Antibodies and Other Reagents

Rabbit polyclonal antibodies against Mff were produced by cloning human Mff cDNA into pET21d, which introduces a carboxy-terminal 6xHis tag. Bacterially expressed protein was purified with nickel-nitrilotriacetic acid agarose using 8 M urea. The protein was further purified with preparative SDS-polyacrylamide gel electrophoresis (PAGE) gels and then used to immunize rabbits. Anti-Fis1 antibodies were purchased from Alexis Laboratories (San Diego, CA), α-tubulin antibodies were from Sigma-Aldrich (St. Louis, MO), Tom20 and c-myc/9E10 were from Santa Cruz Biotechnology (Santa Cruz, CA), cytochrome c antibodies were from BD Biosciences PharMingen, prohibitin antibodies were from RDI (Flanders, NJ), and DLP1 antibodies were from BD Biosciences Transduction Laboratories (Lexington, KY). Secondary antibodies and horseradish peroxidase conjugates were from Pierce Chemical (Rockford, IL). Fluorescent-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA).

Mito-Tracker Red CMX-Ros (Invitrogen) was used at a final concentration of 20 nM. z-VAD-fmk (BIOMOL Research Laboratories, Plymouth Meeting, PA) was used at a final concentration of 25 μM. Staurosporine (Tocris Cookson, Ellisville, MO) was used at a final concentration of 1 μM. Actinomycin D (Sigma-Aldrich) was used at a final concentration of 10 μM. CCCP (Sigma-Aldrich) was used at a final concentration of 2 μg/ml.

Cell Culture and Transfections

HeLa cells and human embryonic kidney (HEK)293T cells were grown with DMEM and 10% fetal calf serum. HEK293 cells were transfected with Ca-phosphate precipitation. HeLa cells were transfected using Lipofectamine 2000 and Opti-MEM (Invitrogen). Cells were transfected with siRNA duplexes at a concentration of 100 pM, and then they were allowed to grow for an additional 3 d before analysis. For cotransfections with overexpression constructs, the siRNA-transfected cells were split after 3 d and retransfected with 50 pM siRNA oligonucleotides and the expression plasmids. These cells were analyzed by immunofluorescence 18 h later. The effectiveness of siRNA was also monitored by RT-PCR and Western blotting.

Drosophila DS2R+ cells were kindly provided by Buzz Baum (University College London) and Shin-ichi Yanagawa (University of Kyoto) (Yanagawa et al., 1998). These cells were grown with Schneider's medium (Invitrogen) and 10% fetal calf serum at 25°C. The primary screen was done in 384-well plates with clear bottoms and black walls. Two sets of 62 plates containing 0.25 μg of double-stranded RNA (dsRNA) per well were provided by the Drosophila RNAi Screening Center (DRSC; http://flyrnai.org). Each well was seeded with 10⁴ DS2R+ cells in 10 μl of Schneider's medium. Plates were then incubated for 30 min at 25°C, followed by addition of 30 μl of Schneider's medium with 10% fetal bovine serum, penicillin, and streptomycin. These plates were sealed and incubated for 3 d at 25°C. The culture medium was then replaced with 50 μl of fresh medium with 20 nM MitoTracker followed by a 30-min incubation at 25°C, washes with phosphate-buffered saline (PBS), and fixation with 50 μl of chilled methanol:acetone (1:1) for 5 min at −20°C. The fixed cells were washed with PBS, and mounting medium (FluorSave; Calbiochem, San Diego, CA) with 0.3 μM 4,6-diamidino-2-phenylindole and sodium azide was added to each well. Mitochondria were observed with a 20× objective on an Axiovert 200M microscope (Carl Zeiss, Thornwood, NY). Abnormal mitochondrial distributions were classified clumped, punctate, and fragmented. Genes that gave rise to abnormal mitochondrial distributions in two independent wells, and they had no obvious nonmitochondrial functions (e.g., transcription, translation) based on their annotations were retested in a secondary screen. Double-stranded RNA was remade from PCR templates provided by the DRSC. The effects on mitochondrial morphology were tested with the same protocol as in the primary screen except that cells were plated in larger petri dishes with glass bottoms or on glass coverslips. The mitochondria could then be examined with a 100× oil immersion objective.

Immunofluorescence

Cells were grown on glass coverslips, and then they were fixed for 10 min at room temperature with prewarmed 3.7% formaldehyde. Where indicated, 20 nM MitoTracker Red was added 30 min before fixation. Cells were permeabilized for 5 min with 0.2% Triton X-100 in phosphate-buffered saline, blocked for 15–30 min with 1% bovine serum albumin in PBS, followed by an overnight incubation with primary antibodies in blocking buffer, three washes with PBS, and 1 h at room temperature with secondary antibodies. Coverslips were mounted on glass slides with FluorSave (Calbiochem). To quantify apoptosis with Hoechst, cells were grown on coverslips, washed three times with PBS, fixed with 3.7% paraformaldehyde, and stained with Hoechst, before counting apoptotic pycnotic nuclei with fluorescence microscopy. Images were acquired with a 100× oil immersion objective on a Zeiss Axiovert 200M microscope with an ORCA ER charge-coupled device camera (Hamamatsu, Bridgewater, NJ).

Mff, Encoded by C2orf33, Is the Human Homologue of Drosophila CG30404/Tango11

A BLAST search shows that humans have two proteins with substantial homology to CG30404/Tango11. The first protein was named FATE, because it is specifically expressed in fetal and adult testis, but the homology to this protein is limited to its carboxy-terminal half. Moreover, pilot experiments with FATE siRNA showed no effect on mitochondria in HeLa cells or other human cell lines commonly used in our laboratory (data not shown), as was expected because FATE is testis specific (Olesen et al., 2001). The second protein, encoded by C2ORF33 and renamed Mff, shows homology over the entire length of the protein (Figure 1D). The expression pattern of Mff was determined with an RNA blot representing a range of human tissues (Supplemental Figure 1). This blot shows that Mff is highly expressed in heart, kidney, liver, brain, muscle, and stomach and at low levels in other tissues. Because this pattern is compatible with a general mitochondrial function, we focused our analysis on this protein.

The main features of human Mff and Drosophila CG30404/Tango11 sequences are a short amino-terminal repeat, a middle segment with strong coiled coil forming propensity, and a carboxy-terminal hydrophobic segment that most likely serves as membrane anchor. The Mff gene encodes at least nine different isoforms represented by multiple expressed sequence tags (ESTs) in GenBank (Figure 1E). These isoforms are generated by the presence or absence of exon 1 and various combinations of exons 5, 6, and 7, which encode the middle part of the protein. The presence or absence of exon 1 most likely results from the use of alternative promoters. Isoforms lacking exon 1 have an alternative translation initiation codon embedded in exon 2. Alternatively spliced exons 5, 6, and 7 encode parts of the protein that are not conserved in Drosophila. However, other mammalian species have largely identical patterns of alternative splicing, suggesting that at least for mammalian cells sequence variations caused by the presence or absence of exons 5, 6, and 7 are functionally important.

Localization of Mff to Mitochondria

To determine the subcellular localization of Mff, we generated an antibody by immunizing rabbits with bacterially expressed Mff protein. Immunofluorescence analysis with this antibody shows colocalization with MitoTracker (Figure 2, A–C). Transfection with an amino-terminal myc-tagged version of Mff also shows mitochondrial localization (Figure 2, D–F). The mitochondrial staining detected with Mff antibody increases when cells are transfected with an Mff expression construct and decreases when they are transfected with Mff siRNA, confirming that staining with our Mff antibody reflects Mff localization (data not shown). The Mff antibody is also specific on Western blots, because the bands on blots are uniformly diminished in siRNA-transfected cells (Figure 3E). Transfection with a construct that encodes Mff with deletion of the carboxy-terminal hydrophobic segment gives rise to diffuse cytosolic staining, showing that the hydrophobic sequence is required for localization to mitochondria (Supplemental Figure 2). We conclude that Mff is localized to mitochondria and that this localization requires the carboxy-terminal transmembrane segment.

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Figure 2.

Fluorescence and biochemical analysis of Mff localization. (A–C) Immunofluorescence of endogenous protein in HeLa cells. (D–F) Overexpression of myc-tagged Mff. The construct used for this experiment encodes isoform 8, which lacks exons 5, 6, and 7 (Figure 1). A and D show Mff or myc antibody staining, B and E show MitoTracker staining, and C and F show merged images with Mff or myc in green and MitoTracker in red. Bar, 10 μm. (G) Western blots of differential centrifugation fractions prepared from HeLa cells. Mff is present in the low speed supernatant (S1) and the medium speed pellet (P2), which contain mitochondria as shown with Tom20 antibody. Tubulin, which was used to track soluble proteins, is also present as a contaminant in the P2 fraction, but very little Mff is present in the S2 fraction, consistent with mitochondrial localization. The Mff antibody detects bands ranging in size from 25 to 39 kDa, most likely corresponding to different splice variants. (H) Protease protection experiment using the mitochondrial (P2) fraction from bovine brain to determine the topology of Mff. Our Mff antibody detects only a single band of 38-kDa in brain extracts, similar in size to the top band in HeLa cells (see G), suggesting that the repertoire of Mff isoforms is more limited in brain than it is in HeLa cells. The P2 fraction was subjected to increasing amounts of trypsin. Proteins that are exposed to the cytosol, like Tom20, are digested at the lowest concentrations, whereas proteins that are protected by membrane, such as the mitochondrial intermembrane space proteins prohibitin and cytochrome c are still protected at the highest concentration. Solubilization with detergents was used to verify that trypsin is able to digest prohibitin and cytochrome c were it not for protection by membrane (data not shown). Together, these data show that Mff is exposed to the cytosol. (I) Alkaline extraction shows that Mff is anchored in membrane. The P2 fraction from bovine brain extracts was resuspended in carbonate buffer, pH 11.5. Membranes were pelleted by centrifugation at 100,000 × g. Tom20 serves as marker for the membrane fraction, and cytochrome c as marker for the cytosolic fraction. These experiments show that Mff cofractionates with mitochondria, that it is exposed to the cytosol, and that it is a membrane-anchored protein.

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Figure 3.

Effects of Mff siRNA on mitochondrial morphology. (A) HeLa cell transfected with scrambled Mff siRNA oligonucleotides and stained with MitoTracker. These cells show wild-type mitochondrial morphologies. (B) HeLa cell transfected with Mff, (C) with Drp1 siRNA and (D) with Fis1 siRNA oligonucleotides. These transfected cells all show highly connected mitochondria, consistent with defects in mitochondrial fission. Bar, 10 μm. (E) Western blots showing protein levels in siRNA-transfected cells. The blots show reduced levels in HeLa cells transfected with Mff, Drp1 or Fis1 siRNA, in comparison with scrambled (scr.) controlled. The Mff antibody shows two prominent bands (25 and 35 kDa) and several fainter bands that may correspond to different isoforms produced by alternative splicing. Tubulin serves as loading control. The levels of Mff are reduced by 88%, of Drp1 by 93%, and of Fis1 by 82% as determined by densitometry.

To further analyze Mff localization, we fractionated bovine brain extracts with differential centrifugation. Mitochondria and other heavy organelles were enriched in the heavy membrane fraction (P2) as shown with Tom20, which serves as a mitochondrial marker (Figure 2G). This fraction contained almost all Mff protein. The topology of Mff on the membrane was determined with a protease protection experiment using increasing amounts of Trypsin. Mff was digested by trypsin at concentrations that affect the mitochondrial outer membrane protein Tom20, but not the intermembrane space protein cytochrome c or the inner membrane protein prohibitin (Figure 2H). These results show that a substantial part of Mff is exposed to the cytosol, similar to the topology of other tail-anchored proteins, such as Fis1 and Tom20. To verify that Mff truly is membrane anchored, HeLa cell homogenates were subjected to alkaline extraction (pH 11.5). Mff was found almost exclusively in the pellet fraction, as was a known integral membrane protein (Tom20), but not cytochrome c (Figure 2I), from which we conclude that Mff is firmly anchored in membrane. Mff thus belongs to the growing number of tail-anchored membrane proteins.

Mff siRNA Produces Interconnected Mitochondria and Tubular Peroxisomes

To determine the effects of Mff loss of function, HeLa cells were transfected with siRNA oligonucleotides. We tested two independent pairs of oligonucleotides, targeting sequences that are present in all isoforms (exon 2 and exon 8). The two pairs of oligonucleotides were similarly effective. Results are shown with the first pair (exon 2), which reduces the protein levels of Mff by ~88% (Figure 3E). MitoTracker staining of cells transfected with Mff siRNA reveals highly interconnected mitochondrial networks. The networks are strikingly similar to the mitochondrial networks in cells transfected with Drp1 siRNA (Figure 3, B and C). The mitochondria of Mff siRNA-transfected cells had fewer free ends than those of Fis1 siRNA (Figure 3D), even though expression levels of both proteins were reduced to similar extents (Figure 3E). These phenotypes were observed in 94 ± 5% of cells (mean percentages and SD determined with 350–400 cells in each of four independent transfection experiments). We conclude that Mff siRNA affects mitochondrial morphology in ways that are indistinguishable from Drp1 siRNA and stronger than the effects of Fis1 siRNA.

Because Fis1 overexpression induces mitochondrial fission (James et al., 2003; Yoon et al., 2003), we tested whether Mff overexpression also induces fission by transfecting HeLa cells with an Mff expression construct. The percentage of untransfected cells with fragmented mitochondria is 7.7 ± 1.7%, whereas the percentage of transfected cells with fragmented mitochondria is 6.0 ± 3.8%, suggesting that Mff overexpression does not induce fission (mean percentages and SD were determined with 100 200 cells in each of 3 independent transfection experiments). These results are different from those obtained previously with Fis1 overexpression, which does cause fission (James et al., 2003; Yoon et al., 2003), but similar to results obtained with Drp1 overexpression, which does not cause fission (Smirnova et al., 2001). It seems likely that Mff and Drp1 are not rate limiting for fission or are more tightly controlled than Fis1.

Because Fis1 and Drp1 were previously shown to affect peroxisomal morphologies as well, we investigated whether Mff siRNA also affects peroxisomal morphology. The effect on peroxisomes was determined by immunofluorescence with an antibody raised against the peroxisomal protein catalase and counterstaining with MitoTracker to detect mitochondria. Peroxisomal staining in untransfected cells is typified by diffuse punctae (Figure 4B). As shown previously, these punctae are converted to short tubules by transfection with Drp1 siRNA (Figure 4F) (Koch et al., 2003; Li and Gould, 2003). We observe similarly tubular peroxisomes upon transfection with Mff siRNA (Figure 4D). These phenotypes were observed in 84 ± 6% of cells (mean percentages and SD were determined with 350 cells in 3 independent transfection experiments). Counterstaining with MitoTracker Red shows that cells with affected peroxisomes invariably also have affected mitochondria (Figure 4, C and E). Because small amounts of peroxisomal staining might have been obscured by mitochondrial staining, we changed the mitochondrial distribution with Drp1 siRNA to make parts of the cell devoid of mitochondria. These areas show colocalization of myc-tagged Mff with a peroxisomal marker (Supplemental Figure 3). We conclude that there is a small but significant amount of Mff on peroxisomes, similar to Fis1, which is also localized to peroxisomes and mitochondria (Koch et al., 2005; Kobayashi et al., 2007). The dual roles and localizations of Drp1, Fis1, and Mff on peroxisomes and mitochondria suggest that these proteins are part of the same fission apparatus, acting on two different organelles.

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Figure 4.

Effects of Mff siRNA on peroxisomal morphology. (A) HeLa cells transfected with scrambled oligonucleotides showing wild-type mitochondrial morphologies by staining with MitoTracker. (B) The same cells stained with catalase antibody, showing the punctate distribution of peroxisomes. The inset shows an enlargement of the peroxisomal staining. (C and D) HeLa cells transfected with Drp1 siRNA oligonucleotides, showing highly connected mitochondria and elongated peroxisomes. The enlargement shows the peroxisomal defect more clearly. (E and F) Similar patterns were obtained with Mff siRNA oligonucleotides. The boxed areas, enlarged in the bottom right-hand corners of D–F, show close-ups of peroxisomes. Bar, 10 μm.

The Drosophila homologue of Mff (CG30404/Tango11) was also identified in a screen for defects in constitutive secretion, suggesting that Mff might affect the secretory pathway (Bard et al., 2006). We tested whether Mff siRNA also affects secretion in mammalian cells, by using a luciferase assay. We were unable to detect significant differences in the rate of secretion or in Golgi morphology, even when there were large differences in mitochondrial and peroxisomal morphologies (data not shown). The secretion defects that were observed in Drosophila cells may have been due to respiratory defects, because these defects can result from mitochondrial fission defects (Estaquier and Arnoult, 2007). We conclude that Mff affects peroxisomal and mitochondrial morphologies in ways that are indistinguishable from the effects of Drp1 siRNA. It seems likely that Mff, Fis1, and Drp1 all affect different aspects of the pathways that mediate mitochondrial and peroxisomal fission.

Fusion Defects Caused by Dominant-Negative Mitofusin Mutants Are Partially Suppressed by Mff siRNA

It was previously shown that dominant-negative mutations in transfected Drp1 suppress mitochondrial fragmentation in mouse Mfn1 and Mfn2 knockout cell lines (Chen et al., 2003). Similar effects were observed with yeast where it was shown that mutations in Dnm1 are epistatic to mutations in Fzo1 (Fekkes et al., 2000; Mozdy et al., 2000; Tieu and Nunnari, 2000). To determine whether Mff contributes to fission, rather than negatively regulating fusion, we tested the ability of Mff siRNA to suppress a mitochondrial fusion defect. The mitochondrial fusion defect was introduced by transfecting HeLa cells with constructs that express dominant interfering Mitofusin mutants. We used Mfn1(K88T) and Mfn2(K109T), which have mutations in the critical lysines of the G1 consensus motifs in their GTP binding domains (Eura et al., 2003; Santel et al., 2003). Dominant interference most likely occurs through coassembly with endogenous protein complexes. Mitofusin complexes on the mitochondrial outer membrane can be heteromeric or homomeric (Chen et al., 2003) and act in trans with similar complexes on opposing mitochondrial membranes during fusion (Koshiba et al., 2004; Meeusen et al., 2004). Because of these heteromeric complexes, dominant interfering mutants are more effective than siRNA, which is hampered by the partial redundancy of Mfn1 and Mfn2 (Chen et al., 2003).

In this experiment, HeLa cells were first transfected with scrambled controls, Mff, Fis1, or Drp1 siRNA followed by a second transfection 3 d later with the dominant-negative Mitofusin constructs. These constructs also have epitope tags to identify the cells that express dominant-negative Mitofusins. All cells transfected with dominant negative Mitofusin constructs show mitochondrial fragmentation, consistent with a complete block of mitochondrial fusion. These mitochondrial fragments are also clustered near the nucleus, which is most likely due to nonspecific perturbations of mitochondrial outer membranes as seen previously with other overexpressed mitochondrial proteins (Smirnova et al., 2001). Perinuclear clustering was not averted by Mff, Drp1, or Fis1 siRNA (Figure 5, B–D), but there was a notable increase in mitochondrial connectivity in almost all Drp1 siRNA cells and in a small fraction of Mff and Fis1 siRNA-transfected cells (Figure 5E). Cotransfection of Mff and Fis1 siRNA oligonucleotides did not enhance the fission defect, but in our hands cotransfected siRNA oligonucleotides are often not as effective as individual transfections (data not shown). It therefore remains possible that Mff and Fis1 are partially redundant. Alternatively, we may not have achieved low enough levels of Mff and Fis1 to fully block their function or Mff and Fis1 are not absolutely required for fission. We can, nevertheless, conclude that Mff and Fis1 siRNA reverse some of the fragmentation caused by dominant-negative Mitofusins, whereas Drp1 siRNA fully reverses their effects.

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Figure 5.

Reversal of mitochondrial fragmentation caused by dominant-negative Mitofusin expression constructs. (A) HeLa cells were transfected with scrambled Mff siRNA oligonucleotides as a control and with a myc-Mfn2(K109T) expression construct. Mitochondria were detected with MitoTracker (red). Cells expressing mutant Mfn2 were detected with myc antibody (green). These cells invariably had fragmented and often clumped mitochondria, most likely due to overexpression of aberrant mitochondrial outer membrane protein. Similar transfections were done with Mff (B), Drp1 (C), and Fis1 (D) siRNA oligonucleotides along with the myc-Mfn2(K109T) expression construct. The cotransfected cells show clumped mitochondria, but these mitochondria often have thin tubular connections, which were never detected when the myc-Mfn2(K109T) construct was transfected alone. The enlargements in the bottom left corners show these connections more clearly. Bar, 10 μm. The percentages of cells with mitochondrial connections are shown in E. The experiments numbered 1 were done with GFP tagged Mfn1(K88T), and the experiments numbered 2 and 3 were done with myc-Mfn2(K109T). For each point, 250–400 cells were counted.

Mff siRNA Partially Inhibits CCCP-induced Mitochondrial Fragmentation

It was previously shown that loss of mitochondrial membrane potential caused by treatment with the protonophore CCCP induces rapid mitochondrial fission. Within minutes, the mitochondria of cells treated with CCCP are converted to small, dispersed, fragments (Griparic et al., 2007). We used this method to investigate the effects of Drp1, Mff, and Fis1 siRNA on inducible mitochondrial fission (Figure 6, A–H). Cells were transfected with siRNA oligonucleotides and stained with MitoTracker 3 d later, followed by a 60-min incubation with CCCP to induce fragmentation and analysis by fluorescence microscopy.

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Figure 6.

Reversal of fission induced by loss of mitochondrial membrane potential. HeLa cells were transfected with scrambled, Mff, Fis1, or Drp1 siRNA oligonucleotides, respectively (A–D), stained with MitoTracker, and then incubated for 60 min with the membrane-depolarizing drug CCCP at 2 μg/ml to induce mitochondrial fission (E–H). Transfection with Drp1 siRNA inhibits almost all of the induced fragmentation. Transfection with Mff siRNA inhibits some, but not all, of the induced fragmentation, whereas transfection with Fis1 siRNA had only a limited effect. Bar, 10 μm. I shows quantification of these effects. N, highly connected tubular networks; T, mixture of tubules as observed in untreated cells; S, short tubules; and R, round fragments. The percentages are means of three independent experiments with 300–400 cells per data point. The error bars show SD for the three experiments. The bars show values obtained after CCCP induction. Although Drp1 siRNA is the only one that strongly inhibits CCCP-induced fission, the effects of Mff and Fis1 siRNA are significant when the percentages of cells with round fragments are compared with those percentages in cells transfected with scrambled oligonucleotides. An unpaired Student's t test shows p < 0.0005 for Mff (*), p < 0.005 for Fis1 (**), and p < 0.0001 for Drp1 (***). The amounts of Mff protein were reduced by 91%, Fis1 by 93%, and Drp1 by 99% (average values as determined by densitometry of Western blots).

A histogram with the distributions of different mitochondrial morphologies is shown in Figure 6I. The results show that Drp1 siRNA renders mitochondria resistant to CCCP-induced fragmentation (Figure 6, C and D). Mff siRNA renders some mitochondria resistant to induced fragmentation, but a range of transition forms (a mixture of tubule lengths or short tubules) are observed as well (Figure 6, E and F). Much weaker effects are observed with Fis1 siRNA (Figure 6, E and F). Quantitation of these results is shown in Figure 6I. We conclude that Mff siRNA has modest but reproducible inhibitory effects on CCCP-induced fragmentation of mitochondria.

Mff siRNA Inhibits Apoptosis

Mutations in Drp1 and Fis1 siRNA were previously shown to inhibit apoptosis by preventing cytochrome c release from mitochondria (Frank et al., 2001; James et al., 2003; Lee et al., 2004). To test whether Mff siRNA also inhibits these processes, HeLa cells were transfected with scrambled control, Mff, Drp1, and Fis1 siRNA oligonucleotides, and apoptosis was induced with staurosporine 3 d later. The pan caspase inhibitor ZVAD-fmk was included to prevent rounding up and detachment of the cells. Samples of cells were taken at fixed intervals and processed for immunostaining with anti-cytochrome c antibodies. The histogram in Figure 7A shows the percentages of cells with cytochrome c released from mitochondria into the cytosol. Mff siRNA strongly inhibits cytochrome c release in the majority of cells, similar to Drp1 and Fis1 siRNA. These experiments were done with the pan-caspase inhibitor z-VAD-fmk to prevent rounding up of cells and thus underestimating the number of cells that have cytochrome c release. The inhibition of cytochrome c release from mitochondria is not absolute, because at 5 h after induction all cells, whether they are transfected with Mff, Drp1, and Fis1 siRNA or with scrambled oligonucleotides, have cytosolic cytochrome c (data not shown). Comparable results were obtained with actinomycin D, as an alternative apoptosis-inducing agent (Supplemental Figure 4). In a separate experiment, z-VAD-fmk was omitted to determine the effects on further progression of apoptosis. The percentages of cells undergoing apoptosis were determined by counting the numbers of pycnotic nuclei in cells stained with Hoechst. At time points up to 3 h, these percentages are significantly lower in transfections with Mff, Fis1, or Drp1 siRNA oligonucleotides than with scrambled oligonucleotides (Figure 7B). This inhibition is also not absolute, because in due time all cells have pycnotic nuclei (Mff, Fis1, or Drp1 siRNA transfected cells are indistinguishable from cells transfected with scrambled oligonucleotides after 5 h; data not shown). We conclude that Mff siRNA inhibits apoptotic release of cytochrome c and further progression of apoptosis similar to the inhibitory effects of Drp1 and Fis1 siRNA, but these effects are not absolute.

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Figure 7.

Inhibition of apoptosis by Mff siRNA. HeLa cells were transfected with scrambled, Mff, Fis1, and Drp1 siRNA oligonucleotides as indicated, and apoptosis was induced by treating the cells with staurosporine or as control with solvent (dimethyl sulfoxide). Cells were fixed and stained at fixed intervals after the addition of staurosporine as indicated. (A) Effects on cytochrome c release from mitochondria as detected by staining the cells with cytochrome c antibody. Along with Staurosporine, z-VAD-fmk was added to prevent further progression of apoptosis. (B) Effects on the formation of apoptotic nuclei as detected with Hoechst staining. No z-VAD-fmk was added to allow further progression of apoptosis. The percentages are means of three independent experiments, each with 300–400 cells per data point. The error bars show SD for these three experiments. An unpaired Student's t test was used for statistical analysis of data collected after 90 min with staurosporine.

We thank other members of the lab for helpful discussions and critical reading of the manuscript. We also thank Dr. Bernard Mathey-Prevot, Dr. Norbert Perrimon, and other members of the Drosophila RNA Screening Center at Harvard Medical School for the wonderful opportunities provided by the center, for help with the screen, and for help evaluating screening results. This work was supported by American Cancer Society grant RSG-01-147-01-CSM and National Institutes of Health grant GM-051866.