合物介导的，该复合物与动力蛋白尾部以及MT相互作用，并显着增加动力蛋白的持续合成能力。动力蛋白沿MT的沿着运动也受到大量MT相关蛋白（MAP）的严重影响，这些蛋白在稳定神经元中的MT高速公路中也起着关键作用。神经系统疾病的清单与缺乏 动力蛋白/动力蛋白依赖性转运包括亨廷顿氏病、帕金森氏病、阿尔茨海默氏病、小头畸形、无脑畸形、佩里综合征和脊髓-小脑共济失调，仅举几例，强调了该系统的生物医学相关性。 利用cryoEM，我计划阐明引起细胞内货物装载和运输的结构-功能关系 由动力蛋白-动力肌动蛋白复合物沿着MAP稳定的MT。我们已经获得了动力蛋白的低分辨率3D结构，并开发了用于分析动力蛋白柔性组分的算法。在接下来的几年里，我们将解决的分子细节dynactin招聘动力蛋白与近原子精度，概述了具体的相互作用，影响货物拴系和促进动力蛋白的持续合成能力沿着MT。同时，我们将探讨MAP诱导的MT稳定化所涉及的结构基序。由此产生的货物运输系统的机制框架将为导致上述疾病的已知突变提供结构背景，突出显示适当神经功能所需的关键相互作用或构象开关。然后，这些特定的动力蛋白和动力肌动蛋白组分将通过cryoEM使用重组表达系统进一步探测，以确定功能废除的确切原因，为未来旨在恢复突变亚基的天然行为的工作提供基础。通过这些研究，我们将进一步开发和优化一种新的分子标记技术，用于EM蛋白质定位，这将在该领域产生深远的影响。

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.