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

摘要 各种激素对肝脏和其他组织中氧化代谢和其他线粒体功能的影响 细胞质[Ca2 +]振荡传播到线粒体。Ca2+被释放到 细胞质从内质网（ER）通过IP3受体（IP3Rs），其中，基于研究结果 从我们和其他人，暴露线粒体在ER-线粒体接触位点（ERMCs）高[Ca2 +]纳米域 以获得低亲和力线粒体Ca 2+摄取位点的活化。ERMC也在其他 这些过程包括脂质代谢、细胞器动力学和自噬。我们的研究揭示了 通过栓系蛋白支持ERMCs。我们已经创造了合成膜连接器来测量和干扰 ERMCs和活细胞中的局部细胞器间通讯，并提供了局部钙和反应性的线索， 氧物种（ROS）信号传导。我们还向数百个实验室提供了这些试剂 它们共同显示了细胞间接触在一系列范式中的作用， 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-线粒体偶联与包括肝脏在内的一系列器官疾病（例如脂肪肝）的关系。 然而，一些根本性的问题仍然没有答案。ERMC动态地进行重组，以满足 细胞的需求不断变化，但ERMCs如何形成和溶解还有待研究。 测定IP3R介导的[Ca2 +]波动可能提供一种控制接触形成的方法， 细胞质[Ca2 +]的升高通过Ca2+敏感的Miro阻止线粒体运动靠近ER 蛋白质，以及IP3R和Miro蛋白，都被认为是细胞器间的组分。 配合物然而，互动的伙伴和机制是难以捉摸的。我们假设IP3R和 Miros以同种型特异性和Ca2+依赖性方式介导ERMC形成，以调节 肝细胞的生理功能。目的#1和2将使用新的遗传和 显微成像工具包，使我们能够具体和系统地测量和扰动ERMC 形成元素。肝脏遗传扰动的影响将通过新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 也将作为IP3R的系链形成伙伴进行专门测试。最后，一个主要的限制，在该领域的 细胞器间通讯研究是对纳米尺度几何结构的定量评价 膜接触仍然困难。在目标#3中，我们将开发和表征测量方法， 在二维和三维电子显微镜数据中描述细胞器界面几何形状，并揭示结构 与钙转移最相关的特征。本论文的工作将对ERMC的分子机制进行探讨 动力学及其生理相关性，并将继续努力创造分子工具和方法 这使得许多研究人员能够探索ER和线粒体或其他细胞器的局部通信。

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.