isolation of enriched mitochondrial fractions from cell cultures and small animal tissue samples for 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.

从细胞培养物和小动物组织样品中分离富集的线粒体组分，用于凝胶活性测定

首先，将填充的细胞沉淀重悬于等于细胞沉淀体积 7 倍的低渗缓冲液中。将细胞悬液转移到 Potter-Dounce 匀浆器中，在冰上孵育 2 分钟，让细胞膨胀。通过在均质器中执行 8 到 10 次冲程来打破细胞膜，该匀浆器与以 600 RPM 旋转的电机驱动的特氟龙杵相连。向细胞悬液中加入等体积的高渗缓冲液，以产生等渗环境。将匀浆转移到 10 至 15 毫升的试管中。在 4 摄氏度下以 1， 000G 离心 5 分钟，然后将上清液收集到 1.5 毫升聚丙烯管中。要收集线粒体粗馏分，请在微量离心机中以 16， 000G 的速度在 4 摄氏度下离心 2 分钟。弃去上清液，用 0.5 毫升缓冲液重悬富含线粒体的沉淀 A.将两个试管的内容物合二为一，如前所述离心。最后一次离心后，将沉淀重悬于 300 微升缓冲液中 A.使用 Bradford 测定定量线粒体蛋白浓度。用剪刀剪下 20 到 30 毫克的动物组织。在过滤器的帮助下，在匀浆缓冲液中洗涤组织 3 到 4 次，注意避免丢失较小的碎片。将含有缓冲液的组织块转移到匀浆器中。对于肝脏、脾脏和肾脏组织，使用 LVM Potter 和电动特氟龙杵以 600 RPM 的速度进行 4 到 6 次上下冲程。

isolation-enriched-mitochondrial-fractions-from-cell-cultures-small

Research

JoVE Journal

Biochemistry

Isolation of Enriched Mitochondrial Fractions from Cell Cultures and Small Animal Tissue Samples for Gel Activity Assays

134 次观看.  University of Zaragoza. 首先，将填充的细胞沉淀重悬于等于细胞沉淀体积 7 倍的低渗缓冲液中。将细胞悬液转移到 Potter-Dounce 匀浆器中，在冰上孵育 2 分钟，让细胞膨胀。通过在均质器中执行 8 到 10 次冲程来打破细胞膜，该匀浆器与以 600 RPM 旋转的电机驱动的特氟龙杵相连。向细胞悬液中加入等体积的高渗缓冲液，以产生等渗环境。将匀浆转移到 10 至 15 毫升的试管中。在 4 摄氏度下以 1， 0...

视频: 从细胞培养物和小动物组织样品中分离富集的线粒体组分，用于凝胶活性测定

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 ...