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

Mitochondriale Fraktionierung in kleinen Gewebeproben für die superkomplexe Analyse

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.

Autor im Rampenlicht: Enthüllung der Dynamik des oxidativen Phosphorylierungssystems und der mitochondrialen Rolle bei Gesundheit und Krankheit

Isolierung von Mitochondrien für die mitochondriale Superkomplexanalyse aus kleinen Gewebe- und Zellkulturproben 

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.

Isolierung von angereicherten mitochondrialen Fraktionen aus Zellkulturen und kleintierischen Gewebeproben für Gelaktivitätsassays

Zu Beginn resuspendieren Sie das gepackte Zellpellet in einem Volumen hypotonen Puffers, das dem Siebenfachen des Zellpelletvolumens entspricht. Übertragen Sie die Zellsuspension in einen Potter-Dounce-Homogenisator und inkubieren Sie sie zwei Minuten lang auf Eis, damit die Zellen aufquellen können. Brechen Sie die Zellmembranen, indem Sie 8 bis 10 Hübe im Homogenisator ausführen, der mit einem motorbetriebenen Teflonstößel gekoppelt ist, der sich mit 600 U/min dreht.Fügen Sie der Zellsuspension ein gleiches Volumen an hypertonischem Puffer hinzu, um eine isotonische Umgebung zu erzeugen. Füllen Sie das Homogenat in ein 10- bis 15-Milliliter-Röhrchen. Zentrifugieren Sie fünf Minuten lang bei 1.000 g bei vier Grad Celsius und sammeln Sie den Überstand in einem 1,5-Milliliter-Polypropylen-Röhrchen.Um die mitochondriale Rohfraktion zu sammeln, zentrifugieren Sie in einer Mikrofuge bei 16.000 g zwei Minuten lang bei vier Grad Celsius. Den Überstand verwerfen und das mit Mitochondrien angereicherte Pellet mit 0,5 Millilitern Puffer A resuspendieren.Kombinieren Sie den Inhalt von zwei Röhrchen in einem und zentrifugieren Sie wie zuvor gezeigt. Nach der letzten Zentrifugation resuspendieren Sie das Pellet in 300 Mikrolitern Puffer A. Quantifizieren Sie die mitochondriale Proteinkonzentration mit dem Bradford-Assay.Schneiden Sie mit einer Schere die 20 bis 30 Milligramm tierisches Gewebe ab. Waschen Sie das Tuch drei- bis viermal mit Hilfe eines Siebs in Homogenisierungspuffer und achten Sie darauf, dass die kleineren Stücke nicht verloren gehen. Übertragen Sie die Gewebestücke mit Puffer in den Homogenisator.Führen Sie für Leber-, Milz- und Nierengewebe vier bis sechs Auf- und Abbewegungen im LVM Potter mit einem motorbetriebenen Teflonstößel bei 600 U/min durch.

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

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 ...