Brooks et al. 10.1073/pnas.0703976104. |
Fig. 6. Flow cytometric analysis of apoptosis after TUNEL staining. HeLa cells were cotransfected with MitoRed and dn-Drp1, Bcl-2, or empty vector. The cells were treated with 20 mM cisplatin for 16 h in the presence or absence of 100 mM VAD or untreated as control. The cells were then subjected to TUNEL assay. TUNEL staining (green FITC signal) in transfected cells (red MitoRed signal) were analyzed by flow cytometry as described in SI Methods. Shown in the figure are results of a representative assay: 8% TUNEL-positive cells in the control group (A) (Control/vector), 32% in the cisplatin-treated group transfected with empty vector (A) (Cisplatin/vector), 8% in the cisplatin-treated group transfected with dominant-negative Drp1 (B) (Cisplatin/dn-Drp1), 9% in the cisplatin-treated group with 100 mM VAD (C) (Cisplatin/VAD), and 16% in the cisplatin-treated group transfected with Bcl-2 (D) (Cisplatin/Bcl-2).
Fig. 7. Effects of stably transfected Bcl-2 and transiently transfected Bcl-XL on mitochondrial fragmentation during apoptosis. (A) Wild-type (NA) and Bcl-2 stably transfected RPTC cells were transfected with MitoRed and then treated with 10 mM azide for 3 h, 1 mM STS for 4 h, or 20 mM cisplatin for 16 h. (B) RPTC cells were transiently transfected with MitoRed and Bcl-2, Bcl-XL, or empty vector (NA). The cells were then treated with 10 mM azide for 3 h. Mitochondrial morphology was examined by fluorescence microscopy to quantify the percentages of cells that had mitochondrial fragmentation. Data are means ± SD of three separate experiments. *, significantly different from untreated cells (Control); #, significantly different from treated wild-type cells (NA).
Fig. 8. Effects of mitochondrion- and ER-targeted Bcl-2 on mitochondrial fragmentation during apoptosis. RPTC cells were cotransfected with MitoRed and wild-type, mitochondrially-targeted, or ER-targeted Bcl-2. The cells were then treated with 10 mM azide for 3 h. Transfected cells with MitoRed-labeled mitochondria were examined by fluorescence microscopy to determine the percentages of cells that had fragmented mitochondria. Data are means ± SD of four separate experiments. *, significantly different from the untreated (Control) group; #, significantly different from the treated vector-transfection (vector) group.
Fig. 9. Mitochondrial morphology in wild-type, Bax-knockout, Bak-knockout, and double-knockout mouse embryonic fibroblasts. (A) Bax and Bak expression. Whole-cell lysates were collected from the cells of indicated genotypes to analyze Bax and Bak expression by immunoblotting. (B) Mitochondrial fragmentation under control conditions. The cells were transfected with MitoRed to fluorescently label mitochondria for microscopic examination. Cells with fragmented mitochondria were counted to determine the percentage of mitochondrial fragmentation. Data are means ± SD of four separate experiments. (C) Mitochondrial length. MitoRed-transfected cells were examined by confocal fluorescence microscopy. Mitochondria were traced to measure their length by using the LSM51 Image Examiner software. Approximately 20 well separated mitochondria were measured in each cell. More than 10 cells were measured for each cell line. Data shown in this figure are average length of >200 mitochondria in each cell line. (D) Mitochondrial morphology in representative cells. Images are shown of further magnified mitochondria (Lower) of the defined areas in the cells.
Fig. 10. Bak (and not Bax) deficiency blocks mitochondrial fragmentation in transformed baby mouse kidney cells. Wild-type, Bax-knockout (Bax-/-), Bak-knockout (Bak-/-), and double-knockout (DKO) BMK cells were transfected with MitoRed and then treated with 10 mM azide for 3 h, 1 mM STS for 4 h, or 20 mM cisplatin for 16 h. Mitochondrial morphology was examined by fluorescence microscopy to quantify the percentages of cells showing mitochondrial fragmentation. Data are means ± SD of three separate experiments. *, significantly different from untreated cells (Control); #, significantly different from treated wild-type cells (wt).
Fig. 11. Bak deficiency inhibits mitochondrial fragmentation in primary cultures of brain cortical neurons. Primary neurons were isolated from the brain cortex of newborn wild-type or Bak-knockout mice and cultured for 3 days. The neuronal cultures were untreated (Control) or treated with 50 mM glutamate, 2 mM camptothecin, or 20 mM cisplatin for 24 h. VAD was included at 100 mM to prevent secondary effects of apoptosis. After treatment, the cells were stained with MitoTracker. Mitochondrial morphology was examined by fluorescence microscopy to count the cells with mitochondrial fragmentation. Data are presented as means ± SD of three separate experiments. *, significantly different from the control group.
Fig. 12. Colocalization of mitochondrial fragmentation and cytochrome c release in Bak- or Bax-knockout cells. Bax-knockout (Bax-/-) or Bak-knockout (Bak-/-) MEFs were transfected with MitoRed and then treated with 10 mM azide for 3 h or 1 mM staurosporine (STS) for 4 h. The cells were fixed for cyt.c immunofluorescence as described in SI Methods. Mitochondrial morphology shown by MitoRed and cyt.c localization as shown by immunofluorescence were examined in the same cells by fluorescence microscopy. The percentages were determined for the following three groups of cells: (i) cells that released cyt.c yet maintained filamentous mitochondria, (ii) cells that released cyt.c and had fragmented mitochondria, and (iii) cells that fragmented mitochondria. Approximately 150 MitoRed-transfected cells were evaluated for each condition.
Fig. 13. Staurosporine-induced mitochondrial fragmentation in Bak- or Bax-reconstituted cells. Bax/Bak double knockout MEFs were cotransfected with MitoRed and GFP, GFP-Bax, or GFP-Bak for 16 h. The cells were then incubated for another 4 h with control medium or 1 mM STS. Mitochondrial fragmentation in transfected cells was examined by fluorescence microscopy. Data are means ± SD of four separate experiments.
Fig. 14. Coimmunoprecipitation analysis of Bak-Mfn1/2 interaction during cisplatin treatment. HeLa cells were untreated or treated with 20 mM STS for 16 h. Whole lysates were collected for immunoprecipitation using an anti-Bak antibody. The resultant immunoprecipitates were analyzed for Mfn1, Mfn2, and Bak by immunoblotting.
Fig. 15. Mitofusins are not precipitated by nonimmune serum from HeLa cells or by anti-Bak antibody from Mfn1/2-deficient MEFs. (A) Whole-cell lysates were collected from HeLa, wild-type (wt), Mfn1-deficient, or Mfn2-deficient MEF cells for immunoblot analysis. (B) Lysates of HeLa, wild-type (wt), Mfn1-deficient, or Mfn2-deficient MEF cells were subjected to immunoprecipitation with anti-Bak antibody (a-Bak) or nonimmune serum (NIS). The immunoprecipitates along with HeLa cell lysate (Input) were analyzed for Mfn2 and Bak by immunoblotting.
Fig. 16. FRET analysis of Bak/Bax interaction with mitofusins. pECFP-Bak or pECFP-Bax was cotransfected with either pEYFP-Mfn1 or pEYFP-Mfn2 into Bax/Bak double-knockout MEFs. The cells were fixed for acceptor photobleaching FRET analysis using a Zeiss LSM 510 confocal microscope as described in SI Methods. As negative control, pECFP-Src and pEYFP were cotransfected and, as positive control, pECFP-Src-EYFP was transfected for FRET analysis. Data are means ± SD of the results from 15-20 cells in each condition.
Fig. 17. Colocalization of transfected YFP-mitofusins with CFP-Bak. Bax/Bak double-knockout MEFs were cotransfected with pECFP-Bak and either pEYFP-Mfn1 or pEYFP-Mfn2. Fluorescence images were collected by confocal microscopy.
SI Methods
Cell lines.
Mouse embryonic fibroblasts (MEF) of various Bax/Bak genotypes were obtained from Dean Tang (University of Texas M. D. Anderson Cancer Center, Houston, TX) and Shivendra Singh (University of Pittsburgh, Pittsburgh, PA). The cells were originally from Stanley Korsmeyer (Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA). MEFs were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS, 10 mM nonessential amino acids, and 1% antibiotics. Transformed baby mouse kidney (BMK) cells were obtained from Eileen White (Rutgers University, Piscataway, NJ) and maintained in DMEM supplemented with 5% FBS and 1% antibiotics. HeLa cells were purchased from American Type Tissue Collection (ATCC) and maintained in MEM supplemented with 10% FBS, 0.1 mM nonessential amino acids, 2 mM L-glutamine, and 1% antibiotics. The rat kidney proximal tubular cell (RPTC) line was originally obtained from Ulrich Hopfer (Case Western Reserve University, Cleveland, OH). RPTC cells stably transfected with Bcl-2 were generated in our previous work (1). The cells were maintained in Ham's F-12/DMEM supplemented with 10% FBS, 5 mg/ml transferrin, 5 mg/ml insulin, 1 ng/ml EGF, 4 mg/ml dexamethasone, and 1% antibiotics.
Isolation and Culture of Primary Cortical Neurons.
The brain cortex was dissected from wild-type or Bak-knockout newborn mice and minced in Hanks' solution. The minced cortical tissues were digested with 0.25% trypsin for 10 min at 37°C, followed by vigorous pipetting to release cells. DNase was then added to digest the DNA released from damaged cells. Subsequently, an equal volume of plating medium (DMEM/F-12 supplemented with 10% FBS, 1 mM glutamine, 1% antibiotics, and 1% N2 supplement) was added to stop trypsin digestion. The mixture was centrifuged for 5 min at 1,000 × g to collect the cell pellet. The cells were washed once with the plating medium and then passed through a 40-mm mesh cell strainer. The cell number was counted after trypan blue staining. Isolated cells were plated at 2 ´ 105 per well in 12-well plates with poly-D-lysine-coated coverslips. After attaching, the cells were maintained for 3 days in the Neurobasal medium (Gibco) supplemented with 0.5 mM glutamine, 1% antibiotics, and 2% B27 supplement. During the culture period, most cells showed the growth and extension of axons, a characteristic of neurons.
Plasmids.
The coding regions of Bax and Bak were amplified from mRNA of rat kidney proximal tubular cells by RT-PCR. Briefly, mRNA was extracted from the cells with Tri Reagent according to the manufacturer's instructions (Molecular Research Center, Cincinnati, OH). Reverse transcription was conducted by using the SuperScript first-strand synthesis kit, following the manufacturer's instruction (Invitrogen, Carlsbad, CA). The coding sequence of Bax was then PCR amplified by using the primer pair: forward 5'-CCCAAGCTTGCCACCATGGACGGGTCCGGGGAGCAG-3' and reverse 5'-CGGGGTACCTCAGCCCATCTTCTTCCAGATG-3'. The coding sequence of Bak was PCR amplified using the primer pair: forward 5'-CCCAAGCTTGCCACCATGGCATCBGGACAAGGACCAG-3' and reverse 5'-CGCGGATCCTCATGATCTGAAGAATCTGTGTAC-3'. The primers introduced HindIII and KpnI digestion sites for Bax as well as HindIII and BamH I digestion sites for Bak. The PCR products were then cloned into pEGFP-C3 (BD Clontech, Palo Alto, CA) by using these enzyme digestion sites. Expression of GFP-Bax and GFP-Bak was confirmed by fluorescence microscopy and immunoblot analysis. Murine Bak and the Bak (L75E) mutant were from Emily H.-Y. Cheng (Washington University School of Medicine, St. Louis, MO). Drp1 and dn-Drp1(K38A) were from Alexander van der Bliek (University of California, Los Angeles, CA) and were subcloned into pcDNA3.1 for this study. Bcl-XL was provided by Xiao-Ming Yin (University of Pittsburgh, Pittsburgh, PA) and was subcloned into pcDNA3.1 for this study. Bcl-2 plasmids for targeted expression in mitochondria or ER were kindly provided by David W. Andrews (McMaster University Health Sciences Centre, Ontario, Canada).
Transfection.
HeLa and MEF cells were plated at ~50% confluence for transfection with 0.5 mg of plasmid DNA by using Metafectene according to the manufacturer's instruction (Biontex, Germany). For Bax or Bak reconstitution experiments, double-knockout MEF cells were plated at ~90% confluence for transfection by using Metafectene. BMK cells were plated at ~90% confluence for transfection with 0.5 mg of plasmid DNA by using Metafectene. Primary cultures of isolated kidney tubular epithelial cells were plated at ~70% confluence for transfection with 1.0 mg of plasmid DNA by using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen). RPTC and Bcl-2 stably transfected RPTC cells were plated at ~50% confluence for transfection with 1.0 mg of plasmid DNA by using Lipofectamine Plus reagent (Invitrogen). To visualize mitochondria, cells were transfected with 0.1 mg of pDsRed2-Mito (BD Clontech) to express the red fluorescent MitoRed protein in mitochondria to label the organelles.
Apoptotic Treatment.
In the majority of experiments, apoptosis was induced by azide, staurosporine (STS), or cisplatin as described (2). Azide blocks cellular respiration and induces ATP depletion in glucose-free medium, leading to mitochondrial outer membrane permeabilization and release of cytochrome c. When the azide-treated cells are returned to glucose-containing medium, the cells develop apoptotic morphology. STS is a general protein kinase inhibitor and induces apoptosis in a variety of cells. Cisplatin is a widely used cancer therapy drug that induces DNA damage and apoptosis (3). In this study, cells were incubated with 10 mM azide in glucose-free Krebs Ringer bicarbonate solution for indicated time. After the incubation, cells were either fixed for immunostaining and microscopic examination or returned to glucose-containing culture medium for 1 h to observe apoptosis. For STS treatment, cells were incubated with 1 mM STS for indicated time and fixed for immunostaining and microscopic examination. For cisplatin treatment, cells were incubated with 20 mM cisplatin in cell culture medium for indicated time. After treatment, cells were fixed, immunostained, and examined by fluorescence and confocal microscopy. In the study of primary neurons, the cells were treated with 50 mM glutamate, 2 mM camptothecin, or 20 mM cisplatin for 24 h.
Analysis of Apoptosis.
(1) Morphological analysis. Apoptosis was evaluated by counting cells with typical apoptotic morphology as described (2, 3). Briefly, after experimental incubation, cells were stained with Hoechst 33342 and then examined by phase-contrast and fluorescence microscopy. Apoptotic cells were identified by typical morphology including cellular condensation, formation of apoptotic bodies, and condensation and fragmentation of the nucleus. Approximately 200 cells were examined in each 35-mm dish to determine the percentage of apoptotic cells. (2)
Flow Cytometric Analysis.
Cells were cotransfected with MitoRed and a gene of interest. After treatment, the cells were fixed and stained with the TUNEL assay kit containing terminal deoxynucleotidyl transferase and FITC-labeled dUTP (in situ Cell Death Detection kit; Roche Applied Science, Indianapolis, IN). TUNEL staining (green FITC signal) in transfected cells (red MitoRed signal) were analyzed by flow cytometry using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). For each sample, 10,000 events were counted.
Mitochondrial Morphology.
In the majority of the experiments, cells were transfected with MitoRed to fluorescently label mitochondria for fluorescence microscopy. Mitochondrial morphology in individual cells was evaluated. Fragmented mitochondria were shortened (<1 mm), punctate, and sometimes rounded, whereas filamentous mitochondria showed a long thread-like tubular structure. Consistent with earlier studies, the mitochondria within a cell were often either filamentous or fragmented. In rare cases of mixed morphologies, we classified the cells based on the majority (>70%) of mitochondria. In some experiments, cell morphology and cyt.c release after immunofluorescence staining were also evaluated in MitoRed-transfected cells. Apoptosis was indicated by typical apoptotic morphology. Cyt.c release was indicated by the loss of mitochondrial cyt.c staining and the appearance of cyt.c in the cytosol. For each sample, several random fields of cells (³100 cells per dish) were evaluated for mitochondrial morphology, apoptosis, and cyt.c release. In the study of primary neurons, mitochondrial morphology was examined after MitoTracker staining. Briefly, at the end of the experiment, 0.5 mM MitoTracker CM-H2XRos (Molecular Probes, Eugene, OR) was added to the cells for 30 min. The cells were then washed twice with fresh medium and fixed for 1 h with 3% paraformaldhyde in culture medium and then mounted on slides with Antifade (Molecular Probes) for fluorescence microscopy.
FRET Assay.
FRET assay was conducted as described (4), using the acceptor photobleaching method that detects the increase of donor fluorescence when the acceptor is selectively photobleached. Briefly, Bak and Bax were subcloned into pECFP-C1, and Mfn1 and 2 were subcloned into pEYFP-C1. pECFP-Bak or pECFP-Bax were cotransfected with pEYFP-Mfn1 or pEYFP-Mfn2 into HeLa cells for 24 h. As negative control, pECFP-Src and pEYFP were cotransfected to express ECFP-Src and EYFP separately. As positive control, pECFP-Src-EYFP was transfected to express the ECFP-Src-EYFP fusion protein. The control plasmids were kindly provided by Roger Y. Tsien (University of California at San Diego La Jolla, CA). After transfection, the cells were fixed with 4% paraformaldehyde and subjected to FRET analysis using a Zeiss LSM 510 confocal microscope. In FRET analysis, a region of interest (ROI 1) was selected and photobleached by applying 100% intensity of 514 nm laser. FRET efficiency was calculated by using the formula: FRET = (Dpost - Dpre)/Dpost, where Dpost and Dpre represent the donor (ECFP) emission intensities before and after the photobleaching. FRET efficiency was also measured in a nonphotobleached region (ROI 2) of the same cell as an in situ control.
Cellular Fractionation.
To analyze the release of mitochondrial cytochrome c, cells were fractionated by using low concentrations of digitonin, which selectively permeabilize the plasma membrane while leaving the mitochondrial membrane intact (1-3). Briefly, cells were incubated for 2-5 min with 0.05% digitonin in an isotonic sucrose buffer. The digitonin-soluble portion was collected as the cytosolic fraction for immunoblot analysis.
Immunoblot Analysis.
Electrophoresis and blotting were conducted using NuPAGE Gel Systems (Invitrogen). Briefly, samples of 25 mg of protein were resolved by SDS/PAGE under reducing condition. The resolved proteins were transferred onto PVDF membranes, which were then incubated with primary antibody overnight at 4°C and horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Antigens on the blots were revealed by incubation with chemiluminescent substrates (Pierce, Rockford, IL).
Antibodies.
Antibodies were from the following sources: polyclonal anti-Bak from Upstate Biotechnology (Lake Placid, NY); monoclonal anti-Bcl-2 and polyclonal anti-Bax from Santa Cruz Biotechnology (Santa Cruz, CA); polyclonal anti-Mfn1 from Margaret T. Fuller at Stanford University School of Medicine (Stanford, CA); polyclonal anti-Mfn2 from Sigma (St. Louis, MO); polyclonal anti-Fis1 from Alexis (San Diego, CA), monoclonal anti-cytochrome c (7H8.2C12 and 6H2.B4) and anti-Drp1 from BD Pharmingen (San Diego, CA); monoclonal anti-Myc from Invitrogen; all secondary antibodies from Jackson ImmunoResearch (West Grove, PA).
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