analyzing mitochondrial supercomplexes in mammalian tissue fractions via in-gel activity assays

用于超复合物分析的小组织样品中的线粒体分离

Supercomplexes (SCs) are supramolecular associations between individual respiratory chain complexes1,2. Since the initial identification of SCs and the description of their composition by the group of Sch&#228;gger2,3, later confirmed by other groups, it was established that they contain respiratory complexes I, III, and IV (CI, CIII, and CIV, respectively) in different stoichiometries. Two main populations of SCs can be defined, those containing CI (and either CIII alone or CIII and CIV) and with very high molecular weight (MW, starting ~1.5 MDa for the smaller SC: CI + CIII2) and those containing CIII and CIV but not CI, with much smaller size (such as CIII2 + CIV with ~680 kDa). These SCs coexist in the inner mitochondrial membrane with free complexes, also in different proportions. Thus, while CI is mostly found in its associated forms (that is, in SCs: ~80% in bovine heart and more than 90% in many human cell types)3, CIV is very abundant in its free form (more than 80% in bovine heart), with CIII showing a more balanced distribution (~40% in its more abundant free form, as a dimer, in bovine heart).
While their existence is now generally accepted, their precise role is still under debate4,5,6,7,8,9,10. According to the plasticity model, different proportions of SCs and individual complexes can exist depending on the cell type or the metabolic status1,7,11. This dynamic nature of the assembly would allow cells to regulate the use of available fuels and the efficiency of the oxidative phosphorylation (OXPHOS) system in response to environmental changes4,5,7. SCs could also contribute to control the reactive oxygen species generation rate and participate in the stabilization and turnover of individual complexes4,12,13,14. Modifications of the SC assembly status have been described in association with different physiological and pathological situations15,16 and with the aging process17.
Thus, changes in the SC patterns have been described in yeast depending on the carbon source used for growth2&#160;and in cultured mammalian cells when glucose is substituted by galactose4. Modifications have also been reported in mouse liver after fasting8 and in astrocytes when mitochondrial fatty acid oxidation is blocked18. In addition, a decrease or alterations in SCs and OXPHOS have been found in Barth syndrome19, heart failure20, several metabolic21 and neurological22,23,24 disorders, and different tumors25,26,27,28. Whether these alterations in SC assembly and levels are a primary cause or represent secondary effects in these pathological situations is still under investigation15,16. Different methodologies can give information about the assembly and function of SCs; these include activity measurements8,29, ultrastructural analysis30,31, and proteomics32,33. A useful alternative that is increasingly being employed and is the starting point for some of the previously mentioned methodologies is the direct determination of SC assembly status by Blue native (BN) electrophoresis developed for this purpose by Sch&#228;gger&#39;s group34,35.
This approach requires reproducible and efficient procedures to obtain and solubilize mitochondrial membranes and can be complemented by other techniques such as in-gel activity analysis (IGA), second-dimension electrophoresis, and western blot (WB). A limitation in the studies on SC dynamics by BN electrophoresis can be the amount of starting cells or tissue samples. We present a series of protocols for the analysis of SC assembly and function, adapted from Sch&#228;gger&#39;s group methods, that can be applied to fresh or frozen cell or tissue samples starting from as little as 20 mg of tissue.

作者聚焦：揭示氧化磷酸化系统动力学和线粒体在健康和疾病中的作用

从小组织和细胞培养样品中分离线粒体用于线粒体超复合物分析 

Our research is focused on better understanding mitochondrial biogenesis, in particular, the OXPHOS System organization and Regulation, and also on defining the role that mitochondria play in diseases from cancer to neurodegenerative disorders. Mitochondria perform multiple and essential roles in the cell, conditioning its whole metabolism and survival. We're slowly increasing our knowledge of mitochondrial complexity, and its contribution to cell adaptation mechanisms in health and disease.We have contributed to establish a new organization model for the OXPHOS system, the plasticity model that integrates previous proposals and assumes the coexistence of super complexes and free complexes in different proportions to optimize its function and allow cellular adaptations. Our protocol allows starting from small tissues or cell culture samples to obtain enough mitochondrial protein amounts to analyze super complexes assembly and status and function by blue native electrophoresis or other techniques. We can do this reproducibly in a short time with a simple equipment and relatively with low costs.

通过凝胶内活性测定分析哺乳动物组织组分中的线粒体超复合物

首先将从哺乳动物细胞培养物或组织获得的线粒体组分重悬于蓝色天然样品缓冲液中，以获得约 10 毫克/毫升的蛋白质浓度。为了溶解线粒体膜，添加洋地黄皂苷以获得每克线粒体蛋白 4 克洋地黄皂苷的比例。轻轻移液混匀，并在冰上孵育 5 分钟。将悬浮液放入微量离心机中，在 20， 000 G 下在 4 摄氏度下离心 25 分钟，以去除不溶性物质。将上清液收集在新管中，然后加入 5% 考马斯蓝 G-250，相当于初始重悬体积的三分之一，并通过移液混合。将冷阴极 A 缓冲液加入电泳仪的上腔室，将阳极缓冲液加入下腔室。将 30 至 100 μg 线粒体蛋白加载到聚丙烯酰胺凝胶上的孔中。电泳后，将凝胶放入塑料盒中。加入足够体积的适当凝胶活性测定溶液以覆盖凝胶，并在室温下轻轻摇动孵育，避光。当适当的条带形成时，去除检测溶液。用蒸馏水洗涤凝胶两次，然后用 40% 甲醇和 10% 乙酸固定 30 分钟。凝胶活性测定分析揭示了小鼠和人类细胞中不同的超级复合物组装模式。在小鼠细胞中观察到游离复合物 1，而在人线粒体中未检测到。复合物 4 模式在人类细胞系中相似，但由于 SCAF1 的突变变体，在小鼠细胞系中有所不同。

analyzing-mitochondrial-supercomplexes-mammalian-tissue-fractions-via

Research

JoVE Journal

Biochemistry

Analyzing Mitochondrial Supercomplexes in Mammalian Tissue Fractions via In-Gel Activity Assays

83 次观看.  University of Zaragoza. 首先将从哺乳动物细胞培养物或组织获得的线粒体组分重悬于蓝色天然样品缓冲液中，以获得约 10 毫克/毫升的蛋白质浓度。为了溶解线粒体膜，添加洋地黄皂苷以获得每克线粒体蛋白 4 克洋地黄皂苷的比例。轻轻移液混匀，并在冰上孵育 5 分钟。将悬浮液放入微量离心机中，在 20， 000 G 下在 4 摄氏度下离心 25 分钟，以去除不溶性物质。将上清液收集在新管中，然后加...

视频: 通过凝胶内活性测定分析哺乳动物组织组分中的线粒体超复合物

Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples 

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial electron transport chain organization, giving rise to the proposal of the "plasticity model." This model postulates the coexistence of different proportions of SCs and complexes depending on the tissue or the cellular metabolic status. The dynamic nature of the assembly in SCs would allow cells to optimize the use of available fuels and the efficiency of electron transfer, minimizing reactive oxygen species generation and favoring the ability of cells to adapt to environmental changes. 
More recently, abnormalities in SC assembly have been reported in different diseases such as neurodegenerative disorders (Alzheimer's and Parkinson's disease), Barth Syndrome, Leigh syndrome, or cancer. The role of SC assembly alterations in disease progression still needs to be confirmed. Nevertheless, the availability of enough amounts of samples to determine the SC assembly status is often a challenge. This happens with biopsy or tissue samples that are small or have to be divided for multiple analyses, with cell cultures that have slow growth or come from microfluidic devices, with some primary cultures or rare cells, or when the effect of particular costly treatments has to be analyzed (with nanoparticles, very expensive compounds, etc.). In these cases, an efficient and easy-to-apply method is required. This paper presents a method adapted to obtain enriched mitochondrial fractions from small amounts of cells or tissues to analyze the structure and function of mitochondrial SCs by native electrophoresis followed by in-gel activity assays or western blot.

This protocol describes a technique for the analysis of respiratory supercomplexes when only small amounts of samples are available.

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial ...