Bu çalışma, Ministerio de Ciencia e Innovación'dan (https://ciencia.sede.gob.es/) "PGC2018-095795-B-I00" hibe numarası ve Diputación General de Aragón'dan (DGA) (https://www.aragon.es/) PF-S ve RM-L'ye "Grupo de Referencia: E35_17R" hibe numarası ve "LMP220_21" hibe numarası ile desteklenmiştir.

Süper Kompleks Analiz için Küçük Doku Örneklerinde Mitokondriyal Fraksiyonlama

<ol>
	<li>Acin-Perez, R., Fernandez-Silva, P., Peleato, M. L., Perez-Martos, A., Enriquez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+active+mitochondrial+supercomplexes.">Respiratory active mitochondrial supercomplexes.</a> Mol Cell. 32 (4), 529-539 (2008).</li><li>Schagger, H., Pfeiffer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supercomplexes+in+the+respiratory+chains+of+yeast+and+mammalian+mitochondria.">Supercomplexes in the respiratory chains of yeast and mammalian mitochondria.</a> EMBO J. 19 (8), 1777-1783 (2000).</li><li>Schagger, H., Pfeiffer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ratio+of+oxidative+phosphorylation+complexes+I-V+in+bovine+heart+mitochondria+and+the+composition+of+respiratory+chain+supercomplexes.">The ratio of oxidative phosphorylation complexes I-V in bovine heart mitochondria and the composition of respiratory chain supercomplexes.</a> J Biol Chem. 276 (41), 37861-37867 (2001).</li><li>Acin-Perez, R., Enriquez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+function+of+the+respiratory+supercomplexes:+the+plasticity+model.">The function of the respiratory supercomplexes: the plasticity model.</a> Biochim Biophys Acta. 1837 (4), 444-450 (2014).</li><li>Cogliati, S., Cabrera-Alarcon, J. L., Enriquez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+functional+role+of+the+electron+transport+chain+supercomplexes.">Regulation and functional role of the electron transport chain supercomplexes.</a> Biochem Soc Trans. 49 (6), 2655-2668 (2021).</li><li>Genova, M. L., Lenaz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+role+of+mitochondrial+respiratory+supercomplexes.">Functional role of mitochondrial respiratory supercomplexes.</a> Biochim Biophys Acta. 1837 (4), 427-443 (2014).</li><li>Kohler, A., Barrientos, A., Fontanesi, F., Ott, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functional+significance+of+mitochondrial+respiratory+chain+supercomplexes.">The functional significance of mitochondrial respiratory chain supercomplexes.</a> EMBO Rep. 24 (11), e57092 (2023).</li><li>Lapuente-Brun, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supercomplex+assembly+determines+electron+flux+in+the+mitochondrial+electron+transport+chain.">Supercomplex assembly determines electron flux in the mitochondrial electron transport chain.</a> Science. 340 (6140), 1567-1570 (2013).</li><li>Milenkovic, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preserved+respiratory+chain+capacity+and+physiology+in+mice+with+profoundly+reduced+levels+of+mitochondrial+respirasomes.">Preserved respiratory chain capacity and physiology in mice with profoundly reduced levels of mitochondrial respirasomes.</a> Cell Metab. 35 (10), 1799-1813 (2023).</li><li>Vercellino, I., Sazanov, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+assembly,+regulation+and+function+of+the+mitochondrial+respiratory+chain.">The assembly, regulation and function of the mitochondrial respiratory chain.</a> Nat Rev Mol Cell Biol. 23 (2), 141-161 (2022).</li><li>Moreno-Loshuertos, R., Fern&#225;ndez-Silva, P., Ostojic, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Clinical Bioenergetics. , 3-60 (2021).</li><li>Fernandez-Vizarra, E., Ugalde, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+assembly+of+the+mitochondrial+respiratory+chain.">Cooperative assembly of the mitochondrial respiratory chain.</a> Trends Biochem Sci. 47 (12), 999-1008 (2022).</li><li>Javadov, S., Jang, S., Chapa-Dubocq, X. R., Khuchua, Z., Camara, A. K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+respiratory+supercomplexes+in+mammalian+cells:+structural+versus+functional+role.">Mitochondrial respiratory supercomplexes in mammalian cells: structural versus functional role.</a> Journal of Molecular Medicine. 99 (1), 57-73 (2021).</li><li>Lopez-Fabuel, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+I+assembly+into+supercomplexes+determines+differential+mitochondrial+ROS+production+in+neurons+and+astrocytes.">Complex I assembly into supercomplexes determines differential mitochondrial ROS production in neurons and astrocytes.</a> Proc Natl Acad Sci U S A. 113 (46), 13063-13068 (2016).</li><li>Mukherjee, S., Ghosh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+of+mitochondrial+respiratory+chain+assembly+and+its+relation+to+mitochondrial+diseases.">Molecular mechanism of mitochondrial respiratory chain assembly and its relation to mitochondrial diseases.</a> Mitochondrion. 53, 1-20 (2020).</li><li>Nesci, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+supramolecular+structure+of+the+mitochondrial+oxidative+phosphorylation+system:+implications+for+pathology.">Molecular and supramolecular structure of the mitochondrial oxidative phosphorylation system: implications for pathology.</a> Life (Basel). 11 (3), 242 (2021).</li><li>Frenzel, M., Rommelspacher, H., Sugawa, M. D., Dencher, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ageing+alters+the+supramolecular+architecture+of+OxPhos+complexes+in+rat+brain+cortex.">Ageing alters the supramolecular architecture of OxPhos complexes in rat brain cortex.</a> Exp Gerontol. 45 (7-8), 563-572 (2010).</li><li>Morant-Ferrando, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid+oxidation+organizes+mitochondrial+supercomplexes+to+sustain+astrocytic+ROS+and+cognition.">Fatty acid oxidation organizes mitochondrial supercomplexes to sustain astrocytic ROS and cognition.</a> Nat Metab. 5 (8), 1290-1302 (2023).</li><li>McKenzie, M., Lazarou, M., Thorburn, D. R., Ryan, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+respiratory+chain+supercomplexes+are+destabilized+in+Barth+Syndrome+patients.">Mitochondrial respiratory chain supercomplexes are destabilized in Barth Syndrome patients.</a> J Mol Biol. 361 (3), 462-469 (2006).</li><li>Rosca, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+mitochondria+in+heart+failure:+decrease+in+respirasomes+and+oxidative+phosphorylation.">Cardiac mitochondria in heart failure: decrease in respirasomes and oxidative phosphorylation.</a> Cardiovasc Res. 80 (1), 30-39 (2008).</li><li>Ramirez-Camacho, I., Garcia-Nino, W. R., Flores-Garcia, M., Pedraza-Chaverri, J., Zazueta, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alteration+of+mitochondrial+supercomplexes+assembly+in+metabolic+diseases.">Alteration of mitochondrial supercomplexes assembly in metabolic diseases.</a> Biochim Biophys Acta Mol Basis Dis. 1866 (12), 165935 (2020).</li><li>Gonzalez-Rodriguez, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+mitochondrial+complex+I+induces+progressive+parkinsonism.">Disruption of mitochondrial complex I induces progressive parkinsonism.</a> Nature. 599 (7886), 650-656 (2021).</li><li>Novack, G. V., Galeano, P., Castano, E. M., Morelli, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+supercomplexes:+physiological+organization+and+dysregulation+in+age-related+neurodegenerative+disorders.">Mitochondrial supercomplexes: physiological organization and dysregulation in age-related neurodegenerative disorders.</a> Front Endocrinol (Lausanne). 11, 600 (2020).</li><li>Ramirez-Camacho, I., Flores-Herrera, O., Zazueta, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relevance+of+the+supramolecular+arrangements+of+the+respiratory+chain+complexes+in+human+diseases+and+aging.">The relevance of the supramolecular arrangements of the respiratory chain complexes in human diseases and aging.</a> Mitochondrion. 47, 266-272 (2019).</li><li>Hollinshead, K. E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+Supercomplexes+Promote+Mitochondrial+Efficiency+and+Growth+in+Severely+Hypoxic+Pancreatic+Cancer.">Respiratory Supercomplexes Promote Mitochondrial Efficiency and Growth in Severely Hypoxic Pancreatic Cancer.</a> Cell Rep. 33 (1), 108231 (2020).</li><li>Ikeda, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+supercomplex+assembly+promotes+breast+and+endometrial+tumorigenesis+by+metabolic+alterations+and+enhanced+hypoxia+tolerance.">Mitochondrial supercomplex assembly promotes breast and endometrial tumorigenesis by metabolic alterations and enhanced hypoxia tolerance.</a> Nat Commun. 10 (1), 4108 (2019).</li><li>Kamada, S., Takeiwa, T., Ikeda, K., Horie, K., Inoue, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+roles+of+COX7RP+and+mitochondrial+oxidative+phosphorylation+in+breast+cancer.">Emerging roles of COX7RP and mitochondrial oxidative phosphorylation in breast cancer.</a> Front Cell Dev Biol. 10, 717881 (2022).</li><li>Marco-Brualla, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+the+ND2+subunit+of+mitochondrial+complex+I+are+sufficient+to+confer+increased+tumorigenic+and+metastatic+potential+to+cancer+cells.">Mutations in the ND2 subunit of mitochondrial complex I are sufficient to confer increased tumorigenic and metastatic potential to cancer cells.</a> Cancers (Basel). 11 (7), 1027 (2019).</li><li>Moreno-Loshuertos, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+hot+can+mitochondria+be?+Incubation+at+temperatures+above+43+degrees+C+induces+the+degradation+of+respiratory+complexes+and+supercomplexes+in+intact+cells+and+isolated+mitochondria.">How hot can mitochondria be? Incubation at temperatures above 43 degrees C induces the degradation of respiratory complexes and supercomplexes in intact cells and isolated mitochondria.</a> Mitochondrion. 69, 83-94 (2023).</li><li>Vonck, J., Schafer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supramolecular+organization+of+protein+complexes+in+the+mitochondrial+inner+membrane.">Supramolecular organization of protein complexes in the mitochondrial inner membrane.</a> Biochim Biophys Acta. 1793 (1), 117-124 (2009).</li><li>Althoff, T., Mills, D. J., Popot, J. L., Kuhlbrandt, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arrangement+of+electron+transport+chain+components+in+bovine+mitochondrial+supercomplex+I1III2IV1.">Arrangement of electron transport chain components in bovine mitochondrial supercomplex I1III2IV1.</a> EMBO J. 30 (22), 4652-4664 (2011).</li><li>Cogliati, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+super-assembly+of+respiratory+complexes+III+and+IV.">Mechanism of super-assembly of respiratory complexes III and IV.</a> Nature. 539 (7630), 579-582 (2016).</li><li>Gonzalez-Franquesa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass-spectrometry-based+proteomics+reveals+mitochondrial+supercomplexome+plasticity.">Mass-spectrometry-based proteomics reveals mitochondrial supercomplexome plasticity.</a> Cell Rep. 35 (8), 109180 (2021).</li><li>Wittig, I., Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+and+applications+of+blue-native+and+clear-native+electrophoresis.">Features and applications of blue-native and clear-native electrophoresis.</a> Proteomics. 8 (19), 3974-3990 (2008).</li><li>Wittig, I., Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Native+electrophoretic+techniques+to+identify+protein-protein+interactions.">Native electrophoretic techniques to identify protein-protein interactions.</a> Proteomics. 9 (23), 5214-5223 (2009).</li><li>Garcia-Cazarin, M. L., Snider, N. N., Andrade, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+isolation+from+skeletal+muscle.">Mitochondrial isolation from skeletal muscle.</a> J Vis Exp. (49), e2452 (2011).</li><li>Lai, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mitochondrial+subpopulations+from+skeletal+muscle:+Optimizing+recovery+and+preserving+integrity.">Isolation of mitochondrial subpopulations from skeletal muscle: Optimizing recovery and preserving integrity.</a> Acta Physiol (Oxf). 225 (2), e13182 (2019).</li><li>Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Native+electrophoresis+for+isolation+of+mitochondrial+oxidative+phosphorylation+protein+complexes.">Native electrophoresis for isolation of mitochondrial oxidative phosphorylation protein complexes.</a> Methods Enzymol. 260, 190-202 (1995).</li><li>Wittig, I., Braun, H. P., Schagger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+PAGE.">Blue native PAGE.</a> Nat Protoc. 1 (1), 418-428 (2006).</li><li>Chomyn, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet-mediated+transformation+of+mtDNA-less+human+cells:+analysis+of+phenotypic+variability+among+clones+from+normal+individuals--and+complementation+behavior+of+the+tRNALys+mutation+causing+myoclonic+epilepsy+and+ragged+red+fibers.">Platelet-mediated transformation of mtDNA-less human cells: analysis of phenotypic variability among clones from normal individuals--and complementation behavior of the tRNALys mutation causing myoclonic epilepsy and ragged red fibers.</a> Am J Hum Genet. 54 (6), 966-974 (1994).</li><li>Moreno-Loshuertos, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+reactive+oxygen+species+production+explain+the+phenotypes+associated+with+common+mouse+mitochondrial+DNA+variants.">Differences in reactive oxygen species production explain the phenotypes associated with common mouse mitochondrial DNA variants.</a> Nat Genet. 38 (11), 1261-1268 (2006).</li><li>Fern&#225;ndez-Vizarra, E., Fern&#225;ndez-Silva, P., Enr&#237;quez, J. A., Celis, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Biology (Third Edition). , 69-77 (2006).</li><li>Cogliati, S., Herranz, F., Ruiz-Cabello, J., Enr&#237;quez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digitonin+concentration+is+determinant+for+mitochondrial+supercomplexes+analysis+by+BlueNative+page.">Digitonin concentration is determinant for mitochondrial supercomplexes analysis by BlueNative page.</a> Biochim Biophys Acta Bioenerg. 1862 (1), 148332 (2021).</li></ol>

Yazarlar herhangi bir çıkar çatışması beyan etmemektedir.

Burada açıklanan protokollerde tanıtılan metodolojik uyarlamalar, mitokondriyal kompleks aktiviteleri korurken (yeterli miktarda numunenin mevcudiyeti tehlikeye girdiğinde çok önemlidir) kayıpları önlemeyi ve verimi artırmayı ve dokunun veya hücre hattının beklenen SC modelini yeniden üretmeyi amaçlamaktadır (bkz. Şekil 2C). Bu amaçla ve SK'leri düzgün bir şekilde tespit etmek için yüksek bir mitokondriyal saflık gerekmediğinden, bu mümkün olduğunda adım, süre...

Süper kompleksler (SK'ler), bireysel solunum zinciri kompleksleriarasındaki supramoleküler ilişkilerdir 1,2. SC'lerin ilk tanımlanması ve bileşimlerinin Schägger 2,3 grubu tarafından tanımlanmasından bu yana, daha sonra diğer gruplar tarafından onaylandığından, farklı stokiyometrilerde solunum kompleksleri I, III ve IV (SIRASIYLA CI, CIII ve CIV) içerdikleri tespit edilmiştir. İki ana SK popülasyonu tanımlanabilir, CI içerenler (ve tek başına CIII veya CIII ve CIV) ve çok yüksek moleküler ağırlığa sahip olanlar (MW, daha küçük SC için ~ 1.5 MDa'dan başlar: CI + CIII2) ve CIII ve CIV içerenler ancak CI içermeyenler, çok daha küçük boyutlu olanlar (~ 680 kDa'lı CIII2 + CIV gibi). Bu SC'ler, iç mitokondriyal zarda da farklı oranlarda serbest komplekslerle bir arada bulunur. Bu nedenle, CI çoğunlukla ilişkili formlarında bulunurken (yani, SC'lerde: sığır kalbinde ~% 80 ve birçok insan hücre tipinde% 90'dan fazla)3, CIV serbest formunda çok bol miktarda bulunur (sığır kalbinde% 80'den fazla), CIII daha dengeli bir dağılım gösterir (daha bol serbest formunda ~% 40, bir dimer olarak, sığır kalbinde).
Varlıkları artık genel olarak kabul edilse de, kesin rolleri hala tartışılmaktadır 4,5,6,7,8,9,10. Plastisite modeline göre, hücre tipine veya metabolik duruma bağlı olarak farklı oranlarda SK'ler ve bireysel kompleksler mevcut olabilir 1,7,11. Düzeneğin bu dinamik doğası, hücrelerin çevresel değişikliklere yanıt olarak mevcut yakıtların kullanımını ve oksidatif fosforilasyon (OXPHOS) sisteminin verimliliğini düzenlemesine izin verecektir 4,5,7. SK'ler ayrıca reaktif oksijen türlerinin üretim hızının kontrolüne katkıda bulunabilir ve bireysel komplekslerin stabilizasyonuna ve cirosuna katılabilir 4,12,13,14. SC montaj durumundaki modifikasyonlar, farklı fizyolojik ve patolojik durumlarla15,16 ve yaşlanma süreci17 ile ilişkili olarak tanımlanmıştır.
Bu nedenle, SC modellerindeki değişiklikler, büyüme2 için kullanılan karbon kaynağına bağlı olarak mayada ve glikoz galaktoz4 ile ikame edildiğinde kültürlenmiş memeli hücrelerinde tanımlanmıştır. Oruç tuttuktan sonra fare karaciğerinde8 ve mitokondriyal yağ asidi oksidasyonubloke edildiğinde astrositlerde de değişiklikler bildirilmiştir 18. Ek olarak, Barth sendromu19, kalp yetmezliği20, çeşitli metabolik21 ve nörolojik 22,23,24 bozukluklarda ve farklı tümörlerde SK'lerde ve OXPHOS'ta bir azalma veya değişiklikler bulunmuştur 25,26,27,28. SK montajındaki ve seviyelerindeki bu değişikliklerin bu patolojik durumlarda birincil neden olup olmadığı veya ikincil etkileri temsil edip etmediği hala araştırılmaktadır15,16. Farklı metodolojiler, SC'lerin montajı ve işlevi hakkında bilgi verebilir; Bunlar arasında aktivite ölçümleri 8,29, ultrastrüktürel analiz30,31 ve proteomik32,33 yer alır. Giderek daha fazla kullanılan ve daha önce bahsedilen metodolojilerin bazıları için başlangıç noktası olan yararlı bir alternatif, Schägger'in grubu34,35 tarafından bu amaç için geliştirilen Blue native (BN) elektroforezi ile SC montaj durumunun doğrudan belirlenmesidir.
Bu yaklaşım, mitokondriyal zarları elde etmek ve çözündürmek için tekrarlanabilir ve verimli prosedürler gerektirir ve jel içi aktivite analizi (IGA), ikinci boyut elektroforezi ve western blot (WB) gibi diğer tekniklerle tamamlanabilir. BN elektroforezi ile SK dinamiği üzerine yapılan çalışmalarda bir sınırlama, başlangıç hücrelerinin veya doku örneklerinin miktarı olabilir. SC montajı ve fonksiyonunun analizi için, Schägger'in grup yöntemlerinden uyarlanan, 20 mg kadar küçük bir dokudan başlayarak taze veya donmuş hücre veya doku örneklerine uygulanabilen bir dizi protokol sunuyoruz.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetic acid</td>
			<td>PanReac</td>
			<td>131008</td>
			<td></td>
		</tr>
		<tr>
			<td>Aminocaproic acid</td>
			<td>Fluka Analytical</td>
			<td>7260</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma-Aldrich</td>
			<td>A2383</td>
			<td></td>
		</tr>
		<tr>
			<td>Bis Tris</td>
			<td>Acrons Organics</td>
			<td>327721000</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford assay</td>
			<td>Biorad</td>
			<td>5000002</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Blue G-250</td>
			<td>Serva</td>
			<td>17524</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Blue R-250</td>
			<td>Merck</td>
			<td>1125530025</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytochrome c</td>
			<td>Sigma-Aldrich</td>
			<td>C2506</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamino&#160; benzidine (DAB)</td>
			<td>Sigma-Aldrich</td>
			<td>D5637</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitonin</td>
			<td>Sigma-Aldrich</td>
			<td>D5628</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>PanReac</td>
			<td>131669</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Fatty acids free BSA</td>
			<td>Roche</td>
			<td>10775835001</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>PanReac</td>
			<td>A1067</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer Teflon pestle</td>
			<td>Deltalab</td>
			<td>196102</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I2399</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>PanReac</td>
			<td>121512</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>PanReac</td>
			<td>121509</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Sigma-Aldrich</td>
			<td>M4125</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Labkem</td>
			<td>MTOL-P0P</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>PanReac</td>
			<td>131404</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Trans-Blot Cell</td>
			<td>BioRad</td>
			<td>1703930</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma-Aldrich</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr>
			<td>MTCO1 Monoclonal Antibody</td>
			<td>Invitrogen</td>
			<td>459600</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>NADH</td>
			<td>Roche</td>
			<td>10107735001</td>
			<td></td>
		</tr>
		<tr>
			<td>NativePAGE 3 to 12% Mini Protein Gels</td>
			<td>Invitrogen</td>
			<td>BN1001BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>NativePAGE Cathode Buffer Additive (20x)</td>
			<td>Invitrogen</td>
			<td>BN2002</td>
			<td></td>
		</tr>
		<tr>
			<td>NativePAGE Running Buffer (20x)&#160;</td>
			<td>Invitrogen</td>
			<td>BN2001</td>
			<td></td>
		</tr>
		<tr>
			<td>NDUFA9 Monoclonal Antibody</td>
			<td>Invitrogen</td>
			<td>459100</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitroblue tetrazolium salt (NBT)</td>
			<td>Sigma-Aldrich</td>
			<td>N6876</td>
			<td></td>
		</tr>
		<tr>
			<td>Pb(NO3)2</td>
			<td>Sigma-Aldrich</td>
			<td>228621</td>
			<td></td>
		</tr>
		<tr>
			<td>PDVF Membrane</td>
			<td>Amersham</td>
			<td>10600023</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenazine methasulfate (PMS)</td>
			<td>Sigma-Aldrich</td>
			<td>P9625</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce ECL Substrate</td>
			<td>Thermo Scientific</td>
			<td>32106</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Merck</td>
			<td>PMSF-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>SDHA Monoclonal Antibody</td>
			<td>Invitrogen</td>
			<td>459200</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium succinate</td>
			<td>Sigma-Aldrich</td>
			<td>S2378</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin/penicillin</td>
			<td>PAN biotech</td>
			<td>P06-07100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S3089</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>PanReac</td>
			<td>A2264</td>
			<td></td>
		</tr>
		<tr>
			<td>UQCRC1 Monoclonal Antibody</td>
			<td>Invitrogen</td>
			<td>459140</td>
			<td></td>
		</tr>
		<tr>
			<td>XCell&#160;SureLock Mini-Cell</td>
			<td>Invitrogen</td>
			<td>&#160;EI0001</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of mitochondria for mitochondrial supercomplex analysis from small tissue and cell culture samples 

Son on yıllarda, solunum süper komplekslerinin (SC'ler) varlığı hakkında toplanan kanıtlar, mitokondriyal elektron taşıma zinciri organizasyonu hakkındaki anlayışımızı değiştirdi ve "plastisite modeli" önerisine yol açtı. Bu model, dokuya veya hücresel metabolik duruma bağlı olarak farklı oranlarda SK'lerin ve komplekslerin bir arada bulunduğunu varsayar. SC'lerdeki düzeneğin dinamik doğası, hücrelerin mevcut yakıtların kullanımını ve elektron transferinin verimliliğini optimize etmesine, reaktif oksijen türlerinin oluşumunu en aza indirmesine ve hücrelerin çevresel değişikliklere uyum sağlama yeteneğini desteklemesine izin verecektir. 
Daha yakın zamanlarda, nörodejeneratif bozukluklar (Alzheimer ve Parkinson hastalığı), Barth Sendromu, Leigh sendromu veya kanser gibi farklı hastalıklarda SC montajında anormallikler bildirilmiştir. SK montaj değişikliklerinin hastalığın ilerlemesindeki rolünün hala doğrulanması gerekmektedir. Bununla birlikte, SC montaj durumunu belirlemek için yeterli miktarda numunenin mevcudiyeti genellikle bir zorluktur. Bu, küçük veya çoklu analizler için bölünmesi gereken biyopsi veya doku örneklerinde, yavaş büyüyen veya mikroakışkan cihazlardan gelen hücre kültürlerinde, bazı birincil kültürlerde veya nadir hücrelerde veya belirli maliyetli tedavilerin etkisinin analiz edilmesi gerektiğinde (nanopartiküller, çok pahalı bileşikler vb.) olur. Bu durumlarda verimli ve uygulaması kolay bir yöntem gerekir. Bu makale, mitokondriyal SC'lerin yapısını ve işlevini doğal elektroforez ve ardından jel içi aktivite deneyleri veya western blot ile analiz etmek için küçük miktarlarda hücre veya dokudan zenginleştirilmiş mitokondriyal fraksiyonlar elde etmek için uyarlanmış bir yöntem sunmaktadır.

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.

Yazar Gündemi: Sağlık ve Hastalıkta Oksidatif Fosforilasyon Sistem Dinamiklerini ve Mitokondriyal Rolleri Ortaya Çıkarmak

Küçük Doku ve Hücre Kültürü Örneklerinden Mitokondriyal Süperkompleks Analizi için Mitokondri İzolasyonu 

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.

Yukarıda açıklanan protokollere göre elde edilen mitokondri verimleri, hücre hattı veya doku tipi, numunelerin doğası (yani taze veya donmuş dokular kullanılıyorsa) veya homojenizasyon işleminin verimliliği gibi çeşitli faktörlere bağlı olarak değişir. Farklı hücre dizilerinden ve dokulardan beklenen mitokondri verimleri Tablo 2'de toplanmıştır. Mitokondriyal fraksiyonlar elde edildikten sonra, bir sonraki adım, ham mitokondriyal numunenin çözündürülmesi ve BN-PAGE ile elek...

Read this Scientific Article Protocol about Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples at JoVE.com

Bu protokol, sadece küçük miktarlarda numune mevcut olduğunda solunum süper komplekslerinin analizi için bir tekniği tanımlar.

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

Araştırmamız, mitokondriyal biyogenezi, özellikle de OXPHOS Sistem organizasyonunu ve Düzenlemesini daha iyi anlamaya ve ayrıca mitokondrinin kanserden nörodejeneratif bozukluklara kadar hastalıklarda oynadığı rolü tanımlamaya odaklanmıştır. Mitokondri, hücrede çok sayıda ve temel rol oynar, tüm metabolizmasını ve hayatta kalmasını şartlandırır. Mitokondriyal karmaşıklık ve bunun sağlık ve hastalıkta hücre adaptasyon mekanizmalarına katkısı hakkındaki bilgimizi yavaş yavaş artırıyoruz.OXPHOS sistemi için yeni bir organizasyon modeli, önceki önerileri entegre eden ve işlevini optimize etmek ve hücresel adaptasyonlara izin vermek için süper komplekslerin ve serbest komplekslerin farklı oranlarda bir arada bulunduğunu varsayan plastisite modeli oluşturulmasına katkıda bulunduk. Protokolümüz, küçük dokulardan veya hücre kültürü örneklerinden başlayarak, süper kompleksleri, montajı ve durumu ve işlevi mavi doğal elektroforez veya diğer tekniklerle analiz etmek için yeterli mitokondriyal protein miktarları elde etmeye izin verir. Bunu basit bir ekipmanla kısa sürede tekrarlanabilir şekilde ve nispeten düşük maliyetlerle yapabiliyoruz.

isolation-mitochondria-for-mitochondrial-supercomplex-analysis-from

Research

JoVE Journal

Biochemistry

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

635 Görüntüleme.  University of Zaragoza. Araştırmamız, mitokondriyal biyogenezi, özellikle de OXPHOS Sistem organizasyonunu ve Düzenlemesini daha iyi anlamaya ve ayrıca mitokondrinin kanserden nörodejeneratif bozukluklara kadar hastalıklarda oynadığı rolü tanımlamaya odaklanmıştır. Mitokondri, hücrede çok sayıda ve temel rol oynar, tüm metabolizmasını ve hayatta kalmasını şartlandırır. Mitokondriyal karmaşıklık ve bunun sağlık ve hastalıkta hücre adaptasyon mekanizmalarına katkısı hakkındaki bil...

Video: Yazar Gündemi: Sağlık ve Hastalıkta Oksidatif Fosforilasyon Sistem Dinamiklerini ve Mitokondriyal Rolleri Ortaya Çıkarmak

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.