isolation of enriched mitochondrial fractions from cell cultures and small animal tissue samples for gel activity assays

Mitochondrial Fractionation in Small Tissue Samples for Supercomplex Analysis

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.

Author Spotlight: Unveiling Oxidative Phosphorylation System Dynamics and Mitochondrial Roles in Health and Disease

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

Выделение обогащенных митохондриальных фракций из клеточных культур и образцов тканей мелких животных для анализа активности геля

Для начала суспендируйте упакованную клеточную гранулу в объеме гипотонического буфера, равном семикратному объему клеточной гранулы. Перенесите клеточную суспензию в гомогенизатор Potter-Dounce и инкубируйте на льду в течение двух минут, позволяя клеткам набухнуть. Разорвите клеточные мембраны, выполнив от 8 до 10 движений в гомогенизаторе, соединенном с тефлоновым пестиком с приводом от двигателя, вращающимся со скоростью 600 об/мин.Добавьте равный объем гипертонического буфера в клеточную суспензию для создания изотонической среды. Перелейте гомогенат в пробирку объемом от 10 до 15 миллилитров. Центрифугируйте при 1000G в течение пяти минут при четырех градусах Цельсия и соберите надосадочную жидкость в полипропиленовую пробирку объемом 1,5 миллилитров.Чтобы собрать митохондриальную сырую фракцию, центрифугируйте в микрофуге при температуре 16 000 G в течение двух минут при четырех градусах Цельсия. Выбросьте надосадочную жидкость и повторно суспендируйте обогащенную митохондриями гранулу с 0,5 миллилитрами буфера А. Объедините содержимое двух пробирок в одну и центрифугируйте, как было показано ранее. После последнего центрифугирования повторно суспендируйте гранулу в 300 микролитрах буфера А. Количественно определите концентрацию митохондриального белка с помощью анализа Брэдфорда.С помощью ножниц отрежьте от 20 до 30 миллиграммов ткани животного. Промойте салфетку три-четыре раза в гомогенизационном буфере с помощью ситечка, стараясь не потерять более мелкие кусочки. Переложите кусочки салфетки с буфером в гомогенизатор.Для работы с печенью, селезенкой и почечными тканями выполните от четырех до шести движений вверх и вниз в LVM Potter с помощью тефлонового пестика с моторным приводом со скоростью 600 об/мин.

Research

JoVE Journal

Biochemistry

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

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

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

Анализ митохондриальных суперкомплексов во фракциях тканей млекопитающих с помощью анализа активности в геле