2.1 Cell lines and reagents
Mouse embryonic fibroblasts (MEF) from transgenic mice over-expressing MsrA were kindly provided by Hang Zhao (Laboratory of Biochemistry, National Heart, Lung, and Blood Institute). Generation of these mice will be described elsewhere. Early-to-middle passage (6-20) cells were grown in Dulbecco's modified Eagle's medium containing 4.5 g/L glucose, supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and penicillin and streptomycin and maintained in a humidified 5% CO2 atmosphere. Glucose oxidase (Type X-S from Aspergillus niger; # G7141), rotenone, and antimycin A were from Sigma.
2.2 Antibodies and immunofluorescence labeling
Polyclonal antiserum against MsrA was generated from rabbits immunized with recombinant mouse MsrA purified from E. coli. This recombinant MsrA lacked the N-terminal 20 amino acids that encode the mitochondrial leader peptide. A monoclonal antibody against cytochrome c was from BD Pharmingen (# 556432) and used at 1:500 dilution. Anti-Mitochondrial Heat Shock Protein 70 monoclonal antibody was from Affinity Bioreagents (now Thermo Scientific; # MA3-028) and diluted 1:250. Alexa Fluor 488- and 594-conjugated species-specific secondary antibodies were from Invitrogen and used at a 1:500 dilution.
Cells were grown on 12 mm glass coverslips until approximately 50-75% confluent and treated as indicated. They were directly fixed for 20-30 min at room temperature with 2% formaldehyde in phosphate buffered saline (PBS). Fixation with 3.7% formaldehyde in PBS or 3.7% formaldehyde in 0.1 M sodium phosphate, pH 7.2, 150 mM NaCl gave similar results. After fixation, cells were rinsed three times with 10% FBS in PBS. Some cells were treated after fixation with 0.1% Triton X-100 for 1 min, followed by three rinses in PBS prior to rinsing in FBS/PBS. The cells were then incubated with primary antibody or serum diluted in 10% FBS/PBS containing 0.2% saponin for 1 hr at room temperature. The cells were then washed three times over a 15 min period with 10% FBS/PBS and incubated with fluorochrome-conjugated secondary antibodies (diluted in 10% FBS/PBS plus 0.2% saponin) for 1 hr at room temperature. Cells were again rinsed for 10 min with 10% FBS/PBS and one final rinse with PBS before mounting on a glass slide with Fluoromount G (Southern Biotech) containing 2.5 μg/mL DAPI (4′,6-diamidino-2-phenylindole) to stain nuclei. For treatment of cells with hydrogen peroxide, either a single bolus of hydrogen peroxide (250 μM) was added to cells or alternatively, hydrogen peroxide was generated enzymatically by glucose oxidase (25-125 mU/mL), which produces hydrogen peroxide in a more chronic manner via oxidation of glucose in the cell-culture medium. Similar results were observed for both.
Images were obtained using a LSM 510 confocal microscope (Carl Zeiss, Thornwood, NY) with a 63× 1.3 numerical aperture PlanApo oil-immersion objective. Optical sections were less than 1.5 μm thick. After acquisition, images were handled using Adobe Photoshop (Adobe Systems).
2.3 Cell Fractionation and Western blotting
MEFs were swollen in ice-cold hypotonic buffer (10 mM Hepes, pH 7.5, 5 mM MgCl2, 40 mM KCl, 1 mM phenylmethylsulfonyl fluoride, protease inhibitor cocktail) for 30 min. Cells were disrupted by passage 5 times through a 26 gauge needle. The broken cells were centrifuged at 800 × g for 8 min to separate the pellet and the supernatant. The supernatant was removed carefully without disrupting the pellet and centrifuged at 9,000 × g for 15 min. The resulting supernatant was saved as the cytosolic fraction. The high spin pellet was washed twice with PBS for the mitochondrial fraction. Both fractions were separated by SDS-PAGE (reducing), followed by heating at 95°C for 5 min. Electrophoresis was performed on 10-20% Tris-Glycine gels (Invitrogen). Proteins were transferred onto nitrocellulose membranes (Invitrogen), and incubated with primary [MsrA, mHSP70, and α-tubulin (Santa Cruz)] and secondary [Alexa Fluor 680 goat anti-rabbit IgG (Invitrogen, A21109) and IRDye800CW (Rockland)] antibodies for 1 hr at room temperature. Membranes were washed three times with 0.1% Tween 20 in 1 × PBS, then visualized by scanning with an Odyssey infrared scanner with 700 and 800 nm channels (LI-COR Biosciences, Lincoln, NE).
2.4 Transmission electron microscopy
Cells were grown on 60 mm Permanox dishes (Nunc Nalgene) until approximately 75-90% confluent, treated as indicated, then directly fixed in 2.5% glutaraldehyde, 1% paraformaldehyde in 0.12 M sodium cacodylate buffer, pH 7.4. Cells were fixed for 30 min at room temperature, and then kept at 4°C overnight in fixative. Cells were post-fixed with osmium tetroxide, stained en bloc with uranyl acetate, ethanol dehydrated, and Epon embedded. Chemicals were from Electron Microscopy Sciences. Thin sections were cut parallel to the adherent surface, stained with uranyl acetate and lead citrate, and viewed with a JEM-1200EX electron microscope (JEOL USA) equipped with an AMT XR-60 digital camera (Advanced Microscopy Techniques).