Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells
Abstract
Many cellular processes require the proper cooperation between mitochondria and the endoplasmic reticulum (ER). Several recent works show that their functional interactions rely on dynamic structural contacts between both organelles. Such contacts, called mitochondria-associated membranes (MAMs), are crucial for the synthesis and intracellular transport of phospholipids, as well as for intracellular Ca(2+) signaling and for the determination of mitochondrial structure. Although several techniques are available to isolate mitochondria, only few are specifically tuned to the isolation of MAM, containing unique regions of ER membranes attached to the outer mitochondrial membrane and mitochondria without contamination from other organelles (i.e., pure mitochondria). Here we provide optimized protocols to isolate these fractions from tissues and cells. These procedures require 4-5 h and can be easily modified and adapted to different tissues and cell types.
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The Percoll gradient in the purification protocol eliminated contaminants from the endoplasmic reticulum and the proteasome.
“The Percoll gradient included in the purification protocol eliminated contaminants from the endoplasmic reticulum and the proteasome (Supplementary Fig. 1a)62.”
From Anti-mitochondrial autoantibodies in systemic lupus erythematosus and their association with disease manifestations (2019) · cites this paper as [62]
Mitochondria isolated from mouse liver or Hep-G2 cells using a combination of published protocols were highly purified, with low cytosolic and nuclear protein content and enrichment of VDAC, cytochrome C, and TOMM22.
“We used a combination of previously published protocols to obtain highly purified mitochondria61,62.”
From Anti-mitochondrial autoantibodies in systemic lupus erythematosus and their association with disease manifestations (2019) · cites this paper as [62]
Cites
Mitofusin 2 tethers endoplasmic reticulum to mitochondria
de Brito, O. M., & Scorrano, L. (2008) · Nature · doi:10.1038/nature07534 · Cited by 2,442 · see citing works
Rizzuto, R., Pinton, P., Carrington, W., Fay, F. S., Fogarty, K. E., Lifshitz, L. M., Tuft, R. A., & Pozzan, T. (1998) · Science · doi:10.1126/science.280.5370.1763 · Cited by 2,257 · see citing works
Self-citationHayashi, T., & Su, T.-P. (2007) · Cell · doi:10.1016/j.cell.2007.08.036 · Cited by 1,826 · see citing works
Chaperone-mediated coupling of endoplasmic reticulum and mitochondrial Ca2+ channels
Szabadkai, G., Bianchi, K., Várnai, P., De Stefani, D., Wieckowski, M. R., Cavagna, D., Nagy, A. I., Balla, T., & Rizzuto, R. (2006) · The Journal of Cell Biology · doi:10.1083/jcb.200608073 · Cited by 1,413 · see citing works
Self-citationOrganelle isolation: functional mitochondria from mouse liver, muscle and cultured filroblasts
Frezza, C., Cipolat, S., & Scorrano, L. (2007) · Nature Protocols · doi:10.1038/nprot.2006.478 · Cited by 1,188 · see citing works
Phospholipid synthesis in a membrane fraction associated with mitochondria.
Vance, J. E. (1990) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(19)39106-9 · Cited by 1,153 · see citing works
Zinser, E., Sperka-Gottlieb, C. D. M., Fasch, E.-V., Kohlwein, S. D., Paltauf, F., & Daum, G. (1991) · Journal of Bacteriology · doi:10.1128/jb.173.6.2026-2034.1991 · Cited by 705 · see citing works
PACS‐2 controls endoplasmic reticulum–mitochondria communication and Bid‐mediated apoptosis
Simmen, T., Aslan, J. E., Blagoveshchenskaya, A. D., Thomas, L., Wan, L., Xiang, Y. K., Feliciangeli, S. F., Hung, C., Crump, C. M., & Thomas, G. (2005) · The EMBO Journal · doi:10.1038/sj.emboj.7600559 · Cited by 593 · see citing works
Pinton, P., Rimessi, A., Marchi, S., Orsini, F., Migliaccio, E., Giorgio, M., Contursi, C., Minucci, S., Mantovani, F., Wieckowski, M., et al. (2007) · Science · doi:10.1126/science.1135380 · Cited by 494 · see citing works
Self-citationRapizzi, E., Pinton, P., Szabadkai, G., Wieckowski, M. R., Vandecasteele, G., Baird, G., Tuft, R. A., Fogarty, K. E., & Rizzuto, R. (2002) · The Journal of Cell Biology · doi:10.1083/jcb.200205091 · Cited by 453 · see citing works
Self-citationStructural and functional link between the mitochondrial network and the endoplasmic reticulum
Giorgi, C., Stefani, D. D., Bononi, A., Rizzuto, R., & Pinton, P. (2009) · The International Journal of Biochemistry & Cell Biology · doi:10.1016/j.biocel.2009.04.010 · Cited by 375 · see citing works
Self-citationHigo, T., Hattori, M., Nakamura, T., Natsume, T., Michikawa, T., & Mikoshiba, K. (2005) · Cell · doi:10.1016/j.cell.2004.11.048 · Cited by 374 · see citing works
Regulating ankyrin dynamics: Roles of sigma-1 receptors
Hayashi, T., & Su, T. (2001) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.98.2.491 · Cited by 331 · see citing works
Achleitner, G., Gaigg, B., Krasser, A., Kainersdorfer, E., Kohlwein, S. D., Perktold, A., Zellnig, G., & Daum, G. (1999) · European Journal of Biochemistry · doi:10.1046/j.1432-1327.1999.00658.x · Cited by 300 · see citing works
Mendes, C. C. P., Gomes, D. A., Thompson, M., Souto, N. C., Góes, T. S., Goes, A. M., Rodrigues, M. Â., Gomez, M. V., Nathanson, M. H., & Leite, M. F. (2005) · Journal of Biological Chemistry · doi:10.1074/jbc.m506623200 · Cited by 270 · see citing works
Shiao, Y., Lupo, G., & Vance, J. E. (1995) · Journal of Biological Chemistry · doi:10.1074/jbc.270.19.11190 · Cited by 261 · see citing works
Copeland, D. E., & Dalton, A. J. (1959) · The Journal of Cell Biology · doi:10.1083/jcb.5.3.393 · Cited by 261 · see citing works
Roderick, H. L., Lechleiter, J. D., & Camacho, P. (2000) · The Journal of Cell Biology · doi:10.1083/jcb.149.6.1235 · Cited by 240 · see citing works
Differential Modulation of SERCA2 Isoforms by Calreticulin
John, L. M., Lechleiter, J. D., & Camacho, P. (1998) · The Journal of Cell Biology · doi:10.1083/jcb.142.4.963 · Cited by 212 · see citing works
Gaigg, B., Simbeni, R., Hrastnik, C., Paltauf, F., & Daum, G. (1995) · Biochimica et Biophysica Acta (BBA) - Biomembranes · doi:10.1016/0005-2736(94)00287-y · Cited by 200 · see citing works
Lebiedzińska, M., Szabadkai, G., Jones, A. W. E., Duszyński, J., & Wieckowski, M. R. (2009) · The International Journal of Biochemistry & Cell Biology · doi:10.1016/j.biocel.2009.02.017 · Cited by 197 · see citing works
Self-citationMannella, C. A., Buttle, K., Rath, B. K., & Marko, M. (1998) · BioFactors · doi:10.1002/biof.5520080309 · Cited by 167 · see citing works
Connections between mitochondria and endoplasmic reticulum in rat liver and onion stem
Morr�, D. J., Merritt, W. D., & Lembi, C. A. (1971) · PROTOPLASMA · doi:10.1007/bf01286410 · Cited by 164 · see citing works
Outet mitochondrial membrane continuous with endoplasmic reticulum
Franke, W. W., & Kartenbeck, J. (1971) · PROTOPLASMA · doi:10.1007/bf01286409 · Cited by 161 · see citing works
Role of Sigma-1 Receptor C-terminal Segment in Inositol 1,4,5-Trisphosphate Receptor Activation
Wu, Z., & Bowen, W. D. (2008) · Journal of Biological Chemistry · doi:10.1074/jbc.m802099200 · Cited by 136 · see citing works
A rapidly sedimenting fraction of rat liver endoplasmic reticulum
Lewis, J. A., & Tata, J. R. (1973) · Journal of Cell Science · doi:10.1242/jcs.13.2.447 · Cited by 107 · see citing works
Ardail, D., Gasnier, F., Lermé, F., Simonot, C., Louisot, P., & Gateau-Roesch, O. (1993) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(19)74483-4 · Cited by 106 · see citing works
Lebiedzińska, M., Duszyński, J., Rizzuto, R., Pinton, P., & Wieckowski, M. R. (2009) · Archives of Biochemistry and Biophysics · doi:10.1016/j.abb.2009.03.007 · Cited by 94 · see citing works
Self-citationVance, J. E., Stone, S. J., & Faust, J. R. (1997) · Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism · doi:10.1016/s0005-2760(96)00153-1 · Cited by 69 · see citing works
Camici, O., & Corazzi, L. (1997) · Molecular and Cellular Biochemistry · doi:10.1023/a:1006889328983 · Cited by 32 · see citing works
Wieckowski, M. R., Szabadkai, G., Wasilewski, M., et al. (2006) · Biochemical and Biophysical Research Communications · doi:10.1016/j.bbrc.2006.07.072 · Cited by 27 · see citing works
Self-citation
Flagged first, then by citations
Cited by
Sorted by citations
A role for mitochondria in nlrp3 inflammasome activation
Zhou, R., Yazdi, A. S., Menu, P., & Tschopp, J. (2011) · Nature · doi:10.1038/nature09663 · Cited by 5,602 · see citing works
VDAC oligomers form mitochondrial pores to release mtDNA fragments and promote lupus-like disease
Kim, J., Gupta, R., Blanco, L. P., Yang, S., Shteinfer-Kuzmine, A., Wang, K., Zhu, J., Yoon, H. E., Wang, X., Kerkhofs, M., et al. (2019) · Science · doi:10.1126/science.aav4011 · Cited by 793 · see citing works
Mancina, R. M., Dongiovanni, P., Petta, S., Pingitore, P., Meroni, M., Rametta, R., Borén, J., Montalcini, T., Pujia, A., Wiklund, O., et al. (2016) · Gastroenterology · doi:10.1053/j.gastro.2016.01.032 · Cited by 676 · see citing works
Low-dose metformin targets the lysosomal AMPK pathway through PEN2
Ma, T., Tian, X., Zhang, B., Li, M., Wang, Y., Yang, C., Wu, J., Wei, X., Qu, Q., Yu, Y., et al. (2022) · Nature · doi:10.1038/s41586-022-04431-8 · Cited by 644 · see citing works
Betz, C., Stracka, D., Prescianotto-Baschong, C., Frieden, M., Demaurex, N., & Hall, M. N. (2013) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1302455110 · Cited by 538 · see citing works
Hedskog, L., Pinho, C. M., Filadi, R., Rönnbäck, A., Hertwig, L., Wiehager, B., Larssen, P., Gellhaar, S., Sandebring, A., Westerlund, M., et al. (2013) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1300677110 · Cited by 478 · see citing works
Yoo, H. C., Park, S. J., Nam, M., Kang, J., Kim, K., Yeo, J. H., Kim, J.-K., Heo, Y., Lee, H. S., Lee, M. Y., et al. (2020) · Cell Metabolism · doi:10.1016/j.cmet.2019.11.020 · Cited by 449 · see citing works
Tubbs, E., Theurey, P., Vial, G., Bendridi, N., Bravard, A., Chauvin, M.-A., Ji-Cao, J., Zoulim, F., Bartosch, B., Ovize, M., et al. (2014) · Diabetes · doi:10.2337/db13-1751 · Cited by 411 · see citing works
Wu, S., Lu, Q., Ding, Y., Wu, Y., Qiu, Y., Wang, P., Mao, X., Huang, K., Xie, Z., & Zou, M.-H. (2019) · Circulation · doi:10.1161/circulationaha.118.033552 · Cited by 298 · see citing works
Cholesterol Stabilizes TAZ in Hepatocytes to Promote Experimental Non-alcoholic Steatohepatitis
Wang, X., Cai, B., Yang, X., Sonubi, O. O., Zheng, Z., Ramakrishnan, R., Shi, H., Valenti, L., Pajvani, U. B., Sandhu, J., et al. (2020) · Cell Metabolism · doi:10.1016/j.cmet.2020.03.010 · Cited by 204 · see citing works
PDK4 augments ER-mitochondria contact to dampen skeletal muscle insulin signaling during obesity
Thoudam, T., Ha, C.-M., Leem, J., Chanda, D., Park, J.-S., Kim, H.-J., Jeon, J.-H., Choi, Y.-K., Liangpunsakul, S., Huh, Y. H., et al. (2019) · Diabetes · doi:10.2337/db18-0363 · Cited by 207 · see citing works
Theurey, P., Tubbs, E., Vial, G., Jacquemetton, J., Bendridi, N., Chauvin, M.-A., Alam, M. R., Le Romancer, M., Vidal, H., & Rieusset, J. (2016) · Journal of Molecular Cell Biology · doi:10.1093/jmcb/mjw004 · Cited by 175 · see citing works
Artificial Mitochondria Transfer: Current Challenges, Advances, and Future Applications
Caicedo, A., Aponte, P. M., Cabrera, F., Hidalgo, C., & Khoury, M. (2017) · Stem Cells International · doi:10.1155/2017/7610414 · Cited by 150 · see citing works
Issop, L., Fan, J., Lee, S., Rone, M. B., Basu, K., Mui, J., & Papadopoulos, V. (2015) · Endocrinology · doi:10.1210/en.2014-1503 · Cited by 147 · see citing works
Gutiérrez, T., & Simmen, T. (2018) · Cell Calcium · doi:10.1016/j.ceca.2017.05.015 · Cited by 104 · see citing works
Haj-Mirzaian, A., Amiri, S., Amini-Khoei, H., Hosseini, M.-J., Haj-Mirzaian, A., Momeny, M., Rahimi-Balaei, M., & Dehpour, A. R. (2017) · Neuroscience · doi:10.1016/j.neuroscience.2017.10.023 · Cited by 100 · see citing works
Nox4 regulates InsP3 receptor‐dependent Ca2+ release into mitochondria to promote cell survival
Beretta, M., Santos, C. X., Molenaar, C., Hafstad, A. D., Miller, C. C., Revazian, A., Betteridge, K., Schröder, K., Streckfuß‐Bömeke, K., Doroshow, J. H., et al. (2020) · The EMBO Journal · doi:10.15252/embj.2019103530 · Cited by 93 · see citing works
Becker, Y., Loignon, R.-C., Julien, A.-S., Marcoux, G., Allaeys, I., Lévesque, T., Rollet-Labelle, E., Benk-Fortin, H., Cloutier, N., Melki, I., et al. (2019) · Scientific Reports · doi:10.1038/s41598-019-40900-3 · Cited by 75 · see citing works
Tubbs, E., & Rieusset, J. (2016) · Journal of Visualized Experiments · doi:10.3791/54899 · Cited by 60 · see citing works
Wang, W.-X., Prajapati, P., Nelson, P. T., & Springer, J. E. (2020) · Molecular Neurobiology · doi:10.1007/s12035-020-01937-y · Cited by 49 · see citing works
Takeda, K., Uda, A., Mitsubori, M., Nagashima, S., Iwasaki, H., Ito, N., Shiiba, I., Ishido, S., Matsuoka, M., Inatome, R., et al. (2021) · Communications Biology · doi:10.1038/s42003-021-01720-2 · Cited by 32 · see citing works
The anti-viral dynamin family member MxB participates in mitochondrial integrity
Cao, H., Krueger, E. W., Chen, J., Drizyte-Miller, K., Schulz, M. E., & McNiven, M. A. (2020) · Nature Communications · doi:10.1038/s41467-020-14727-w · Cited by 25 · see citing works
Seibenhener, M. L., Zhao, T., Du, Y., Calderilla-Barbosa, L., Yan, J., Jiang, J., Wooten, M. W., & Wooten, M. C. (2013) · Behavioural Brain Research · doi:10.1016/j.bbr.2013.04.006 · Cited by 22 · see citing works
p66Shc aging protein in control of fibroblasts cell fate
Suski, J. M., Karkucinska-Wieckowska, A., Lebiedzinska, M., Giorgi, C., Szczepanowska, J., Szabadkai, G., Duszynski, J., Pronicki, M., Pinton, P., & Wieckowski, M. R. (2011) · International Journal of Molecular Sciences · doi:10.3390/ijms12085373 · Cited by 22 · see citing works
Self-citationChatel, B., Ducreux, S., Harhous, Z., Bendridi, N., Varlet, I., Ogier, A. C., Bernard, M., Gondin, J., Rieusset, J., Westerblad, H., et al. (2021) · Disease Models & Mechanisms · doi:10.1242/dmm.048981 · Cited by 8 · see citing works
- Authors
- Wieckowski, M. R., Giorgi, C., Lebiedzinska, M., Duszynski, J., & Pinton, P.
- Affiliations
- Instytut Biologii Doświadczalnej im. Marcelego Nenckiego (PL)
- Vita-Salute San Raffaele University (IT)
- University of Ferrara (IT)
- Corresponding author
- Wieckowski, Mariusz R
- Pinton, Paolo
- Venue
- Nature Protocols
- Published
- 2009-10-08
- Volume
- 4
- Issue
- 11
- Pages
- 1582-1590
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- OpenAlex
- W2094999489
- Cited by
- 979
- References
- 31
- Citations by year
- 2026: 71 · 2025: 83 · 2024: 96 · 2023: 79 · 2022: 70 · 2021: 80 · 2020: 73 · 2019: 69 · 2018: 61 · 2017: 68 · 2016: 45 · 2015: 41 · 2014: 38 · 2013: 47 · 2012: 28
- FWCI
- 7.51
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- Institutional Research Information System University of Ferrara (University of Ferrara) (submittedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
- Life Sciences › Neuroscience › Sensory Systems › Ion Channels and Receptors
- Health Sciences › Medicine › Epidemiology › Autophagy in Disease and Therapy
- Keywords
- mitochondria-associated membranes, mitochondrial isolation, endoplasmic reticulum, mitochondrial outer membrane, phospholipid synthesis, mitochondrial structure, subcellular fractionation
- MeSH terms
- Animals, Cell Fractionation, Cells, Cultured, Endoplasmic Reticulum, Humans, Intracellular Membranes, Mitochondria, Specimen Handling, Mitochondrial Membranes
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