ER-mitochondrial communication in calcium signaling, energy metabolism and liver disease

钙信号传导、能量代谢和肝脏疾病中的内质网线粒体通讯

基本信息

批准号：
10785141
负责人：
GYORGY CSORDAS
金额：
$ 14.59万
依托单位：
THOMAS JEFFERSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2026-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10785141
关键词：
3-Dimensional Affinity Autophagocytosis BCL2L1 gene Binding Calcium Calcium Signaling Cell Line Cell Respiration Cell Survival Cells Cellular biology Clustered Regularly Interspaced Short Palindromic Repeats Communication Communication Research Competence Complex Computer software Coupling Cytoplasm Data Data Set Dependence Descriptor Dimensions Disease Electron Microscopy Elements Endoplasmic Reticulum Energy Metabolism Evaluation Fatty Liver Genetic Geometry Health Hela Cells Hepatic Hepatocyte Hormones Image Impairment Laboratories Linear Models Link Liver Liver diseases Mammalian Cell Measures Mediating Medicine Membrane Metabolic Metabolism Methods Mitochondria Modeling Molecular Molecular Analysis Molecular Target Morphology Movement Neurons Nucleotides Obesity Organ Organelles Outer Mitochondrial Membrane Pharmaceutical Preparations Phenotype Physiological Process Production Protein Isoforms Proteins Proteomics Quantitative Evaluations Reactive Oxygen Species Reagent Regulation Research Personnel Role Scheme Scientist Signal Transduction Site Specificity Structure Structure-Activity Relationship Techniques Testing Tissues Vesicle Work cell type functional disability imaging approach immune function in vivo imaging lipid biosynthesis lipid metabolism microscopic imaging mitochondrial dysfunction mitochondrial membrane mouse model nano nanoscale non-alcoholic fatty liver non-alcoholic fatty liver disease novel protein complex receptor scaffold software development tool uptake

项目摘要

ABSTRACT The effects of various hormones on oxidative metabolism and other mitochondrial functions, in the liver and other tissues, are mediated by cytoplasmic [Ca2+] oscillations propagated to the mitochondria. Ca2+ is released to the cytoplasm from the endoplasmic reticulum (ER) through the IP3 receptors (IP3Rs), which, based on findings from us and others, expose mitochondria at ER-mitochondria contact sites (ERMCs) to high [Ca2+] nanodomains to attain activation of the low-affinity mitochondrial Ca2+ uptake sites. ERMCs were also recognized in other processes including lipid metabolism, organelle dynamics and autophagy. Our work has revealed the physical support of ERMCs by tethering proteins. We have created synthetic membrane linkers to measure and perturb ERMCs and local inter-organelle communication in live cells, and provided clues to local calcium and reactive oxygen species (ROS) signaling. We have also provided these reagents to several hundred laboratories worldwide, which together have showed the role of interorganellar contacts in a range of paradigms including metabolism, vesicle dynamics, neuronal and immune functions and linked structural or functional impairments of the ER-mitochondrial coupling to an array of disorders across organs including the liver (e.g. fatty liver). However, fundamental questions remain unanswered. ERMCs are dynamically restructured to meet the continuously changing demands of the cell, but how ERMCs are formed and dissolved is yet to be determined. IP3R-mediated fluctuations in [Ca2+] might provide a means to control contact formation, given that elevations of cytoplasmic [Ca2+] stop mitochondrial movements close to the ER through the Ca2+-sensing Miro proteins, and both the IP3R and Miro proteins, have been implicated as components of interorganellar complexes. However, the interaction partners and mechanisms are elusive. We hypothesize that IP3Rs and Miros mediate ERMC formation in an isoform-specific and Ca2+-dependent manner to regulate physiological functions of hepatocytes. Aims#1&2 will test this hypothesis using novel genetic and microscopic imaging toolkits that will enable us to specifically and systematically measure and perturb ERMC forming elements. The effect of genetic perturbations in the liver will be tested by novel, in vivo imaging approaches. The interactomes of IP3R and Miro will be evaluated primarily by unbiased proteomics, but Bcl-xL will also be specifically tested as a tether forming partner for the IP3R. Finally, a major limitation in the field of inter-organellar communication research is that quantitative evaluation of the geometry of nanometer scale membrane contacts remains difficult. In Aim#3 we will develop and characterize methods of measuring and describing organelle interface geometry in 2-and 3D electron microscopy data and uncover the structural features most relevant to calcium transfer. The proposed work will explore the molecular mechanisms of ERMC dynamics and their physiological relevance and will continue our efforts in creating molecular tools and methods that allow many investigators to explore the local communications of ER and mitochondria or other organelles.

抽象的各种激素对肝脏和其他器官中氧化代谢和其他线粒体功能的影响组织中，由传播到线粒体的细胞质 [Ca2+] 振荡介导。 Ca2+ 被释放到细胞质从内质网 (ER) 通过 IP3 受体 (IP3R)，根据研究结果我们和其他人将 ER-线粒体接触位点 (ERMC) 的线粒体暴露于高 [Ca2+] 纳米域以激活低亲和力线粒体 Ca2+ 吸收位点。 ERMC 也在其他领域得到认可过程包括脂质代谢、细胞器动力学和自噬。我们的工作揭示了身体通过束缚蛋白支持 ERMC。我们创建了合成膜连接器来测量和扰动 ERMC 和活细胞中的局部细胞器间通讯，并为局部钙和反应性提供线索氧物质（ROS）信号传导。我们还向数百个实验室提供了这些试剂在世界范围内，它们共同展示了细胞间接触在一系列范式中的作用，包括新陈代谢、囊泡动力学、神经元和免疫功能以及相关的结构或功能损伤 ER-线粒体与包括肝脏在内的一系列器官疾病的耦合（例如脂肪肝）。然而，基本问题仍未得到解答。 ERMC 进行动态重组以满足细胞的需求不断变化，但 ERMC 是如何形成和溶解的仍有待研究决定。 IP3R 介导的 [Ca2+] 波动可能提供一种控制接触形成的方法，因为细胞质 [Ca2+] 的升高阻止线粒体通过 Ca2+ 感应 Miro 靠近 ER 运动蛋白质，以及 IP3R 和 Miro 蛋白质，已被认为是细胞间质的组成部分复合物。然而，互动伙伴和机制却难以捉摸。我们假设 IP3R 和 Miros 以异构体特异性和 Ca2+ 依赖性方式介导 ERMC 形成，以调节肝细胞的生理功能。目标 #1 和 2 将使用新的遗传和显微成像工具包将使我们能够专门、系统地测量和扰动 ERMC 形成要素。肝脏中遗传扰动的影响将通过新颖的体内成像进行测试接近。 IP3R 和 Miro 的相互作用组将主要通过无偏蛋白质组学进行评估，但 Bcl-xL 还将作为 IP3R 的系绳形成伙伴进行专门测试。最后，该领域的一个主要限制细胞器间通讯研究是对纳米尺度几何结构的定量评价膜接触仍然很困难。在目标#3中，我们将开发和描述测量和表征的方法描述 2D 和 3D 电子显微镜数据中的细胞器界面几何形状并揭示结构与钙转移最相关的特征。拟议的工作将探索 ERMC 的分子机制动力学及其生理相关性，并将继续努力创建分子工具和方法这使得许多研究人员能够探索内质网和线粒体或其他细胞器的局部通讯。