materials  polyclonal anti - caspase 8 ( catalog # 559932 ) and 
  monoclonal anti - caspase 8 ( catalog # 551242 ) antibodies were from bd 
  pharmingen . mouse monoclonal anti - rab5 ( catalog # sc46692 ) , monoclonal 
  anti - p85 ( catalog # sc1637 ) , and rabbit polyclonal anti - rab7 ( catalog 
  # sc10767 ) antibodies were from santa cruz biotechnology . polyclonal anti - eea1 
  ( catalog # ab50313 ) and polyclonal anti - m6pr ( catalog # ab32815 ) antibodies were 
  from abcam ( cambridge , ma ) . goat anti - rabbit and goat anti - mouse antibodies 
  coupled to horseradish peroxidase ( hrp ) and monoclonal anti - actin antibody 
  ( catalog # a5316 ) were from bio - rad . alexa fluor 488- and alexa fluor 
  555-labeled secondary antibodies , alexa fluor 488-labeled transferrin 
  ( catalog # t13342 ) , bodipy fl low density lipoprotein ( ldl ; catalog 
  # l3483 ) , and to - pro-3 ( catalog # t3605 ) were from molecular probes 
  ( eugene , or ) . the fugene 6 transfection reagent was from roche diagnostics . 
  the purelink quick plasmid miniprep ( catalog # k2100 - 10 ) and 
  purelink hipure plasmid filter purification ( catalog # k2100 - 17 ) kits 
  were from invitrogen . cell medium and antibiotics were from cellgro mediatech , 
  inc ( herndon , va ) . fetal bovine serum ( catalog # sh30070.03 ) was from hyclone 
  ( logan , ut ) . peroxidase from hrp and the peroxidase substrate 2,2 
  azino - bis(3-ethylbenzthiazoline-6-sulfonic acid ) ( abts ) were from sigma . chemiluminescent 
  substrate ( catalog # 34078 ) and protein a / g beads were from pierce . cell culture293 t and a549 cells were cultured in dulbecco 's 
  modified eagle 's medium supplemented with 10% fetal bovine serum and 
  antibiotics ( 10,000 units / ml penicillin , 10 g / ml streptomycin ) at 37 
  c , 5% co2 . the previously described human neuroblastoma cells 
  nb7 and nb7c8 were cultured in rpmi with 10% fetal bovine serum and 
  antibiotics ( 20 ) . lentiviral - mediated down - regulation of caspase 8 in a549 cells by short 
  hairpin rna was previously described 
  ( 25 ) . for transient 
  transfection experiments in hek293 t , nb7 , and nb7c8 cells , the reagent 
  fugene was used following the instructions provided by the 
  manufacturer . wild type rab5 and the 
  mutants rab5/s34n and rab5/q79l were subcloned from pet3c vector ( a kind gift 
  from dr . s. schmid , the scripps research institute ) into pcdna3.1(+ ) by pcr 
  using the primers 5-cgggatccatggctaatcgaggagcaaca-3 and 
  5-cccgcggccgcttagttactacaacactgact-3 and the restriction sites 
  bamhi / noti . rab5 binding domain  
  comprising the last 73 amino acids of rabaptin-5 ( residues 789862 ) was 
  obtained by pcr using human rabaptin5 cloned into pcmv - sport6 ( invitrogen ) as 
  a template and the primers 
  5-cccgcggccgctcatgtctcaggaagctggt-3and 
  5-cggtcgacaagctaaggctaccgttgaaca-3. the pcr product was digested 
  and ligated into pgex-6p1 ( ge healthcare ) by using the restriction sites 
  sali / noti . human caspase 8 was available in the pcdna3.1(+ ) and pegfp - n2 
  vectors , as previously described 
  ( 13 , 
  19 ) . the caspase 8 mutants 
  c360a and y380f were previously reported and cloned into pegfp - n2 vector 
  ( 13 ) . bovine flag - tagged wild 
  type p85 , p85/r274a ( mutant defective for rab - gap activity ) , and 
  p85/r368a / r649a ( double mutant r , defective in both sh2 domains 
  via point mutations ) were previously described 
  ( 8) . cells were grown for 24 h on glass 
  coverslips and subjected or not to treatment as indicated . after rinsing with 
  pbs , cells were fixed in pbs , 4% paraformaldehyde ( 10 min ) , permeabilized with 
  0.1% triton x-100 ( 5 min ) , and blocked with 2% bsa in pbs ( 30 min ) at room 
  temperature . cells were then incubated with either monoclonal anti - rab5 igg 
  ( 1:50 ) , polyclonal anti - rab7 igg ( 1:50 ) , polyclonal anti - m6pr igg ( 1:50 ) , or 
  polyclonal anti - eea1 ( 1:250 ) primary antibodies followed by incubation with 
  the respective alexa fluor conjugated secondary antibodies ( 1:250 ) . samples were then washed and 
  mounted onto slides with the vectashield hard - set mounting media ( vector 
  laboratories , burlingame , vt ) and visualized on a nikon eclipse c1 confocal 
  microscope with a 1.4 na 60 oil immersion lens using minimum pinhole 
  ( 30 m ) . images were captured using ez - c1 3.50 imaging software and 
  analyzed using national institutes of health imagej software . image analysis ; measurement of the incidence of cells with peripheral 
  rab5pictures were randomly chosen , and cells were comparatively 
  defined as positive for periphery rab5 staining  or 
   negative for periphery rab5 staining  based on the high or low 
  relative staining for rab5 at cell periphery . then the percentage of 
   positive cells  was calculated with respect to the total cell 
  number . total cell number was counted by nuclear staining ( topro3 ) . for 
  quantification of peripheral rab5 , pictures were randomly chosen , and 
  round - shaped cells were picked for individual analysis . cell periphery was 
  defined as the area between the cell surface and 23 m beneath ( in 
  cells ranging between 20 and 25 m in diameter ) . relative levels of rab5 were calculated 
  within this area by using the  radial profile plugin  of the 
  imagej software . a total of six cells per picture , five pictures per 
  experiment in a total of three independent experiments was analyzed . for 
  analysis , the red channel ( rab5 staining ) was adjusted to a window / level of 
  160 pixels , and then a radial profile was drawn around the cell ( outer 
  circle ) . thereafter , a second radial profile ( inner circle ) was drawn with a 
  radius of 3/4 of the outer circle . finally , an arbitrary estimation of the 
  percentage of peripheral rab5 was calculated as % peripheral rab5 = 100 - ( no . 
  of pixels first , both red and green channels to be analyzed were adjusted to a 
  window / level of 160 pixels , and the integrated intensity of pixels for each 
  channel was measured . thereafter , the integrated intensity of pixels 
  co - localized was measured by using the  co - localization rgb 
  plugin  of imagej . cell extracts were prepared in radioimmune 
  precipitation assay buffer ( 100 mm tris base , 150 mm 
  nacl , 1 mm edta , 1% deoxycholic acid , 1% triton x-100 , 0.1% sds , 50 
  mm naf ) containing protease inhibitors , boiled in laemmli buffer , 
  and separated by sds - page on 10 or 12% acrylamide minigels ( bio - rad ) by 
  loading 25 g of total protein per lane and transferred to nitrocellulose . blots were blocked with 5% milk in 0.1% tween , pbs and then probed with 
  anti - actin ( 1:5000 ) , anti - rab5 ( 1:500 ) , anti - rab7 ( 1:300 ) , anti - eea1 ( 1:1000 ) , 
  or anti - m6pr ( 1:2500 ) antibodies . bound antibodies were detected with 
  horseradish peroxidase - conjugated secondary antibodies and the ecl system . transient transfections  cells were grown for 24 h in 
  complete medium at 5070% confluence . transfections were performed with 
  10 g of the indicated plasmids by using the fugene system according 
  to the manufacturer 's instructions . post - transfection , extracts were obtained 
  and subjected to either immunoprecipitation or pulldown analysis . cell extracts were prepared in a buffer 
  containing 20 mm tris , ph 7.4 , 150 mm nacl , 1% nonidet 
  p-40 , and protease inhibitors . supernatants obtained after centrifugation 
  ( 13,000  g , 5 min , 4 c ) were used for immunoprecipitation 
  assays ( 500 g of total protein per assay ) with protein a / g 
  beads - immobilized antibody . the immunoprecipitated samples were solubilized in 
  laemmli buffer , boiled , and separated by sds - page and analyzed by western 
  blotting as indicated above . the rab5 pulldown assay was 
  performed as previously described 
  ( 27 , 
  28 ) with some modifications . after treatment , cell extracts were prepared in lysis buffer containing 25 
  mm hepes , ph 7.4 , 100 mm nacl , 5 mm 
  mgcl2 , 1% nonidet p-40 , 10% glycerol , 1 mm 
  dithiothreitol , and protease inhibitors . extracts were incubated by 5 min on 
  ice and clarified by centrifugation ( 10,000  g , 1 min , 4 
  c ) . post - nuclear supernatants were used immediately for pulldown assays 
  by adding 150 g of precoated beads . gsh beads were precoated with 100 
  g of gst - r5bd by 1 h at 4 c . pulldown incubations were carried out by 
  15 min in a rotating shaker at 4 c . thereafter , beads were collected and 
  washed 3 times with lysis buffer containing 0.01% nonidet p-40 and protease 
  inhibitors . samples were solubilized in laemmli buffer , boiled , and separated 
  by sds - page and analyzed by western blotting as indicated above . endosome 
  fractionation and hrp uptake assays were performed as previously described 
  ( 29 , 
  30 ) . briefly , cells were grown 
  in 15-cm plates for 24 h in complete medium . cell monolayers were washed in 
  pbs and incubated at 37 c with 5 ml of 2 mg / ml hrp in minimal essential 
  medium supplemented with 10 mm glucose and 10 mm hepes , 
  ph 7.4 ( internalization medium ) , at the indicated times . incubations were 
  stopped on ice by washing cells thoroughly with pbs at 4 c . cells were 
  scraped , collected by centrifugation at 1500 rpm at 4 c for 5 min in a 
  sorvall rt 6000d centrifuge . cells were then washed with 10 ml of ice - cold 
  pbs , centrifuged at 1700 rpm at 4 c for 5 min and finally resuspended in 
  homogenization buffer ( 250 mm sucrose , 1 mm edta , 3 
  mm imidazole , ph 7.4 ) , centrifuged at 2000 rpm at 4 c for 10 
  min . the cell pellet was resuspended in 500 l of homogenization buffer 
  containing protease inhibitors and homogenized by 8 passages through a 
  22-gauge needle in a 1-ml syringe ( bd biosciences ) . post - nuclear 
  supernatants were obtained by centrifuging homogenates 10 min at 3000 rpm at 4 
  c in a bench eppendorf centrifuge . the post - nuclear supernatants ( pns ) was brought to 40.6% sucrose by mixing 400 l of pns with 600 l of 60% 
  sucrose in homogenization buffer buffer . a gradient was prepared by overlaying 
  1 ml of homogenate , 40.6% sucrose , 2 ml of 35% sucrose , 1.5 ml of 30% sucrose , 
  400 l homogenization buffer ( 8% sucrose ) in a sw55ti centrifuge tube . the 
  gradient was centrifuged at 36,000 rpm ( 125,000  g ) for 66 min 
  at 4 c in a beckman l765 ultracentrifuge . endosome fractions were 
  collected as follows ; early endosomes at the 30/35% sucrose interface and late 
  endosomes at the 8/30% sucrose interface . for protein screening , 45 l of 
  each fraction was mixed with sample buffer and loaded onto 10% acrylamide 
  minigels . to measure the relative amount of endocytosed hrp and the 
  distribution in endosome fractions , 210 l of each fraction was 
  incubated with 2,2 azino - bis(3-ethylbenzthiazoline-6-sulfonic acid ) 
  ( sigma ) and read at 405 nm according to the manufacturer 's protocol . nb7 
  and nb7c8 cells were grown at confluence in complete medium for 24 h. 
  thereafter , cells were detached with trypsin , centrifuged , re - suspended with 
  0.2% bsa in minimal essential medium supplemented with 10 mm 
  glucose , 10 mm hepes , ph 7.4 ( internalization medium ) , and 
  incubated for 30 min at 37 c . for internalization experiments , 250 l 
  of aliquots of cell suspensions ( 5  10 cells ) were placed 
  in 1000-l test tubes . cells were centrifuged and resuspended with 0.2% bsa 
  in internalization medium containing either 30 g/l alexa fluor 
  488-labeled transferrin or 5 g/l bodipy fl ldl at 4 c . incubations were carried out at 37 c and stopped on ice at different time 
  points as indicated . thereafter , cells were washed with ice - cold pbs , fixed 
  with 0.5% paraformaldehyde , and resuspended with 2% bsa in pbs . for recycling 
  experiments , cells were incubated with 30 g/l of alexa fluor 
  488-labeled transferrin as indicated above for 15 min . then samples were 
  immediately placed in ice , washed with ice - cold pbs , re - suspended with 0.2% 
  bsa in internalization medium , and incubated at different times at 37 c . samples were 
  analyzed by fluorescence - activated cell sorter ( facscalibur ) using the 
  cellquest pro software . statistical analysis where pertinent , results were compared 
  using unpaired t tests of at least three independent experiments or 
  as indicated . caspase 8 induces accumulation of rab5 endosomes at cell 
  periphery  emerging evidence suggests that caspase 8 influences 
  cellular adhesion and migration 
  ( 22 ) likely via activation of 
  rac at the cell periphery ( 15 , 
  16 ) . interestingly , rac 
  function is spatially regulated by the endocytic protein rab5 during cell 
  migration ( 23 ) . to explore 
  this potential link between caspase 8 and endocytic machinery , we first 
  evaluated the cellular distribution of several endocytic markers in cells 
  expressing or deficient for caspase 8 . to this end we used the previously 
  reported neuroblastoma cells nb7 , which lack endogenous caspase 8 and those 
  stably reconstituted for  physiological  levels of caspase 8 
  ( i.e. higher expression than nb5 but lower than nb16 ) 
  ( 19 , 
  20 ) . we assessed the 
  distribution of early endosomal markers ( eea1 and rab5 ) and late endosomal or 
  lysosomal proteins ( rab7 and the cation - independent mannose-6 phosphate 
  receptor ( m6pr ) ) in this model . interestingly , the localization of rab5 
  differed in cells expressing caspase 8 relative to caspase 8-deficient cells , 
  with accumulation of rab5 in the cell periphery 
  ( fig . this 
  altered distribution was selective to rab5 , with no changes in expression or 
  distribution of eea1 , rab7 , and m6pr ( data not shown ) , suggesting that at 
  physiological levels of expression caspase 8 did not influence all aspects of 
  the endocytic pathway . we were unable to determine whether overexpression of 
  caspase 8 could further augment this effect or could induce changes to 
  secondary endosomal pathways , as overexpression of caspase 8 induces apoptosis 
  ( data not shown ) . figure 1.caspase 8 induces accumulation of rab5 positive early endosomes at the 
  cell periphery . a , localization of rab5 in neuroblastoma cells . nb7 neuroblastoma cells stably transfected with caspase 8 ( nb7c8 ) were 
  cultured for 24 h on glass coverslips and analyzed via confocal microscopy . rab5 was detected with a mouse monoclonal antibody ( red channel ) and 
  nuclei were stained with topro-3 ( dna , blue channel ) . representative 
  pictures are shown for parental ( caspase 8 -/- , left panel ) 
  and stably reconstituted cells ( caspase 8 + , right panel ) . the percentage of cells with 
  redistributed rab5 was determined as described under  experimental 
  procedures . values obtained from each experiment were averaged from at 
  least 10 pictures per sample ( 1050 cells per field ) . data shown 
  represent the average of five independent experiments ( mean  s.e . ) . 
  , comparison with nb7 ( p < 0.001 ) . neuroblastoma cells 
  lacking ( - ) , reconstituted for ( + ) , or reconstituted and silenced ( via short 
  hairpin rna ( shrna ) for c8 ( s ) ) expression of caspase 8 ( see 
  the inset ) were analyzed by immunofluorescence , and the percentage of 
  peripheral rab5 was calculated relative to total rab5 using the imagej 
  software with the radial profile plugin as described under d , a549 carcinoma cells subjected to lentiviral delivery of 
  either empty vector ( a549 ) or caspase 8 short hairpin rna ( a549/shc8 ) to 
  suppress caspase 8 expression ( inset , the doublet band 
  corresponds to a and b isoforms ) were analyzed by immunofluorescence as 
  described above . the incidence of cells with peripheral accumulation of rab5 
  was calculated as in b. data are representative of three independent 
  experiments , each one performed in triplicate ( mean  s.e . ) . 
  , e , rab5 was detected with a 
  mouse monoclonal antibody ( red channel ) , eea1 was detected with a 
  rabbit polyclonal antibody ( green channel ) , and nuclei were stained 
  with to - pro-3 ( blue channel ) . caspase 8 induces accumulation of rab5 positive early endosomes at the 
  cell periphery . a , localization of rab5 in neuroblastoma cells . nb7 neuroblastoma cells stably transfected with caspase 8 ( nb7c8 ) were 
  cultured for 24 h on glass coverslips and analyzed via confocal microscopy . rab5 was detected with a mouse monoclonal antibody ( red channel ) and 
  nuclei were stained with topro-3 ( dna , blue channel ) . representative 
  pictures are shown for parental ( caspase 8 -/- , left panel ) 
  and stably reconstituted cells ( caspase 8 + , right panel ) . values obtained from each experiment were averaged from at 
  least 10 pictures per sample ( 1050 cells per field ) . data shown 
  represent the average of five independent experiments ( mean  s.e . ) . 
  , comparison with nb7 ( p < 0.001 ) . neuroblastoma cells 
  lacking ( - ) , reconstituted for ( + ) , or reconstituted and silenced ( via short 
  hairpin rna ( shrna ) for c8 ( s ) ) expression of caspase 8 ( see 
  the inset ) were analyzed by immunofluorescence , and the percentage of 
  peripheral rab5 was calculated relative to total rab5 using the imagej 
  software with the radial profile plugin as described under  experimental 
  procedures .  , d , a549 carcinoma cells subjected to lentiviral delivery of 
  either empty vector ( a549 ) or caspase 8 short hairpin rna ( a549/shc8 ) to 
  suppress caspase 8 expression ( inset , the doublet band 
  corresponds to a and b isoforms ) were analyzed by immunofluorescence as 
  described above . the incidence of cells with peripheral accumulation of rab5 
  was calculated as in b. data are representative of three independent 
  experiments , each one performed in triplicate ( mean  s.e . ) . e , rab5 was detected with a 
  mouse monoclonal antibody ( red channel ) , eea1 was detected with a 
  rabbit polyclonal antibody ( green channel ) , and nuclei were stained 
  with to - pro-3 ( blue channel ) . the peripheral accumulation of rab5 ( red channel ) is evident . in neuroblastoma cells lacking caspase 8 , rab5 was homogenously distributed 
  throughout the cell body , with a minor accumulation at the cell periphery 
  ( fig . the accumulation of rab5 at the cell periphery was 
  dependent upon expression of caspase 8 , as suppression of caspase 8 with short 
  hairpin rna ablated the accumulation of rab5 in the periphery 
  ( fig . 1c ) . to further 
  evaluate the extent of rab5 accumulated at the cell periphery , we measured the 
  relative levels of rab5 within the area located within a distance of 23 
  m of peripheral edge of the cell ( this distance represents 25% of the 
  cell ; supplemental fig . as expected , a 2-fold increase of peripheral rab5 
  was associated to expression of caspase 8 
  ( fig . these 
  observations were confirmed in parallel studies expressing gfp - tagged caspase 
  8 in nb7 cells ( supplemental fig . overall co - localization of rab5 with 
  early ( eea1 ) or late endocytic proteins ( rab7 , m6pr ) was not generally 
  affected by expression of caspase 8 ( supplemental fig . 2 ) , confirming rab5 as 
  a specific target . to extend these observations we also used a549 carcinoma cells in which 
  endogenous caspase 8 was silenced ( fig . caspase 8 
  expression was associated with peripheral accumulation of rab5 , whereas loss 
  of caspase 8 promoted a more homogenous distribution throughout the cell 
  ( fig . . the 
  peripheral accumulation of rab5 did not result from protein stabilization , as 
  no significant differences in rab5 protein levels were associated with caspase 
  8 expression ( supplemental fig . these data indicate that 
  caspase 8 influences localization of rab5 endosomes within the cell . importantly , most rab5 ( 95% ) was found associated to membrane 
  fractions , as revealed by subcellular fractionation ( data not shown ) . only a slight variation 
  in membrane - associated rab5 was detected in caspase 8-expressing cells ( 93 or 
  97% total rab5 was found associated to membranes in cells lacking or 
  expressing caspase 8 , respectively ) . because most rab5 is membrane - associated 
  regardless the caspase 8 status , it would appear that relevant changes are in 
  subcellular distribution of membrane - associated rab5 . the possibility that 
  caspase 8 affects rab5 shuttling between different early endosome populations 
  or between early endosomes and the plasma membrane is unexpected and 
  potentially important . internalization assays ( 37 c by 30 min unless otherwise indicated ) 
  were performed to analyze the distribution of hrp within late and early 
  endosome fractions . hrp activity was measured in each case and expressed as 
  percentage of total activity . data represent the average of three independent 
  experiments ( mean  s.e . ) . a , neuroblastoma lacking caspase 8 
  ( - ) and stably reconstituted for caspase 8 ( + ) ; , p < 
  0.05 . b , carcinoma cells expressing caspase 8 ( a549/control , + ) or 
  those with expression of caspase 8 suppressed ( a549/shc8 ( s ) ) were 
  similarly compared ; # , p < 0.054 . internalization assays ( 37 c by 30 min unless otherwise indicated ) 
  were performed to analyze the distribution of hrp within late and early 
  endosome fractions . hrp activity was measured in each case and expressed as 
  percentage of total activity . data represent the average of three independent 
  experiments ( mean  s.e . ) . a , neuroblastoma lacking caspase 8 
  ( - ) and stably reconstituted for caspase 8 ( + ) ; , p < 
  0.05 . b , carcinoma cells expressing caspase 8 ( a549/control , + ) or 
  those with expression of caspase 8 suppressed ( a549/shc8 ( s ) ) were 
  similarly compared ; # , p < 0.054 . caspase 8 alters the targeting of cargo from early to late 
  endosomes  because rab5 is involved in trafficking of cargo from the 
  plasma membrane to early and sorting endosomes 
  ( 2 ) , we evaluated whether 
  caspase 8-mediated re - distribution of rab5 influenced this process by tracking 
  the accumulation of fluid - phase hrp in early and late endosomes . 4 ) revealed that the 
  accumulation of hrp in late endosomes was substantially decreased in caspase 
  8-expressing neuroblastoma cells compared with caspase 8-deficient controls 
  ( fig . because substantial accumulation of hrp in late endosomes is 
  observed only after 1015 min of internalization ( data not shown ) , we 
  assayed late endosomes at 30 min internalization . similar trends were obtained 
  after 10 min of internalization , although variation was higher 
  ( fig . similarly , we observed an increased accumulation of hrp in late 
  but not early endosomes from caspase 8-deficient carcinoma cells when compared 
  with caspase 8-expressing control cells 
  ( fig . the overall 
  internalization rate was independent of caspase 8 expression in both carcinoma 
  and neuroblastoma cells ( data not shown and see below ) , implicating caspase 8 
  in regulating the delivery of cargo between the early and late endosome . to test this possibility directly we tracked the internalization and 
  localization of two differentially sorted cargos ; that is , transferrin 
  ( i.e. cargo destined for recycling ) and ldl ( a cargo destined for 
  degradation ) . we observed no difference in uptake of transferrin or ldl nor 
  did we observe any changes in transferrin recycling as a function of caspase 8 
  expression ( fig . the distribution of internalized transferrin was similar in these cells and 
  co - localized to the same degree with eea1 and rab5 regardless of caspase 8 
  expression ( data not shown ) . by contrast , the intracellular distribution of 
  internalized ldl was markedly different when caspase 8 was expressed . among 
  caspase 8-deficient cells , ldl was detected throughout the cell body 
  ( fig . 3b , upper 
  left panel ) , whereas in caspase 8-reconstituted cells a considerable 
  retention of ldl was seen at the cell periphery , even 30 min of 
  post - internalization ( fig . 
  3b , upper right panel ) . a partial 
  co - localization of ldl with rab5 was observed in caspase 8-expressing or 
  -deficient cells , but this co - localization increased when caspase 8 was 
  present ( fig . 3 , b and 
  c ) . these data demonstrated that expression of caspase 8 
  influenced the localization and fate of cargo destined to late endosomes . in 
  fact , we observed the accumulation of ldl in rab7-positive late endosomes 
  ( fig . importantly , caspase 8 did not affect expression or 
  distribution of these protein markers for late endosomes 
  ( fig . 4 and data not shown ) . taken together , these results suggest that caspase 8 regulates the movement of 
  cargo from early ( rab5 ) to late ( rab7 ) endosome compartments . caspase 8 catalytic activity is not required to influence rab5 
  localization  proteolytic activity is required for the induction of 
  apoptosis by caspase 8 but is dispensable for other activities , such as the 
  promotion of cell migration 
  ( 13 , 
  14 , 
  16 ) . both apoptosis and cell 
  migration are dependent upon endocytic processes , and it was therefore unclear 
  whether proteolytic activity was required for caspase 8 to regulate the 
  rab5-mediated trafficking . therefore , we tested a neuroblastoma cell line 
  stably reconstituted for a catalytically inactive mutant of caspase 8 ( caspase 
  8/c360a ) . interestingly , this mutant displayed an apparent accumulation of 
  rab5 at the cell periphery similar to cells expressing native caspase 8 
  ( fig . moreover , 
  the expression of catalytically inactive caspase 8 resulted in decreased 
  accumulation of hrp tracer in late , but not early , endosomes 
  ( fig . thus , the 
  mutant recapitulated the caspase 8 phenotype indicating that caspase 8 
  activity is not required for regulation of rab5-mediated endosomal 
  targeting . increased rab5 gtp loading in cells expressing caspase 8the 
  processes of early endosome fusion , early - to - late endosome conversion , and 
  subcellular localization depend on proper rab5 activation / inactivation in time 
  and space , which is governed by cycling between gtp binding and subsequent 
  hydrolysis to gdp ( for review , see refs . 
  1 and 
  2 ) . we assessed whether caspase 
  8 influenced rab5 gtp loading using a pulldown approach with a fusion protein 
  that selectively binds gtp - loaded rab5 ( gst - r5bd ) 
  ( 32 ) . rab5 mutant rab5/q79l 
  ( gtpase - deficient ) but not the gdp binding  dominant negative  
  rab5/s34n ( 26 ) mutant 
  ( fig . importantly , we detected increased levels of gtp - bound rab5 among 
  cells expressing caspase 8 relative to cells lacking caspase 8 
  ( fig . densitometry confirmed that gtp loading of endogenous rab5 was 
  significantly ( 2.5-fold ) increased in caspase 8-expressing cells 
  ( fig . 5 ) , 
  leading us to conclude that caspase 8 expression influences rab5 activity as 
  well as localization . figure 3.ldl is retained at periphery of cells expressing caspase 8 . 
 a , uptake of ldl or transferrin ( top and middle 
  panels ) among neuroblastoma cells lacking caspase 8 ( -c8 ) or 
  stably reconstituted for caspase 8 ( + c8 ) at times as indicated . b , the uptake of ldl ( ldl - bodipy - fl , green 
  channel ) was assessed after 15 min at 37 c as described under 
   experimental procedures . samples were analyzed via confocal 
  microscopy , and rab5 was detected with a mouse monoclonal antibody ( red 
  channel ) ; dna is counterstained with to - pro-3 ( blue channel ) . representative images are shown for neuroblastoma - deficient for caspase 8 
  ( caspase 8-) or reconstituted for caspase 8 expression ( caspase 
  8 + ) . c , analysis of the 
  co - localization of rab5 and ldl - bodipy - fl . analysis was performed by using the 
  imagej software as indicated under  experimental procedures . data 
  represent the average of three independent experiments ( mean  s.e . ) . 
  , p < ldl is retained at periphery of cells expressing caspase 8 . 
 a , uptake of ldl or transferrin ( top and middle 
  panels ) among neuroblastoma cells lacking caspase 8 ( -c8 ) or 
  stably reconstituted for caspase 8 ( + c8 ) at times as indicated . b , the uptake of ldl ( ldl - bodipy - fl , green 
  channel ) was assessed after 15 min at 37 c as described under 
   experimental procedures . samples were analyzed via confocal 
  microscopy , and rab5 was detected with a mouse monoclonal antibody ( red 
  channel ) ; dna is counterstained with to - pro-3 ( blue channel ) . representative images are shown for neuroblastoma - deficient for caspase 8 
  ( caspase 8-) or reconstituted for caspase 8 expression ( caspase 
  8 + ) . c , analysis of the 
  co - localization of rab5 and ldl - bodipy - fl . analysis was performed by using the 
  imagej software as indicated under  experimental procedures . data 
  represent the average of three independent experiments ( mean  s.e . ) . 
  , p < rab5 activity is regulated by caspase 8 via p85we 
  next sought to evaluate the molecular mechanism by which caspase 8 could 
  influence rab5 gtp loading and function . interestingly , the sh2 domains of 
  p85 were recently shown to interact with a phosphorylated tyrosine 
  ( tyr-380 ) located in a peptide loop between the small and large subunits of 
  the catalytic domain of caspase 8 . interactions between sh2 domains and this 
  tyrosine do not require caspase 8 catalytic activity 
  ( 13 , 
  16 ) . moreover , p85 acts 
  as a rab - gap , promoting rab5 hydrolysis of gtp 
  ( 8) . therefore , we sought to 
  determine whether p85 was a critical intermediate in the caspase 
  8-regulated rab5 function . we first evaluated whether caspase 8 and p85 interacted within our 
  cells . indeed , p85 co - precipitated with caspase 8 from cells stably 
  ( fig . 6b ) , caspase 8 induced an 8-fold increase in gtp 
  loading of an exogenous rab5 reporter ( fig . importantly , p85 expression overcame the 
  capacity of caspase 8 to promote rab5 gtp loading 
  ( fig . 6d and 
  supplemental fig . therefore , we sought to evaluate whether caspase 8 and 
  p85 act reciprocally to control rab5 activity . to test this possibility 
  we used gfp - tagged caspase 8 mutants which were catalytically inactive ( c360a ) 
  or unable to interact with p85 ( y380f ) 
  ( 16 ) . neuroblastoma cells 
  stably transfected with these mutants confirmed that catalytic activity was 
  not required for promotion of rab5 gtp loading 
  ( fig . however , 
  mutation of the sh2 binding site that disrupted p85 association ( y380f ) 
  abrogated the ability of caspase 8 to increase rab5 gtp loading 
  ( fig . the findings were extended using hek293 t cells transiently transfected with 
  either gfp alone ( control ) or gfp - tagged caspase 8 , caspase 8/c360a , or 
  caspase 8/y380f . as in the neuroblastoma cells , expression of both wild type 
  caspase 8-gfp or caspase 8/c360a - gfp , but not caspase 8/y380f - gfp , increased 
  rab5-gtp levels ( supplemental fig . these data raised the notion that 
  p85 association with caspase 8 might influence its ability to bind rab5 
  and thereby promote gtp hydrolysis . to address this , we expressed a p85/r274a mutant ( gap - deficient ) and 
  a p85/r368a / r649a double mutant ( termed r ) which lacked 
  sh2-tyrosine binding activity 
  ( 8) in neuroblastoma cells 
  expressing caspase 8 . although expression 
  of wild type p85 suppressed rab gtp loading , this required rab5-gap 
  activity , as the p85/r274a mutant could not overcome the caspase 
  8-mediated effect . similarly , phosphotyrosine binding activity of p85 
  was critical to overcoming caspase 8-induced gtp loading , as loss of caspase 8 
  binding in the p85/r mutants permitted caspase 8-induced rab5 
  gtp loading ( fig . 7d ) . 
  based on these results , we propose a simple initial model wherein caspase 8 
  may increase rab5 gtp loading by interfering with rab5 gap activity of 
  p85 via sh2 interactions with tyr-380 of caspase 8 
  ( fig . a and b , uptake of ldl ( green channel ) was 
  performed and assessed with respect to delivery to late endosomes . late 
  endosome were identified by antibodies to rab7 ( a ) and m6pr 
  ( b ) as indicated ( green channels ) , and dna was 
  counterstained with to - pro-3 ( blue channel ) . representative images 
  are shown for cells deficient in caspase 8 ( caspase 8-) or 
  reconstituted for caspase 8 ( caspase 8 + ) . c , colocalization analysis of rab7 or m6pr and 
  ldl - bodipy - fl . data represent the average of three independent experiments 
  ( mean  s.e . ) . a and b , uptake of ldl ( green channel ) was 
  performed and assessed with respect to delivery to late endosomes . late 
  endosome were identified by antibodies to rab7 ( a ) and m6pr 
  ( b ) as indicated ( green channels ) , and dna was 
  counterstained with to - pro-3 ( blue channel ) . representative images 
  are shown for cells deficient in caspase 8 ( caspase 8-) or 
  reconstituted for caspase 8 ( caspase 8 + ) . c , colocalization analysis of rab7 or m6pr and 
  ldl - bodipy - fl . data represent the average of three independent experiments 
  ( mean  s.e . ) . , in both cases p < 0.05 . figure 5.proteolytic activity of caspase 8 is not required for altered rab5 
  localization and late endosome targeting of cargo . a , 
  neuroblastoma cells deficient for caspase 8 ( caspase 8 -/- ) or stably 
  reconstituted for active ( caspase 8 + ) or inactive caspase 8 
  expression ( caspase 8 c360a ) were analyzed for rab5 localization . rab5 was detected with a mouse monoclonal antibody ( red channel ) and 
  dna stained with to - pro-3 ( blue channel ) . the incidence of cells with peripheral accumulation of rab5 was 
  calculated as described in fig . the result is representative of three independent 
  experiments ( mean  s.e . ) . b , hrp accumulation in cells deficient for 
  caspase 8 ( caspase 8 -/- ) or stably reconstituted for active 
  ( caspase 8 + ) or inactive caspase 8 ( caspase 8 c360a ) was 
  analyzed as described above . data were averaged from three independent 
  experiments ( mean  s.e . ) . , proteolytic activity of caspase 8 is not required for altered rab5 
  localization and late endosome targeting of cargo . a , 
  neuroblastoma cells deficient for caspase 8 ( caspase 8 -/- ) or stably 
  reconstituted for active ( caspase 8 + ) or inactive caspase 8 
  expression ( caspase 8 c360a ) were analyzed for rab5 localization . rab5 was detected with a mouse monoclonal antibody ( red channel ) and 
  dna stained with to - pro-3 ( blue channel ) . the incidence of cells with peripheral accumulation of rab5 was 
  calculated as described in fig . the result is representative of three independent 
  experiments ( mean  s.e . ) . , b , hrp accumulation in cells deficient for 
  caspase 8 ( caspase 8 -/- ) or stably reconstituted for active 
  ( caspase 8 + ) or inactive caspase 8 ( caspase 8 c360a ) was 
  analyzed as described above . data were averaged from three independent 
  experiments ( mean  s.e . ) . , the role of p85 in caspase 8-mediated regulation of rab5 was 
  reflected in the localization of this protein . expression of wild type , but 
  not p85/r274a or p85/r , prevented the accumulation of 
  peripheral rab5 in caspase 8-expressing cells ( supplemental fig . conversely , expression of the rab - gap deficient p85 
  allows the accumulation of rab5 in the periphery of caspase 8-negative cells 
  ( supplemental fig . 7a , upper panels ) . finally , unlike wild type 
  ( fig . 5 ) , 
  caspase 8/y380f did not induce accumulation of rab5 at the cell periphery 
  ( supplemental fig . a , rab5-gtp levels 
  were assessed using a gst - r5bd pulldown assay as described under 
   experimental procedures .  left panel , hek293 t cells were 
  transiently transfected with wild type ( wt ) rab5 , rab5/s34n , or 
  rab / q79l mutants . after 24 h cells were lysed , and gtp - loaded rab5 was pulled 
  down with gst - r5bd . right panel , neuroblastoma cells lacking or 
  expressing caspase 8 were subjected to similar analysis . b , rab5-gtp 
  levels were normalized to total input rab5 and quantified by scanning 
  densitometry of three independent experiments ( mean  s.e . ) . 
  , c , 
  caspase 8 immunoprecipitated ( ip ) from confluent cultures of 
  neuroblastoma cells was assessed for the presence of associated p85 as 
  detected by immunoblotting . d , 
  rab - gtp levels were measured as in a , among neuroblastoma cells 
  ectopically expressing rab5 . nb7 lacking or expressing caspase 8 were either 
  co - transfected with rab5 and empty vector or rab5 and p85. cell lysates 
  were generated 24 h post - transfection , and rab5-gtp levels were measured . upper panel , a representative western blotting from three independent 
  experiments is shown ; the lower graph shows an average from three 
  independent experiments by scanning densitometry ( mean  s.e . ) . 
  , p < a , rab5-gtp levels 
  were assessed using a gst - r5bd pulldown assay as described under 
   experimental procedures .  left panel , hek293 t cells were 
  transiently transfected with wild type ( wt ) rab5 , rab5/s34n , or 
  rab / q79l mutants . after 24 h cells were lysed , and gtp - loaded rab5 was pulled 
  down with gst - r5bd . right panel , neuroblastoma cells lacking or 
  expressing caspase 8 were subjected to similar analysis . b , rab5-gtp 
  levels were normalized to total input rab5 and quantified by scanning 
  densitometry of three independent experiments ( mean  s.e . ) . 
  , c , 
  caspase 8 immunoprecipitated ( ip ) from confluent cultures of 
  neuroblastoma cells was assessed for the presence of associated p85 as 
  detected by immunoblotting . d , 
  rab - gtp levels were measured as in a , among neuroblastoma cells 
  ectopically expressing rab5 . nb7 lacking or expressing caspase 8 were either 
  co - transfected with rab5 and empty vector or rab5 and p85. cell lysates 
  were generated 24 h post - transfection , and rab5-gtp levels were measured . upper panel , a representative western blotting from three independent 
  experiments is shown ; the lower graph shows an average from three 
  independent experiments by scanning densitometry ( mean  s.e . ) . 
  , p < caspase 8 has been associated with endocytic vesicles during execution of 
  the apoptotic pathway 
  ( 1012 ) 
  and has been implicated in endosome remodeling after a pro - apoptotic stimuli 
  during the type ii extrinsic pathway 
  ( 33 ) . in these cases , we report a novel role for caspase 
  8 in controlling rab5 localization , activity , and function . the presence of 
  caspase 8 in a variety of cells was shown to modify early endosome topology ; 
  in particular , the accumulation of rab5-positive endosomes at the cell 
  periphery . in agreement with this we observed concurrent changes in cargo 
  transport from early to late endosomes and found that these events were 
  associated with caspase 8-dependent increases in the levels of gtp - bound 
  rab5 . our results are somewhat surprising with respect to previous reports using 
  overexpression strategies to map rab5 activity 
  ( 34 ) . we did not observe 
  formation of enlarged early endosomes by caspase 8 ( despite the fact that 
  caspase 8 increased gtp loading of rab5 ) . compared with the previously 
  reported enlargement of early endosomes by expression of the rab / q79l mutant 
  ( 26 ) , the caspase 8 impact on 
  rab5 function was less pronounced . we could duplicate the prior reported 
  effects by expression of rab5/q79l , but not rab5/s34n , which led to formation 
  of enlarged early endosomes in our cells . this approach revealed a substantial 
  increase in co - localization of rab5 with eea1 ( data not shown ) . although 
  informative , the concern with any overexpression system is that some of the 
  effects observed may be exaggerated relative to a cell 's normal physiology . 
  indeed , we observed no increase of eea1/rab5 colocalization among cells 
  expressing caspase 8 ( supplemental fig . , such differences in 
  localization may reflect the observation that caspase 8 also activates the 
  small gtpase rac ( 15 ) . thus , 
  it remains possible that coordinate rac and rab5 activation results in 
  peripheral retention of early endosomes , which might not be observed after 
  simple expression of active rab5 . nonetheless , our data are consistent with a 
  model proposing a dynamic and reversible assembly of rab5 domains in early 
  endosomes with a cycling - dependent  handoff  to rab7 
  ( 35 , 
  36 ) . in contrast to the role of caspase 8 during apoptosis , no proteolytic 
  activity was required to modify endosomal targeting . these data differ from 
  caspase-3 , a related enzyme that has been reported to alter transport via a 
  proteolysis - dependent mechanism 
  ( 37 ) . nonetheless , recent 
  evidence supports non - apoptotic functions for caspase 8 that are independent 
  of catalytic activity , with several studies supporting a role for caspase 8 in 
  cell migration and invasion 
  ( 1316 , 
  22 ) . in fact , caspase 8 was 
  found to be phosphorylated on tyrosine 380 in these studies . a , rab5 gtp loading was assessed by using the gst - r5bd pulldown assay 
  among neuroblastoma cells expressing active caspase 8 , inactive caspase 
  8(c360a ) , or a caspase 8 mutant deficient for phosphorylation at the tyr-380 
  sh2 binding site . arrows indicate the relative mass of 
  caspase 8 or the nonphosphorylatable caspase 8 mutant . b , data were evaluated from three independent experiments 
  by scanning densitometry ( mean  s.e . ) . , p < 
  0.005 ; , p < 0.05 ; # , p < 0.05 ; # # , 
  p < 0.1 . c , caspase 8 was 
  immunoprecipitated ( ip ) from confluent cultures of expressing 
  gfp - tagged caspase 8 constructs . caspase 8 and p85 were detected by 
  western blotting in immunoprecipitates ( upper panel ) and 25 g of 
  whole cell lysates ( wcl , lower panel ) . d , neuroblastoma 
  cells expressing caspase 8 were co - transfected with either rab5 or pcdna3.1 
  ( empty vector control ) or with rab5 and flag - tagged  wild type p85 , rab - gap deficient p85 ( p85/r274a ) , or sh2-inactive p85 
  ( p85/r368a / r649a ( r ) . rab5 ( upper panel ) , flag , and caspase 8 ( lower 
  panel ) were detected by western blotting . numbers indicate 
  rab5-gtp levels normalized to total rab5 by densitometry averaged from two 
  independent experiments . a , rab5 gtp loading was assessed by using the gst - r5bd pulldown assay 
  among neuroblastoma cells expressing active caspase 8 , inactive caspase 
  8(c360a ) , or a caspase 8 mutant deficient for phosphorylation at the tyr-380 
  sh2 binding site . arrows indicate the relative mass of 
  caspase 8 or the nonphosphorylatable caspase 8 mutant . b , data were evaluated from three independent experiments 
  by scanning densitometry ( mean  s.e . ) . , p < 
  0.005 ; , p < 0.05 ; # , p < 0.05 ; # # , 
  p < 0.1 . c , caspase 8 was 
  immunoprecipitated ( ip ) from confluent cultures of expressing 
  gfp - tagged caspase 8 constructs . caspase 8 and p85 were detected by 
  western blotting in immunoprecipitates ( upper panel ) and 25 g of 
  whole cell lysates ( wcl , lower panel ) . d , neuroblastoma 
  cells expressing caspase 8 were co - transfected with either rab5 or pcdna3.1 
  ( empty vector control ) or with rab5 and flag - tagged  wild type  
  p85 , rab - gap deficient p85 ( p85/r274a ) , or sh2-inactive p85 
  ( p85/r368a / r649a ( r ) . after 24 h , rab5 ( upper panel ) , flag , and caspase 8 ( lower 
  panel ) were detected by western blotting . numbers indicate 
  rab5-gtp levels normalized to total rab5 by densitometry averaged from two 
  independent experiments . figure 8.model showing the role of caspase 8 in regulating rab5 activity . a 
  simple mechanism by which caspase 8 expression may influence rab5-gtp levels 
  and endosomal maturation via its ability to bind to p85 is shown . . a 
  simple mechanism by which caspase 8 expression may influence rab5-gtp levels 
  and endosomal maturation via its ability to bind to p85 is shown . it is not yet clear whether caspase 8 phosphorylation blocks caspase 
  catalytic activity directly ; however , it does create an sh2 binding site bound 
  by src family kinases ( 13 ) and 
  by the sh2 domains of p85 
  ( 16 ) . this sh2 binding site 
  has been shown to be critical for recruitment of caspase 8 to the cell 
  periphery ( 13 ) . thus , caspase 
  8 maturation and proteolytic activity could be regulated conformationally or 
  via sequestration after sh2 binding . in this case we observe a robust 
  association with p85 , a known rab - gap , and caspase 8 , which is 
  abrogated in a non - phosphorylated y380f mutant . the adaptor protein p85 
  is a known regulator of endocytosis that can act through a number of 
  mechanisms . here we show that increased expression of p85 overcomes the 
  effect of caspase 8 expression , decreasing rab5 gtp loading ; however , this 
  requires both p85 rab - gap activity and sh2 binding function . together 
  the results suggest a model in which caspase 8 is located proximal to 
  p85 and binds to it in a manner that influences its ability to regulate 
  rab5 ( fig . it is clear that alterations in endocytosis can affect a number of 
  different cell pathways . in particular , cell migration and delivery of cell 
  surface receptors are directly controlled by mechanisms involving rab5 
  activation and re - localization 
  ( 23 ) . our observation that 
  caspase 8 influences endosome dynamics may provide mechanistic insights into 
  how this protease regulates cell motility 
  ( 13 , 
  16 ) . the recently reported 
  role of rab5 in guiding rac signaling has clear implications for cell 
  migration ( 23 ) . an intricate 
  relationship between rab5 and the integrin trafficking 
  ( 18 , 
  38 ) suggests coordinated 
  signaling among endocytic , cytoskeletal , and apoptotic pathway during cell 
  migration . these results provide a new basis to begin to understand the 
  molecular mechanisms by which caspase 8 impacts tumor cell biology .
caspase 8 is a cysteine protease that initiates apoptotic signaling via the 
  extrinsic pathway in a manner dependent upon association with early endosomes . 
  previously , we identified caspase 8 as an effector of migration , promoting 
  motility in a manner dependent upon phosphorylation on tyr-380 by src family 
  kinases and its subsequent association with src homology 2 domain - containing 
  proteins . here 
 we demonstrate the regulation of the small gtpase rab5 , which 
  mediates early endosome formation , homotypic fusion , and maturation by caspase 
  8 . 
 regulation requires the tyr-380 phosphorylation site but not caspase 
  proteolytic activity . 
 tyr-380 is essential for interaction with the src 
  homology 2 domains of p85 , a multifunctional adaptor for 
  phosphatidylinositol 3-kinase , that possesses rab - gap activity . 
 interaction 
  between caspase 8 and p85 promotes rab5 gtp loading , alters endosomal 
  trafficking , and results in the accumulation of rab5-positive endosomes at the 
  edge of the cell . 
 conversely , caspase 8-dependent gtp loading of rab5 is 
  overcome by increased expression of p85 in a rab - gap - dependent manner . 
 thus , we demonstrate a novel function for caspase 8 as a modulator of 
  p85 rab - gap activity and endosomal trafficking .