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 组织，是由细胞质[钙]振荡传播到线粒体。将CA2发布给 细胞质从内质网(ER)通过IP3受体(IP3Rs)，这是基于发现 来自我们和其他人的，将内质网-线粒体接触点(ERMC)的线粒体暴露在高[钙]纳米结构域中 激活线粒体低亲和力的钙摄取位点。ERMCs在其他国家也得到认可 过程包括脂类代谢、细胞器动力学和自噬。我们的研究揭示了 系留蛋白对ERMCs的支持。我们已经创造了合成膜连接物来测量和扰动 ERMCs和活细胞内局部细胞器间的通讯，并提供了局部钙和反应的线索 氧物种(ROS)发出信号。我们还向数百个实验室提供了这些试剂 全世界，它们共同显示了细胞器间联系在一系列范例中的作用，包括 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害 内质网-线粒体耦合到包括肝脏在内的一系列器官紊乱(例如脂肪肝)。 然而，根本问题仍然没有得到回答。ERMC被动态重组以满足 对细胞的需求不断变化，但ERMCs是如何形成和溶解的还有待研究 下定决心。IP3R介导的[Ca~(2+)]波动可能提供一种控制接触形成的手段，因为 胞浆内[Ca~(2+)]升高通过钙敏感的Miro阻止线粒体靠近内质网的运动 蛋白质以及IP3R和MIRO蛋白都被认为是细胞器间的组成部分。 复合体。然而，互动伙伴和机制是难以捉摸的。我们假设IP3R和 Miros以异构体特异性和钙依赖的方式调节ERMC的形成 肝细胞的生理功能。AIMS#1和2将使用新的基因和 显微成像工具包，将使我们能够专门和系统地测量和干扰ERMC 形成元素。基因突变对肝脏的影响将通过新的活体成像进行测试。 接近了。IP3R和MIRO的相互作用将主要通过无偏倚蛋白质组学进行评估，但Bclxl 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是 细胞器间通讯研究是对纳米尺度几何形状的定量评价 膜接触仍然很困难。在目标3中，我们将开发和描述测量和 在二维和三维电子显微镜数据中描述细胞器界面几何并揭示结构 与钙转移最相关的特征。这项拟议的工作将探索ERMC的分子机制 动力学及其生理相关性，并将继续努力创造分子工具和方法 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。

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.