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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要复杂的分隔成具有专用功能的膜封闭细胞器是真核细胞。细胞器彼此紧密并列，形成膜接触位点通信节点，对许多细胞功能至关重要。尽管它们无处不在，但留下的却很少了解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站点使得不可能使用传统的生化方法来纯化它们并开始经典的使用 X 晶体学、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，我们提出整合新颖的尖端显微镜模式，以直接观察细胞器内部的相互作用不受干扰的细胞环境。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分别是内质网、线粒体和溶酶体。该接触位点的蛋白质将被标记 iPSC 内，它们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使以高亚细胞精度检测这些接触成为可能。在荧光的引导下定位，薄电子透明窗口将在这些细胞内进行微机械加工。随后的冷冻电子断层扫描、次断层平均和深度分类将实现三维结构确定这些接触位点。我们的工作将有助于回答一个重要的生物学问题：ER-线粒体如何- 溶酶体接触位点标记线粒体裂变，这是维持线粒体健康供应所必需的过程细胞。在此期间建立的自动化样品制备、数据收集和处理的管道项目将作为未来现场结构研究的变革蓝图。该项目将奠定基础对新兴的、尚未开发的和复杂的领域进行高分辨率结构表征膜接触位点以及了解其在健康和疾病中的功能的机制基础。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框架直接在细胞、分离的组织和类器官中的蛋白质结构。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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：
发表时间：
2024-02-04
期刊：
影响因子：
0
作者：
Chen, Brandon;Lynn;Jadhav, Pankaj;Halligan, Benjamin S;Rossiter, Nicholas J;Guerra, Rachel M;Koshkin, Sergei;Koo, Imhoi;Morlacchi, Pietro;Hanna, David A;Lin, Jason;Banerjee, Ruma;Pagliarini, David J;Patterson, Andrew D;M
通讯作者：
M

Structure of the human heparan-α-glucosaminide N-acetyltransferase (HGSNAT).

人乙酰肝素-α-氨基葡萄糖N-乙酰转移酶（HGSNAT）的结构。