analyzing mitochondrial supercomplexes in mammalian tissue fractions via in-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 

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.

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

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

Research

JoVE Journal

Biochemistry

65 ビュー.  University of Zaragoza. まず、哺乳類の細胞培養物または組織から得られたミトコンドリア画分を青色の天然サンプルバッファーに再懸濁して、1ミリリットルあたり約10ミリグラムのタンパク質濃度を得ます。ミトコンドリア膜を可溶化するためには、ジギトニンを添加して、ミトコンドリアタンパク質1グラム当たり4グラムのジギトニンの比率を得る。穏やかなピペッティングで混合し、氷上...