cells and culture conditions  the following cells were used : 
  bhk21 ( c-13 ) fibroblasts ( baby hamster kidney clone 13 , american type culture 
  collection ) , bhk21-tta / anti - clathrin heavy chain ( chc ) cell line 
  ( 30 ) , cav-1-ko and cav-1-wt 
  mouse endothelioma cell lines 
  ( 31 ) , c2c12 mouse myoblasts 
  ( 32 ) , and 3t3 mouse 
  fibroblasts ( invitrogen ) .
the cells were cultured in dulbecco 's modified 
  eagle 's medium ( high glucose , with sodium pyruvate and l - glutamine ) 
  ( paa , pasching , austria ) supplemented with 10 or 20% ( the latter for c2c12 
  cells only ) fetal calf serum ( paa , pasching , austria ) , 2 mm 
  l - glutamine ( invitrogen ) , 50 g / ml gentamicin ( paa , pasching , 
  austria ) . for the cav-1-ko and cav-1 wild type ( wt ) endothelioma cell lines , 
 
the growth media was additionally supplemented with 1% nonessential amino 
  acids ( sigma ) , 1% sodium pyruvate ( invitrogen ) , and 2 mm 
  2-mercaptoethanol diluted in phosphate - buffered saline ( invitrogen ) . for the 
  bhk21-tta / anti - chc cell line ,
the following additives were added : 10% 
  tetracycline - negative fetal calf serum ( paa , pasching , austria ) , 0.2 mg / ml 
  geneticin ( g418 , invitrogen ) , 0.5 g / ml puromycin ( sigma ) , 2 g / ml 
  tetracycline ( sigma ) . for the induction of chc antisense rna expression , 
 
peptides , proteins , and fluorophores  peptides 
  5,6-carboxytetramethylrhodamine ( tamra)-tat ( grkkrrqrrr ) and 
  tamra - ptd4 ( yaraarqara ) 
  ( 29 ) were synthesized as 
  d - isomers and coupled directly to tamra at the n terminus ( peptide 
  specialty laboratories gmbh , heidelberg , germany ) .
peptides and labels were 
  diluted in rpmi 1640 medium without phenol red ( paa , pasching , austria ) and 
  applied at 10 m final concentration to the cells . at lower 
  concentrations ,
as we have shown previously 
  ( 8) , only the endocytic mode of 
  uptake was detected ( supplemental fig .
s1 ) . to monitor chc expression and 
  function , transferrin ( tf ) conjugated to alexa fluor 633 ( invitrogen ) was 
  added as a marker for clathrin - dependent endocytosis .
nonreactive forms of the 
  fluorophores fitc ( fluorescein isothiocyanate ) and tamra were generated by 
  incubation with tris buffer ( indicated by an asterisk ) and used as a small 
  molecule to monitor the generation of pores during the transduction event .
to 
  control for the complete inhibition of endocytosis at 4 c , the globular 
  protein tat - biotin - streptavidin ( tat - bt - sav ) labeled with the fluorophore 
  cyanine dye was used as an additional marker for fluid - phase uptake 
  ( supplemental fig .
live cell uptake experiments  for all experiments either 
  8-well -slides or 35-mm -dishes ( ibidi , martinsried , germany ) were 
  used .
the cells were seeded onto the observation chambers and incubated 
  overnight at 37 c with 5% co2 .
the peptides tamra - tat and 
  tamra - ptd4 ( 10 m ) , the fluorophores fitc * and tamra * 
  ( 10 m ) , and tf ( 10 g / ml ) were diluted in rpmi 1640 medium 
  ( paa , pasching , austria ) without phenol red and used at the indicated 
  concentrations .
special care was taken to ensure that the volume of the 
  peptide solution above the cells was comparable in the two different 
  observation chambers and that the exchange against the appropriate peptide 
  ( label , marker ) dilutions was performed very gently .
immediately after 
  addition of the peptide ( label , marker ) to the cells , time lapses over 60 min 
  ( with time intervals of one image per min ) were recorded .
for the uptake experiments at 4 c a custom - built cooling chamber was 
  used .
the height of this cooling chamber occupying the cooling flow and the 
  radius of the loophole at the bottom were optimized to guarantee a constant 
  temperature exchange between the 35-mm -dish observation chamber and the 
  cool water flux .
the temperature was regulated by a thermostat , and the exact 
  temperature of 4 c inside the medium above the cells was verified by 
  measurements with a thermometer before and after the 1-h time lapses .
microscopy , image acquisition , and analysis  confocal optical 
  sections were acquired with a zeiss confocal laser scanning microscope , lsm510 
  meta , mounted on an axiovert 200 m inverted microscope equipped with a live 
  cell microscope incubation cage ( okolab , ottaviano , italy ) using a 63 
  plan - apochromat na1.4 oil immersion , phase - contrast objective .
the microscope 
  incubation cage maintained a humidified atmosphere of 5% co2 and 37 
  c , which was used throughout except for the low temperature experiments . 
 
for all settings the main beam splitter was hft uv/488/543/633 , and the 
  specific parameters for the single fluorophores were as follows : fitc excited 
  at 488 nm , detected with a 500530-nm bandpass filter ; tamra - tat and 
  tamra - fluorophore excited at 543 nm , detected with a 565615-nm bandpass 
  filter , transferrin - alexa fluor 633 excited with 633 nm , detected with a 
  650-nm long pass filter .
phase contrast images were recorded with excitation 
  at 488 nm and detection in the transmission channel .
the laser power for 
  observation was 1% ( 488 nm , 25 milliwatts ) , 7% ( 543 nm , 1 milliwatt ) , and 25% 
  ( 633 nm , 5 milliwatts ) .
settings were adjusted in a way that image pixels were 
  not over- or underexposed with the range indicator function in the zeiss laser 
  scanning microscopy software version 3.2 . to ensure that weak intracellular 
  fluorescence signals of the peptides were not missed , a set of overexposed 
  images was additionally collected . for the quantification of transduction , 100150 cells per 
  transduction experiment
were evaluated to obtain the percentage of transduced 
  cells ( indicated by nucleolar appearance of the labeled peptide ) , and the 
  kinetics of tat transduction was further characterized by the earliest time 
  point when transduction could be detected within a field of view ( initiation 
  time of transduction ) .
western blotting  for western blot analysis of the cav-1-ko 
  and wt cells , half a million cells were counted , resuspended in 100 l of 
  phosphate - buffered saline , and boiled in laemmli sample buffer for 10 min , and 
  cell lysates were analyzed by sds - page followed by blotting onto 
  polyvinylidene difluoride membranes .
signals were detected with the following 
  primary antibodies : rabbit anti - cav-1 polyclonal antibody ( lifespan 
  biosciences , inc . ,
seattle , wa ) and mouse anti - cav-2 and anti - cav-3 monoclonal 
  antibodies ( 1:2000 dilution , bd transduction laboratories ) .
anti - rabbit 
  igg - hrp ( sigma ) and anti - mouse igg - hrp ( enhanced chemiluminescence , amersham 
  biosciences ) were used as secondary antibodies .
immunoreactive signals were 
  visualized using enhanced chemiluminescence plus detection solution ( amersham 
  biosciences ) and recorded using a luminescence imager ( luminescent image 
  analyzer las-1000 , fuji photo film , tokyo , japan ) .
because of their particularly high transduction ability , which solely 
  depends on a minimal number of arginines , rrps play a special role among cpps 
  ( 18 , 
  3335 ) . 
 
to clarify the role of endocytosis in the uptake mode of cpps with low 
  molecular weight ( lmw ) cargos into living cells , we investigated the 
  occurrence and extent of transduction of tat and ptd4 as cpps with 
  high and low transduction frequency , respectively 
  ( 8) . the intracellular 
  distribution of peptides in living cells was analyzed by laser scanning 
  confocal microscopy .
to unravel the relevance of endocytic routes for the 
  uptake and intracellular distribution of peptides , endocytic pathways were 
  specifically inhibited by genetic approaches or were blocked in ensemble by 
  incubation of cells at low temperature .
role of clathrin - mediated endocytosis in cpp 
  uptake  clathrin - dependent endocytosis represents a major endocytic 
  pathway . for example
, transferrin is taken up exclusively by this route , and 
  several enveloped viruses 
  ( 36 ) , such as equine arteritis 
  virus ( 37 ) , exploit this route 
  ( 38 ) . as an early step of this 
  route , upon binding of an extracellular ligand to specific cell - surface 
  receptors ,
clathrin together with other adapter proteins builds an endocytic 
  coat at the plasma membrane ( fig . 
 
2a ) . the coated membrane buds and pinches off to form a 
  cargo - filled vesicle ( fig . 
 
clathrin - dependent endocytic uptake of tat has been repeatedly reported as 
  a possible mechanism for cpp entry 
  ( 23 , 
  39 , 
  40 ) . to clarify the 
  contribution of clathrin - dependent endocytosis in the uptake mode of 
  arginine - rich cpps
, we used the bhk21-tta / anti - chc 
  ( 30 ) cell line .
this cell line 
  expresses antisense chc rna under the control of a tetracycline - responsive 
  element ( fig .
more specifically , the transcription activator ( tta ) is composed of the dna 
  binding domain of tetracycline repressor protein and a c - terminal activation 
  domain of vp16 ( herpes simplex virus protein ) that functions as a strong 
  transcription activator ( 41 ) . 
 
the presence of tetracycline prevents binding of the transactivator tta to the 
  operator sequence and thus transcription of antisense rna . in the absence of 
 
tetracycline the transactivator tta binds to its operator sequence and 
  activates the transcription of antisense rna . as a consequence ,
the synthesis 
  and functionality of chc protein is significantly reduced , thereby suppressing 
  clathrin - dependent endocytosis 
  ( 30 ) .
the absence of 
  tetracycline for 2 days was reported to inhibit 90% of transferrin 
  internalization , and the expression of the chc protein was reduced to 10% over 
  6 days in the absence of tetracycline 
  ( 30 ) . therefore , to explore to 
  what extent transduction of tat depends on clathrin - dependent endocytosis , 
  uptake of tamra - tagged tat and ptd4
was investigated in the 
  presence ( + tet ) and absence ( tet ) of tetracycline over 6 days .
to 
  simultaneously control the level of clathrin - dependent endocytosis , 
  internalization of alexa fluor 633-labeled transferrin was monitored 
  ( fig .
the control cells ( + tet cells ) are shown on the left panel of 
  fig . 2c and 
  supplemental fig . s3 .
tat was homogeneously distributed throughout the 
  cytoplasm and accumulated in the nucleolus 
  ( fig .
2c , 
  arrowheads ) and therefore displayed the uptake mode of transduction . in 
  addition , the labeled peptide was also present in cytoplasmic vesicles .
ptd4 applied at the same concentration and monitored at 
  identical confocal microscope settings did not show any 
  transduction - associated cytoplasmic localization comparable with tat .
however , 
  we observed the presence of ptd4-containing vesicles 
  ( fig . 2c , left 
  side , lower panel ) .
fluorescent transferrin was internalized at high 
  rates and enriched in the trans - golgi network 
  ( 42 ) .
s3a , right panel ) was verified by the 
  suppression of uptake of transferrin .
tat displayed the same intracellular 
  distribution regarding the vesicular uptake as well as transduction , indicated 
  by similar intensities inside the nucleolar compartment compared with the 
  control ( + tet ) cells ( fig . 
  2c and supplemental fig .
similar to the control 
  cells , no diffuse intracellular occurrence was observed for ptd4 , 
  but in contrast to + tet cells the vesicular internalization of ptd4 
  was almost completely abolished .
the 
  clathrin coat is required for membrane invagination , and for the scission of 
  clathrin - coated vesicles dynamin is needed .
b , schematic 
  representation of the tet - off system , allowing a conditional knockdown of chc 
  in the bhk21-tta / anti - chc cell line . the binding of the transcriptional 
  activator tta to an operator sequence in the absence of tetracycline 
  ( tet ) results in activation of transcription of chc - antisense 
  rna and thereby repression of the chc mrna translation .
c , confocal 
  optical sections of living cells during incubation with the fluorescent cpps 
  tat ( upper panel ) or ptd4 ( bottom panel ) in the 
  presence of the transferrin ( tf ) as a marker for clathrin - dependent 
  endocytosis .
each panel displays high magnification images of the phase 
  contrast ( pc ) and the fluorescently labeled compound to show the 
  details of their intracellular distribution and low magnification images to 
  highlight the frequency of cpp transduction and tf internalization ( see also 
  supplemental fig .
transduction experiments were performed in the presence ( left panel ) 
  and absence ( right panel ) of tetracycline .
although uptake of tf is 
  nearly abolished after tetracycline removal over a period of 6 days , the tat 
  cpp is still capable of reaching all intracellular compartments ( diffuse , 
  nonvesicular fluorescence , and accumulation inside nucleoli ) , indicating that 
  this mode of uptake is not influenced by clathrin - dependent endocytosis . 
 
vesicular uptake of the cpp tat was still detected under tet conditions 
  albeit at reduced levels .
scale bar , 10 m for high and 20 m 
  for low magnification images .
the 
  clathrin coat is required for membrane invagination , and for the scission of 
  clathrin - coated vesicles dynamin is needed .
b , schematic 
  representation of the tet - off system , allowing a conditional knockdown of chc 
  in the bhk21-tta / anti - chc cell line . the binding of the transcriptional 
  activator tta to an operator sequence in the absence of tetracycline 
  ( tet ) results in activation of transcription of chc - antisense 
  rna and thereby repression of the chc mrna translation .
c , confocal 
  optical sections of living cells during incubation with the fluorescent cpps 
  tat ( upper panel ) or ptd4 ( bottom panel ) in the 
  presence of the transferrin ( tf ) as a marker for clathrin - dependent 
  endocytosis .
each panel displays high magnification images of the phase 
  contrast ( pc ) and the fluorescently labeled compound to show the 
  details of their intracellular distribution and low magnification images to 
  highlight the frequency of cpp transduction and tf internalization ( see also 
  supplemental fig .
transduction experiments were performed in the presence ( left panel ) 
  and absence ( right panel ) of tetracycline .
although uptake of tf is 
  nearly abolished after tetracycline removal over a period of 6 days , the tat 
  cpp is still capable of reaching all intracellular compartments ( diffuse , 
  nonvesicular fluorescence , and accumulation inside nucleoli ) , indicating that 
  this mode of uptake is not influenced by clathrin - dependent endocytosis . 
 
vesicular uptake of the cpp tat was still detected under tet conditions 
  albeit at reduced levels .
scale bar , 10 m for high and 20 m 
  for low magnification images .
we confirmed that tat transduction was observed in most cells by acquiring 
  low magnification images ( fig . 
  2c and supplemental fig .
s3 ) . to control for any 
  potential side effects of tetracycline , the parental bhk21 cells 
  ( 43 , 
  44 ) were incubated with the 
  cpps and transferrin in the presence and absence of tetracycline .
however , no 
  difference of peptide transduction and vesicle formation was observed ( data 
  not shown ) .
therefore , we conclude that clathrin - dependent endocytosis is not 
  required for transduction of the rrp tat fused to an lmw cargo .
role of caveolin - mediated endocytosis in cpp uptake  besides 
  the classical clathrin - mediated endocytic pathway ,
caveolae - mediated 
  endocytosis is one of the main endocytic entry routes into living cells 
  ( 22 , 
  45 ) .
for example , it is 
  exploited by bacterial toxins and by simian virus 40 
  ( 36 ) .
caveolae are 
  flask - shaped , small ( 5070 nm diameter ) invaginations in the plasma 
  membrane ( fig .
3a ) 
  that constitute membrane domains enriched in cholesterol and sphingolipids , 
  called lipid rafts ( 46 ) . 
  caveolae are characterized by the presence of the integral membrane protein 
  caveolin-1 and are involved in the intracellular transport of lipid 
  raft - associated molecules 
  ( 47 ) .
this pathway has been 
  repeatedly reported as an uptake route for cpps into the cells 
  ( 24 , 
  25 ) .
former studies used 
  fluorescently labeled -subunit of cholera toxin as a marker to monitor 
  caveolar uptake .
however , the pathway chosen by cholera toxin subunit  
  depends on the cell type ( 48 ) 
  and hence may not be a faithful indicator for a single internalization 
  pathway . to specifically inhibit only the caveolin - dependent route and to 
  prevent the potential side effects caused by chemical inhibitors of 
  endocytosis , we made use of an endothelial heart cell line generated from a 
  knock - out ( ko ) mouse deficient for caveolin ( cav-1 ) and the respective wild 
  type ( wt ) cell line .
as reported previously 
  ( 31 ) , in the absence of 
  caveolin-1 , caveolin-2 protein is degraded .
in contrast to wt cells , no cav-1 and cav-2 were 
  detected in the ko cells , and in addition , antibodies directed against muscle 
  cell - specific cav-3 gave a much weaker signal in the extract of ko cells 
  compared with those from wild type cells . to elucidate if caveolae - dependent endocytosis plays a role in the uptake 
  mode of tat with the lmw tamra , we applied tat 
  ( fig .
3c , middle 
  panel ) to the medium of wild type cells and cav-1-ko cells . to control 
  whether the tagged fluorophore supports peptide internalization , the uptake of 
  tamra * alone at the same concentration as the peptides was studied 
  ( fig .
confocal optical sections are displayed at 
  high and low magnification . because of the large size of these cells , it was 
  not possible to show a higher number of cells per field and at the same time 
  keep imaging parameters constant throughout all experiments .
tat also became 
  internalized by an endocytic route , as indicated by the punctated 
  intracellular fluorescence .
in contrast to tat , the ptd4 peptide 
  entered the cells only by the endocytic mode and could not be detected freely 
  inside the cyto- and nucleoplasm and the nucleolus 
  ( fig .
tamra was excluded from the cytoplasm and 
  intracellular compartments providing strong evidence that peptide uptake is 
  not aided by the fluorophore .
based on these results , we conclude that 
  caveolae - mediated endocytosis is not involved in the uptake mode of 
  transduction of tat conjugated to an lmw cargo .
a , schematic diagram illustrating structural features of flask - shaped 
  caveolae , which are lined by caveolin .
caveolae - mediated endocytosis is driven 
  by a coat made of the integral membrane proteins caveolin-1 , -2 , or -3 . 
 
b , western blot 
  analysis of the integral membrane proteins caveolin ( cav ) -1 , -2 , and -3 in wt 
  and caveolin 1 ko endothelioma cells .
c , confocal optical sections of living cells during 
  incubation with the fluorescent cpps tat ( upper panel ) or 
  ptd4 ( mid panel ) and the fluorophore tamra * ( bottom 
  panel ) .
each panel displays images of the phase contrast ( pc ) 
  and the peptide or fluorophore fluorescence at high magnification to display 
  their intracellular distribution and at low magnification to highlight the 
  frequency of cpp transduction in wt and cav-1-ko cells ( see also supplemental 
  fig .
whereas the amphipathic control peptide ptd4 and the 
  fluorophore tamra * were not transduced , the cpp tat was homogeneously 
  distributed in the cytoplasm , and it reached the nucleus where it accumulated 
  inside the nucleolar compartment ( marked by arrowheads ) .
scale bar , 10 
  m for high and 20 m for low magnification images .
a , schematic diagram illustrating structural features of flask - shaped 
  caveolae , which are lined by caveolin .
caveolae - mediated endocytosis is driven 
  by a coat made of the integral membrane proteins caveolin-1 , -2 , or -3 . 
 
b , western blot 
  analysis of the integral membrane proteins caveolin ( cav ) -1 , -2 , and -3 in wt 
  and caveolin 1 ko endothelioma cells .
c , confocal optical sections of living cells during 
  incubation with the fluorescent cpps tat ( upper panel ) or 
  ptd4 ( mid panel ) and the fluorophore tamra * ( bottom 
  panel ) .
each panel displays images of the phase contrast ( pc ) 
  and the peptide or fluorophore fluorescence at high magnification to display 
  their intracellular distribution and at low magnification to highlight the 
  frequency of cpp transduction in wt and cav-1-ko cells ( see also supplemental 
  fig .
whereas the amphipathic control peptide ptd4 and the 
  fluorophore tamra * were not transduced , the cpp tat was homogeneously 
  distributed in the cytoplasm , and it reached the nucleus where it accumulated 
  inside the nucleolar compartment ( marked by arrowheads ) . both cpps 
  displayed vesicular uptake in wt and cav-1-ko cells .
scale bar , 10 
  m for high and 20 m for low magnification images .
. a , 
  schematic overview of the different pathways of endocytic internalization that 
  are suppressed at 4 c : clathrin- and caveolin - dependent endocytosis , 
  clathrin- and caveolin - independent endocytosis , and macropinocytosis . 
 
c , confocal optical sections of living cells during 
  incubation with the fluorescent cpps tat ( upper panel ) or 
  ptd4 ( lower panel ) in the presence of the fluorophore 
  fitc * as a small molecule marker to control for membrane pores or damage . each 
  panel displays high magnification images of the phase contrast ( pc ) 
  and the fluorescently labeled peptide or fluorophore to show their uptake and 
  intracellular distribution and low magnification images to highlight the 
  frequency of transduction at 37 and 4 c ( see also supplemental fig .
the transduction 
  experiments were performed in bhk21 cells kept at 37 and 4 c . although no 
  intracellular vesicles were found at 4 c , the transduction of tat 
  ( nonvesicular , diffuse fluorescence with accumulation inside nucleoli ) 
  remained unchanged both at 37 and 4 c .
in contrast , the amphipathic 
  control peptide ptd4 and fluorophore fitc * were not transduced at 
  37 c nor at 4 c .
scale bars are 10 m for high and 20 
  m for low magnification images .
. a , 
  schematic overview of the different pathways of endocytic internalization that 
  are suppressed at 4 c : clathrin- and caveolin - dependent endocytosis , 
  clathrin- and caveolin - independent endocytosis , and macropinocytosis . 
 
c , confocal optical sections of living cells during 
  incubation with the fluorescent cpps tat ( upper panel ) or 
  ptd4 ( lower panel ) in the presence of the fluorophore 
  fitc * as a small molecule marker to control for membrane pores or damage . each 
  panel displays high magnification images of the phase contrast ( pc ) 
  and the fluorescently labeled peptide or fluorophore to show their uptake and 
  intracellular distribution and low magnification images to highlight the 
  frequency of transduction at 37 and 4 c ( see also supplemental fig .
the transduction 
  experiments were performed in bhk21 cells kept at 37 and 4 c . although no 
  intracellular vesicles were found at 4 c , the transduction of tat 
  ( nonvesicular , diffuse fluorescence with accumulation inside nucleoli ) 
  remained unchanged both at 37 and 4 c .
in contrast , the amphipathic 
  control peptide ptd4 and fluorophore fitc * were not transduced at 
  37 c nor at 4 c .
scale bars are 10 m for high and 20 
  m for low magnification images .
uptake of cpps upon shutting off endocytic pathways  because 
  neither clathrin- nor caveolin - dependent endocytosis inhibits cpp uptake and 
  in both cases we could still find tat - containing vesicles concomitantly with 
  freely available cytoplasmic tat peptide , we tried next to inhibit all 
  endocytic pathways simultaneously . for this purpose
, we followed the internalization of tat and 
  ptd4 at low temperature , 4 c . 
 
4a depicts all 
  potential endocytic uptake routes in cells that are expected to be inhibited 
  under such conditions .
previous reports addressing the uptake of arginine - rich 
  cpps with lmw cargos into living cells are inconsistent .
although some reports 
  insisted on the inability of cpps to penetrate cells at low temperature and 
  hence endocytosis would be required for internalization 
  ( 49 ) , according to other 
  reports , cpp uptake is not inhibited at 4 c 
  ( 14 , 
  19 , 
  20 ) . 
 
4b displays the 
  experimental setup for assessing the transduction ability of tat and 
  ptd4 in c2c12 mouse myoblasts ( data not shown ) and in bhk21 hamster 
  fibroblasts in the presence ( 37 c ) and absence ( 4 c ) of endocytosis 
  by time - lapse confocal microscopy . to make sure that potential membrane 
  lesions generated by low temperature conditions would not corrupt the 
  transduction assay , the fluorophore fitc * was applied simultaneously with the 
  tamra - labeled peptides to the cells . in case of severe membrane damage or pore 
  formation induced by the peptides , the 389-da - sized fitc * molecule should also 
  be detectable intracellularly .
s5 reveal that even at 4 c 
  tat entered living cells and distributed over the cytoplasm and nucleus , where 
  it accumulated inside nucleoli . at the same time the fluorophore fitc * did not 
  gain access to intracellular compartments indicating that the plasma membrane 
  was not compromised .
the complete obstruction of endocytosis was further 
  confirmed by the absence of fluorescently labeled vesicles at 4 c . 
 
vesicles were not observed for the cpp tat nor for ptd4 . 
  furthermore , uptake of the globular tat fusion protein tat - bt - sav that is 
  restricted to endocytosis ( 8) 
  was also blocked on the level of the plasma membrane at 4 c ( supplemental 
  fig .
these results prove unambiguously that arginine - rich cpps like tat 
  are capable of reaching intracellular compartments of living cells by a 
  mechanism that is independent of endocytosis .
however , the data also clearly 
  show that a minimal number of arginines is crucial to permit transduction .
assembly of 
  confocal optical sections of living cells with time intervals of 15 min 
  derived from the 60-min time lapses ( see supplemental movies s1s10 ) 
  displaying the kinetics of tat uptake in the different cell lines .
all cell 
  lines showed an unchanged transduction behavior in the control cells and the 
  cells that were inhibited for a distinct or all pinocytic events .
but the 
  average entry time point of the tat peptide is cell type - specific .
the 
  last column summarizes the transduction frequencies of tat after 60 
  min of incubation in cells , where distinct or all endocytic pathways were 
  suppressed .
transduction frequency was scored by counting the percentage of 
  cells showing the intracellular freely available peptide ( see 
  fig . 1 , b and 
  c ) .
assembly of 
  confocal optical sections of living cells with time intervals of 15 min 
  derived from the 60-min time lapses ( see supplemental movies s1s10 ) 
  displaying the kinetics of tat uptake in the different cell lines .
all cell 
  lines showed an unchanged transduction behavior in the control cells and the 
  cells that were inhibited for a distinct or all pinocytic events .
but the 
  average entry time point of the tat peptide is cell type - specific .
the 
  last column summarizes the transduction frequencies of tat after 60 
  min of incubation in cells , where distinct or all endocytic pathways were 
  suppressed .
transduction frequency was scored by counting the percentage of 
  cells showing the intracellular freely available peptide ( see 
  fig . 1 , b and 
  c ) .
scale bar , 10 m . frequency and initiation time of tat transduction  to 
  quantify our results on the transduction ability in the absence of a distinct 
  pathway or all pathways of endocytosis , we evaluated the percentages of 
  transduced cells after 60 min of incubation with the cpp tat and the 
  initiation time of transduction ( fig . 
  5 and supplemental movies s1s10 ) .
the control cells ( + tet , 
  day 0 ) showed a transduction frequency of 83% .
while over the first 3 days 
  after induction of the conditional knockdown of chc ( tet ) the 
  transduction percentages were reduced to 68% , they recovered back to 84% 
  transduction frequencies by day 6 , although the internalization of 
  fluorescently labeled transferrin was reduced by 90% .
the frequency of 
  transduced cells was identical in cav-1-deficient and isogenic wt cells ( 54% ) , 
  although in these cells it was lower than in the bhk21-tta / anti - chc cells . 
 
finally , the inhibition of all potential pinocytic pathways at 4 c 
  determined in bhk21 fibroblasts revealed that the transduction of the tat 
  peptide is diminished about 12% in comparison with transduction occurring in 
  cells kept at 37 c , but still 63% of the cells displayed nucleolar 
  accumulation of tat . in bhk21-tta / anti - chc cells ,
the transduction mode of uptake initiated 
  between 15 and 30 min of peptide addition after chc knockdown as well as in 
  control cells .
the kinetics of peptide uptake in cav-1-ko and wild type cells 
  displayed an uptake initiation at 45 min after application of tat .
the fastest 
  uptake of tat occurred in bhk21 fibroblasts within 1 min of addition to the 
  medium and independent of temperature .
a summary of the uptake kinetics of tat 
  peptide in all conditions tested is presented in 
  fig .
the frequency and initiation time of tat transduction was 
  unchanged within a given cell type independent of endocytosis . however
, both 
  parameters were cell type - specific , suggesting that the membrane composition 
  influences the velocity of transduction .
despite the controversy and uncertainty regarding the uptake mechanism , the 
  property of cpps to deliver nonpermeable molecules into living cells makes 
  them attractive vectors to be used in biological sciences as well as in 
  medicine and biotechnology .
former studies used chemical inhibitors of 
  endocytosis to assign the uptake of cpps to a particular endocytic pathway . 
  however
, the potential side effects and lack of specificity of such inhibitors 
  make these studies difficult to interpret .
either the chemical compounds 
  affect more than one specific pathway , like methyl--cyclodextrin that 
  affects both lipid raft ( 26 ) 
  and caveolin - coated vesicle formation 
  ( 50 ) hence also 
  caveolin - dependent endocytosis , or they have other side effects that may 
  impact the import of rrps , e.g. chlorpromazine was shown to interface 
  with a number of ca - dependent signaling pathways 
  ( 51 ) and to bind to dopamine 
  receptors ( 19 , 
  52 ) .
thus , we have used 
  genetically modified systems or physical methods to clarify the role of 
  endocytosis in the translocation of rrps .
the different phenotypes shown in fig . 
  1 permit at least two explanations for the occurrence of free rrps 
  inside the cytoplasm and nucleoplasm .
either rrps become endocytosed and a 
  portion of the peptide stored in vesicles gets released into the cytoplasm or 
  a second nonendocytic entry route that allows rrps to directly cross the 
  plasma membrane has to be considered . earlier reports associated uptake of rrps with and without attached cargos 
  to distinct endocytic pathways 
  ( 16 , 
  2328 , 
  39 , 
  40 , 
  49 ) .
however , our results 
  unambiguously demonstrate that the transduction of tat into living cells is 
  not dependent on any endocytic or pinocytic events .
we could exclude the 
  pathway of clathrin - mediated endocytosis by a carefully controlled knockdown 
  experiment .
also caveolin - mediated endocytosis was not involved in tat 
  translocation , because caveolin knock - out cells showed an identical 
  transduction frequency to the wild type cells .
most importantly , tat was not 
  excluded from cells that were gently transferred to 4 c , a state where 
  all potential endocytic pathways are inhibited .
it is noteworthy to mention that the amount of tat transduced into a cell 
  varies between cells within a single experiment , but its intracellular 
  distribution does not .
this phenomenon was observed in every cell type and was 
  independent of which endocytic route was down - regulated .
( fig . 
  2 , 
  3 , 
  4 , 
  5 and supplemental fig . 
 
s3s5 ) . comparing the transduction frequencies of the specific cell types , a 
  variation from 68 to 84% for the bhk21-tta / anti - chc cell line , over 
  6375% in bhk21 cells , and to 54% in the mouse endothelioma cells
can be 
  found , suggesting that the particular membrane composition of different cell 
  types impacted on transduction ( fig . 
 
this was further corroborated by the observation that the 
  average initiation time of transduction was cell type - specific , but it did not 
  change upon the inhibition of endocytosis within a given cell type .
all 
  experiments were conducted in addition to ptd4 as a representative 
  cpp ( 29 ) with a reduced number 
  of arginines compared with tat . at the same concentration ,
this peptide did 
  not gain access to the intracellular compartments in a freely diffusing form 
  and was internalized exclusively by endocytosis .
this could be explained by 
  the fact that a minimal number of six arginines is required to permit 
  transduction ( 18 ) . instead
although not capable of performing transduction , the cpp 
  ptd4 was endocytosed more efficiently than non - cpp compounds added 
  to the medium , e.g. the fluorophore tamra * alone 
  ( fig .
recent mechanistic studies with artificial membrane systems described the 
  formation of pores as a consequence of the interaction with intermediate 
  concentrations of the rrp tat 
  ( 53 ) .
however , the fluorophore 
  fitc * , concomitantly applied to the cells together with the tat peptide , 
  remained outside of the cells , whereas tat transduced selectively into the 
  cells ( fig .
our data indicate that tat cpp internalization is independent 
  of endocytosis and occurs without disruption of the cell membrane .
these 
  properties and its high intracellular bioavailability make tat cpp a very 
  effective tool to deliver small compounds into living cells .
 arginine - rich peptides are a subclass of cell - penetrating peptides that are 
  taken up by living cells and can be detected freely diffusing inside the 
  cytoplasm and nucleoplasm . 
 this phenomenon has been attributed to either an 
  endocytic mode of uptake and a subsequent release from vesicles or to direct 
  membrane penetration ( transduction ) . to distinguish between both 
  possibilities 
 , we have blocked endocytic pathways suggested to be involved in 
  uptake of cell - penetrating peptides . 
 we have then monitored by confocal 
  microscopy the uptake and distribution of the cell - penetrating transactivator 
  of transcription ( tat ) peptide into living mammalian cells over time . 
 to 
  prevent side effects of chemical inhibitors , we used genetically engineered 
  cells as well as different temperature . 
 we found that a knockdown of 
  clathrin - mediated endocytosis and a knock - out of caveolin - mediated endocytosis 
  did not affect the ability of tat to enter cells . 
 in addition , the tat peptide 
  showed the same intracellular distribution throughout the cytoplasm and 
  nucleus as in control cells . 
 even incubation of cells at 4 c did not 
  abrogate tat uptake nor change its intracellular distribution . 
 we therefore 
  conclude that this distribution results from tat peptide that directly 
  penetrated ( transduced ) the plasma membrane . 
 the formation of nonselective 
  pores is unlikely , because simultaneously added fluorophores were not taken up 
  together with the tat peptide . in summary , although the frequency and kinetics 
  of tat transduction varied between cell types , it was independent of 
  endocytosis . 
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
Supplementary Material