In-situ architecture of membrane contact sites mediating organelle fission

项目总结 复杂的分隔成具有专用功能的膜封闭细胞器是 真核细胞。细胞器彼此紧密对置，形成膜接触部位，这些部位 用于通信的节点，对许多蜂窝功能至关重要。尽管它们无处不在，但几乎没有留下 知道这些接触的分子成分是如何组织成功能信号通道的 不同的细胞器。这在很大程度上是因为这些联系人的动态性质和低细胞拷贝数 站点使得不可能使用传统的生化方法来提纯它们并开始经典的 用X-结晶学、核磁共振和单粒子冷冻电子显微镜进行结构-功能研究。在这里，我们 建议集成新的尖端显微镜模式，以直接在 不受干扰的蜂窝环境。我们将在一个由Proteins组成的联系站点上建立我们的工作流程 内质网、线粒体和溶酶体。这个接触点的蛋白质将被标记 在IPSCs内，这些细胞将在显微镜网格上分化为神经元。低温共聚焦显微镜 温度将能够以高亚细胞精度检测这些接触。在荧光的引导下 局部化，薄薄的电子透明窗口将在这些细胞内进行微机械加工。随后的低温- 电子断层扫描、亚断层图像平均和深度分类将使三维结构成为可能 确定这些接触点。我们的工作将有助于回答一个重要的生物学问题：内质网线粒体是如何- 溶酶体接触部位标记线粒体进行分裂，这一过程对于维持线粒体的健康供应至关重要。 牢房。在此期间建立的自动化样品准备、数据收集和处理的管道 该项目将成为未来现场结构研究的变革性蓝图。这一项目将为 关于新兴的、未被开发的和复杂的 膜接触部位以及了解其在健康和疾病中的作用的机制基础。这 该项目将为其他地方对研究感兴趣的研究人员提供急需的指导框架 蛋白质结构直接存在于细胞、分离的组织和有机体中。

PROJECT SUMMARY Sophisticated compartmentalization into membrane enclosed organelles of dedicated function is a hallmark of eukaryotic cells. Organelles come in close apposition to each other forming membrane contact sites that are nodes for communication, critical for many cellular functions. Despite their ubiquitous presence, little remains known about how the molecular components of these contact organize into functional signaling conduits between disparate organelles. This is largely because the dynamic nature and low cellular copy number of these contact sites make it impossible to use conventional biochemical approaches to purify them and embark on classical structure-function studies using X-crystallography, NMR, and single particle cryo-electron microscopy. Here, we propose integration of novel cutting-edge microscopy modalities to visualize organellar interactions directly within unperturbed cellular context. We will establish our workflow on one contact site formed by proteins on endoplasmic reticulum, mitochondria, and lysosomes, respectively. Proteins at this contact site will be tagged within iPSCs, which will be differentiated into neurons on microscopy grids. Confocal microscopy at cryogenic temperatures will enable detection of these contacts with high subcellular precision. Guided by the fluorescence localization, thin electron-transparent windows will be micromachined inside these cells. Subsequent cryo- electron tomography, subtomogram averaging and deep-classification will enable three-dimensional structure determination of these contact sites. Our work will help answer a vital biological question: how ER-mitochondria- lysosome contact site marks mitochondria for fission, a process essential for maintaining their healthy supply in the cell. The pipelines for automated sample preparation, data collection, and processing established during this project will serve as a transformative blueprint for future structural studies in-situ. This project will lay a foundation towards high-resolution structural characterization of the emerging, underexplored and complex field of membrane contact sites and a mechanistic basis for understanding their function in health and disease. This project will provide much needed directional framework to researchers elsewhere who are interested in studying protein structures directly in cells, isolated tissues and organoids.