Investigating the relationship between respiratory chain organisation and mitochondrial morphology by electron cryo-tomography

线粒体在公共领域很突出，突发新闻经常报道新发现的线粒体功能与其主要作用之间的联系-利用储存在食物中的能量以ATP的形式为细胞生存提供动力。越来越多的证据表明，线粒体功能障碍与衰老和帕金森氏症等神经退行性疾病有关。呼吸体是一个巨大的分子机器，它在线粒体内膜上进行细胞呼吸。它由四个络合物(I-IV)组成，它们将电子从NADH和琥珀酸转移到分子氧。通过这个过程获得的能量被用来将质子泵过线粒体内膜，从而产生用于产生ATP的膜电位。在所有影响能量代谢的线粒体疾病中，大约50%可以追溯到复合体I的一个亚基的突变。在令人兴奋的新数据中，我们发现复合体I的辅助蛋白在呼吸系统的稳定性中起着关键作用。击倒这种蛋白质对呼吸、产生活性氧物种(ROS)和降低内膜电位显示出额外的有害影响。通过尖端的成像技术，我们还观察到线粒体的形态被扰动，这可能对网络的形成和内膜蛋白质复合体的结构产生额外的影响。在这个多学科的项目中，包括荧光光学显微镜和高分辨率电子冷冻显微镜(CryoEM)在内的最先进的成像方法将被用来研究线粒体网络的破坏以及对我们的复合体I突变体的蛋白质结构的影响。这将与生化实验相结合，探索电子转移装置的结构和功能完整性。这些将包括分析呼吸超复合体的组装状态以及它们的电子转移、质子泵和氧气吸收活动。还将评估它们限制ROS过度(和破坏性)生产的能力。所获得的知识将用于阐明和指导与复杂的i功能障碍相关的疾病的进一步研究。

Mitochondria are prominent in the public domain, with breaking news stories often reporting on newly discovered links between mitochondrial function and their primary role- harnessing the energy stored in food to provide the power for cell survival in the form of ATP. There is increasing evidence that relates mitochondrial dysfunction with ageing and neurodegenerative disorders, such as Parkinson's disease. The respirasome is a massive molecular machine that carries out cellular respiration in the mitochondrial inner membrane. It is comprised of four complexes (I-IV), which transfer electrons from NADH and succinate to molecular oxygen. Energy gained through this process is used to pump protons across the inner mitochondrial membrane, leading to a membrane potential that is used to generate ATP. Approximately 50% of all mitochondrial disorders affecting energy metabolism can be traced back to mutations in one of the subunits of complex I.In exciting new data, we have discovered that an accessory protein of complex I plays a key role in the stability of the respirasome. Knock-down of this protein shows additional deleterious effects on respiration, production of reactive oxygen species (ROS) and reduction of the inner membrane potential. By cutting-edge imagining techniques, we have also observed that mitochondrial morphology is perturbed, which likely has additional consequences on network formation and the structure of protein complexes in the inner membrane.In this multi-disciplinary project, state of-the-art imaging methods including fluorescence light microscopy and high-resolution electron cryomicroscopy (cryoEM), will be used to investigate the break-down of mitochondrial networks and the consequence on protein structures in our complex I mutants. This will be combined with biochemical experiments to explore the structural and functional integrity of the electron transfer apparatus. These will include analysis of the assembled state of the respiratory super-complexes alongside their electron transfer, proton-pumping and oxygen uptake activities. Their ability to restrict excessive (and damaging) production of ROS will also be evaluated. Knowledge gained will be used to shed light on, and guide the further study of, diseases associated with complex I dysfunction.