programmed cell death ( pcd ) plays an important role in sculpting tissues during animal development ( adams and cory , 2002 ; danial and korsmeyer , 2004 ) . the molecular regulators that are central to this process seem to be evolutionarily conserved from worms to mammals ( danial and korsmeyer , 2004 ; horvitz , 1999 ) and include autocatalytic initiator caspases , trans - activable effector caspases , cytosolic activating factors ( apaf-1 ) , and multidomain bcl-2 proteins ( danial and korsmeyer , 2004 ; horvitz , 1999 ) . the proapoptotic bcl-2-family proteins oligomerize and permeabilize mitochondria , releasing intermembrane space components such as cytochrome - c and smac / diablo into the cytosol , where they activate initiator caspases by an atp - dependent mechanism ( danial and korsmeyer , 2004 ) . initiator caspases trans - activate effector caspases that cleave multiple cellular substrates , resulting in dna degradation , nuclear condensation , and loss of cell integrity ( danial and korsmeyer , 2004 ; wang , 2001 ) . mitochondrial outer - membrane permeabilization has been proposed to depend on the mitochondrial fission and fusion machinery ( perfettini et  al . , 2005 ; youle and karbowski , 2005 ) . consistent with this , mitochondria undergo dramatic fragmentation very close in time to cytochrome - c release during mammalian cell death ( frank et  al . , 2001 ; mancini et  al . , 1997 ) . furthermore , an increase in mitochondrial fragmentation and a block in mitochondrial fusion are essential for cell death progression ( frank et  al . , 2001 ; karbowski et  al . , 2002 ; yu et  al . , 2005 ) . in normal cells , the balance in the rates of mitochondrial fission  and fusion regulated by dynamin - related protein-1 ( drp-1 ) , fis-1 and endophilin ( fission ) , or mitofusins and opa-1 ( fusion ) maintains the dynamic , interconnected mitochondrial tubules ( meeusen and nunnari , 2005 ; okamoto and shaw , 2005 ; yaffe , 1999 ) . an increase in recruitment of drp-1 to the mitochondria accentuates staurosporine , lipid , and free oxygen radical stress - induced mitochondrial outer - membrane permeabilization ( breckenridge et  al . moreover , multiple apoptotic stimuli induce mitochondrial recruitment of the proapoptotic bcl-2-family protein , bax , to drp-1 and mitofusin-2-positive putative mitochondrial fragmentation sites ( karbowski et  al . , 2002 ; 2005 ) in a fis-1-dependent manner ( lee et  al . , 2004 ) , consistent with a role for mitochondrial fission and fusion machinery in cell death . in drosophila , rhg - family proteins ( richardson and kumar , 2002 ) , genotoxic stresses , and protein synthesis inhibitors ( chew et  al . , 2004 ; zimmermann et  al . , 2002 ) antagonize drosophila inhibitor of apoptosis protein-1 ( diap-1)-mediated inhibition of the activation of the apical caspase dronc in an ark- ( drosphila apaf-1 ) and atp - dependent manner ( mills et  al . , 2006 ) , leading to effector caspase activation and cell death . cytochrome - c has been shown to be differentially displayed from the mitochondria  during cell death ( varkey et  al . , 1999 ) . knockdown  of drosophila cytochrome - c did not affect cell death triggered by genotoxic stress in vitro ( means et  al . , 2006 ) and ex vivo ( zimmermann et  al . , 2002 ) or developmental stimuli in vivo ( dorstyn et  al . , 2004 ) , although certain nonapoptotic caspase activation pathways utilized during sperm individualization were affected ( arama et  al . , 2003 ) . furthermore , mitochondrial morphology during drosophila pcd has not been previously reported . in this study , we show that multiple apoptotic stimuli result in mitochondrial fragmentation upstream of caspase activation , phosphatidylserine exposure , and nuclear condensation in drosophila cells . while etoposide induced mitochondrial fragmentation , c6-ceramide resulted in an increase in mitochondrial contiguity prior to its fragmentation . drp-1 mutant or rnai - treated s2r cells are considerably protected from multiple apoptotic stimuli , consistent with reduced mitochondrial fragmentation . thus , mitochondrial remodeling plays an important role in modifying the propensity of cells to undergo pcd in drosophila . precisely timed ecdysone pulses induce reaper and hid expression in the drosophila larval midgut ( 0 hr after puparium formation [ apf ] ) or the salivary gland ( 10 hr apf ) and trigger developmental pcd ( yin and thummel , 2005 ) . mitochondria , visualized by using matrix - targeted gfp ( mito - gfp ) ( pilling et  al . , 2006 ) in acridine orange - positive , dying prepupal midgut cells ( 1 hr apf , figure  1aiii ) and salivary glands ( 4 hr apf , figure  1biii ) , were remarkably fragmented , unlike third - instar larval ( 4 hr apf ) mitochondria . quantification revealed a dramatic decrease in the prepupal mitochondrial cross - sectional area ( csa ; midgut [ 0.7 m from 1.4 m , figure  1a ] and salivary gland [ figure  s1a , see the supplemental data available with this article online ] ) and a significant increase in the number of mitochondria per cell ( figure  s1a ) . moreover , ecdysone - induced mitochondrial fragmentation was mimicked ex vivo on third - instar larval wing discs by using 1 mm ecdysone ( kilpatrick et  al . , 2005 ) for 2 hr ( figure  1c ; figure  s1b ) . in addition , overexpression of hid resulted in mitochondrial fragmentation in acridine orange - positive eye disc cells ( figure  s1c ) . thus , mitochondria in drosophila tissues fragment during pcd , as has been reported in c. elegans ( jagasia et  al . , 2005 ) and mammalian cells ( frank et  al . , , mitochondrial morphology was temporally characterized in etoposide- , actinomycin - d- , cycloheximide- , or c6-ceramide ( a lipid cell death mediator)-treated ( walter and hajnoczky , 2005 ) larval hemocytes ( sriram et  al . , 2003 ) and the s2r cell line . a 3- to 4-fold increase in nuclear condensation ( 6 hr , figure  1h ) was preceded by effector caspase activation ( 5 hr , figures 2a2biv ) and phosphatidylserine ( ps ) exposure in propidium iodide ( pi)-negative hemocytes ( 6 hr , figures 2a2bv ) . these cells subsequently ( 10 hr ) became characteristically blebbed ( figures 2a2bvi ) and pi permeable ( figures 1d1eiv , top inset ) . the number of etoposide - treated apoptotic hemocytes increased with time ( figure  1 g ) . interestingly , mitochondrial fragmentation ( figures 1dii and 1 g ) , as confirmed by quantifying functionally isolated mitochondria ( figure  s2 and movie s1 ) ( collins et  al . , 2002 ) at 3 hr , preceded the onset of ps exposure ( figure 2av ) or  nuclear damage ( figures 1 g and 1h ) . quantification showed an increase in the number of mitochondria and  the contribution of fragmented mitochondria to the mitochondrial csa ( figures s3b , s3d , and s3e ) . mitochondrial fragmentation was also observed in cycloheximide- or actinomycin - d - treated , mito - yfp - transfected s2r cells ( 6 hr , figures s3 g and s3h ) . surprisingly , mitochondria in c6-ceramide - treated ( 3060 min ) hemocytes that had normal nuclei were highly contiguous ( open arrowheads , figure  1eii ) . quantifying functionally isolated mitochondrial csa per cell showed a significant increase in the contribution of tubular or extensively tubular mitochondria in these cells when compared with untreated cells ( figure  s3f ) . however , by 4 hr , these extensively tubular mitochondria underwent fragmentation in fitc - annexin v ( anv)-negative hemocytes that had normal nuclei ( figures 1eiii and 1i ) , similar to what was observed with genotoxic stress ( figure  1dii ) . therefore , genotoxic stresses triggered mitochondrial fragmentation , while the lipid cell death mediator induced increased mitochondrial contiguity and subsequent fragmentation prior to ps exposure , nuclear condensation , and finally plasma membrane permeability during drosophila cell death . in hemocytes incubated with an apoptotic stimulus , mitochondrial fragmentation ( 34 hr ) preceded any detectable effector caspase activation ( 5 hr [ figures 2a and 2biv ] ) . furthermore , inhibiting caspases with zvad - fmk or by overexpressing diap-1 ( diap-1 + ) ( hay et  al . , 1995 ) did not affect mitochondrial fragmentation ( figure  2c ) , although hemocyte death was inhibited ( figures 2f2h ) , as revealed by a lack of apoptotic markers ( figures 2d and 2e ; zvad - fmk ; data not shown ) . in addition , overexpression of dcp-1 , a drosophila effector caspase , did not affect mitochondrial morphology ( figure  s4 ) . the drp-1 mutants used to study the role of mitochondrial  remodeling during drosophila pcd are functional null alleles , drp-1 ( gly293ser mutation ) , picked in a forward screen for genes affecting neurotransmission and drp-1 , a p element insertion between the first two exons of drp-1 ( 13510 in this study ) and a hypomorph , nrd ( arg278trp mutation ; 3665 in this study ) ( rikhy et  al . , 2007 ; verstreken et  al . , 2005 ) . drp-1 , 13510 , and the deficiency df exel6008 were second - instar larval lethal ( data not shown ) ; however , drp-1 yielded bang - sensitive escapers ( verstreken et  al . , 2005 ) . the hypomorphic trans - allelic combination of 3665/13510 was third - instar larval lethal , although it yielded a few temperature - sensitive adults ( rikhy et  al . , 2007 ) . a genomic duplication of drp-1 ( dp [ 2;1 ] js13 [ rikhy et  al . , 2007 ] ) completely rescued the lethality associated with drp-1 , 13510 , and 3665/13510 . mitochondria in drp-1 and 3665/13510 hemocytes were extensively tubular when compared with wild - type mitochondria ( open arrowheads , figures 3bi3biii ) . quantifying mitochondrial morphology revealed a 2-fold decrease in the number of mitochondria and a significant increase in the contribution of tubular and extensively tubular  mitochondria to the total mitochondrial csa in drp-1 mutant hemocytes when compared with wild - type cells ( figure  s5 ) . interestingly , 13510/+ hemocytes ( figures 3biv and 3ci ) or eye disc cells ( figure  s5 ) displayed a dominant mitochondrial fission defect that was completely rescued by a genomic duplication of drp-1 ( figure  3cii ) . the mitochondrial fission defect in mutant cells could result from a reduced mitochondrial association of drp-1 ( figure  s6 ) . an increase in mitochondrial contiguity due to a loss of drp-1 function was also confirmed by measuring fluorescence recovery after photobleaching ( frap ) of mito - yfp ( collins et  al . , 2002 ) in drp-1 rnai - treated s2r cells that had extensively tubular mitochondria ( figure  3d ) . relative frap of mito - yfp in a defined mitochondrial region in drp-1 rnai - treated cells was significantly ( p < 0.01 ) higher than that observed in mock rnai - treated cells ( figure  3f ) . drp-1 mutant hemocytes were protected from etoposide - induced death up to at least 10 hr ( figures 4a and 4b ) , as revealed by a lack of caspase activation , ps exposure , or pi permeability in the majority ( 80% ) of these cells ( figures 4fi , 4fii , 4fv , and 4fvi ) . furthermore , drp-1 mutant and dsrna - treated s2r cells were significantly protected ( p < 0.01 ) from cycloheximide- , actinomycin - d- ( figures 4c and 4d ) , or uv - b - induced ( figure consistent with increased protection , mitochondria in the majority ( 98% ) of etoposide - treated drp-1 hemocytes failed to fragment ( 6 hr ; compare figure  4gii with figure  4gi ) . interestingly , mitochondria in etoposide - treated 3665/13510 hemocytes revealed a tubular , yet beaded and swollen intermediate in mitochondrial fragmentation by 4 hr ( figure  4giii ) that yielded some fragmented mitochondria in few ( 25% ) cells later ( 6 hr ; data not shown ) . therefore , reduced ( drp-1 ) or delayed ( 3665/13510 ) mitochondrial fragmentation decreased effector caspase activation and protected cells from genotoxic stress . moreover , an increase in expression of drp-1 in hemocytes resulted in enhancement of etoposide - induced cell death ( figure  s8 ) . the majority ( 70% ) of the c6-ceramide - treated drp-1 hemocytes did not show effector caspase activation or ps exposure and displayed significant ( p < 0.05 ) protection ( figures 4e , 4fiii , and 4fvii ) , similar to what was observed with etoposide , although hemocytes derived from the weaker allelic combination , 13510/3665 , were apoptotic ( figures 4fiv and 4fviii ) . unlike 13510/3665 mitochondria , drp-1 mitochondria failed to fragment ( figure  4hi ) , consistent with an essential role for drp-1-mediated mitochondrial fragmentation during apoptosis in drosophila . moreover , developmental pcd in drp-1 mutant larvae was considerably reduced , as revealed by the enlarged central nervous system and a prominently elongated ventral ganglion ( figure  4i ) , similar to other pcd - defective mutants reported ( mills et  al . , 2006 ) . during metamorphosis , the first ecdysone pulse triggers mitochondrial fragmentation in prepupal tissues , although it is after the second ecdysone pulse that salivary gland histolysis occurs . it is likely that diap-1 inhibits caspases in these cells that have fragmented mitochondria until it is downregulated at the transcriptional level or degraded after the second ecdysone pulse ( yin and thummel , 2005 ) . the data presented here show involvement of mitochondrial fragmentation for ark - mediated dronc activation during cell death . the rhg - family proteins that localize to the mitochondria ( claveria et  al . , 2002 ; haining et  al . , 1999 ; olson et  al . , 2003 ) might activate drp-1-mediated mitochondrial fragmentation . this could result in exposure of cytochrome - c ( varkey et  al . , 1999 ) or release of peanut ( gottfried et  al . , 2004 ) , which antagonize diap-1-mediated suppression of dronc however , as drosophila pcd was unaffected upon knockdown of cytochrome - c ( dorstyn et  al . , 2004 ) , mitochondrial fragmentation in drosophila and mammalian cells would increase mitochondrial surface area and perhaps the concentration of bulky head group lipids on the outer mitochondrial membrane , facilitating recruitment of proapoptotic proteins . drp-1 might organize sites for drosophila bcl-2-family protein debcl function on mitochondria ( dorstyn et  al . , 2002 ) that are similar to mitochondrial sites of bax recruitment in mammalian cells ( karbowski et  al . , 2002 ) . these results provide the first , to our knowledge , evidence that drp-1-mediated mitochondrial fragmentation upstream of effector caspase activation modifies apoptotic sensitivity ( figure  4j ) . thus , mitochondrial fragmentation , like caspase activation , plays a conserved and unifying role in diverse cell death pathways from worms to mammals ( frank et  al . although the function of the highly contiguous mitochondria during lipid - induced cell death remains poorly understood , this study brings to the forefront a modulatory role for mitochondrial remodeling in determining the susceptibility of drosophila cells to death . media and chemicals were obtained from gibco - brl or sigma - aldrich unless otherwise specified . secondary antibodies ( jackson immunoresearch laboratories ) were conjugated to fluorescent dyes as recommended by the manufacturer . 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 ) . third - instar larval tissues were dissected in schneider insect medium supplemented with 10% non - heat - inactivated fetal bovine serum and 1 g / 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 ( 20c ) , 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 . 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 m ) , cycloheximide ( 0.5 g / ml ) , or c6-ceramide ( 20 m or 40 m ; matreya , inc . ) diluted in 1 medium 1 ( m1 ) supplemented with 2 mg / ml glucose ( imaging medium [ i m ] ) at 20c for the indicated times prior to staining nuclei with 5 g / ml hoechst-33342 ( 5 min ) . hemocytes were preincubated ( 30 min ) with 50 m caspase inhibitor-1 ( zvad - fmk ; calbiochem ) in i m before incubation with apoptotic stimuli in the presence of 50 m 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  10 s2r cells were cotransfected with 1 g sirnai ( dharmacon , inc . ) and 0.25 g 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 . hemocytes were incubated with 10 m caspace substrate ( fitc - vad - fmk , promega ) for 20 min ( 20c ) or stained with pi ( 20 g / ml ) , anv ( 2 g / ml ) ( santa cruz biotechnology ) , and hoechst-33342 ( 5  g / 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 . ) 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 i m supplemented with 1.5 mg / ml bsa ( im+bsa ) for 20 min ( 20c ) and were imaged . s2r cells were grown in schneider 's medium supplemented with 10% fetal bovine serum ( gemini bio - products ) at 25c . a total of 1  10 cells were plated and transfected with 1 g eyfp - mito vector ( lajeunesse et  al . , 2004 ) by using 5 l cellfectin transfection reagent ( invitrogen ) . mitochondria were selected by using defined thresholds post background correction and were quantified by using metamorph ( molecular devices corporation ) . third - instar larvae were exposed ( 90 s ) on a dual intensity transilluminator ( tm20 ; uvp , inc . ) a total of 6  hr later , wing discs were dissected and stained with 3 g / ml acridine orange in pbs ( 5 min ) , washed , and imaged on the confocal microscope . 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 = 510 ) . s2r cells that were cotransfected with 1 g sirnai ( dharmacon , inc . ) and 1 g 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 19942012 ) , and sicontrol nontargeting sirna # 1 ( dharmacon , inc . ) , a mitochondrial region of interest ( roi , 1.75 m 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 12981317 ) and the 3 primer 5-tgcttggtgttgatgtaggc-3 ( designed to nm_134850 bases 14901509 ) 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 ) .
summarythe role of mitochondria in drosophila programmed cell death remains unclear , although certain gene products that regulate cell death seem to be evolutionarily conserved . 
 we find that developmental programmed cell death stimuli in vivo and multiple apoptotic stimuli ex  vivo induce dramatic mitochondrial fragmentation upstream of effector caspase activation , phosphatidylserine exposure , and nuclear condensation in drosophila cells . unlike genotoxic stress , a lipid cell death mediator induced an increase in mitochondrial contiguity prior to fragmentation of the mitochondria . using genetic mutants and rnai - mediated knockdown of drp-1 , we find that drp-1 not only regulates mitochondrial fission in normal cells , but mediates mitochondrial fragmentation during programmed cell death . 
 mitochondria in drp-1 mutants fail to fragment , resulting in hyperplasia of tissues in vivo and protection of cells from multiple apoptotic stimuli ex vivo . 
 thus , mitochondrial remodeling is capable of modifying the propensity of cells to undergo death in drosophila .