Media and chemicals were obtained from GIBCO-BRL or Sigma-Aldrich unless otherwise specified. Fluorescent dyes for tagging and Mitotracker dyes were purchased from Molecular Probes. Secondary antibodies (Jackson ImmunoResearch Laboratories) were conjugated to fluorescent dyes as recommended by the manufacturer.
Drosophila stocks were grown at 25°C in cornmeal agar bottles. drp-1 mutants, Collagen-GAL4, and UAS-Mito-GFP were kindly provided by Hugo J. Bellen (Baylor College of Medicine, TX), K.S. Krishnan (DBS, TIFR, Mumbai), Charles R. Dearolf (Duke University, NC), and William Saxton (Indiana University, IN).
Treatment of Tissues and Cells Ex Vivo
Third-instar larval tissues were dissected in Schneider insect medium supplemented with 10% non-heat-inactivated fetal bovine serum and 1 μg/ml bovine pancreatic insulin (SCM). They were washed in 1× PBS (pH 7.4) and incubated with 1 mM water-soluble ecdysone (AG Scientific, Inc.) diluted in PBS for 2 hr (20°C), fixed with 4% formaldehyde (30 min), permeabilized with 0.37% Igepal (13 min), and labeled with Alexa 568 Phalloidin (1:200; Molecular Probes) for 30 min. The tissues were mounted beneath a coverslip and imaged. Mitochondria were selected based on a threshold and were quantified by using Metamorph (Molecular Devices Corporation).
Hemocytes derived from late third-instar larvae and S2R+
cells were plated in 35 mm coverslip-bottom dishes. Hemocytes at ~1 hr postdissection were incubated with etoposide (10 μM), cycloheximide (0.5 μg/ml), or C6
-ceramide (20 μM or 40 μM; Matreya, Inc.) diluted in 1× Medium 1 (M1) supplemented with 2 mg/ml glucose (imaging medium [IM]) at 20°C for the indicated times prior to staining nuclei with 5 μg/ml Hoechst-33342 (5 min). Hemocytes were preincubated (30 min) with 50 μM Caspase inhibitor-1 (zVAD-fmk; Calbiochem) in IM before incubation with apoptotic stimuli in the presence of 50 μM zVAD-fmk in order to inhibit caspases. Immunofluorescence staining was carried out as described earlier (Sriram et al., 2003
), by using affinity-purified rabbit anti-Drp-1 antibody (1:200), mouse Biotin antibody (1:500), and appropriate secondary antibodies (1:500). Drp-1 punctae were selected by using a threshold on background corrected images and were quantified.
A total of 1 × 106 S2R+ cells were cotransfected with 1 μg siRNAi (Dharmacon, Inc.) and 0.25 μg pAVW vector (expressing EYFP only) (Drosophila Genomics Resource Centre, Indiana University), by using Cellfectin transfection reagent (Invitrogen). After 5 days, cells were treated with prescribed concentrations of actinomycin-D or cycloheximide for 18 hr, and surviving YFP-expressing cells in five randomly selected fields were counted (n = 5; total cells = 14,020). Counts were normalized to the untreated control; error bars represent standard error of the mean for normalized data.
Detection of Active Caspases and PI, AnV Staining
Hemocytes were incubated with 10 μM caspACE substrate (FITC-VAD-FMK, Promega) for 20 min (20°C) or stained with PI (20 μg/ml), AnV (2 μg/ml) (Santa Cruz Biotechnology), and Hoechst-33342 (5 μg/ml) diluted in M1 supplemented with 2.5 mM calcium chloride and 2 mg/ml glucose (20 min) and were imaged.
Tissues and cell cultures in 35 mm coverslip-bottom dishes were imaged by using 60× or 100×, 1.4 NA, oil-emersion objectives on a Nikon TE 2000-U epi-fluorescent inverted microscope with optimized dichroics and filters and a Cascade 512B EM-CCD camera (Photometrics) controlled by Metamorph (Molecular Devices Corporation). Confocal images were acquired by using either a 60×, 1.4 NA or a 100×, 1.45 NA objective on a Biorad MRC 1024 or Zeiss LSM 510 (Carl Zeiss MicroImaging, Inc.) microscope equipped with optimized dichroics and filters. Images were processed by using Metamorph (Molecular Devices Corporation) and Adobe Photoshop software.
Tubulin-GAL4 or Collagen-GAL4 was used to express Mito-GFP in multiple tissues by using the GAL4-UAS system (Brand and Perrimon, 1993
). Hemocytes were incubated with 100 nM Mitotracker diluted in IM supplemented with 1.5 mg/ml BSA (IM+BSA) for 20 min (20°C) and were imaged. S2R+
cells were grown in Schneider's medium supplemented with 10% fetal bovine serum (Gemini Bio-Products) at 25°C. A total of 1 × 106
cells were plated and transfected with 1 μg EYFP-Mito vector (LaJeunesse et al., 2004
) by using 5 μl Cellfectin transfection reagent (Invitrogen). Mitochondria were selected by using defined thresholds post background correction and were quantified by using Metamorph (Molecular Devices Corporation).
UV-B Induced Apoptosis in Drosophila Third-Instar Larvae
Third-instar larvae were exposed (90 s) on a dual intensity transilluminator (TM20; UVP, Inc.) fitted with 6 UV-B tubes (20W × 6). A total of 6 hr later, wing discs were dissected and stained with 3 μg/ml acridine orange in PBS (5 min), washed, and imaged on the confocal microscope.
TMRM Fluorescence-Loss Assay
Hemocytes were incubated with 10 nM TMRM diluted in SCM or IM+BSA (15 min) and were exposed to 555 nm wavelength light on a wide-field microscope. Time-lapse images (0.2 s) were acquired every 1 s until TMRM was released from all the mitochondria. Individual mitochondria were outlined and quantified by using Metamorph (Molecular Devices Corporation) (n = 5–10).
S2R+ cells that were cotransfected with 1 μg siRNAi (Dharmacon, Inc.) and 1 μg EYFP-Mito by using Cellfectin transfection reagent (Invitrogen) according to the manufacturer's instructions were analyzed 5 days posttransfection. Drp siRNA was targeted to 5′-ACACTCCGGTTCACAATAA-3′ (NM_134850 bases 1994–2012), and siCONTROL nontargeting siRNA #1 (Dharmacon, Inc.) was used as a control. For FRAP, a mitochondrial region of interest (ROI, ~1.75 μm in diameter) was selected and bleached (after 4 s, indicated by a solid bar) with high-intensity 488 nm wavelength light (~1 s), and fluorescence in the ROI was measured for an additional 25 s.
RNA was extracted from transfected S2R+
cells by using the RNeasy Mini Kit (QIAGEN), and RT-PCR was conducted by using the QIAGEN OneStep RT-PCR kit (QIAGEN). The Drp-specific 5′ primer 5′-TCATTCACGAGGAGATGCAG-3′ (designed to NM_134850 bases 1298–1317) and the 3′ primer 5′-TGCTTGGTGTTGATGTAGGC-3′ (designed to NM_134850 bases 1490–1509) were used to amplify a 208 bp product. Ribosomal protein 49 (rp49) primers (5′-ATGACCATCCGCCCAGCATAC-3′ and 5′-GAGAACGCAGGCGACCGTTGG-3′) amplified a 391 bp fragment between bases 1 and 391 of the coding region (GenBank accession number Y13939) (Ge et al., 2004