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Isolation of functional pure mitochondria by superparamagnetic microbeads
Analytical Biochemistry 389 (2009) 1–5
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Isolation of functional pure mitochondria by superparamagnetic microbeads Hue-Tran Hornig-Do a,*,1, Gritt Günther b,1, Maria Bust a, Patricia Lehnartz a, Andreas Bosio b, Rudolf J. Wiesner a,c,d a
Institute of Vegetative Physiology, Medical Faculty, University of Köln, 50931 Köln, Germany Miltenyi Biotec, 51429 Bergisch Gladbach, Germany c Center for Molecular Medicine Cologne (CMMC), University of Köln, 50931 Köln, Germany d Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases (CECAD), University of Cologne, Zulpicher Strasse 47, 50674 Cologne, Germany b
a r t i c l e
i n f o
Article history: Received 30 September 2008 Available online 11 March 2009 Keywords: Mitochondria Magnetic separation Flow cytometry
a b s t r a c t Isolation of mitochondria by current methods relies mainly on their physicochemical properties. Here we describe an alternative approach to obtain functional mitochondria from human cells in a fast, reproducible, and standardized procedure. The new approach is based on superparamagnetic microbeads conjugated to anti-TOM22 antibody. The bead conjugates label the cytoplasmic part of the human mitochondrial membrane protein TOM22 and, thus, allow for a gentle isolation of mitochondria in a high gradient magnetic field. By comparing the MACS (magnetic cell separation) approach with mitochondria isolation methods using differential centrifugation and ultracentrifugation we demonstrate that the MACS approach provides the highest yield of isolated mitochondria. The quality, enrichment, and purity of mitochondria isolated with this protocol are comparable to mitochondria obtained using the ultracentrifuge method, and a typical separation procedure takes only approximately 1 to 2 h from initial cell homogenization. Mitochondria isolated with the new approach are sufficient for protein import, blue native gel electrophoresis, and other mitochondrial assays. Ó 2009 Elsevier Inc. All rights reserved.
Mitochondria play a central role in many cellular functions, including bioenergetics, apoptosis, and the metabolism of lipids, iron, nucleotides, and amino acids. As mounting evidence has implicated mitochondria as key participants in degenerative diseases [1,2] and aging [3], mitochondria have become the subject of intense study in numerous fields, including biomedical research, drug discovery, and proteomics. Because experimental conditions can be precisely controlled in isolated mitochondria compared with intact cells or tissues, isolated organelles provide a unique tool to investigate not only mechanisms of apoptosis, reactive oxygen species (ROS)2 production, and biogenetics but also mitochondrial DNA (mtDNA), mitochondrial RNA (mtRNA), and mitochondrial protein synthesis. Among various approaches for mitochondria purification, differential centrifugation * Corresponding author. Fax: +49 221 478 3538. E-mail address: [email protected] (H.-T. Hornig-Do). 1 Shared first authorship. 2 Abbreviations used: ROS, reactive oxygen species; mtDNA, mitochondrial DNA; mtRNA, mitochondrial RNA; DC, differential centrifugation; UC, ultracentrifugation; MACS, magnetic cell separation; TOM22, 22-kDa translocase of outer mitochondrial membrane; PBS, phosphate-buffered saline; EDTA, ethylenediaminetetraacetic acid; BSA, bovine serum albumin; IgG1, immunoglobulin G1; APC, allophycocyanin; FACS, fluorescence-activated cell sorter; EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TFAM, mitochondrial transcription factor A; RCR, respiratory control ratio; COXI, cytochrome c oxidase subunit I; BN–PAGE, blue native gel electrophoresis. 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.02.040
(DC) is the most frequently used protocol due to its quick and inexpensive nature. This method, developed during the 1950s [4] and modified in many ways [5–8], provides a mitochondrial fraction in which integrity and functionality of the organelle are maintained. Other purification protocols that also result in highly pure mitochondrial fractions, such as preparation of an isopycnic gradient followed by ultracentrifugation (UC) [9,10], are time-consuming and expensive and require access to an ultracentrifuge. Here we describe a fast and easy method to obtain pure functional mitochondria with high yield from human cultured cells. The new method is based on magnetic sorting, which was originally developed for the separation of cells [11] but has also been successfully used to purify cell compartments such as Golgi vesicles [12], endosomes [13], lysosomes [14], nuclei [15], and plasma membranes [16]. Here we compared the new approach, MACS (magnetic cell separation), with the DC and UC methods by testing the organelles’ performance using different assays for purity and function. Materials and methods Mitochondria isolation with superparamagnetic microbeads Anti-TOM22 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) were developed for the magnetic isolation of mitochon-
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Isolation of mitochondria by superparamagnetic microbeads / H.-T. Hornig-Do et al. / Anal. Biochem. 389 (2009) 1–5
dria from human cells. Microbeads are a colloidal suspension of extremely small (50 nm diameter) superparamagnetic particles. Conjugated to a monoclonal anti-TOM22 antibody, the microbeads bind specifically to the 22-kDa translocase of outer mitochondrial membrane (TOM22) on the surface of human mitochondria and, thereby, magnetically label the organelles. The labeled mitochondria can then be efficiently isolated in the magnetic field of a MACS Separator. Different human cell lines were harvested at approximately 90% confluency and washed twice with phosphate-buffered saline (PBS). Cells (1 107) were lysed in 1 ml of ice-cold PBS, including Complete Protease Inhibitor Cocktail Tablets (Roche, Germany), by shearing through a needle approximately 20 times. With respect to different cell sizes, different needle diameters were used for cell disruption: 26G for 293 HEK cells, 29G for HeLa cells, and 27G for osteosarcoma cells. Glass homogenizers were employed for large numbers of cells. After cell disruption, an aliquot of the lysate was examined for trypan blue exclusion to ensure that 80% of the cells were lysed. For magnetic labeling, the crude cell lysate was incubated with 25 ll of anti-TOM22 MicroBeads for 15 to 60 min at 4 °C. Subsequently, the suspension was loaded onto a preequilibrated MACS Column (Miltenyi Biotec), which was placed in the magnetic field of a MACS Separator (Miltenyi Biotec). Columns were washed three times with 3 ml of PEB buffer (PBS [pH 7.2], 2 mM ethylenediaminetetraacetic acid [EDTA], and 0.5% bovine serum albumin [BSA]). After removing the column from the magnetic field, retained mitochondria were eluted with 5 ml of PEB buffer. Following centrifugation at approximately 13,000g for 1 min, the mitochondrial pellet was washed twice with 0.32 M sucrose, 1 mM EDTA, and 10 mM Tris–HCl and was finally resuspended in the appropriate incubation buffer. Staining and FACS analysis of isolated mitochondria Isolated mitochondria from approximately 1 105 cells were resuspended in 100 ll of cold PEB buffer prefiltered through a 0.22-lm membrane. Mitochondria were incubated with a primary antibody (e.g., anti-TOM22 monoclonal mouse antibody or antiTOM22–biotin, Miltenyi Biotec) for 10 min at 4 °C. Washing was performed by the addition of 1 ml of cold PEB buffer to the mitochondria and subsequent centrifugation for 2 min at 13,000g at 4 °C. The supernatant was aspirated except for the last 25 ll (no pellet was visible due to the low amount of starting material). Mitochondria were resuspended, and the volume was adjusted to 100 ll with cold PEB buffer. Then mitochondria were incubated in the dark with the secondary antibody (e.g., rat anti-mouse immunoglobulin G1 (IgG1)–allophycocyanin (APC) or anti-biotin– APC, Miltenyi Biotec) for 10 min at 4 °C. After three washing and centrifugation steps, mitochondria were resuspended in 1 ml of cold PEB buffer and fluorescence measurement was performed on a fluorescence-activated cell sorter (FACS) (FACSCalibur, Becton–Dickinson, Heidelberg, Germany). Data were analyzed using CellQuest software (Becton–Dickinson). Mitochondria isolated from stably transfected 293 HEK cells expressing the enhanced green fluorescent protein (EGFP) fused to a mitochondrial targeting sequence were directly analyzed using a FACS. Mitochondria isolation using DC and UC Mitochondria were purified by DC, as described in detail previously [7]. Purification of mitochondria using a Percoll/Metrizamide gradient and UC was described in detail previously [10]. Because Metrizamide was not available, Nycodenz (Serva, Heidelberg, Germany) was used to prepare a Percoll/Nycodenz gradient.
Antibodies used in Western blot analysis Mouse monoclonal antibodies used were from the following: against 70 kDa subunit of complex II (Molecular Probes, Eugene, OR, USA), against core 2 subunit of complex III (Molecular Probes), against subunit I of complex IV (Molecular Probes), against b-actin (Sigma, Taufkirchen, Germany), against cytochrome c (BD Biosciences, San Jose, CA, USA), against 22 kDa translocase of outer mitochondrial membrane (Miltenyi Biotec), against KDEL (Abcam, Cambridge, UK), against Rab4 (BD Biosciences), against lamin A/C (BD Biosciences), and against Golgin-97 (Molecular Probes). A rabbit antiserum was used against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling, Danvers, MA, USA), and a rabbit antiserum had previously been raised against recombinant human mitochondrial transcription factor A (TFAM) [17]. The following secondary antibodies were used: peroxidase-conjugated goat anti-mouse IgG (Perbio Science, Bonn, Germany) and peroxidase conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, Cambridgeshire, UK). Results Mitochondria isolation using the MACS approach To test whether mitochondria could be successfully isolated with the MACS protocol, we generated a 293-reporter HEK cell line persistently expressing EGFP fused to a mitochondrial targeting sequence derived from the precursor of subunit VIII of human cytochrome c oxidase. Import of EGFP into, and fluorescence labeling of mitochondria in the stably transfected 293 HEK reporter cell line was verified by fluorescence microscopy (data not shown). An aliquot of the crude reporter cell lysate before mitochondria separation, the flow-through and washes, and eluted mitochondria were analyzed with a FACS. Mitochondria could be detected as green fluorescence in the crude reporter cell lysate (Fig. 1A). In the flow-through and wash fractions, a marginal EGFP signal was detected (Fig. 1B), whereas the mitochondrial fraction exhibited a strong EGFP signal overlapping with the original fraction (Fig. 1C and D). These data strongly indicate that mitochondria can be efficiently isolated by the MACS approach. To find out the advantages and disadvantages of the new approach, we compared the MACS protocol with the DC and UC methods, focusing on relevant parameters of mitochondria preparations such as yield, quality, and purity of the isolated organelles. Yield of isolated mitochondrial fractions The amount of mitochondria isolated from 293 HEK cells using the MACS protocol was twofold higher than that using DC (see Fig. S1A in supplementary material). In comparison with the UC method, the MACS approach yielded four times more mitochondria from osteosarcoma cells (see Fig. S1B). These results clearly indicate that the highest yield in two different cell types was achieved with the MACS protocol. Quality of mitochondrial preparations Well-coupled mitochondria are a prerequisite to achieving reliable reproducible results in nearly all functional assays. Coupling of isolated mitochondria reflecting the quality of the preparations was measured using an oxygen electrode. Respiratory control ratios (RCRs) higher than 3 are well accepted to indicate tightly coupled mitochondria [18]. All three methods provided tightly coupled mitochondria (see Fig. S1C in supplementary material). However, mitochondria isolated with the MACS protocol were found to be
Isolation of mitochondria by superparamagnetic microbeads / H.-T. Hornig-Do et al. / Anal. Biochem. 389 (2009) 1–5
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Fig. 1. FACS analysis of mitochondria isolated from transfected 293 HEK cells using the MACS protocol. Mitochondria in transfected 293 HEK cells express EGFP fused to a mitochondrial targeting sequence and, therefore, are fluorescently labeled. The green line displays the specific fluorescence of mitochondria, whereas the gray area represents the autofluorescence of mitochondria from untransfected 293 HEK cells. Compared with the crude cell lysate before isolation (A), the mitochondrial fraction (C) shows a significant enrichment of mitochondria, whereas hardly any signal was detectable in the flow-through and wash fractions (B). An overlay of the crude cell lysate and mitochondrial fraction (D) confirms the specific enrichment of mitochondria obtained with the MACS protocol.
better coupled than those obtained using DC, whereas RCRs of mitochondria isolated using the new approach and UC were similar. Enrichment of isolated mitochondria Western blot analysis revealed that the amount of mitochondrial proteins, such as cytochrome c oxidase subunit I (COXI, inner
membrane), TOM22 (outer membrane), mitochondrial transcription factor A (TFAM, nucleoid protein), and cytochrome c (attached to outer leaflet of inner membrane), were strongly increased in the mitochondrial fraction obtained using the MACS protocol (Fig. 2A). This indicates a significant enrichment of mitochondria in the eluted fraction. Moreover, FACS analysis of the mitochondrial fractions obtained by the three methods showed that 59% of the mito-
Fig. 2. (A) Enrichment of mitochondria. The amounts of subunit I of complex IV (COXI), 22-kDa translocase of outer mitochondrial membrane (TOM22), mitochondrial transcription factor TFAM (TFAM), and cytochrome c (Cyt c) in whole cell lysate were compared with equal amounts of protein from the mitochondrial fraction isolated from HeLa cells using the MACS protocol. (B) The amounts of b-actin, GAPDH, Rab4, and Golgin-97, representing proteins from cytoskeleton, cytosol, Golgi apparatus, and endosome, respectively, in whole cell lysate were compared with the equal protein amounts from the mitochondrial fraction isolated from HeLa cells using the MACS protocol. (C) Amounts of COXI (mitochondria), KDEL (endoplasmic reticulum), and lamins A and C (nucleus) in the mitochondrial fractions obtained using DC, the MACS approach, and UC.
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Isolation of mitochondria by superparamagnetic microbeads / H.-T. Hornig-Do et al. / Anal. Biochem. 389 (2009) 1–5
chondrial fraction of the DC preparation was TOM22 positive, whereas the mitochondrial fraction of the MACS preparation contained 89% TOM22 positive events and did not differ significantly from the mitochondrial fraction of the UC preparation, which showed 88% TOM22 positive events. Purity of mitochondrial fractions Another important aim of mitochondria preparation is to obtain highly pure organelles because the presence of contaminants might interfere with the analyses and the interpretation of results. Common relevant contaminants of mitochondria preparations are compounds of endoplasmic reticulum, endosomes, Golgi apparatus, nucleus, and cytosol, which can be monitored by Western blot analysis using antibodies specific for organelle/cell compartmentspecific marker proteins. b-Actin, GAPDH, Golgin-97, and Rab4, representing proteins from cytoskeleton, cytosol, Golgi apparatus, and endosome, respectively, could not be detected in the MACS preparation (Fig. 2B) or in other preparations (data not shown) but could be detected in the whole cell lysate. Mitochondria isolated by the MACS protocol showed fewer contaminants from endoplasmic reticulum and nucleus, detected with the antibodies against KDEL and lamins A and C, respectively, compared with the mitochondrial fraction obtained by the DC method. Mitochondria prepared by the UC method contained similar amounts of contaminants from endoplasmic reticulum and nucleus (Fig. 2C). Thus, the purity of mitochondria isolated with these latter two methods was comparable. Mitochondrial protein import An antibody against TOM22, a component of the translocase of the outer mitochondrial membrane (TOM), is used for purification of mitochondria in the MACS protocol. Because translocation of preproteins across the mitochondrial outer membrane is mediated by the TOM complex, targeting of TOM22 with antibodies might disturb protein import into mitochondria. Thus, we performed an import assay [19] with mitochondrial fractions isolated using the DC and MACS protocols and compared the efficiency of the import of the TFAM protein. As shown in Fig. 3, the 29-kDa TFAM precursor was partially converted into a 24-kDa protein in the presence of mitochondria. The imported protein was resistant to proteinase K treatment, whereas the 29-kDa precursor was accessible to diges-
Fig. 3. Import of TFAM into mitochondria isolated from HeLa cells using the MACS approach or DC. Mitochondria were incubated for 1 h with 35S-labeled TFAM. Import was studied by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and fluorography. Where indicated, CCCP was added during the incubation and proteinase K was added after the incubation. The first lane shows an aliquot of reticulocyte lysate, whereas the other lanes contain protein from sedimented mitochondria. p, precursor protein; m, imported mature protein. Import efficiency was calculated by (amount of imported TFAM) / (amount of precursor protein + imported protein).
tion. In the presence of the uncoupler CCCP, the import of TFAM was completely inhibited. The relative amount of imported TFAM into mitochondria isolated with the MACS approach was approximately 34% under our assay conditions and did not differ significantly from the imported amount obtained using the DC protocol. Therefore, an inhibitory effect of the used antibody on mitochondrial protein import could be excluded. FACS analysis of isolated mitochondria from HeLa wild-type and HeLa rho-0 cells PicoGreen was shown to stain mtDNA in living cells [20]. However, interference with the nuclear signal limits the use of PicoGreen alone. Here we used PicoGreen to easily distinguish between mitochondria containing mtDNA and mtDNA-free mitochondria isolated from HeLa wild-type and HeLa rho-0 cells, respectively. Mitochondria isolated from HeLa wild-type cells were TOM22 and PicoGreen positive, whereas PicoGreen fluorescence was heavily reduced in mitochondria obtained from HeLa rho-0 cells (Fig. 4).
Fig. 4. PicoGreen staining of mitochondria derived from HeLa wild-type and HeLa rho-0 cells. Both cell lines were stained with PicoGreen (2 ll/ml culture medium, Molecular Probes) for 1 h at 37 °C. Culture medium was replaced by dye-free medium. After a 20-min incubation step, cells were lysed and mitochondria were isolated using the MACS approach. Purified mitochondria were counterstained with anti-TOM22–biotin and anti-biotin–APC and were analyzed using a FACS.
Isolation of mitochondria by superparamagnetic microbeads / H.-T. Hornig-Do et al. / Anal. Biochem. 389 (2009) 1–5
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starting material (1 106 cells) but also permits the isolation of huge quantities of mitochondria from large amounts of material. Mitochondria isolated using the MACS approach were successfully tested in various assays such as protein import and BN–PAGE. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ab.2009.02.040. References
Fig. 5. Respiratory chain complexes from 143 B osteosarcoma cells (WT) and cybrid cells containing a truncation mutation in COXI (DCOX). Mitochondria of WT isolated with UC and mitochondria DCOX obtained using the MACS protocol were resolved by BN–PAGE followed by Western blot and were detected with monoclonal antibodies against 70-kDa subunit for complex II, core 2 for complex III, and COXI for complex IV. A mixture of high-molecular-weight purified proteins was used as molecular mass marker: thyroglobulin, 669 kDa; ferritin monomer, 440 kDa; catalase, 232 kDa; lactate dehydrogenase, 140 kDa; albumin, 66 kDa.
Analysis of membrane protein complexes To find out whether mitochondria isolated with the MACS protocol are capable of further analysis of the protein composition of the respiratory chain, we subjected mitochondria obtained using the UC or MACS protocol to blue native gel electrophoresis (BN– PAGE), and complexes and subcomplexes were identified by Western blot analysis [21,22]. Mitochondria from 143 B osteosarcoma cells (WT cybrid) were isolated with the UC method. The MACS protocol was used to obtain mitochondria from cybrid cells containing a truncation mutation in COXI (DCOX). As shown previously [23], in contrast to WT cybrid cells, no complex IV was found in DCOX cybrid cells because assembly of this complex was severely disturbed (Fig. 5). The expression of complexes II and III, which are not affected by the mutation, did not differ significantly in both cybrid cell lines, demonstrating that mitochondria isolated with the MACS approach are suitable for BN–PAGE. Conclusions The ultimate goal of a mitochondrial isolation protocol is to obtain organelles as pure and functional as possible. For some applications, such as measurement of OXPHOS enzyme activity, crude mitochondrial fractions obtained using DC are sufficient. For other purposes, such as analysis of the mitochondrial proteome, one has the choice of different approaches to obtain highly pure mitochondria. Here we have developed a new approach that provides functional intact mitochondria with a very high yield in a fast, reproducible, and standardized procedure. Furthermore, the quality and purity of the obtained mitochondrial fraction using the MACS approach are comparable to those using the UC method, which also is time-consuming and needs access to an ultracentrifuge. Our method is generally applicable to a variety of different human cell lines such as 293 HEK, HeLa, and 143 B osteosarcoma cybrid cells. Another great advantage of the method presented here is that the approach can be performed with very small amounts of
[1] D.C. Wallace, Mitochondrial diseases in man and mouse, Science 283 (1999) 1482–1488. [2] N. Sergeant, A. Wattez, M. Galvan-Valencia, A. Ghestem, J.P. David, J. Lemoine, P.E. Sautiere, J. Dachary, J.P. Mazat, J.C. Michalski, J. Velours, R. Mena-Lopez, A. Delacourte, Association of ATP synthase a-chain with neurofibrillary degeneration in Alzheimer’s disease, Neuroscience 117 (2003) 293–303. [3] A. Trifunovic, Mitochondrial DNA and ageing, Biochim. Biophys. Acta 1757 (2006) 611–617. [4] C. De Duve, B.C. Pressman, R. Gianetto, R. Wattiaux, F. Appelmans, Tissue fractionation studies: VI. Intracellular distribution patterns of enzymes in ratliver tissue, Biochem. J. 60 (1955) 604–617. [5] D. Rickwood, M.T. Wilson, V.M. Darley-Usmar, Mitochondria: A Practical Approach, IRL Press, Oxford, UK, 1987. [6] J.A. Enriquez, A. Perez-Martos, M.J. Lopez-Perez, J. Montoya, In organello RNA synthesis system from mammalian liver and brain, Methods Enzymol. 264 (1996) 50–57. [7] E. Fernandez-Vizarra, M.J. Lopez-Perez, J.A. Enriquez, Isolation of biogenetically competent mitochondria from mammalian tissues and cultured cells, Methods 26 (2002) 292–297. [8] C. Frezza, S. Cipolat, L. Scorrano, Organelle isolation: functional mitochondria from mouse liver, muscle, and cultured fibroblasts, Nat. Protoc. 2 (2007) 287– 295. [9] B. Storrie, E.A. Madden, Isolation of subcellular organelles, Methods Enzymol. 182 (1990) 203–225. [10] N.K. Scheffler, S.W. Miller, A.K. Carroll, C. Anderson, R.E. Davis, S.S. Ghosh, B.W. Gibson, Two-dimensional electrophoresis and mass spectrometric identification of mitochondrial proteins from an SH-SY5Y neuroblastoma cell line, Mitochondrion 1 (2001) 161–179. [11] S. Miltenyi, W. Muller, W. Weichel, A. Radbruch, High gradient magnetic cell separation with MACS, Cytometry 11 (1990) 231–238. [12] C.V. Mura, M.I. Becker, A. Orellana, D. Wolff, Immunopurification of Golgi vesicles by magnetic sorting, J. Immunol. Methods 260 (2002) 263–271. [13] L.A. Perrin-Cocon, P.N. Marche, C.L. Villiers, Purification of intracellular compartments involved in antigen processing: a new method based on magnetic sorting, Biochem. J. 338 (1999) 123–130. [14] O. Diettrich, K. Mills, A.W. Johnson, A. Hasilik, B.G. Winchester, Application of magnetic chromatography to the isolation of lysosomes from fibroblasts of patients with lysosomal storage disorders, FEBS Lett. 441 (1998) 369–372. [15] A.P. Kausch, T.P. Owen Jr., S. Narayanswami, B.D. Bruce, Organelle isolation by magnetic immunoabsorption, BioTechniques 26 (1999) 336–343. [16] E.L. Lawson, J.G. Clifton, F. Huang, X. Li, D.C. Hixson, D. Josic, Use of magnetic beads with immobilized monoclonal antibodies for isolation of highly pure plasma membranes, Electrophoresis 27 (2006) 2747–2758. [17] K. Weber, D. Ridderskamp, M. Alfert, S. Hoyer, R.J. Wiesner, Cultivation in glucose-deprived medium stimulates mitochondrial biogenesis and oxidative metabolism in HepG2 hepatoma cells, Biol. Chem. 383 (2002) 283–290. [18] D. Chretien, P. Rustin, T. Bourgeron, A. Rotig, J.M. Saudubray, A. Munnich, Reference charts for respiratory chain activities in human tissues, Clin. Chim. Acta 228 (1994) 53–70. [19] H.L. Garstka, W.E. Schmitt, J. Schultz, B. Sogl, B. Silakowski, A. Perez-Martos, J. Montoya, R.J. Wiesner, Import of mitochondrial transcription factor A (TFAM) into rat liver mitochondria stimulates transcription of mitochondrial DNA, Nucleic Acids Res. 31 (2003) 5039–5047. [20] N. Ashley, D. Harris, J. Poulton, Detection of mitochondrial DNA depletion in living human cells using PicoGreen staining, Exp. Cell Res. 303 (2005) 432– 446. [21] L.G. Nijtmans, N.S. Henderson, I.J. Holt, Blue native electrophoresis to study mitochondrial and other protein complexes, Methods 26 (2002) 327–334. [22] I. Wittig, H.P. Braun, H. Schagger, Blue native PAGE, Nat. Protoc. 1 (2006) 418– 428. [23] M. D’Aurelio, C.D. Gajewski, G. Lenaz, G. Manfredi, Respiratory chain supercomplexes set the threshold for respiration defects in human mtDNA mutant cybrids, Hum. Mol. Genet. 15 (2006) 2157–2169.
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Isolation of functional pure mitochondria by superparamagnetic microbeads Hue-Tran Hornig-Do a,*,1, Gritt Günther b,1, Maria Bust a, Patricia Lehnartz a, Andreas Bosio b, Rudolf J. Wiesner a,c,d a
Institute of Vegetative Physiology, Medical Faculty, University of Köln, 50931 Köln, Germany Miltenyi Biotec, 51429 Bergisch Gladbach, Germany c Center for Molecular Medicine Cologne (CMMC), University of Köln, 50931 Köln, Germany d Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases (CECAD), University of Cologne, Zulpicher Strasse 47, 50674 Cologne, Germany b
a r t i c l e
i n f o
Article history: Received 30 September 2008 Available online 11 March 2009 Keywords: Mitochondria Magnetic separation Flow cytometry
a b s t r a c t Isolation of mitochondria by current methods relies mainly on their physicochemical properties. Here we describe an alternative approach to obtain functional mitochondria from human cells in a fast, reproducible, and standardized procedure. The new approach is based on superparamagnetic microbeads conjugated to anti-TOM22 antibody. The bead conjugates label the cytoplasmic part of the human mitochondrial membrane protein TOM22 and, thus, allow for a gentle isolation of mitochondria in a high gradient magnetic field. By comparing the MACS (magnetic cell separation) approach with mitochondria isolation methods using differential centrifugation and ultracentrifugation we demonstrate that the MACS approach provides the highest yield of isolated mitochondria. The quality, enrichment, and purity of mitochondria isolated with this protocol are comparable to mitochondria obtained using the ultracentrifuge method, and a typical separation procedure takes only approximately 1 to 2 h from initial cell homogenization. Mitochondria isolated with the new approach are sufficient for protein import, blue native gel electrophoresis, and other mitochondrial assays. Ó 2009 Elsevier Inc. All rights reserved.
Mitochondria play a central role in many cellular functions, including bioenergetics, apoptosis, and the metabolism of lipids, iron, nucleotides, and amino acids. As mounting evidence has implicated mitochondria as key participants in degenerative diseases [1,2] and aging [3], mitochondria have become the subject of intense study in numerous fields, including biomedical research, drug discovery, and proteomics. Because experimental conditions can be precisely controlled in isolated mitochondria compared with intact cells or tissues, isolated organelles provide a unique tool to investigate not only mechanisms of apoptosis, reactive oxygen species (ROS)2 production, and biogenetics but also mitochondrial DNA (mtDNA), mitochondrial RNA (mtRNA), and mitochondrial protein synthesis. Among various approaches for mitochondria purification, differential centrifugation * Corresponding author. Fax: +49 221 478 3538. E-mail address: [email protected] (H.-T. Hornig-Do). 1 Shared first authorship. 2 Abbreviations used: ROS, reactive oxygen species; mtDNA, mitochondrial DNA; mtRNA, mitochondrial RNA; DC, differential centrifugation; UC, ultracentrifugation; MACS, magnetic cell separation; TOM22, 22-kDa translocase of outer mitochondrial membrane; PBS, phosphate-buffered saline; EDTA, ethylenediaminetetraacetic acid; BSA, bovine serum albumin; IgG1, immunoglobulin G1; APC, allophycocyanin; FACS, fluorescence-activated cell sorter; EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TFAM, mitochondrial transcription factor A; RCR, respiratory control ratio; COXI, cytochrome c oxidase subunit I; BN–PAGE, blue native gel electrophoresis. 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.02.040
(DC) is the most frequently used protocol due to its quick and inexpensive nature. This method, developed during the 1950s [4] and modified in many ways [5–8], provides a mitochondrial fraction in which integrity and functionality of the organelle are maintained. Other purification protocols that also result in highly pure mitochondrial fractions, such as preparation of an isopycnic gradient followed by ultracentrifugation (UC) [9,10], are time-consuming and expensive and require access to an ultracentrifuge. Here we describe a fast and easy method to obtain pure functional mitochondria with high yield from human cultured cells. The new method is based on magnetic sorting, which was originally developed for the separation of cells [11] but has also been successfully used to purify cell compartments such as Golgi vesicles [12], endosomes [13], lysosomes [14], nuclei [15], and plasma membranes [16]. Here we compared the new approach, MACS (magnetic cell separation), with the DC and UC methods by testing the organelles’ performance using different assays for purity and function. Materials and methods Mitochondria isolation with superparamagnetic microbeads Anti-TOM22 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) were developed for the magnetic isolation of mitochon-
2
Isolation of mitochondria by superparamagnetic microbeads / H.-T. Hornig-Do et al. / Anal. Biochem. 389 (2009) 1–5
dria from human cells. Microbeads are a colloidal suspension of extremely small (50 nm diameter) superparamagnetic particles. Conjugated to a monoclonal anti-TOM22 antibody, the microbeads bind specifically to the 22-kDa translocase of outer mitochondrial membrane (TOM22) on the surface of human mitochondria and, thereby, magnetically label the organelles. The labeled mitochondria can then be efficiently isolated in the magnetic field of a MACS Separator. Different human cell lines were harvested at approximately 90% confluency and washed twice with phosphate-buffered saline (PBS). Cells (1 107) were lysed in 1 ml of ice-cold PBS, including Complete Protease Inhibitor Cocktail Tablets (Roche, Germany), by shearing through a needle approximately 20 times. With respect to different cell sizes, different needle diameters were used for cell disruption: 26G for 293 HEK cells, 29G for HeLa cells, and 27G for osteosarcoma cells. Glass homogenizers were employed for large numbers of cells. After cell disruption, an aliquot of the lysate was examined for trypan blue exclusion to ensure that 80% of the cells were lysed. For magnetic labeling, the crude cell lysate was incubated with 25 ll of anti-TOM22 MicroBeads for 15 to 60 min at 4 °C. Subsequently, the suspension was loaded onto a preequilibrated MACS Column (Miltenyi Biotec), which was placed in the magnetic field of a MACS Separator (Miltenyi Biotec). Columns were washed three times with 3 ml of PEB buffer (PBS [pH 7.2], 2 mM ethylenediaminetetraacetic acid [EDTA], and 0.5% bovine serum albumin [BSA]). After removing the column from the magnetic field, retained mitochondria were eluted with 5 ml of PEB buffer. Following centrifugation at approximately 13,000g for 1 min, the mitochondrial pellet was washed twice with 0.32 M sucrose, 1 mM EDTA, and 10 mM Tris–HCl and was finally resuspended in the appropriate incubation buffer. Staining and FACS analysis of isolated mitochondria Isolated mitochondria from approximately 1 105 cells were resuspended in 100 ll of cold PEB buffer prefiltered through a 0.22-lm membrane. Mitochondria were incubated with a primary antibody (e.g., anti-TOM22 monoclonal mouse antibody or antiTOM22–biotin, Miltenyi Biotec) for 10 min at 4 °C. Washing was performed by the addition of 1 ml of cold PEB buffer to the mitochondria and subsequent centrifugation for 2 min at 13,000g at 4 °C. The supernatant was aspirated except for the last 25 ll (no pellet was visible due to the low amount of starting material). Mitochondria were resuspended, and the volume was adjusted to 100 ll with cold PEB buffer. Then mitochondria were incubated in the dark with the secondary antibody (e.g., rat anti-mouse immunoglobulin G1 (IgG1)–allophycocyanin (APC) or anti-biotin– APC, Miltenyi Biotec) for 10 min at 4 °C. After three washing and centrifugation steps, mitochondria were resuspended in 1 ml of cold PEB buffer and fluorescence measurement was performed on a fluorescence-activated cell sorter (FACS) (FACSCalibur, Becton–Dickinson, Heidelberg, Germany). Data were analyzed using CellQuest software (Becton–Dickinson). Mitochondria isolated from stably transfected 293 HEK cells expressing the enhanced green fluorescent protein (EGFP) fused to a mitochondrial targeting sequence were directly analyzed using a FACS. Mitochondria isolation using DC and UC Mitochondria were purified by DC, as described in detail previously [7]. Purification of mitochondria using a Percoll/Metrizamide gradient and UC was described in detail previously [10]. Because Metrizamide was not available, Nycodenz (Serva, Heidelberg, Germany) was used to prepare a Percoll/Nycodenz gradient.
Antibodies used in Western blot analysis Mouse monoclonal antibodies used were from the following: against 70 kDa subunit of complex II (Molecular Probes, Eugene, OR, USA), against core 2 subunit of complex III (Molecular Probes), against subunit I of complex IV (Molecular Probes), against b-actin (Sigma, Taufkirchen, Germany), against cytochrome c (BD Biosciences, San Jose, CA, USA), against 22 kDa translocase of outer mitochondrial membrane (Miltenyi Biotec), against KDEL (Abcam, Cambridge, UK), against Rab4 (BD Biosciences), against lamin A/C (BD Biosciences), and against Golgin-97 (Molecular Probes). A rabbit antiserum was used against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling, Danvers, MA, USA), and a rabbit antiserum had previously been raised against recombinant human mitochondrial transcription factor A (TFAM) [17]. The following secondary antibodies were used: peroxidase-conjugated goat anti-mouse IgG (Perbio Science, Bonn, Germany) and peroxidase conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch, Cambridgeshire, UK). Results Mitochondria isolation using the MACS approach To test whether mitochondria could be successfully isolated with the MACS protocol, we generated a 293-reporter HEK cell line persistently expressing EGFP fused to a mitochondrial targeting sequence derived from the precursor of subunit VIII of human cytochrome c oxidase. Import of EGFP into, and fluorescence labeling of mitochondria in the stably transfected 293 HEK reporter cell line was verified by fluorescence microscopy (data not shown). An aliquot of the crude reporter cell lysate before mitochondria separation, the flow-through and washes, and eluted mitochondria were analyzed with a FACS. Mitochondria could be detected as green fluorescence in the crude reporter cell lysate (Fig. 1A). In the flow-through and wash fractions, a marginal EGFP signal was detected (Fig. 1B), whereas the mitochondrial fraction exhibited a strong EGFP signal overlapping with the original fraction (Fig. 1C and D). These data strongly indicate that mitochondria can be efficiently isolated by the MACS approach. To find out the advantages and disadvantages of the new approach, we compared the MACS protocol with the DC and UC methods, focusing on relevant parameters of mitochondria preparations such as yield, quality, and purity of the isolated organelles. Yield of isolated mitochondrial fractions The amount of mitochondria isolated from 293 HEK cells using the MACS protocol was twofold higher than that using DC (see Fig. S1A in supplementary material). In comparison with the UC method, the MACS approach yielded four times more mitochondria from osteosarcoma cells (see Fig. S1B). These results clearly indicate that the highest yield in two different cell types was achieved with the MACS protocol. Quality of mitochondrial preparations Well-coupled mitochondria are a prerequisite to achieving reliable reproducible results in nearly all functional assays. Coupling of isolated mitochondria reflecting the quality of the preparations was measured using an oxygen electrode. Respiratory control ratios (RCRs) higher than 3 are well accepted to indicate tightly coupled mitochondria [18]. All three methods provided tightly coupled mitochondria (see Fig. S1C in supplementary material). However, mitochondria isolated with the MACS protocol were found to be
Isolation of mitochondria by superparamagnetic microbeads / H.-T. Hornig-Do et al. / Anal. Biochem. 389 (2009) 1–5
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Fig. 1. FACS analysis of mitochondria isolated from transfected 293 HEK cells using the MACS protocol. Mitochondria in transfected 293 HEK cells express EGFP fused to a mitochondrial targeting sequence and, therefore, are fluorescently labeled. The green line displays the specific fluorescence of mitochondria, whereas the gray area represents the autofluorescence of mitochondria from untransfected 293 HEK cells. Compared with the crude cell lysate before isolation (A), the mitochondrial fraction (C) shows a significant enrichment of mitochondria, whereas hardly any signal was detectable in the flow-through and wash fractions (B). An overlay of the crude cell lysate and mitochondrial fraction (D) confirms the specific enrichment of mitochondria obtained with the MACS protocol.
better coupled than those obtained using DC, whereas RCRs of mitochondria isolated using the new approach and UC were similar. Enrichment of isolated mitochondria Western blot analysis revealed that the amount of mitochondrial proteins, such as cytochrome c oxidase subunit I (COXI, inner
membrane), TOM22 (outer membrane), mitochondrial transcription factor A (TFAM, nucleoid protein), and cytochrome c (attached to outer leaflet of inner membrane), were strongly increased in the mitochondrial fraction obtained using the MACS protocol (Fig. 2A). This indicates a significant enrichment of mitochondria in the eluted fraction. Moreover, FACS analysis of the mitochondrial fractions obtained by the three methods showed that 59% of the mito-
Fig. 2. (A) Enrichment of mitochondria. The amounts of subunit I of complex IV (COXI), 22-kDa translocase of outer mitochondrial membrane (TOM22), mitochondrial transcription factor TFAM (TFAM), and cytochrome c (Cyt c) in whole cell lysate were compared with equal amounts of protein from the mitochondrial fraction isolated from HeLa cells using the MACS protocol. (B) The amounts of b-actin, GAPDH, Rab4, and Golgin-97, representing proteins from cytoskeleton, cytosol, Golgi apparatus, and endosome, respectively, in whole cell lysate were compared with the equal protein amounts from the mitochondrial fraction isolated from HeLa cells using the MACS protocol. (C) Amounts of COXI (mitochondria), KDEL (endoplasmic reticulum), and lamins A and C (nucleus) in the mitochondrial fractions obtained using DC, the MACS approach, and UC.
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Isolation of mitochondria by superparamagnetic microbeads / H.-T. Hornig-Do et al. / Anal. Biochem. 389 (2009) 1–5
chondrial fraction of the DC preparation was TOM22 positive, whereas the mitochondrial fraction of the MACS preparation contained 89% TOM22 positive events and did not differ significantly from the mitochondrial fraction of the UC preparation, which showed 88% TOM22 positive events. Purity of mitochondrial fractions Another important aim of mitochondria preparation is to obtain highly pure organelles because the presence of contaminants might interfere with the analyses and the interpretation of results. Common relevant contaminants of mitochondria preparations are compounds of endoplasmic reticulum, endosomes, Golgi apparatus, nucleus, and cytosol, which can be monitored by Western blot analysis using antibodies specific for organelle/cell compartmentspecific marker proteins. b-Actin, GAPDH, Golgin-97, and Rab4, representing proteins from cytoskeleton, cytosol, Golgi apparatus, and endosome, respectively, could not be detected in the MACS preparation (Fig. 2B) or in other preparations (data not shown) but could be detected in the whole cell lysate. Mitochondria isolated by the MACS protocol showed fewer contaminants from endoplasmic reticulum and nucleus, detected with the antibodies against KDEL and lamins A and C, respectively, compared with the mitochondrial fraction obtained by the DC method. Mitochondria prepared by the UC method contained similar amounts of contaminants from endoplasmic reticulum and nucleus (Fig. 2C). Thus, the purity of mitochondria isolated with these latter two methods was comparable. Mitochondrial protein import An antibody against TOM22, a component of the translocase of the outer mitochondrial membrane (TOM), is used for purification of mitochondria in the MACS protocol. Because translocation of preproteins across the mitochondrial outer membrane is mediated by the TOM complex, targeting of TOM22 with antibodies might disturb protein import into mitochondria. Thus, we performed an import assay [19] with mitochondrial fractions isolated using the DC and MACS protocols and compared the efficiency of the import of the TFAM protein. As shown in Fig. 3, the 29-kDa TFAM precursor was partially converted into a 24-kDa protein in the presence of mitochondria. The imported protein was resistant to proteinase K treatment, whereas the 29-kDa precursor was accessible to diges-
Fig. 3. Import of TFAM into mitochondria isolated from HeLa cells using the MACS approach or DC. Mitochondria were incubated for 1 h with 35S-labeled TFAM. Import was studied by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and fluorography. Where indicated, CCCP was added during the incubation and proteinase K was added after the incubation. The first lane shows an aliquot of reticulocyte lysate, whereas the other lanes contain protein from sedimented mitochondria. p, precursor protein; m, imported mature protein. Import efficiency was calculated by (amount of imported TFAM) / (amount of precursor protein + imported protein).
tion. In the presence of the uncoupler CCCP, the import of TFAM was completely inhibited. The relative amount of imported TFAM into mitochondria isolated with the MACS approach was approximately 34% under our assay conditions and did not differ significantly from the imported amount obtained using the DC protocol. Therefore, an inhibitory effect of the used antibody on mitochondrial protein import could be excluded. FACS analysis of isolated mitochondria from HeLa wild-type and HeLa rho-0 cells PicoGreen was shown to stain mtDNA in living cells [20]. However, interference with the nuclear signal limits the use of PicoGreen alone. Here we used PicoGreen to easily distinguish between mitochondria containing mtDNA and mtDNA-free mitochondria isolated from HeLa wild-type and HeLa rho-0 cells, respectively. Mitochondria isolated from HeLa wild-type cells were TOM22 and PicoGreen positive, whereas PicoGreen fluorescence was heavily reduced in mitochondria obtained from HeLa rho-0 cells (Fig. 4).
Fig. 4. PicoGreen staining of mitochondria derived from HeLa wild-type and HeLa rho-0 cells. Both cell lines were stained with PicoGreen (2 ll/ml culture medium, Molecular Probes) for 1 h at 37 °C. Culture medium was replaced by dye-free medium. After a 20-min incubation step, cells were lysed and mitochondria were isolated using the MACS approach. Purified mitochondria were counterstained with anti-TOM22–biotin and anti-biotin–APC and were analyzed using a FACS.
Isolation of mitochondria by superparamagnetic microbeads / H.-T. Hornig-Do et al. / Anal. Biochem. 389 (2009) 1–5
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starting material (1 106 cells) but also permits the isolation of huge quantities of mitochondria from large amounts of material. Mitochondria isolated using the MACS approach were successfully tested in various assays such as protein import and BN–PAGE. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ab.2009.02.040. References
Fig. 5. Respiratory chain complexes from 143 B osteosarcoma cells (WT) and cybrid cells containing a truncation mutation in COXI (DCOX). Mitochondria of WT isolated with UC and mitochondria DCOX obtained using the MACS protocol were resolved by BN–PAGE followed by Western blot and were detected with monoclonal antibodies against 70-kDa subunit for complex II, core 2 for complex III, and COXI for complex IV. A mixture of high-molecular-weight purified proteins was used as molecular mass marker: thyroglobulin, 669 kDa; ferritin monomer, 440 kDa; catalase, 232 kDa; lactate dehydrogenase, 140 kDa; albumin, 66 kDa.
Analysis of membrane protein complexes To find out whether mitochondria isolated with the MACS protocol are capable of further analysis of the protein composition of the respiratory chain, we subjected mitochondria obtained using the UC or MACS protocol to blue native gel electrophoresis (BN– PAGE), and complexes and subcomplexes were identified by Western blot analysis [21,22]. Mitochondria from 143 B osteosarcoma cells (WT cybrid) were isolated with the UC method. The MACS protocol was used to obtain mitochondria from cybrid cells containing a truncation mutation in COXI (DCOX). As shown previously [23], in contrast to WT cybrid cells, no complex IV was found in DCOX cybrid cells because assembly of this complex was severely disturbed (Fig. 5). The expression of complexes II and III, which are not affected by the mutation, did not differ significantly in both cybrid cell lines, demonstrating that mitochondria isolated with the MACS approach are suitable for BN–PAGE. Conclusions The ultimate goal of a mitochondrial isolation protocol is to obtain organelles as pure and functional as possible. For some applications, such as measurement of OXPHOS enzyme activity, crude mitochondrial fractions obtained using DC are sufficient. For other purposes, such as analysis of the mitochondrial proteome, one has the choice of different approaches to obtain highly pure mitochondria. Here we have developed a new approach that provides functional intact mitochondria with a very high yield in a fast, reproducible, and standardized procedure. Furthermore, the quality and purity of the obtained mitochondrial fraction using the MACS approach are comparable to those using the UC method, which also is time-consuming and needs access to an ultracentrifuge. Our method is generally applicable to a variety of different human cell lines such as 293 HEK, HeLa, and 143 B osteosarcoma cybrid cells. Another great advantage of the method presented here is that the approach can be performed with very small amounts of
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