Methods in cell biology 80, mitochondria, 2nd ed l pon, e schon (AP, 2007)

148 Kurenai Tanji and Eduardo Bonilla 12 Rinse the slides with dH2O several times 13 Counterstain the slides briefly with hematoxylin 14 Rinse the slides with dH2O, dehydrate through ascending ethanol series, and clear in xylene 15 Mount the slides with synthetic resin (Permount) IV In Situ Hybridization to mtDNA ISH is a technique that permits the precise cellular localization and identification of cells that express a particular nucleic acid sequence The essence of this method is the hybridization of a nucleic acid probe with a specific nucleic acid sequence found in a tissue section ISH has been used extensively in human pathologic conditions to correlate mitochondrial abnormalities with the presence of mutated mtDNAs, an analysis that provides strong support for a pathogenic role of a specific mitochondrial genotype As the method relies on sequence homology, it has been applied mainly to diVerentiate mtDNAs with large-scale deletions from wild-type sequences in samples from patients with KSS or isolated OM In these studies, two probes have been used to determine the distribution of wild-type and D-mtDNAs: one probe, in the undeleted region, hybridizes to both wild-type and D-mtDNAs (the ‘‘common’’ probe); the other, correspond to mtDNA sequences located within the deletion, hybridizes only to wild-type mtDNAs (the ‘‘wild-type’’ probe) Typical results on serial muscle sections showed abundant hybridization signal (focal accumulations of mtDNAs) with the common probe, but not with the wild-type probe, in COX-deficient RRF of the patients (Bonilla et al., 1992; Moraes et al., 1992) These observations indicated that the predominant species of mtDNAs in COX-deficient RRF of KSS and OM patients are D-mtDNAs Furthermore, the concentration of D-mtDNAs in these RRF reached the required threshold level to impair the translation of the mitochondrial genome because they were characterized immunohistochemically by a lack or a marked reduction of the mtDNA-encoded COX II polypeptide (Bonilla et al., 1992; Moraes et al., 1992) An important extension of ISH to mtDNA is regional ISH, which has been applied to determine the spatial distribution of multiple D-mtDNAs in samples from patients with Mendelian-inherited OM These are disorders characterized by progressive external ophthalmoplegia (PEO) and mitochondrial myopathy In Mendelian OM, hundreds of diVerent deletions coexist within the same muscle in aVected family members Thus, as opposed to the single deletions found in sporadic KSS and OM, Mendelian OM is associated with multiple D-mtDNAs that are apparently generated over the life span of the individual The genetic defects in these disorders cause mutations in nuclear gene products that regulate the mitochondrial nucleotide pool (DiMauro and Bonilla, 2004) ISH of serial muscle sections from these patients, in which a diVerent mtDNA regional probe was used on each section, showed specific, and diVerent, RRFs losing hybridization Optical Imaging Techniques to Visualize Mitochondria 149 signal with each specific probe, while the remaining RRFs hybridized intensely These observations have provided the strongest evidence to date that each RRF in Mendelian-inherited OM contains a clonal expansion of a single species of D-mtDNA (Moslemi et al., 1996; Vu et al., 2000) Although ISH utilizing RNA probes is more widely used in typical cell and molecular biology applications, most of our recent experience derives from the use of digoxigenin (DIG)-labeled DNA probes to visualize mtDNA (Manfredi et al., 1997; Vu et al., 2000) We describe here a method that we employ for the identification of RRF (Fig 4) and for the detection of depletion of mtDNA on muscle-frozen sections Although tissue samples can be frozen or fixed and paraYn-embedded, we described a procedure optimized for frozen sections that has been tested thoroughly on muscle sections from patients with mitochondrial myopathies Because of the focal pattern of distribution of mutant and wild-type mtDNAs in muscle fibers, one must make sure that serial sections above and below the ones used for ISH are characterized histochemically by staining for COX and SDH activities (Fig 4) The size of the labeled probe is very important and should be optimized to promote specificity and tissue penetration Sizes between 300 and 400 nucleotides are optimum, even though we have obtained excellent results with 500-nucleotide probes DNA probes can be prepared in diVerent ways, but we routinely use PCR-generated DIG-labeled mtDNA probes Kits and polymerases for DNA labeling are available from most molecular biology companies It is important that prior to performing ISH experiments, the concentration of the probe be determined by dot blot and that the specificity of the probe be tested by Southern blot Method Collect 8-mm-thick frozen sections on poly(L-lysine)-coated (0.1%) slides Fix the sections with 4% paraformaldehyde for 30 at RT Rinse the slides with PBS containing 5-mM MgCl2 (PBS–MgCl2, pH 7.4) three times for at RT Incubate the sections with 5-mg/ml proteinase K for h at 37  C Place the slides in PBS–MgCl2 at  C for to remove the proteinase K Acetylate the sections by incubating in 0.1-M triethanolamine containing 0.25% acetic anhydride for 10 at RT Treat the slides with 5-mg/ml RNase (DNase-free) in RNase buVer (50-mM NaCl and 10-mM Tris–HCl, pH 8) for 30 at 37  C Rinse the slides briefly with PBS–MgCl2 Dehydrate the sections through ascending series of ethanol (optional) 10 Incubate with hybridization buVer without probe (prehybridization) for 1–2 h at 37  C (In our experience, this step can be eliminated.) Hybridization solution: 50% deionized formamide, 20-mM Tris–HCl, 0.5-mM NaCl, 10-mM EDTA, 0.02% Ficoll, 0.02% polyvinylpyrollidone, 0.12% BSA, 150 Kurenai Tanji and Eduardo Bonilla Fig Cellular localization of mtDNA by ISH Serial muscle sections from a patient with MERRF were stained for SDH (A) and COX (B) activity, and for mtDNA localized by ISH using digoxigeninlabeled probes (C) The ISH signal is seen as the red material A control section subjected to ISH without the denaturing step (D) shows no hybridization signal Note the strong mtDNA signal in an RRF (asterisk) characterized by increased SDH activity and lack of COX activity Bar ¼ 50 m Optical Imaging Techniques to Visualize Mitochondria 11 12 13 14 15 16 17 18 19 20 21 151 0.05% salmon sperm DNA, 0.05% total yeast RNA, 0.01% yeast tRNA, and 10% dextran sulfate Denature the probe/hybridization solution (20 ng/ml) at 92  C for 10 and immediately place the solution on ice until it is applied to the sections Blot oV excess hybridization solution and apply probe/hybridization solution to the sections Denature the sections covered with the hybridization solution at 92  C for 10–15 Hybridize overnight at 42  C Rinse the slides briefly with 2Â SSC (3-M NaCl, 0.3-M sodium citrate, pH 7.0) at RT followed by rinsing twice with 1Â SSC for 15 at 45  C, and once with 0.2Â SSC for 30 at 45  C Rinse the slides with PBS for at RT Incubate the slides with 1% BSA for h at RT Incubate the slides with alkaline phosphatase-conjugated anti-DIG antibody (1:1000–1:5000) for h at RT Rinse the slides with PBS three times for each, at RT Incubate the slides with color-substrate solution (SIGMA FASTTM Fast Red TR/Naphthol AS-MX Alkaline phosphatase substrate tablets) until the desired strength of the signal is obtained Mount the sections with glycerol-PBS, 1:1 Acknowledgments This work was supported by grants from the National Institutes of Health (NS11766 and PO1HD32062) References Ackrell, B A C (2002) Cytopathies involving 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M., Brown, G K., Brown, R M., et al (1998) SURF-1, encoding a factor involved in the biogenesis of cytochrome c oxidase, is mutated in Leigh syndrome Nat Genet 20, 337–343 CHAPTER Assay of Mitochondrial ATP Synthesis in Animal Cells and Tissues Cristofol Vives-Bauza, Lichuan Yang, and Giovanni Manfredi Department of Neurology and Neuroscience Weill Medical College of Cornell University New York, New York 10021 I Introduction II ATP Synthesis Assays III Methodological Considerations A Cell Permeabilization (Detergent Titration in Cultured Cells) B ATP Detection by Luciferase–Luciferin C Specificity of the Assay IV Experimental Procedures A Measurement of ATP Synthesis in Cultured Cells B ATP Synthesis in Mitochondria Isolated from Animal Tissues V Measurement of High-Energy Phosphates in Animal Tissue and Cultured Cells by HPLC A Apparatus and Reagents B Preparation of Biological Samples C Chromatography D Standard Curves E Measurement of Creatine, Phosphocreatine, and Phosphorylated Nucleotides References I Introduction Mitochondria are the major cellular source of adenosine triphosphate (ATP) synthesis Its oxidative phosphorylation system generates 36 molecules of ATP per molecule of glucose, as opposed to only molecules of ATP generated by METHODS IN CELL BIOLOGY, VOL 80 Copyright 2007, Elsevier Inc All rights reserved 155 0091-679X/07 $35.00 DOI: 10.1016/S0091-679X(06)80007-5 156 Cristofol Vives-Bauza et al glycolysis in the cytoplasm Therefore, the measurement of mitochondrial ATP synthesis can be considered a pivotal tool in understanding many important characteristics of cellular energy metabolism, both in the normal physiological state and in pathological conditions such as mitochondrial disorders In the first section of this chapter, we will discuss approaches to measure ATP synthesis from mammalian cells and tissues In the second section, we will discuss procedures to estimate the steady-state content of ATP and other high-energy phosphates by high-performance liquid chromatography (HPLC) II ATP Synthesis Assays Two methodological issues need to be addressed when measuring mitochondrial ATP synthesis The first one is how to deliver reaction substrates to the mitochondria Because of the low permeability of the plasma membrane to some hydrophilic substrates, such as adenosine diphosphate (ADP), some investigators prefer to analyze isolated mitochondria (Tatuch and Robinson, 1993; Tuena de Gomez-Puyou et al., 1984; Vazquez-Memije et al., 1996) However, for ADP phosphorylation to take place, mitochondria need to be maintained intact and in a coupled state Therefore, ATP synthesis can only be measured on freshly isolated mitochondria Moreover, the isolation of highly coupled mitochondria from cultured cells involves delicate and time-consuming procedures, and the results are sometimes inconsistent, leading to a potential lack of reproducibility Measurement of ATP synthesis on whole cells requires a permeabilization step to allow for the penetration of hydrophilic substrates through biological membranes Cell membranes can be permeabilized with detergents such as saponin (Kunz et al., 1993) or digitonin (Houstek et al., 1995; Wanders et al., 1993, 1994, 1996) In addition, the use of permeabilized cells rather than isolated mitochondria reduces substantially the number of cells required for each assay The second issue is how to detect and quantify ATP Fluorimetry is a commonly used method to detect ATP produced by isolated mitochondria (Houstek et al., 1995; Tatuch and Robinson, 1993; Wanders et al., 1993, 1994, 1996) Another method employed on isolated mitochondria is the incorporation of [32]Pi into ADP and its subsequent transfer to glucose-6-phosphate by hexokinase, followed by extraction of unincorporated [32]Pi and measurement of radioactivity in a scintillation counter (Tuena de Gomez-Puyou et al., 1984) Typically, for steady-state ATP measurements based on fluorescence or radioassays, isolated mitochondria or permeabilized cells are incubated with appropriate substrates followed by a lysis extraction in strong acid conditions to inactivate cellular ATPases With this approach, in order to obtain kinetic measurements of ATP synthesis, replicate tests have to be run at various time intervals Alternatively, it would be convenient to perform measurements of ATP synthesis, which not require repeated sampling and which allow for kinetic measurements to be performed on single samples In this chapter, we describe a rapid kinetic approach Assay of Mitochondrial ATP Synthesis in Animal Cells and Tissues 157 to monitor continuous ATP synthesis in mammalian cells that takes advantage of the luciferase–luciferin system Firefly luciferase is widely used as a reporter for gene expression to study promoter regulation in mammalian cells, but its bioluminescence properties have also been used to measure ATP content in isolated mitochondria (Lemasters and Hackenbrock, 1973; Strehler and Totter, 1952; Wibom et al., 1990, 1991) and in permeabilized cells (James et al., 1999; Maechler et al., 1998; Ouhabi et al., 1998) The reaction catalyzed by luciferase is: Luciferase ỵ Luciferin ỵ ATP ! Luciferase - Luciferyl - AMP ỵ PPi Luciferase - Luciferyl - AMP ỵ O2 ! Luciferase ỵ Oxyluciferin ỵ AMP ỵ CO2 ỵ hv The reaction produces a flash of yellow-green light, with a peak emission at 560 nm, whose intensity is proportional to the amount of substrates in the reaction mixture (Deluca, 1976) III Methodological Considerations A Cell Permeabilization (Detergent Titration in Cultured Cells) Although more convenient than isolating coupled mitochondria, permeabilization procedures also require standardization InsuYcient permeabilization could result in an underestimation of ATP synthesis due to lack of available substrates Conversely, excessive permeabilization leads to mitochondrial membrane damage and uncoupling For these reasons, it is important to establish the optimal amount of detergent needed per unit of cell protein We use digitonin as the detergent of choice because it results in plasma membrane permeabilization at concentrations that not aVect mitochondrial membranes significantly Alternatively, saponin may be used as a detergent (Kunz et al., 1993) particularly in muscle cells Due to variation in membrane cholesterol content, the parameters for digitonin treatment need to be optimized for each cell type In our experience, the digitonin concentration at which HeLa and COS-7 cells (1-mg/ml cell proteins) become permeable to substrates and achieve the maximal ATP synthesis rate is 50 mg/ml, while N2A mouse neuroblastoma cells require 25 mg/ml and HEK 293T (HEK, human embryonic kidney cells) cells require 75 mg/ml (Fig 1) B ATP Detection by Luciferase–Luciferin A number of variables need to be taken into consideration in setting up a luminescence assay First, depending on the ATP concentrations, firefly luciferase shows two diVerent rates of light production, possibly due to the binding of substrates at two diVerent catalytic sites At high concentrations of ATP, a short flash ... ATP–luminescence curve is constructed by measuring flash luminescence derived from ATP solutions containing 0-, 0.0 5-, 0. 1-, 0. 5-, 1-, 5-, and 10-mM ATP in buVer A plus 10 ml of buVer B, using a single... the cells are rinsed and maintained in glucose-free buVer to deplete most of the residual intracellular glucose By assaying luminescence with 160 Cristofol Vives-Bauza et al and without inhibitors... combine: 20-ml hydrazine buVer (0.1-M Tris, 0.4-M hydrazine, 0.4-mM EDTA, 10-mM MgSO4, pH 8.5) 200-ml NAD, 80 mg/ml 200-ml LDH, 50 mg/10 ml in distilled water Pipette ml of the above mixture into