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

描述（由申请人提供）：细胞器、膜泡和蛋白质沿着轴突内的微管（MT）细胞骨架进行长距离的主动运输对我们神经系统的功能至关重要。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——运动蛋白（将货物运送到轴突尖端）和动力蛋白（在逆行方向上移动）。虽然基于动力蛋白的运输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。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物介导的，该复合物与动力蛋白尾部和mt相互作用，并显著增加动力蛋白的加工能力。动力蛋白沿着MT的运动也受到一系列MT相关蛋白（MAPs）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。这张神经系统疾病的清单与缺乏

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.