In-situ architecture of membrane contact sites mediating organelle fission

项目摘要 复杂的区室化成具有专门功能的膜封闭细胞器是 真核细胞细胞器彼此紧密并列，形成膜接触部位， 用于通信的节点，对于许多蜂窝功能至关重要。尽管它们无处不在， 我们知道这些接触的分子成分如何组织成功能性信号传导管道， 不同的细胞器这主要是因为这些接触的动态性质和低细胞拷贝数 这些地点使得不可能使用常规的生物化学方法来纯化它们并开始进行经典的 使用X-晶体学、NMR和单粒子低温电子显微镜进行结构-功能研究。这里我们 提出了新的尖端显微镜模式的整合，以直观的细胞器相互作用，直接在 未受干扰的细胞环境。我们将在一个由蛋白质形成的接触位点上建立我们的工作流程， 内质网、线粒体和溶酶体。该接触位点的蛋白质将被标记 在iPSC内，它将在显微镜网格上分化为神经元。低温共焦显微镜 温度将使得能够以高亚细胞精度检测这些接触。在荧光的引导下 定位，薄的电子透明窗口将在这些细胞内微加工。随后的低温- 电子断层扫描、亚断层图像平均和深层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：ER-线粒体- 溶酶体接触位点标志着线粒体的分裂，这是一个维持线粒体健康供应的重要过程， 牢房在此期间建立的用于自动化样品制备、数据收集和处理的管道 该项目将作为未来现场结构研究的变革蓝图。这个项目将奠定基础 对新兴的、探索不足的和复杂的化学领域进行高分辨率的结构表征， 膜接触位点和了解其在健康和疾病中的功能的机械基础。这 该项目将为其他有兴趣研究的研究人员提供急需的方向性框架 直接在细胞、分离的组织和类器官中的蛋白质结构。

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.