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

高分辨率 3D 成像描述轴突运输的分子基础

基本信息

批准号：
8756639
负责人：
Gabriel C Lander
金额：
$ 284.25万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-30 至 2019-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8756639
关键词：
Active Biological Transport Algorithms Alzheimer&apos s Disease Axon Axonal Transport Behavior Binding Cerebellar Ataxia Complex Cryoelectron Microscopy Cytoskeleton Disease Dynein ATPase Future Huntington Disease Intracellular Transport Kinesin Label Link Mediating Membrane Microcephaly Microtubule Stabilization Microtubule-Associated Proteins Microtubules Molecular Motor Movement Multiprotein Complexes Mutate Mutation Names Nervous System Physiology Nervous system structure Neurodegenerative Disorders Neurofibrillary Tangles Neurons Nutrient Organelles Parkinson Disease Play Proteins Recombinants Resolution Role Science Structure Structure-Activity Relationship Syndrome System Tail Techniques Three-Dimensional Imaging Vesicle Work base combat dynactin flexibility lissencephaly nervous system disorder novel public health relevance three dimensional structure

项目摘要

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.

描述（由申请人提供）：沿轴突中微管（MT）细胞骨架的细胞器，膜囊泡和蛋白质的主动运输对我们的神经系统的功能至关重要。这是两种类型的电动机 - 驱动蛋白（将货物运送到轴突尖端和逆行方向移动的动力蛋白）来完成这款ATP燃料的重要细胞质成分的穿梭。虽然基于驱动蛋白的运输的分子基础具有良好的特征，但我们对更复杂的动力蛋白运动的理解严重缺乏。货物结合到动力蛋白是由一种名为Dynactin的多蛋白复合蛋白介导的，该复合蛋白与Dynein尾巴和MT相互作用，并显着提高了动力蛋白的加工性。沿MT的动力蛋白运动也受到多种与MT相关蛋白（MAP）的严重影响，这些蛋白（MAP）在稳定神经元中MT高速公路方面也起着关键作用。与缺陷有关的神经疾病清单 Dynein/Dynactin依赖性运输包括亨廷顿，帕金森氏症，阿尔茨海默氏症，小头畸形，Lissencephaly，Perry综合征和Spino-Cerebellar Ataxia，仅举几例，以下几例，以此为例。我计划使用Cryoem，阐明引起细胞内货物的负载和运输的结构功能关系通过沿着地图稳定的MTS的动力蛋白 - 二奈乳蛋白复合物。我们已经获得了Dynactin的低分辨率3D结构，并开发了用于分析动力蛋白柔性成分的算法。在接下来的几年中，我们将以几乎原子的精度来解决Dynactin募集到动力蛋白的分子细节，概述影响货物束缚并促进MTS沿MTS的Dynein加工性的特定相互作用。同时，我们将探索与地图诱导的MT稳定相关的结构基序。该货物运输系统的最终机械框架将为导致上述疾病的已知突变提供结构背景，突出了正确的神经功能功能所需的关键相互作用或构象开关。然后，将使用重组表达系统进一步探测这些特定的动力蛋白和dynactin成分，以确定废除功能的确切原因，为将来的工作提供了旨在恢复突变亚基的本地行为的基础。通过这些研究的过程，我们将进一步开发并优化EM蛋白质定位的新型分子标记技术，该技术将在该领域具有深远的影响。