In-situ architecture of membrane contact sites mediating organelle fission

介导细胞器裂变的膜接触位点的原位结构

• 批准号: 10472370
• 负责人: Shyamal Mosalaganti
• 金额: $ 138.22万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10472370
• 关键词: Architecture; Biochemical; Biological; Cell physiology; Cells; Classification; Communication; Complex; Confocal Microscopy; Cryo-electron tomography; Cryoelectron Microscopy; Crystallography; Data Collection; Detection; Disease; Electrons; Endoplasmic Reticulum; Eukaryotic Cell; Fluorescence; Foundations; Future; Health; In Situ; Lipids; Lysosomes; Mediating; Membrane; Microscopy; Mitochondria; Modality; Molecular; Nature; Neurons; Organelles; Organoids; Preparation; Process; Proteins; Research Personnel; Resolution; Role; Sampling; Signal Transduction; Site; Structure; Temperature; Thinness; Tissues; Work; cryogenics; human disease; induced pluripotent stem cell; insight; interest; novel; particle; protein structure; three dimensional structure

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.

项目摘要 专用功能的膜封闭细胞器的精致隔室化是 真核细胞。细胞器彼此密切相关，形成膜接触站点 通信节点，对于许多细胞功能至关重要。尽管存在着无处不在的存在 知道这些接触的分子成分如何组织到功能信号导管之间 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 网站使得不可能使用常规的生化方法净化它们并启动古典 结构功能研究使用X晶格学，NMR和单个颗粒冷冻电子显微镜。在这里，我们 提出将新型尖端显微镜模态的整合，以直接在内部可视化细胞器相互作用 不受干扰的细胞环境。我们将在由蛋白质形成的一个接触站点上建立工作流程 内质网，线粒体和溶酶体分别。此接触站点的蛋白质将被标记 在IPSC中，将在显微镜网格上分为神经元。在低温下共焦显微镜 温度将使这些接触以高细胞精度检测。由荧光引导 本地化，薄电子透明窗口将在这些单元格内进行微加工。随后的冷冻 电子断层扫描，亚图平均和深度分类将实现三维结构 确定这些接触站点。我们的工作将有助于回答一个至关重要的生物学问题：er-mitochria-如何 溶酶体接触位点标记线粒体的裂变，这是维持其健康供应至关重要的过程 细胞。在此期间建立的自动样品准备，数据收集和处理的管道 项目将作为现场结构研究的变革性蓝图。这个项目将奠定基础 朝向出现的高分辨率结构表征 膜接触地点和理解其在健康和疾病中功能的机理基础。这 项目将为有兴趣学习的其他地方的研究人员提供急需的方向框架 蛋白质结构直接在细胞，分离的组织和类器官中。

项目成果

BRD4-mediated epigenetic regulation of endoplasmic reticulum-mitochondria contact sites is governed by the mitochondrial complex III.

BRD4 介导的内质网-线粒体接触位点的表观遗传调控由线粒体复合物 III 控制。

• DOI: 10.1101/2024.02.02.578646
• 发表时间: 2024
• 期刊: bioRxiv : the preprint server for biology
• 作者: Chen,Brandon;Lynn-Nguyen,TheophilusM;Jadhav,Pankaj;Halligan,BenjaminS;Rossiter,NicholasJ;Guerra,RachelM;Koshkin,Sergei;Koo,Imhoi;Morlacchi,Pietro;Hanna,DavidA;Lin,Jason;Banerjee,Ruma;Pagliarini,DavidJ;Patterson,AndrewD;M
• 通讯作者: M