Determining structure and organization of neurofilaments in situ using cryo-electron tomography

项目摘要 作为神经元细胞骨架网络的主要组成部分，神经丝是必不可少的 轴突的径向生长和传导速度。神经细丝参与了 多发性神经功能障碍的发病机制及其作为轴突损伤的重要生物标志物 神经退行性和创伤性疾病。尽管它们在细胞的结构和功能中起着关键作用 轴突，对神经细丝的分子组装知之甚少。目前，还不清楚是如何 单个神经丝亚单位组装成成熟的细丝，或这些细丝如何与 形成支持轴突生长的细胞骨架网络。 冷冻电子断层扫描(CryoET)可以显示细胞内的蛋白质复合体 三维纳米分辨率。这些蛋白质的详细结构信息可以 通过使用专门的计算技术从断层扫描中获得。在这项建议中，我们的目标是 用CryoET研究轴突内神经丝的结构和组织。 为了实现这一目标，我们将用Cryoet成像初级神经元的轴突，并确定 亚纳米分辨率的神经细丝结构的亚断层图像分类 平均。分子模型将从平均结构中构建以揭示原位 神经细丝的组装。神经细丝的结构和模型将在他们的 细胞环境以了解神经元细胞骨架组织如何随着轴突的生长而进化 更厚了。为了使拟议的工作成为可能，将开发新的计算方法 Cryoet的数据分析贯穿整个过程。 我们相信，这项研究将提供第一次对原位神经丝组装和 加深我们对神经元细胞骨架组织的理解。所获取的结构信息 这项研究将为未来轴突成熟过程的研究以及 各种神经系统疾病的结构基础。

Project Summary As one of the major components of the neuronal cytoskeleton network, neurofilaments are essential for the radial growth and conduction velocity of axons. Neurofilaments are involved in the pathogenesis of multiple neurological disorders and serve as important axonal damage biomarkers in neurodegenerative and traumatic diseases. Despite their critical role in the structure and function of axons, little is known about the molecular assembly of neurofilaments. Currently, it is still unclear how individual neurofilament subunits assemble into mature filaments, or how those filaments interact with each other to form the cytoskeleton network that supports the growth of axons. Cryo-electron tomography (CryoET) allows visualization of protein complexes inside cells at nanometer resolution in three dimensions. Detailed structural information of those proteins can be obtained from the tomogram using specialized computational techniques. In this proposal, we aim to study the structure and organization of neurofilaments within axons using CryoET. To achieve this goal, we will image the axon of primary neurons with CryoET and determine the structures of neurofilaments at subnanometer resolution using subtomogram classification and averaging. Molecular models will be constructed from the averaged structures to reveal the in situ assembly of neurofilaments. The structures and models of neurofilaments will be studied in their cellular context to understand how the neuronal cytoskeleton organization evolves, as the axons grow thicker. To make the proposed work possible, novel computational methods will be developed for CryoET data analysis throughout the process. We believe this research will provide the first structural insight into in situ neurofilament assembly and deepen our understanding of neuronal cytoskeleton organization. The structural information acquired from this research will pave the way for future studies of the axon maturation process, as well as the structural basis of various neurological diseases.