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

项目概要 作为神经元细胞骨架网络的主要组成部分之一，神经丝至关重要 用于轴突的径向生长和传导速度。神经丝参与 多种神经系统疾病的发病机制，并作为重要的轴突损伤生物标志物 神经退行性疾病和创伤性疾病。尽管它们在结构和功能中发挥着关键作用 对于轴突，人们对神经丝的分子组装知之甚少。目前还不清楚如何 单个神经丝亚基组装成成熟的丝，或者这些丝如何与 彼此形成支持轴突生长的细胞骨架网络。 冷冻电子断层扫描 (CryoET) 可以在 三个维度的纳米分辨率。这些蛋白质的详细结构信息可以是 使用专门的计算技术从断层图像中获得。在本提案中，我们的目标是 使用 CryoET 研究轴突内神经丝的结构和组织。 为了实现这一目标，我们将使用 CryoET 对初级神经元的轴突进行成像，并确定 使用亚断层扫描分类和亚纳米分辨率的神经丝结构 平均。分子模型将从平均结构构建，以揭示原位 神经丝的组装。将研究神经丝的结构和模型 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。为了使拟议的工作成为可能，将开发新的计算方法 CryoET 整个过程的数据分析。 我们相信这项研究将为原位神经丝组装和 加深我们对神经元细胞骨架组织的理解。获取的结构信息 这项研究将为未来研究轴突成熟过程以及 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.