Quantitation of DNA Copy Number in Individual Mitochondrial

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Anal. Chem. 2007, 79, 7691-7699

Quantitation of DNA Copy Number in Individual Mitochondrial Particles by Capillary Electrophoresis Marian Navratil, Bobby G. Poe, and Edgar A. Arriaga*

Department of Chemistry, University of Minnesota, 207 Pleasant Street SE, Minneapolis, Minnesota 55455

Here, we present a direct method for determining mitochondrial DNA (mtDNA) copy numbers in individual mitochondrial particles, isolated from cultured cells, by means of capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. We demonstrate that this method can detect a single molecule of PicoGreen-stained mtDNA in intact DsRed2-labeled mitochondrial particles isolated from human osteosarcoma 143B cells. This ultimate limit of mtDNA detection made it possible to confirm that an individual mitochondrial nucleoid, the genetic unit of mitochondrial inheritance, can contain a single copy of mtDNA. The validation of this approach was achieved via monitoring chemically induced mtDNA depletion and comparing the CE-LIF results to those obtained by quantitative microscopy imaging and multiplex realtime PCR analysis. Owing to its sensitivity, the CE-LIF method may become a powerful tool for investigating the copy number and organization of mtDNA in mitochondrial disease and aging, and in molecular biology techniques requiring manipulation and quantitation of DNA molecules such as plasmids. Mitochondria play an important role in a number of cellular processes, including ATP production, iron and calcium homeostasis, and apoptosis signaling. In human cells, mitochondrial DNA (mtDNA) is a 16 569-base-pair-long, double-stranded, circular molecule that is present in hundreds to thousands of copies. It encodes 13 polypeptides involved in oxidative phosphorylation, 2 rRNAs, and 22 tRNAs,1 which combined together represent less than 1% of all gene products found in mitochondria. Despite its relatively small size, this polyploid genome is clearly an Achilles’ heel in cellular function, as hundreds of different mutations in mtDNA have been associated with aging,2,3 diabetes,4 a number of cardiomyopathies, and neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease.5 Variation in the mtDNA * To whom correspondence should be addressed, Phone: 612-624-8024. Fax: 612-626-7541. E-mail: [email protected]. (1) Anderson, S.; Bankier, A. T.; Barrell, B. G.; de Bruijn, M. H.; Coulson, A. R.; Drouin, J.; Eperon, I. C.; Nierlich, D. P.; Roe, B. A.; Sanger, F.; Schreier, P. H.; Smith, A. J.; Staden, R.; Young, I. G. Nature 1981, 290, 457-465. (2) Berdanier, C. D.; Everts, H. B. Mutat. Res. 2001, 475, 169-183. (3) Drew, B.; Leeuwenburgh, C. Acta Physiol. Scand. 2004, 182, 333-341. (4) Reardon, W.; Ross, R. J.; Sweeney, M. G.; Luxon, L. M.; Pembrey, M. E.; Harding, A. E.; Trembath, R. C. Lancet 1992, 340, 1376-1379. (5) Egensperger, R.; Kosel, S.; Schnopp, N. M.; Mehraein, P.; Graeber, M. B. Neuropathol. Appl. Neurobiol. 1997, 23, 315-321. 10.1021/ac0709192 CCC: $37.00 Published on Web 09/19/2007

© 2007 American Chemical Society

copy number also influences the phenotype and affects the onset of a disease.6 While elucidating the organization, distribution, and inheritance of mtDNA at the subcellular level would be highly beneficial to understand the role of mitochondria in disease, these studies have been complicated by the reticular structure of mitochondria. There is a need for single-organelle methods that are able to sample the complex mitochondrial network and quantitate single molecules of mtDNA for monitoring the progression of mitochondrial diseases and aging. Although methods such as real-time PCR,7-9 Southern blotting,10 or fluorescence microscopy,11 have been reported for relative mtDNA quantitation in bulk, information about the absolute mtDNA content in individual organelles is limited. Capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection has been used previously to characterize the properties of individual organelles, including mitochondria,12 nuclei,13 and acidic organelles.14 Following electrophoretic separation, the detection of electrodispersed individual organelles is accomplished using off-column LIF detection in a sheath flow cuvette,15 which, in special situations, allows for single-molecule detection. This report presents a direct method for determining the mtDNA copy number in individual mitochondrial particles based on CE-LIF. As a proof of concept, we used this method to detect chemically induced mtDNA depletion in human osteosarcoma 143B cells, down to extremely low mtDNA copy numbers. At the early stages of mtDNA depletion, the mtDNA content quantitated by CE-LIF exhibited trends similar to those obtained by real-time PCR (qPCR) and live confocal microscopy imaging, but the last two techniques are less adequate to monitor mtDNA levels at the late stages of depletion, where the copy numbers are low. This method’s ability to detect a single mtDNA molecule in individual (6) Bentlage, H. A.; Attardi, G. Hum. Mol. Genet. 1996, 5, 197-205. (7) Bai, R. K.; Perng, C. L.; Hsu, C. H.; Wong, L. J. Ann. N. Y. Acad. Sci. 2004, 1011, 304-309. (8) He, L.; Chinnery, P. F.; Durham, S. E.; Blakely, E. L.; Wardell, T. M.; Borthwick, G. M.; Taylor, R. W.; Turnbull, D. M. Nucleic Acids Res. 2002, 30, e68. (9) Miller, F. J.; Rosenfeldt, F. L.; Zhang, C.; Linnane, A. W.; Nagley, P. Nucleic Acids Res. 2003, 31, e61. (10) Moraes, C. T. Trends Genet. 2001, 17, 199-205. (11) Dellinger, M.; Geze, M. J. Microsc. 2001, 204, 196-202. (12) Presley, A. D.; Fuller, K. M.; Arriaga, E. A. J. Chromatogr., B: Anal. Technol. Biomed. Life Sci. 2003, 793, 141-150. (13) Xiong, G.; Chen, Y.; Arriaga, E. A. Anal. Chem. 2005, 77, 3488-3493. (14) Chen, Y.; Walsh, R. J.; Arriaga, E. A. Anal. Chem. 2005, 77, 2281-2287. (15) Cheng, Y. F.; Wu, S.; Chen, D. Y.; Dovichi, N. J. Anal. Chem. 1990, 62, 496-503.

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biological particles makes CE-LIF the ideal tool for investigating the intracellular mtDNA organization and segregation, as well as perturbations which are believed to contribute directly to the pathogenesis of mitochondrial diseases.16 Last, this method may also find wide application in many fields of molecular and cell biology that require manipulation of mtDNA or other circular forms of DNA such as plasmids. MATERIALS AND METHODS Reagents. Sucrose was purchased from Fluka (Buchs, Switzerland); DMSO and ethanol were purchased from Fisher Scientific (Pittsburgh, PA). Trypsin solution (10×, 5.0 g/L trypsin, 2.0 g/L EDTA‚4Na, 8.5 g/L NaCl), N-(2-hydroxyethyl)(piperazine)N-(ethanesulfonic acid) (HEPES), phosphate-buffered saline (10× PBS, containing 100 mM KH2PO4/Na2HPO4 solution, pH 7.4, 27 mM KCl, 1370 mM NaCl), OPTI-MEM medium, powdered minimum essential medium (MEM), Tris-HCl, gentamicin, uridine, and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma (St. Louis, MO). Fluorescein, PicoGreen, geneticin, λ DNA, DMRIE-C lipofection reagent, and fetal bovine serum were purchased from Invitrogen (Carlsbad, CA). Mitochondria-targeted red fluorescent protein was expressed from a commercially available plasmid, pDsRed2-Mito (Clontech, Mountain View, CA). Mannitol was purchased from Riedel de-Hae¨n (Seelze, Germany). Ethidium bromide was purchased from Bio-Rad (Hercules, CA). Buffers. Capillary electrophoresis buffer contained 250 mM sucrose and 10 mM HEPES, pH 7.4. Buffer M contained 70 mM sucrose, 5 mM HEPES, 5 mM EDTA, and 210 mM mannitol. These buffers were prepared using Milli-Q deionized water, filtered with a 0.22-µm membrane, and adjusted to pH 7.4 with KOH. Cell culture. Human osteosarcoma 143B cell line (ATCC, Manassas, VA) was cultured in 0.22-µm-filtered DMEM medium containing 10% (v/v) fetal bovine serum, 50 µg/mL uridine, and 10 µg/mL gentamicin stored at 4 °C. The high-glucose DMEM medium was used to compensate for the dysfunction of the oxidative phosphorylation system in the late stages of the mtDNA depletion. All cells were cultured in 75-cm2 vented culture flasks at 37 °C and 5% CO2 and split every 3-4 days. For splitting, the cells were rinsed with PBS, lifted with 0.25 g/L trypsin for 5 min, and diluted in fresh growth medium. Construction of Stably Transfected Fluorescent Clones. Human osteosarcoma 143B cells were transfected with the pDsRed2-Mito plasmid, coding for a fusion protein of red fluorescent protein (DsRed2) and the mitochondrial targeting sequence from subunit VIII of cytochrome c oxidase, containing the neomycin/kanamycin resistance gene. The transfection was performed using the lipofection reagent DMRIE-C according to the manufacturer’s instructions. Briefly, the plasmid was suspended in OPTI-MEM medium and incubated with DMRIE-C at room temperature for 30 min. The cells were washed with OPTIMEM before the lipid-DNA complex was layered over the cells. After 6 h, cell culture medium (MEM containing 20% calf serum) was added. At 24-h post transfection, the cells were lifted, seeded onto a 10-cm Petri dish at low density, and cultured in growth medium containing 500 µg/mL geneticin. After 5-6 days, a single colony of the successfully transfected cells was isolated using (16) Chen, X. J.; Butow, R. A. Nat. Rev. Genet. 2005, 6, 815-825.

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cloning rings and subcultured. The stable fluorescent transformants were maintained in DMEM growth medium containing 250 µg/mL geneticin. Mitochondrial DNA Depletion. The 143B cells expressing the DsRed2 fluorescent protein targeted to mitochondria were cultured in the presence of 50 ng/mL ethidium bromide (EtBr) for 1-4 days to deplete the cells of mtDNA. At this concentration, mtDNA replication is inhibited, but the viability of the cell culture remains unaffected.17 Untreated cells were used as a reference. Mitochondrial DNA Labeling with PicoGreen. The stock solution of PicoGreen reagent, as provided by the manufacturer, was diluted 3:1000 in warm growth medium. Adhered 143B cells were labeled with the PicoGreen solution for 60 min at 37 °C and 5% CO2. Following labeling, the excess PicoGreen was removed by washing the cells twice with growth medium. For real-time PCR analysis, PicoGreen labeling was omitted. Sample Preparation for CE-LIF. Cells were harvested by adding 6 mL of a 143B cell suspension (∼8 × 105 cells/mL) to 6 mL of growth medium and centrifuged at 1000g for 5 min. The supernatant was removed, and the cells were rinsed by centrifugation at 1000g and resuspended in 3 mL of buffer M. The cell suspension was disrupted with a nitrogen cavitator (Parr Instruments, Moline, IL) at 500 psi for 15 min at 0 °C. The cell lysate was then centrifuged at 1400g for 10 min to eliminate intact cells, nuclei, and heavy cellular debris. The supernatant containing mitochondria was removed, and mitochondria were sedimented by centrifugation at 16000g for 10 min. The mitochondrial pellet was washed, resuspended in 0.5 mL of CE buffer, and analyzed. The mitochondria sample produced by this isolation procedure is mitochondria-enriched but also contains other contaminating organelles, e.g., peroxisomes. However, these contaminating organelles can be excluded by dual labeling with mitochondriatargeted DsRed2 fluorescent protein and PicoGreen. CE-LIF Setup. The design of the CE system with postcolumn LIF detection depicted in Supporting Information Figure 1 is similar to that of an instrument that has been described previously.18 A 488-nm Ar ion laser line (Melles Griot, Irvine, CA) was used for excitation. Fluorescence from PicoGreen and DsRed2 was separated using a 560-nm long-pass dichroic mirror (Omega Optical, Brattleboro, VT) and measured with two photomultiplier tubes (R1477; Hamamatsu, Bridgewater, NJ) equipped with interference filters transmitting in the range of 502.5-537.5 and 607.5-662.5 nm, respectively. The output from the PMTs was digitized at 200 Hz using a NiDaq I/O board (PCI-MIO-16XE-50, National Instruments, Austin, TX), and the PicoGreen and DsRed2 fluorescence data were saved as a binary file by LabView (National Instruments). The detector was aligned by continuously, electrokinetically injecting a 10-9 M fluorescein solution at -400 V/cm while optimizing the position of the capillary outlet to collect maximum fluorescence. The limit of detection for fluorescein was ∼5 zmol, and the relative standard deviation for the fluorescence intensity of individual fluorescent microspheres was ∼10%. CE-LIF Analysis of Mitochondria. Mitochondria were hydrodynamically injected by siphoning at 7.2 kPa for ∼5 s. Then, the capillary inlet was placed in fresh running buffer and the (17) Nass, M. M. Exp. Cell Res. 1972, 72, 211-222. (18) Duffy, C. F.; Fuller, K. M.; Malvey, M. W.; O’Kennedy, R.; Arriaga, E. A. Anal. Chem. 2002, 74, 171-176.

separation potential was applied. CE-LIF analysis of isolated mitochondria was carried out at -300 V/cm in a 49.4-cm-long, 50-µm-i.d., fused-silica capillary coated with poly(acryloylaminopropanol)19 in order to reduce the interactions of the outer mitochondrial proteins with the capillary wall. The CE-LIF data were analyzed using Igor Pro 5.02 software (Wavemetrics, Lake Oswego, OR). The cross-talk between the PicoGreen and DsRed2 channels, resulting from spectral overlap of their emission spectra, was measured and corrected for using PicoGreen-labeled mitochondria and DsRed2-labeled mitochondria. The cross-talk from the PicoGreen channel into the DsRed2 channel was 9.5%, and conversely, that from the DsRed2 into the PicoGreen channel was 15.7% (data not shown). The fluorescence intensity for each detected event, with an intensity greater than 5× the standard deviation of the background, was tabulated using the in-housewritten procedure PickPeaks. Peaks that occurred simultaneously in both channels were tabulated using an in-house-written procedure. Detection of Individual Mitochondrial Particles by CELIF. Mitochondria that migrate out of the capillary simultaneously could be detected as a single event, with a fluorescence intensity equal to the sum of the intensities of all comigrating mitochondria, which could bias the quantitation. Therefore, the probability of detecting multiple mitochondria as a single event was estimated using the statistical overlap theory (SOT). While SOT was developed to predict the probability of detecting single-component peaks in poorly resolved multicomponent gas chromatography separations,20 it has also been applied to electrophoretic separations by CE-LIF,21 since the temporal distribution of particles is governed by homogeneous statistics. According to SOT, the probability of detecting singlets, doublets, and triplets, for the most crowded region in Figure 1a (750-800 s), was 97.6, 1.2, and 0.014%, respectively, so that the majority of detected events can be considered individual mitochondria. mtDNA Standard Preparation. A 567-bp-long mtDNA standard spanning positions 7743-8310 was amplified from 143B mtDNA using the following primers: 5′-CTAACATCTCAGACGCTCAGG-3′ and 5′-AGTTAGCTTTACAGTGGGCTC-3′. Following PCR, the amplified mtDNA was separated on a 2% agarose gel, imaged with ethidium bromide, and the band was excised. The mtDNA standard was purified from the agarose gel using the High Pure PCR Product Purification Kit (Roche Diagnostics, Indianapolis, IN) and quantitated with Quant-iT PicoGreen dsDNA assay kit (Invitrogen, Carlsbad, CA). Determination of mtDNA Copy Number in Individual Mitochondria. Absolute quantitation of mtDNA by qPCR was performed using the 567-bp-long mtDNA standard. A flowchart illustrating the determination of mtDNA copy number in individual mitochondria is shown in Supporting Information Figure 2. The mitochondrial preparations used for CE-LIF analysis were solubilized (see Sample Preparation for Real-Time PCR Analysis) to release mtDNA from mitochondria (panel A). PCR was performed (panel B) as described below using the MB COX II primers and molecular beacon (see Table 1). The number of mtDNA copies (19) Gelfi, C.; Curcio, M.; Righetti, P. G.; Sebastiano, R.; Citterio, A.; Ahmadzadeh, H.; Dovichi, N. J. Electrophoresis 1998, 19, 1677-1682. (20) Davis, J. M. Anal. Chem. 1997, 69, 3796-3805. (21) Ahmadzadeh, H.; Dua, R.; Presley, A. D.; Arriaga, E. A. J. Chromatogr., A 2005, 1064, 107-114.

per microliter in each sample was determined in quadruplicate using a calibration curve constructed with the mtDNA standard (panel C). The calibration curve ranged from 6 × 106 to 6 copies, and the PCR amplification efficiency determined by analyzing serial dilutions of the standard was 99.8% (panel B). In order to calculate the mtDNA copy number for each detected mitochondrial event by CE-LIF (panel D), the sum of all the PicoGreen fluorescence intensities, as measured by CE-LIF, was divided by the number of mtDNA copies in the CE-LIF injection volume (10.20 ( 0.03 nL), as determined by qPCR (panel G). This gives a factor that relates the PicoGreen fluorescence intensity to the number of mtDNA copies (e.g., 0.0145 ( 0.0015 AU/copy of mtDNA; N ) 4). The mtDNA copy number for each mitochondrion was calculated by dividing the fluorescence intensity of each spike by this factor. Three assumptions were made to calculate the mtDNA copy number in isolated mitochondria: First, PicoGreen binds to mtDNA quantitatively; the PicoGreen reagent was used in excess and it has been reported to detect DNA at concentrations as low as 25 pg/mL, even in the presence of salts or proteins.22 Second, EtBr (50 ng/mL) used for mtDNA depletion does not affect the quantitation of mtDNA; the fluorescence response in the range of 25 pg/mL-25 ng/mL λDNA remained linear even upon addition of 50 ng/mL EtBr (r2 ) 0.9999), and the slope of the calibration curve was only slightly decreased (6.5%; data not shown). Last, the fluorescence of free Picogreen within mitochondria does not interfere with the DNA detection; it has been reported that PicoGreen undergoes 1980-fold fluorescence enhancement upon binding double-stranded DNA, and DNA quantitation over 4 orders of magnitude is possible using the same dye concentration.22 Confocal Microscopy Imaging. For live confocal microscopy imaging, an Olympus IX-81 inverted fluorescence microscope (Melville, NY) equipped with a 120-W xenon lamp, a DS-IX100 disk spinning unit (Olympus America Inc., Melville, NY), and a C9100-01 EM CCD camera (Hamamatsu Corp., Bridgewater, NJ) was used. The 143B cells expressing DsRed2 were grown to low confluence on chambered coverslips (Nalge Nunc International, Rochester, NY), labeled with PicoGreen prior to the microscopic analysis and imaged in an environmental microincubator (Harvard Apparatus, Holliston, MA) to maintain physiological temperature. Confocal images of the cell plane with the highest mitochondria density were captured with an oil immersion 60× objective (NA 1.45; Olympus America Inc.) using TRITC (e. 510-560 nm, 565 nm dichroic, em 570-650 nm; Chroma, Rockingham, VT) and FITC (ex 460-500 nm, 505 nm dichroic, em. 510-560 nm; Chroma) filter cubes with the same exposure times for all of the images (10 s for both fluorophores). Acquisition was performed using SimplePCI 5.3 software (Compix Imaging Systems, Cranberry Township, PA). Analysis of Confocal Images. Images were analyzed using SimplePCI 5.3 software as follows: the green and red components of each two-color image were contrast-enhanced and saved as two separate grayscale images. The fluorescence intensity of the cytosol (area not containing any mitochondria or the nucleus) was measured to calculate the mean background fluorescence (Bg) (22) Singer, V. L.; Jones, L. J.; Yue, S. T.; Haugland, R. P. Anal. Biochem. 1997, 249, 228-238.

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Figure 1. CE-LIF detection of mtDNA in individual mitochondrial particles. Mitochondrial particles isolated from 143B cells expressing the DsRed2 fluorescent protein targeted to mitochondria were labeled with PicoGreen for mtDNA detection. (a) Two traces in the resulting electropherogram represent the PicoGreen signal (upper trace) and the DsRed2 signal (lower trace) and were offset for clarity. (b) is an enlarged view of the 482-500-s detection window where three kinds of events can be seen (*, red-only; ×, green-only; O, coincident). The dashed line represents the detection threshold; the right axis shows the mtDNA copy number, as detected in PicoGreen-labeled mitochondrial particles (upper trace) by CE-LIF. The traces were offset vertically for clarity. (c) mtDNA copy number was calculated for each event in the electropherogram shown in Figure 3a using its green fluorescent intensity and the total mtDNA content determined by real-time PCR. The bars represent the average percent of occurrence of a given number of mtDNA copies per mitochondrial particle. The error bars represent the standard deviation for each bin as calculated from the distributions of five consecutive runs. (d) is a comparison of the mtDNA copy number distributions for mitochondrial particles isolated from cells treated with ethidium bromide for 0-4 days (D0-D4). The individual histograms are shown in Supporting Information Figure 4. Table 1. Primers and Molecular Beacons primers forward β-globin reverse β-globin forward COX II reverse COX II

70,803-70,824 71,049-71,074 7,798-7,820 8,186-8,209

sequence 5′-TTTCCCACCCTTAGGCTGCTG-3′ 5′-GGGAAAGAAAACATCAAGCGTCCCA-3′ 5′-CATCCTAGTCCTCATCGCCCTCC-3′ 5′-GGGCATGAAACTGTGGTTTGCTCC-3′

molecular beacons

position

sequence

β-globina

70,945-70,962 8,072-8,092

CY5-5′-CCAGCGATGGCCTGGCTCACCTGGCGCTGG-BHQ2 FAM-5′-CCAGCGGCCTAATGTGGGGACAGCTCACGCTGG-BHQ1

MB MB COX IIa a

position

The stem sequence is shown in boldface lettering.

and the noise (calculated as the standard deviation of at least 50 individual pixel intensities in an intracellular area that excludes mitochondria and the nucleus). Bg plus 10× the standard deviation was set as the threshold for object selection, and each identified object that was less than 4 pixels in size was excluded from the 7694

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data pool. The selected objects were applied onto the unprocessed original images, and the total area (TA) and total fluorescence intensity (TFI) were measured for both the red (R) and green (G) component. The green/red ratio (G/R) was calculated for each individual cell:

G/R )

TFIG - TAG × BgG TFIR - TAR × BgR

(1)

The average and standard deviation of the G/R ratio reported for each sample was calculated using g10 cells. Nucleoid fluorescence was determined as the total green fluorescence intensity of a selected object minus the area of the object × Bg. Sample Preparation for Real-Time PCR Analysis. Cells were harvested by mixing 0.5 mL of the cell suspension with 0.5 mL of cell culture medium and then centrifuged at 1000g for 5 min. The cells were rinsed, resuspended in 0.5 mL of CE buffer, and counted using a hemocytometer counting chamber (Hausser Scientific Partnership, Horsham, PA). The cells were solubilized by adding 5 µL of a lysis solution, containing 0.5% Triton X-100, 2 mg/mL proteinase K, and 10 mM EDTA, to 45 µL of the cell suspension. The cells were incubated with the lysis solution for 60 min at 37 °C followed by heat inactivation at 95 °C for 2 min, as described previously.23,24 Primer and Molecular Beacon Probe Design and RealTime PCR. Three-color molecular beacon real-time PCR (qPCR) reactions were used to quantitate the mtDNA in whole-cell lysate relative to nuclear DNA. The qPCR assay was performed in triplicate for each sample. Primers and molecular beacons were designed with FastPCR (http://www.biocenter.helsinki.fi/bi/ Programs/fastpcr.htm) to amplify a 357-bp fragment of mtDNA25 and a 271-bp fragment of the nuclear β-globin housekeeping gene. Primers were synthesized at the Microchemical Facility (University of Minnesota), and molecular beacons were purchased from Integrated DNA Technologies (Coralville, IA). Primer and molecular beacon sequences are shown in Table 1. The 25-µL qPCR reaction contained 1 µL of DNA sample, 1 µL of each primer and probe (300 nM final concentration), 6.5 µL of PCR grade water and 12.5 µL of 2×FastStart TaqMan Probe Master Mix with ROX (Roche Diagnostics Corp., Indianapolis, IN). qPCR conditions were as follows: 10 min at 95 °C, followed by 50 cycles of denaturation for 30 s at 95 °C, annealing for 60 s at 60 °C, and extension for 30 s at 72 °C. The amplification reactions were performed using a Stratagene Mx3000P QPCR system (La Jolla, CA). To ensure that different amplification efficiencies did not bias the analysis, five serial dilutions were used to determine the amplification efficiency for each sample and template. The threshold cycle (Ct) values for each of the templates were determined using the amplification-based threshold algorithm of the Mx3000P version 2.0 software (Stratagene) and were used to calculate the mtDNA concentration relative to the concentration of the β-globin gene.

mtDNA ratio ) 2(Ctβ-globin-CtmtDNA) × 2 nDNA

(2)

RESULTS In cultured cells, mitochondria form a complex reticular network of functionally and morphologically heterogeneous or(23) Khrapko, K.; Bodyak, N.; Thilly, W. G.; van Orsouw, N. J.; Zhang, X.; Coller, H. A.; Perls, T. T.; Upton, M.; Vijg, J.; Wei, J. Y. Nucleic Acids Res. 1999, 27, 2434-2441. (24) Melov, S.; Schneider, J. A.; Coskun, P. E.; Bennett, D. A.; Wallace, D. C. Neurobiol. Aging 1999, 20, 565-571. (25) Poe, B. G.; Navratil, M.; Arriaga, E. A. Anal. Biochem. 2007, 362, 193200.

ganelles.26 With the emergence of recent insights into the mitochondrial organization has come a need for new methods to define this heterogeneity, as traditional microscopic techniques are commonly inadequate for differentiating between individual organelles. This fact is documented by an image of human osteosarcoma 143B cells expressing red fluorescent protein DsRed2 (λex ) 563 nm, λem ) 582 nm) targeted to mitochondria (Figure 2a). This fusion protein is exceptionally bright and photostable27 and accumulates specifically in the mitochondrial matrix due to a mitochondrial targeting sequence.28 If, upon cell disruption, the integrity of the inner mitochondrial membrane is permanently compromised, the DsRed2 entrapped in the mitochondrial matrix leaks out, which results in a loss of the red fluorescence signal. This makes the DsRed2 protein a suitable indicator of mitochondrial particle integrity and a marker of particles that have retained molecular information from the original mitochondrial network. Within mitochondria, mtDNA is arranged into protein-DNA assemblies termed nucleoids. Figure 2b displays these assemblies upon labeling with PicoGreen, a green fluorescent nucleic acid dye (λex ) 502 nm, λem ) 522 nm), which binds mtDNA in mitochondria of live cells.29 Although nucleoids usually contain more than one copy of mtDNA, indentifying nucleoids containing a single mtDNA molecule is difficult and their existence remains a matter of debate. In this work, we postulate that intact mitochondrial particles (i.e., those retaining DsRed2) containing mtDNA (i.e., those PicoGreen-labeled) most likely contain intact nucleoids, as these are tightly associated with the inner mitochondrial membrane,30 and it is not expected that they have been disrupted in intact mitochondrial particles. Analysis of mtDNA by CE-LIF. The electrophoretic separation of mitochondrial particles was performed in a poly(acryloylaminopropanol)-coated capillary, and the fluorescent signals of DsRed2 and PicoGreen were captured using a dual-channel LIF detection system as the separated mitochondria migrated out of the capillary (Supporting Information Figure 1). A typical electropherogram (Figure 1a) contains well-defined spikes that correspond to individual particles, rather than broad peaks typical of fluorophores that diffuse freely during conventional CE separations. An enlargement of this electropherogram (Figure 1b) reveals three types of events: (i) spikes that appear only in the DsRed2 channel represent intact mitochondrial particles with no detectable amount of mtDNA (*), (ii) events that are only detected in the PicoGreen channel (×) are composed of fragmented mitochondria that have leaked the DsRed2 protein, and (iii) spikes that appear simultaneously in both channels represent intact mitochondrial particles containing detectable amounts of mtDNA (O). On average, 186 ( 15, 493 ( 37, and 138 ( 22 (N ) 5) type i, ii, and iii events, respectively, were detected in a 10-nL sample plug of a mitochondria-enriched preparation. The extent of mitochondrial fragmentation can be monitored by counting type (26) Collins, T. J.; Berridge, M. J.; Lipp, P.; Bootman, M. D. EMBO J. 2002, 21, 1616-1627. (27) Bevis, B. J.; Glick, B. S. Nat. Biotechnol. 2002, 20, 83-87. (28) Rizzuto, R.; Brini, M.; Pizzo, P.; Murgia, M.; Pozzan, T. Curr. Biol. 1995, 5, 635-642. (29) Ashley, N.; Harris, D.; Poulton, J. Exp. Cell Res. 2005, 303, 432-446. (30) Albring, M.; Griffith, J.; Attardi, G. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 1348-1352.

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Figure 2. Visualizing mtDNA content by confocal microscopy. 143B cells expressing DsRed2 targeted to mitochondria treated with ethidium bromide for 0 (a-c) and 4 (d-f) days were labeled with PicoGreen and imaged by confocal microscopy. Red fluorescence of mitochondrial DsRed2 (a, d) colocalizes with green punctuate fluorescence of PicoGreen-labeled mtDNA (b, e). (c, f) is a superimposition of the green and red signals and the yellow appearance is a result of colocalization of PicoGreen-labeled mtDNA and DsRed2-labeled mitochondria. (c) Arrowheads show mitochondria with no detectable levels of mtDNA. (d) Arrows point to donut-shaped mitochondria typical of F0 cells (completely devoid of mtDNA). Bar, 10 µm.

ii events, i.e., the events detected in the PicoGreen channel only. In these experiments, harsh disruption conditions (i.e., nitrogen cavitation and mechanical shear) were chosen in order to maximize the chances of obtaining mitochondrial particles with smaller dimensions and containing a single nucleoid per particle. Type i events represent regions of the mitochondrial network distant from nucleoids, or mitochondria that did not contain mtDNA in their original subcellular milieu. Last, intact mitochondrial particles containing mtDNA (type iii events) provide a means to investigate mtDNA copy numbers per mitochondrial particle. Quantitation of mtDNA Copy Number in Individual Mitochondrial Particles by CE-LIF. In order to quantitate mtDNA copy number in individual mitochondrial particles, the CE-LIF detector response was calibrated using mtDNA samples previously characterized by qPCR (see Supporting Information Figure 2 for details). The conversion factor that was used to transform the PicoGreen fluorescence response into the copy number (e.g., 0.0145 ( 0.0015 AU/mtDNA copy; N ) 4) did not show much variability (RSD ) 10%). Another indication that the mtDNA quantitation is accurate comes from the fact that the least intense events (∼0.016 V) translated into ∼1 copy of mtDNA (Supporting Information Figure 6). Attention must be paid to mitochondrial aggregates, which usually saturate the detector response and could potentially affect the total green fluorescence and thus bias the conversion factor. However, these aggregates can easily be identified and excluded from the dataset. For each detected event in a CE-LIF run, its peak height in the PicoGreen trace reveals the mtDNA content in the corresponding mitochondrial particle. As shown by the right y-axis of Figure 1b, the peak intensity of the events corresponding to one mtDNA molecule was above the detection threshold. 7696 Analytical Chemistry, Vol. 79, No. 20, October 15, 2007

An independent assessment of the ability of CE-LIF to detect a single molecule of mtDNA is based upon the number of PicoGreen molecules per mtDNA molecule and upon previous reports of the limit of detection (LOD) of postcolumn LIF systems. Given a PicoGreen:dsDNA intercalation ratio of 0.3, which is independent of the base pair distribution,31 one mtDNA molecule (16 569 bp) binds ∼5000 molecules of PicoGreen. This is over 6× more than the LOD of the CE-LIF detector (as determined by 0.5-µm fluorescent beads32). These results further confirm that CE-LIF has sufficient sensitivity to detect a single copy of mtDNA. Based on the CE-LIF measurements, the distribution of the mtDNA copy number per mitochondrial particle ranges from 1 to 22 mtDNA copies per particle for 143B cells (Figure 1c). These results show that single mtDNA molecules are the prevalent species in mitochondrial particles, a value that is distinctively different from the average of 6.2 ( 1.1 (N ) 5) mtDNA copies/ particle. It is interesting to note that, in vivo, nucleoids are discrete and well-separated structures with an average distance of 1.60 ( 0.69 µm (N g 50; cf. Figure 2c), and their average size (0.62 ( 0.21; N g 50) is comparable to the mean size of isolated mitochondrial particles (0.52 ( 0.12 µm; cf. Supporting Information Figure 2). If we assume that the disruption method used gives rise primarily to mitochondrial particles containing a single nucleoid, most of the data shown in Figure 1c represent the mtDNA copy number in individual nucleoids. Thus, the CE-LIF results suggest that there are nucleoids with one mtDNA molecule, and that, on average, there are 6.2 ( 1.1 (N ) 5) mtDNA copies per nucleoid. (31) Schweitzer, C.; Scaiano, J. C. Phys. Chem. Chem. Phys. 2003, 5, 4911-4917. (32) Poe, B. G.; Navratil, M.; Arriaga, E. A. J. Chromatogr., A 2006, 1137, 249255.

Figure 3. Detection of mtDNA content. (a) The mtDNA content expressed as a relative green-to-red fluorescence intensity ratio determined by confocal microscopy of 143B cells expressing DsRed2 targeted to mitochondria and labeled with PicoGreen, during 0-4-day exposure to ethidium bromide. The error bars represent the standard deviation of the green-to-red ratio for each sample determined by analyzing a minimum of 10 cells. (b) The mtDNA content expressed as a relative ratio of mtDNA to nuclear DNA determined upon lysis of whole cells in a three-color multiplex qPCR reaction. The right axis gives absolute values of the mtDNA/nDNA ratio representing the number of mtDNA molecules per cell. The ratio was calculated according to eq 2. The error bars represent the standard deviation of three replicate runs for each sample. (c) The mtDNA content expressed as a relative green-to-red fluorescence intensity ratio determined by CE-LIF analysis of mitochondrial particles isolated from 143B cells treated with ethidium bromide for 0-4 days (black trace). The gray trace represents a mtDNA depletion model in which the sensitivity of CE-LIF was adjusted to be equal to that determined for confocal microscopy (the signal-to-noise ratio for CE-LIF was higher by a factor of 22). The error bars represent the standard deviation of at least three consecutive runs for each sample.

Table 2. Nucleoid Properties Measured by Confocal Microscopy and CE-LIF confocal microscopy

CE-LIF

days of EtBr treatment

average no. of nucleoids per planea

mean nucleoid fluorescence (AU)a

% of mitochondria containing mtDNAb

mean PicoGreen fluorescence (AU)c

0 1 2 3 4

75.3 ( 3.5 23.9 ( 6.0 9.0 ( 6.0 6.0 ( 4.7 0.6 ( 0.8

3300 ( 1400 2600 ( 1500 2300 ( 1400 1000 ( 950 380 ( 540

42.3 ( 2.5 25.1 ( 1.7 16.4 ( 3.0 8.6 ( 3.1 1.2 ( 0.4

0.104 ( 0.025 0.095 ( 0.025 0.070 ( 0.019 0.089 ( 0.023 0.045 ( 0.033

a Determined in a single confocal plane for a minimum of 10 cells. b These values represent the average and standard deviation in at least three consecutive CE-LIF runs. c These values represent the average and standard deviation of nucleoid fluorescence, as detected in isolated mitochondria by CE-LIF (N > 33).

Ethidium Bromide-Induced Depletion of mtDNA. Depletion of mtDNA is a valuable tool in cell biology. For example, mtDNA depletion is required for the construction of cybrids (aka cytoplasmic hybrids) that combine the nuclear background of an mtDNA-depleted cell with the mitochondrial genome of another one). Depletion of mtDNA also represents a clinical manifestation of numerous mtDNA mutations33 and has recently been associated with certain types of cancer34 and the pathogenesis of Alzheimer’s disease.35 Presently, techniques such as confocal fluorescence microscopy or qPCR have been used to monitor the progression of mtDNA depletion. Thus, the comparison of CE-LIF with these established techniques is a good assessment of its ability to monitor chemically induced mtDNA depletion. The mitochondrial morphology underwent dramatic changes as mtDNA depletion progressed (Figure 2): it changed from a highly reticulated structure typical of normal cells (Figure 2a) to a population of small isolated mitochondria packed primarily in the perinuclear space (Figure 2d). The organization of the mitochondria into a population of small distinct organelles is (33) Elpeleg, O.; Mandel, H.; Saada, A. J. Mol. Med. 2002, 80, 389-396. (34) Tseng, L. M.; Yin, P. H.; Chi, C. W.; Hsu, C. Y.; Wu, C. W.; Lee, L. M.; Wei, Y. H.; Lee, H. C. Genes Chromosomes Cancer 2006, 45, 629-638. (35) Rodriguez-Santiago, B.; Casademont, J.; Nunes, V. Eur. J. Hum. Genet. 2001, 9, 279-285.

typical of F0 cells (cells completely devoid of mtDNA), as reported by others.36 These mitochondria also exhibited other structural characteristics typical of F0 cells, such as donut-shaped organelles (Figure 2d, indicated by arrows). In most of the cells, we were unable to visually discern any mtDNA after 4 days of EtBr exposure, while the signal of the nuclear DNA remained unaffected (Figure 2e). A more quantitative description of the changes in the abundance of mtDNA is provided by the ratio of the total green fluorescence associated with nucleoids to the total red DsRed2 fluorescence determined in a given confocal image. Using this parameter, the overall amount of mtDNA decreased with the length of the EtBr treatment (Figure 3a). In addition to a progressive reduction in the total mtDNA content, confocal microscopy showed a significant decrease in the actual number of nucleoids and in the average PicoGreen fluorescence intensity during EtBr treatment (Table 2). Both the PicoGreen fluorescence intensity and the number of mtDNA nucleoids varied widely for the observed cells obtained from a given time point in the EtBr treatment. This is attributed mainly to sampling or cell-to-cell variability rather than to the (36) Gilkerson, R. W.; Margineantu, D. H.; Capaldi, R. A.; Selker, J. M. FEBS Lett. 2000, 474, 1-4.

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irreproducibility of the method, because the relative standard deviation of confocal microscopy measurements is typically less than 10% (data not shown). Unlike Southern blotting, qPCR is capable of detecting a single copy of DNA and the mtDNA content is usually reported relative to nuclear DNA (nDNA).9 In this work, we used a 271-base-pairlong segment of the nuclear β-globin housekeeping gene as an endogenous standard. The primers used for quantitation of mtDNA were designed to amplify a 357-bp fragment of the mitochondrial COX II gene and were detected in a three-color multiplex qPCR reaction. The amplification efficiency for both mtDNA and β-globin was ∼100% over at least 4 orders of magnitude (see Supporting Information Figure 3 for details). The mean relative abundance of mtDNA (n ) 3), expressed as a mtDNA/nDNA ratio (the amount of mtDNA per diploid nuclear genome), was calculated using eq 2 and the respective Ct values, and represents the number of mtDNA molecules per cell. Using the qPCR approach, we monitored the loss of mtDNA caused by EtBr treatment of 143B cells (Figure 3b). The mtDNA copy number per cell dropped from 720 ( 80 to 13 ( 9 after a 4-day exposure to EtBr, which agrees with the previous reports of King and of Wiseman,37,38 who reported up to a 93% decrease in the mtDNA content in 143B cells after 4 days of EtBr-induced mtDNA depletion. Monitoring of mtDNA depletion by CE-LIF revealed a similar trend in the mtDNA copy number (Figure 1d; see Supporting Information Figure 4 for more details). After 4 days of EtBr treatment, only 1.2% of all mitochondrial particles contained detectable levels of mtDNA; a similar decrease in the actual number of nucleoids was also detected by confocal microscopy (Table 2). DISCUSSION This is the first report that describes the determination of the mtDNA copy number in individual, intact mitochondrial particles isolated from mammalian cells (see Figure 1 and Materials and Methods for details on detecting a single copy of mtDNA). Previous reports relied on multiple assumptions to quantitate mtDNA;39,40 upon harvesting individual organelles, these reports used other methods (i.e., microscopy, qPCR) to quantitate the mtDNA content. Herein, the described CE-LIF method combines the separation and detection into a single step and eliminates errors associated with particle harvesting and handling. Southern blotting and qPCR are established methods for determining mtDNA content. While Southern blotting requires large amounts of sample (typically 0.1 pg or more), the sensitivity of qPCR makes it possible to quantitate mtDNA in single cells; however, qPCR cannot be easily applied to determine the mtDNA levels in individual subcellular particles due to stochastic variations associated with single-molecule PCR amplifications, possible losses of mtDNA or PCR reagents to the reaction vials, or contamination caused by exogenous mtDNA. Microscopy has become a powerful technology for studying the structure, distribution, dynamics, and biochemical properties

of mitochondria. While fluorescence microscopy usually offers sufficient lateral resolution to distinguish individual mitochondria and mtDNA,41 quantitative microscopy of individual organelles usually employs a confocal setup to reduce out-of-focus light and the overlap of mitochondria in different focal planes (cf. Figure 2). However, their quantitation is often tedious and limited by low sensitivity and strong background signal. Furthermore, the constant mitochondrial motion in live cells contributes to image blurriness, especially when long exposure times are used (∼10 s), introducing errors and artifacts into the signal. Although singlemolecule imaging techniques such as those using total internal reflectance42 are promising alternatives, the potential pitfalls of confocal microscopy emphasize the need for new methods allowing the rapid, sensitive, and reproducible measurement of the mtDNA content. CE-LIF offers several advantages over the aforementioned techniques. Similar to flow cytometry,32 it combines a low LOD, down to the sub-zeptomole range (