In situ structural cell biology - studying bioenergetics at the nanoscope.

线粒体是真核细胞的发电站，也是细胞能量单位三磷酸腺苷（ATP）再生的主要部位。细胞的生存和功能高度依赖于能量产生与能量消耗的匹配，因此线粒体中ATP的产生受到高度调节。能量生产调节中的扰动是许多人类疾病的一个强烈特征，包括癌症和神经病。ATP的产生是通过五种大膜蛋白（CI-CV）的氧化磷酸化（OXPHOS）作用进行的，这些大膜蛋白（CI-CV）嵌入线粒体高度折叠的内膜（嵴）中。CI-CIV利用富含电子的底物氧化产生的能量来建立质子的跨膜梯度，这被CV用来为ATP再生提供动力。据推测，随着细胞能量需求的增加，内膜的内折叠增加，以增加可容纳更多OXPHOS蛋白的表面积。此外，当细胞能量需求高时，CI-CIV形成的超复合体（SCs）更为普遍，这表明SCs在调节OXPHOS中的作用。此外，已知SCs的组成与civv数有特殊的关系。这是特别有趣的，因为CIV（细胞色素c氧化酶）是这些蛋白链的终端电子受体，通常被认为是OXPHOS的整体调节剂。最近的蛋白质组学分析表明，CIV亚基Cox7A的多个异构体可以控制CIV是作为单体、二聚体还是超复合体的一部分存在，并且异构体的表达水平在细胞类型、发育阶段和健康与疾病之间存在差异。在这个建议中，我们的目标是将细胞的生理能量需求与线粒体分子结构的变化联系起来，即线粒体嵴的折叠量和表面积，以及在不同生长条件下和不同的Cox7A异构体突变体中嵴中超复合体的数量、变异性和组织。为了实现我们的目标，我们将使用我们的合作者Cristina Ugalde博士现有的野生型和突变型人类细胞系，这些细胞系只表达一种Cox7A亚型。这些细胞将在不同的生长条件下生长，包括培养基中的高/低葡萄糖和ATP以及同步的细胞周期。将在不同的时间点采集线粒体，并对线粒体功能、SC形成和生物能量能力进行生化分析。选择变化最大的生长条件进行金刚石光源（DLS）纳米成像。为了分析嵴结构和表面积的变化，细胞将在em网格上生长，并在Diamond的B24软x射线光束线上成像。为了分析嵴膜中超复杂成分和组织的变化，将在Diamond的低温电子显微镜设备（eBIC）收集和分析分离的嵴膜的低温电子断层图。通过将DLS的生物成像技术与实验室生化和生物能量分析相结合，我们将能够确定线粒体膜和蛋白质组织的纳米级变化，这些变化伴随着能量产生的变化——这是理解能量调节和疾病的必要步骤。

Mitochondria are the powerhouse of eukaryotic cells and the principal site for the regeneration of the cellular energy unit adenosine triphosphate (ATP). A cell's survival and function is highly dependent on matching energy production with energy consumption and thus the production of ATP in mitochondria is highly regulated. Perturbance in energy production regulation is a strong feature of many human diseases including cancer and neuropathies. ATP production is carried out via oxidative phosphorylation (OXPHOS) by five large membrane proteins (CI-CV) embedded in the highly folded internal membranes of mitochondria (cristae). CI-CIV harness the energy from the oxidation of electron rich substrates to build up a transmembrane gradient of protons which is used by CV to power ATP regeneration.It is hypothesised that as energy demand of cells increase, the infolding of the inner membrane increases to increase the surface area available to house more OXPHOS proteins. Furthermore, CI-CIV form supercomplexes (SCs) that are more prevalent when cellular energy demand is high suggesting a role for SCs in regulating OXPHOS. In addition, it is known that the composition of SCs varies specifically in relation to CIV number. This is particularly interesting as CIV (cytochrome c oxidase) is the terminal electron acceptor of these protein chains and is often presented as the overall regulator of OXPHOS. Recent proteomics analysis suggested that the multiple isoforms of a CIV subunit Cox7A could control whether CIV exists as a monomer, dimer or part of a supercomplex and that the expression level of the isoforms varies between cell type, developmental stage and health versus disease.In this proposal we aim to link the physiological energy demand of cells with changes in the molecular structure of mitochondria - namely the amount of infolding and surface area of mitochondrial cristae, and the number, variability, and organisation of supercomplex in cristae under different growth conditions and in different Cox7A isoform mutants. To achieve our aim, we will be using existing wildtype and mutant human cell lines from our collaborator Dr. Cristina Ugalde which express only one Cox7A isoform. These cells will be grown under different growth conditions including high/low glucose and ATP in culture media and under synchronized cell cycles. Mitochondria will be harvested at different time points and analysed biochemically for mitochondrial function, SC formation and bioenergetic capabilities. Growth conditions which show greatest variation will be selected for nanoscale imaging at Diamond light source (DLS). To analyse changes in cristae structure and surface area, cells will be grown on EM-grids and imaged at Diamond's B24 soft x-ray beamline. To analyse changes in supercomplex composition and organisation in cristae membranes, cryo-electron tomograms of isolated cristae membranes will be collected and analysed at Diamond's cryo-electron microscopy facility (eBIC).Through the combination of DLS's bioimaging technologies with lab-based biochemical, and bioenergetic profiling we will be able to determine the nanoscopic changes in mitochondrial membrane and protein organisation which accompany changes in energy production - a necessary step for understanding energy regulation and disease.