# 2007 The Authors

Journal compilation# 2007 Blackwell Publishing Ltd

doi: 10.1111/j.1600-0854.2007.00572.xTraffic 2007; 8: 848–866Blackwell Munksgaard

A Comprehensive Model for the Cellular Uptake ofCationic Cell-penetrating Peptides

Falk Duchardt1,†, Mariola Fotin-Mleczek1,†,

Heinz Schwarz2, Rainer Fischer1 and

Roland Brock1,3,*

1Interfaculty Institute for Cell Biology, University ofTubingen, Auf der Morgenstelle 15, 72076 Tubingen,Germany2Max-Planck-Institute for Developmental Biology,Spemannstr. 35, 72076 Tubingen, Germany3Department of Biochemistry, Nijmegen Centre forMolecular Life Sciences, Radboud University NijmegenMedical Centre, PO Box 9101, 6500 HB Nijmegen,The Netherlands*Corresponding author: Roland Brock,r.brock@ncmls.ru.nl†These authors contributed equally to this work.

The plasma membrane represents an impermeable

barrier for most macromolecules. Still some proteins and

so-called cell-penetrating peptides enter cells efficiently.

It has been shown that endocytosis contributes to the

import of these molecules. However, conflicting results

have been obtained concerning the nature of the endo-

cytic process. In addition, there have been new findings

for an endocytosis-independent cellular entry. In this

study, we provide evidence that the Antennapedia-

homeodomain-derived antennapedia (Antp) peptide,

nona-arginine and the HIV-1 Tat-protein-derived Tat pep-

tide simultaneously use three endocytic pathways: mac-

ropinocytosis, clathrin-mediated endocytosis and

caveolae/lipid-raft-mediated endocytosis. Antennapedia

differs from Tat and R9 by the extent by which the

different import mechanisms contribute to uptake. More-

over, at higher concentrations, uptake occurs by a mech-

anism that originates from spatially restricted sites of the

plasma membrane and leads to a rapid cytoplasmic

distribution of the peptides. Endocytic vesicles could

not be detected, suggesting an endocytosis-independent

mode of uptake. Heparinase treatment of cells negatively

affects this import, as does the protein kinase C inhibitor

rottlerin, expression of dominant-negative dynamin and

chlorpromazine. Thismechanism of uptakewas observed

for a panel of different cell lines. For Antp, significantly

higher peptide concentrations and inhibition of endocy-

tosis were required to induce its uptake. The relevance of

these findings for import of biologically active cargos is

shown.

Key words: antennapedia homeodomain, cell-penetrating

peptide, endocytosis, oligo-arginine, TAT

Received 1 February 2006, revised and accepted for publi-

cation 26 March 2007, uncorrected manuscript published

online 29 March 2007, published online 10 May 2007

For most polar molecules, the plasma membrane repre-

sents an impermeable barrier. It is therefore highly remark-

able that some proteins and peptides possess the ability to

cross this border and reach the cytoplasm. Among these

are transcription factors belonging to the homeodomain

family, such as theDrosophila melanogaster Antennapedia

homeodomain protein (1), the HIV-1 Tat protein (2) and

fibroblast growth factors 1 and 2 (3). Peptides that possess

this ability were identified as the protein transduction

domains (PTDs) of aforementioned proteins such as the

Antennapedia-homeodomain-derived antennapedia (Antp)

peptide (4) and the HIV-1 Tat-derived Tat peptide, denoted

here as Tat (5). Alternatively, peptides, among them oligo-

arginine peptides (6), were designed de novo based on

structure activity relationships of PTDs. The attractivity of

these peptides, generally defined as cell-penetrating pepti-

des (CPPs), in biomedical research is a consequence of their

ability to mediate the import of membrane-impermeable,

biologically active molecules such as small interfering

RNA, DNA, peptides or entire proteins into the cells ex

vivo and in whole organisms [for reviews, see Dietz and

Bahr (7) and Snyder and Dowdy (8)].

Initially, the import of these cell-penetrating molecules,

proteins and peptides alike, was considered to occur by

direct permeation of the plasma membrane (9). This model

was based on evidence obtained from cell biological as

well as biochemical and biophysical experiments. More

recently, it was shown that endocytosis plays a major role

in the import of the basic and amphiphilic Antp peptide, the

highly basic and arginine-rich R9 and Tat peptides and in

the import of the HIV Tat protein itself (10–12). No specific

receptor has been implicated in the uptake of these

molecules. Instead, in some cases, the initial association

with the plasma membrane was attributed to multivalent

interactions with cell surface heparan sulfate proteo-

glycans (13,14).

To this point, however, conflicting results were obtained

for the involvement of specific endocytic pathways. Data

were presented that supported a role of macropinocytosis

(15), clathrin-mediated endocytosis (CME) (11) and caveo-

lae/lipid-raft-mediated endocytosis (16,17).

Contrary to this uptake through endocytosis, for a fluores-

cein-labeled Tat peptide, Ziegler et al. observed a rapid

cellular import into fibroblasts that was heparan sulfate

dependent (18). Similarly, Tunnemann et al. reported that

the Tat peptide conjugated to a small peptide cargo enters

cells by a rapid and endocytosis-independent process, with

resemblance to the one described by Ziegler et al., while

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larger conjugates enter cells by endocytosis (19). They

concluded that endocytosis is restricted to high molecular

weight complexes. However, this conclusion is in conflict

with a number of reports observing a vesicular mode of

uptake for the Tat peptide as well (11,20–22).

As an explanation for these apparent discrepancies, in this

article, we provide evidence that the peptides use the

three endocytic pathways simultaneously. Antennapedia

differs from R9 and Tat by the extent that individual

processes contribute to import. Moreover, for R9 and Tat

above a concentration threshold in the lower micromolar

range, both peptides are internalized predominantly

through a process that leads to a rapid distribution of

peptides into the cytoplasm and nucleus. Our observations

suggest that this mechanism is endocytosis independent.

Blockage of macropinocytosis and lipid-raft-dependent

endocytosis lowers this threshold. This rapid cytoplasmic

entry originates from spatially confined zones of the

plasma membrane. For Antp, this uptake was observed

only for cells treated with the inhibitor of lipid-raft-depen-

dent endocytosis methyl-b-cyclodextrin (MbCD) at signifi-

cantly higher peptide concentrations. The analysis of the

mechanistic basis showed that this uptake is heparan sul-

fate dependent and sensitive to the protein kinase C (PKC)

inhibitor rottlerin, the expression of dominant-negative

(DN) dynamin and chlorpromazine (CPZ). The relevance

of these results for the application of CPPs as tools in cell

biological research is shown using a Smac-derived peptide

(23) that potentiates the Fas-dependent induction of

apoptosis.

Results

The intracellular distribution of R9 and Tat is

concentration dependent

For the three cationic CPPs Antp, R9 and Tat (Table 1),

there is a consensus that endocytosis at least strongly

contributes to cellular uptake (24). However, conflicting

evidence has been presented concerning the role of the

individual endocytic pathways. Moreover, some research-

ers observed a rapid entry of peptides into the cytosol for

which endocytosis did not seem to be involved. We

reasoned that the apparent discrepancies were a conse-

quence of the different experimental conditions with

respect to cell lines, incubation times and peptide concen-

trations. For this reason, we performed experiments in the

presence of different peptide concentrations. Experiments

investigating the uptake mechanism of CPP have mostly

used these peptides at lower micromolar concentrations,

while the applications of CPP–peptide conjugates for

interfering with molecular interactions inside the cell have

frequently used these molecules in the mean to upper

micromolar range (25–28). The selected concentration

range for our experiments therefore covered peptide

concentrations used in both types of experiments. As

a cellular model system, we selected HeLa cells because

this cell line has been used in a significant number of

previous reports (11,13,17,20,21,29). All three peptides

were synthesized as fluorescein-labeled analogues. The

cellular uptake was quantified in living cells by flow

cytometry. Trypsinization of cells, required to detach the

cells from the tissue culture plate prior to flow cytometry,

removed peptides merely adsorbed to the outer plasma

membrane (11). In addition, the intracellular peptide distri-

bution was investigated by live cell confocal laser scanning

microscopy.

For Antp, the cellular fluorescence was proportional to the

concentration of peptide in the medium (Figures 1A, 2A).

With respect to the intracellular fluorescence, the cell

population was fully homogeneous for each peptide con-

centration. Neither saturation nor changes in the intracel-

lular distribution of peptide was observed. Antennapedia

was localized predominantly within vesicular structures.

Fluorescence could also be detected in the cytoplasm, and

this fluorescence also increased with peptide concentra-

tion. In contrast, the cellular uptake of R9 and Tat did not

increase linearly with peptide concentration (Figures 1B,C,

2A). Both flow cytometry and fluorescence microscopy

clearly showed that at concentrations higher than 10 mM,

cytoplasmic delivery of peptides was strongly enhanced.

Simultaneously, the cell population became very hetero-

geneous with respect to peptide uptake. Some cells were

fluorescent to the point that they were outside the

detection range. Other cells showed a moderate uptake,

and finally, some cells exhibited only a weak intracellular

fluorescence. This heterogeneity in the level of the cell

population was reflected by a heterogeneity in the distri-

bution of fluorescence on the subcellular level. With in-

creasing peptide concentration, an increasing number of

cells exhibited a clear enrichment of peptide in the nucleus

and cytoplasm, with little vesicular fluorescence, resem-

bling the distribution reported by Tunnemann et al. (19).

To exclude that the highly efficient peptide import was

a result of damage of the plasma membrane, we tested

the membrane integrity using a trypan blue exclusion test

(data not shown) and incubation with peptide in the

presence of propidium iodide (PI) (Figure 1D). Cells

Table 1: Primary structures of the peptides used in this studya

Entry Peptide Sequence

1 Antp Fluo-RQIKIWFQNRRMKWKK-CONH2

2 R9 Fluo-RRRRRRRRR-CONH2

3 Tat Fluo-YGRKKRRQRRR-CONH2

4 Smac–Antp AVPIAQK-RQIKIWFQNRRMKWKK-

eK(Fluo)-CONH2

5 Smac–R9 AVPIAQK-RRRRRRRRR-eK(Fluo)-CONH2

aAll peptides were synthesized as C-terminal peptide amides

(-CONH2). Fluo represents 5(6)-carboxyfluorescein.

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Uptake of Cationic Cell-Penetrating Peptides

coincubated with PI and R9 at a peptide concentration of

20 mM exhibited efficient peptide entry but no PI staining.

However, the subsequent treatment of the same cells

with detergent resulted in a strong PI signal within the

nucleus and in the simultaneous exit of peptide already

2 min after addition of detergent. These two independent

assays clearly show the integrity of the plasma membrane

during peptide incubation. To further exclude toxic effects,

Figure 1: Distinct concentration-depen-

dent phenotypes for the uptake of dif-

ferent CPPs. A–C) HeLa cells were

incubated for 30 min with increasing con-

centrations of the indicated peptides. For

fluorescence microscopy, cells were

washed twice with medium and analyzed

immediately. The scale bars represent

20 mm. For flow cytometry, cells were

washed and harvested with trypsin/EDTA.

D) HeLa cells were incubated with R9

(20 mM) in the presence of PI (5 mg/mL)

for 20 min (upper panels). Then, detergent

(Tween 0.5% v/v) was added to the cells

(bottom panels), and images were acquired

15 min after addition of detergent by dual-

channel confocal laser scanning micro-

scopy. R9 was detected in the fluorescein

channel and PI in the rhodamine channel. E)

HeLa cells were incubated with increasing

concentrations of peptides for 6 h and

subsequently, cell viability was determined

using the MTT assay. EDTA, ethylenedia-

minetetraacetic acid.

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Duchardt et al.

the viability of cells was also determined after incubation

with peptides for 6 h. Using the 3-(4,5-dimethylthiazol-2-yl)-

2,5-diphenyltetrazolium bromide (MTT) test, no toxicity

was observed for peptide concentrations up to 40 mM

(Figure 1E).

R9 and Tat differ from Antp in the concentration

dependence of the uptake mechanism

The surprising observation that for R9 and Tat, different

peptide concentrations led to clearly distinct phenotypes

with respect to intracellular peptide distribution motivated

us to ask whether different import mechanisms may be

responsible for this effect. To test this hypothesis, we

investigated the involvement of the three major endocytic

pathways in peptide uptake in dependence on peptide

concentration. Pharmacological inhibitors were used to

interfere with individual endocytic pathways (30–32).

Chlorpromazine was used for the inhibition of clathrin-

mediated internalization, 5-(N-ethyl-N-isopropyl)amiloride

(EIPA) for inhibition of macropinocytosis and MbCD for

disruption of import through caveolae/lipid rafts. The

specificity of the inhibitors is an issue of debate, also

within the field of CPP uptake (33). For fluorescently

labeled tracer molecules [transferrin, dextran and cholera

toxin beta subunit (CTB)], each of the inhibitors showed

the expected effect (Figure S1). However, for none of the

tracer molecules, import could be fully blocked. All three

inhibitors alone or in combination, applied at concentra-

tions used in this study, had no toxic effect (Figure S2). The

effects of all three inhibitors on the delivery of Antp, R9

and Tat within a concentration range of 2–40 mM were

tested by flow cytometry (Figure 2). The concentration-

dependent peptide import measured in cells not treated

with inhibitor was used as a 100% reference for deter-

mining the reduction in uptake caused by the different

inhibitors (Figure 2A). For Antp, CPZ and EIPA reduced the

cellular fluorescence by about 20% for the entire concen-

tration range. Preincubation of cells with MbCD had no

detectable effect. In contrast, for R9 and Tat, the effect

exerted by each inhibitor was strongly dependent on

peptide concentration. At peptide concentrations <5 mM,

CPZ was without effect. However, with increasing peptide

concentration, the inhibitory activity of CPZ increased,

amounting to about 80% at a peptide concentration of

40 mM. At low peptide concentrations, both MbCD and

EIPA inhibited the import of R9 and Tat only slightly if at

all. Interestingly, at higher peptide concentrations, the

same compounds did not reduce but dramatically

enhanced the uptake of these CPPs. We observed a four-

to sevenfold increase of cellular fluorescence compared

with the fluorescence in the control, not treated with

inhibitors. At a concentration of 40 mM, the fluorescence

was outside the detection range. These results again

confirm a difference between the cellular import of Antp

and one of the other two peptides. Moreover, the

dependence of the inhibitor effects on peptide concen-

tration strongly suggests that the contribution of the

individual uptake mechanisms may in fact depend on

peptide concentration.

To test this hypothesis in more detail, the cellular peptide

distribution was analyzed at low (2 mM) and high (20 mM)

peptide concentrations. For Antp, all three inhibitors led

to distinct phenotypes with respect to cellular peptide

Figure 2: Dependence of the

effect of inhibitors of endocytosis

on peptide concentration. A) Con-

centration dependence of the cellu-

lar uptake of Antp, R9 and Tat. B–D)

Cellular peptide uptake in the pres-

ence of 10 mg/mL CPZ (B), 5 mM

MbCD (C) and 50 mM EIPA (D).

HeLa cells were incubated for 30

min at 378C with the indicated

inhibitor of endocytosis or remained

untreated (A). Then, the cells were

incubated with increasing concen-

trations of CPPs for further 30 min

in the presence or absence of inhibi-

tors, washed, harvested with tryp-

sin/EDTA and analyzed by flow

cytometry. The effect of the inhibi-

tors on cellular fluorescence is

shown relative to the fluorescence

measured in the inhibitor-free

controls incubated with the same

concentration of peptide. EDTA,

ethylenediaminetetraacetic acid.

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Uptake of Cationic Cell-Penetrating Peptides

distribution (Figure 3A). Preincubation of cells with CPZ

strongly decreased the small fraction of peptide present

in the cytoplasm. The peptide was localized in vesicular

structures that were predominantly localized at one

side of the nucleus. The inhibition of caveolae/lipid-raft-

mediated endocytosis by MbCD resulted in the reduction

of cytoplasmic and vesicular fluorescence but simulta-

neously resulted in the enrichment of peptide at the

plasma membrane. The major part of this fluorescence

was resistant to trypsin treatment (not shown), explaining

why in flow cytometry, no effect of MbCD on cellular

fluorescence had been detected. The molecular basis for

this observation, that is close association with the plasma

membrane or incorporation, could not be resolved. Inhib-

ition of macropinocytosis by EIPA also reduced the

internalization of Antp; however, a cytoplasmic as well

as a vesicular staining were detected. In this case,

intense peptide-loaded vesicles were localized predomi-

nantly around the nucleus. Consistent with the results

obtained by flow cytometry, the effects of the three

inhibitors on uptake were independent on peptide con-

centration. To support the observation that the three

endocytic pathways contribute to the uptake of Antp,

we incubated cells with medium containing peptide and

the corresponding tracer: transferrin for CME, CTB for

caveolae/lipid-raft-mediated endocytosis and dextran for

macropinocytosis (Figure 3B). We observed a partial co-

localization of Antp with each tracer. However, the level

of colocalization with dextran and CTB was strongly time

dependent (Figure 3C). At the early stage of endocytosis,

more than 50% of Antp-loaded vesicles were positive for

transferrin and only 20% showed a colocalization with

dextran or CTB, respectively. These observations indicate

that CME is the preferred mode of internalization but not

the exclusive one. However, the internalization of Antp

and transferrin was not fully synchronized. Transferrin

uptake was faster than the one of Antp and less than 20%

of the transferrin vesicles contained detectable levels of

peptide. In contrast, the internalization of dextran and

CTB was slower and 60% of dextran- and CTB-loaded

vesicles contained Antp. With the progression of endo-

cytosis, the fraction of vesicles containing peptide and

dextran/CTB continuously increased. The fusion of

vesicles of different origin and the release of their

contents into sorting/late endosomes is a likely explana-

tion for this observation. At the early time-point, when

one may assume that no merging of vesicles had

occurred, the percentages of the Antp-containing vesicle

populations added up to about 100%, validating the

accuracy of the protocol used for image processing. The

results obtained for Antp at a concentration of 10 mMwere

representative for the whole concentration range.

Figure 3: The involvement of three

endocytic pathways in the uptake of

Antp. A) HeLa cells were treated with the

indicated inhibitors for 30 min at 378C or

remained untreated, followed by incuba-

tion for a further 30 min with Antp (20 mM)

in the absence or presence of the respect-

ive inhibitor. The scale bars represent

20 mm. B) HeLa cells were incubated for

30 min at 378C with medium containing

Antp (10 mM) and a transferrin Alexa Fluor

633 conjugate (10 mg/mL), a dextran Alexa

Fluor 647 conjugate (10 mM) or Alexa Fluor

555-labeled CTB (10 mg/mL). C) Colocali-

zation of Antp and the indicated tracers at

different times of uptake.

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Duchardt et al.

For the uptake of R9 and Tat at low peptide concentrations,

CPZ had no effect (Figure 4). In contrast, at high peptide

concentrations, the same inhibitor completely abolished

the cytoplasmic and nuclear localization of the peptides.

Under the assumption that CPZ is a specific inhibitor of

clathrin-dependent endocytosis, this observation provided

strong evidence that at high concentrations, this endocytic

pathway is a key internalization mechanism used by these

peptides, which leads to their cytoplasmic and nuclear

enrichment. The inhibition of transferrin uptake confirmed

that inhibition of CME is at least one activity exerted by

CPZ on HeLa cells. However, CPZ originally was identified

as an antipsychotic drug (34), acting as an antagonist of

dopamine D2 receptors (35). Moreover, CPZ directly

interacts with calmodulin, thereby also interfering with

a number of Ca2þ-dependent signaling pathways (36).

Given the fact that our further experiments (see below)

supported a nonendocytic uptake mechanism, in the

following we refer to this uptake as CPZ sensitive rather

than taking these results as direct evidence for clathrin-

dependent endocytosis.

At low concentrations, the CPZ-sensitive pathway did not

seem to be involved in the uptake of R9 and Tat. MbCD

and EIPA exerted a slight inhibitory effect, leading to

phenotypes similar to the ones observed for Antp. How-

ever, at high peptide concentrations, the same inhibitors

strongly promoted the cytoplasmic and nuclear distribution

of peptides (Figure 4). This result was surprising but

completely consistent with the data obtained by flow

cytometry (Figure 2C,D). Evidently, at high peptide con-

centrations, the inhibition of endocytosis exerted by these

inhibitors directed the peptides toward a more efficient

compensatory mechanism.

Having observed that for R9 and Tat, inhibitors of endocy-

tosis lower the threshold for the rapid entry of peptide into

the cytoplasm, we decided to investigate the uptake of

Figure 4: Dependence of the effects of in-

hibitors of endocytosis on the concentra-

tion of R9 and Tat. HeLa cells were treated

with the indicated inhibitors for 30 min at 378Cor remained untreated. Then, the cells were

incubated for a further 30 min with either 2 or

20 mM of R9 A) or Tat peptide B) in the

presence or absence of the respective inhibi-

tor. The scale bars represent 20 mm. C)

MbCD-induced cytoplasmic uptake of Antp in

HeLa cells. HeLa cells were treated with

MbCD for 30 min at 378C or remained

untreated. Then, the cells were incubated for

a further 30 min with 100 mM of Antp in the

presence or absence of MbCD. The scale bars

represent 20 mm.

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Uptake of Cationic Cell-Penetrating Peptides

Antp at even higher concentrations. Antennapedia was

applied to HeLa cells at a concentration of 100 mM. In the

absence of inhibitors, in spite of a further increase of

cytoplasmic fluorescence, vesicular staining was still

prominent (Figure 4C). In contrast, in the presence of

MbCD, some cells showed a homogeneous cytoplasmic

and nuclear fluorescence. However, after about 20-min

incubation, morphological changes of the cells were indica-

tive of cell damage. In contrast to R9 and Tat, EIPA was

without effect. Nevertheless, these data indicate that the

differences between Antp and R9 and Tat may be rather

quantitative than qualitative.

To provide direct evidence that application of EIPA and

MbCD promoted the uptake of R9 and Tat through the

CPZ-sensitive rapid uptake mechanism, HeLa cells were

incubated either with MbCD or with EIPA alone or in

combination with CPZ (Figure 5A). R9 was applied at

a concentration of 10 mM. In control cells, at this concen-

tration, the peptide was predominantly localized within

vesicles. Consistent with our hypothesis, at this peptide

concentration, CPZ had no effect on peptide uptake. As

before, preincubation of cells with MbCD or EIPA led to

a pronounced localization of peptides in the cytoplasm

and nucleus. However, this phenotype was completely

Figure 5: MbCD- and EIPA-mediated induction of a rapid cytoplasmic internalization mechanism. A) HeLa cells were treated with

the indicated inhibitors for 30 min at 378C and then incubated with 5 mM of R9 in the presence of the respective inhibitors for a further

30 min. The scale bars represent 20 mm. B) HeLa cells were treated with the indicated inhibitors as described in (A), followed by incubation

with increasing concentrations of R9 or Tat and then harvested with trypsin/EDTA, and the intracellular fluorescence was quantified with

flow cytometry. The error bars represent the mean deviation of three independent experiments. EDTA, ethylenediaminetetraacetic acid.

854 Traffic 2007; 8: 848–866

Duchardt et al.

reversed when cells were treated with either MbCD or

EIPA in combination with CPZ. The primary inhibitory

effects of both MbCD (membrane enrichment) and EIPA

(vesicles distributed around the nucleus) were detected

again. The MbCD-induced promotion of peptide import

and the reversal of this effect by CPZ were confirmed by

flow cytometry (Figure 5B). For R9 and Tat, applied at

concentrations higher than 5 mM, the inhibition of caveolae/

lipid-raft-mediated endocytosis by MbCD enhanced pep-

tide uptake by up to 800 (R9) and 400% (Tat) of controls

not treated with inhibitor. This increase was almost

completely inhibited when cells were treated with a com-

bination of MbCD and CPZ.

The uptake of R9 at high peptide concentrations

originates from spatially restricted membrane regions

Having shown the remarkable efficiency of the CPZ-

sensitive uptake mechanism used by R9 and Tat at high

peptide concentrations, we aimed at obtaining information

about the time–course of this uptake mechanism. Cells

were incubated with R9 (20 mM), and the internalization of

peptide was imaged by time-lapse confocal fluorescence

microscopy, with the peptide still in the medium (Figure

6A). Image acquisition was started about 60 seconds

after addition of peptide. Frames were recorded every

30 seconds.

In some cells, areas of higher fluorescence intensity were

observed that seemed to emerge from spatially restricted

membrane regions. During the next 10 min, further highly

fluorescent zones (one or two per cell) developed that

were the origin of a rapid spreading of fluorescence

throughout the cytoplasm and nucleus. We chose to call

these highly efficient internalization platforms nucleation

zones (NZs). Almost immediately after formation of an NZ,

the peptide was enriched in the nucleus. In agreement

with our previous observation, the cell population became

heterogeneous with regards to the intracellular fluores-

cence. The efficient peptide entry strictly depended on the

formation of the NZs. Cells lacking these structures did not

Figure 6: The rapid uptake of R9 originates from spatially confined internalization platforms. A) Medium containing R9 at

a concentration of 20 mM was added to the cells and uptake of fluorescence followed by time-lapse confocal microscopy at room

temperature with frames recorded every 30 seconds. Four frames recorded at 1, 5, 10 and 15min are shown. B) Time-lapse images for the

fluorescence intensity graph (C) marked with a star. The scale bar corresponds to 20 mm. C) Increase of fluorescence intensities over time

determined for the time series shown in (B) from circular ROI placed into individual cells. To render the comparison of uptake kinetics for

different days insensitive to differences in laser power and fluorescence detection efficiency, rather than in absolute values, the values are

expressed in fold increase in comparison with the intracellular signal after addition of peptide. For the graph marked with a star, the points

corresponding to the individual frames are represented by white diamonds. NZs are marked with arrows. D) NZ-mediated uptake

accompanied by bleb formation. The individual time-points are given in the images. The scale bar corresponds to 10 mm.

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Uptake of Cationic Cell-Penetrating Peptides

exhibit any cytoplasmic or nuclear fluorescence. At micro-

scope settings at which usually endocytic uptake was

followed, for many cells, fluorescence went into satura-

tion. Therefore, further time-lapse recordings were per-

formed in which saturation was carefully avoided

(Figure 6B; Video S1). The uptake kinetics was analyzed

by determining the fluorescence over time for circular

regions of interest (ROI). For nearly all cells showing the

rapid import, the intracellular fluorescence reached a pla-

teau (Figure 6C). However, individual cells strongly varied

in the level of fluorescence at which this plateau was

reached. In addition, there was no correlation between the

uptake kinetics and the increase in fluorescence. In one

case, a strong entry of peptide occurred within 4 min, in

another case, it was within 10 min. A similar variation in

time was observed for cells with a much weaker uptake.

Very remarkably in some cases, import was not continu-

ous. Instead, the increase in cellular fluorescence occurred

in sequential steps, each having similar kinetics and height.

Such a discontinuous stepwise import would be highly

unusual for endocytosis.

A correlation of this increase in cellular fluorescence with

the image sequences showed that for at least some cells,

each step was accompanied with the formation of an NZ

and the dissipation of fluorescence from this NZ into the

cytoplasm (Figure 6B,C). If no NZ was visible during

uptake, this was probably a result of the confocal nature

of the image acquisition that restricted fluorescence

detection to one section through the cell.

In addition, for some cells, after local enrichment of

fluorescence, a bleb-like membrane protrusion was

formed that culminated in the pinching-off of a fluorescent

vesicular structure. An example for a large bleb is shown in

Figure 6D. Concomitant with bleb formation, the rapid

uptake of fluorescence and distribution of fluorescence

into the cytoplasm and nucleus occurred. In some cases,

a fluorescent vesicle was suddenly visible next to an NZ in

only one image frame. It is therefore very likely that in

many of the cases in which no bleb was detected, this was

because of the timing of the image acquisition and/or the

confocality of the detection volume.

The compensatory uptake of R9 in the presence of MbCD

exhibited the same characteristics (not shown). To exclude

(i) that the formation of NZ and rapid cellular uptake were

because of the presence of fluorophore and (ii) that the

cytoplasmic fluorescence was a result of a concentration-

dependent release of proteolytic breakdown products into

the cytoplasm, unlabeled R9 (15 mM) was mixed with

fluorescein-labeled R9 (5 mM) peptide. The formation of

NZs, the import kinetics and the cellular distribution fully

corresponded to the one observed for the fluorescein-

labeled analogue alone (not shown). In addition, the same

formation of NZ, uptake kinetics and distribution were

observed for a tetramethylrhodamine-labeled Tat peptide,

further excluding a role of the fluorophore in our observa-

tions. For MbCD-treated HeLa cells, on incubation with

50 mM Antp, NZ were also observed.

In the NZ, peptides are transiently confined

Fluorescence microscopy had failed to resolve distinct

vesicles at the NZs. As an alternative means to obtain

information about a possible vesicular confinement of the

peptide within the NZ, we compared the mobility of the

peptide within and outside the NZs using fluorescence loss

in photobleaching [FLIP (37)]. The peptide-associated

fluorescein fluorescence was bleached in ROI either inside

or outside the NZ (Figure 7). Bleaching inside the NZ

resulted in the complete destruction of fluorescence not

only in the bleached region but also in the whole cell. In

contrast, bleaching outside the NZ depleted all fluorescein

fluorescence, except the NZ-associated signal, providing

strong evidence for a confinement or association of

peptide within these structures. These FLIP experiments

had been conducted with peptide present in the medium.

To decide whether the NZ-associated fluorescence after

bleaching of fluorescence outside the NZ represented

peptide that was permanently associatedwith a subcellular

structure or only transiently confined peptide, cells were

washed prior to FLIP inside the nucleus. In this case, FLIP

resulted in the depletion of all fluorescence (not shown).

This result confirms that fluorescence inside the NZs

represents a highly dynamic trafficking state of the pep-

tides. This observation also explains why in previous

experiments, in which cells were routinely washed prior

to microscopy, it had been impossible to detect the NZ.

The formation of NZs is CPZ sensitive and dynamin

and heparan sulfate dependent

Having shown that cytoplasmic and nuclear peptide deliv-

ery of R9 and Tat could be inhibited by CPZ, we asked

whether this inhibitor directly affected the development of

NZ. Pretreatment of cells with CPZ abolished the develop-

ment of NZ and the efficient internalization of peptide

(Figure 8A). The rapid import kinetics of the NZ-mediated

Figure 7: In the NZs, peptides are transiently confined. FLIP

inside A) and outside B) an NZ. Fluorescein-labeled peptides were

bleachedwithin the indicated areas (ROIs) for 60 seconds with the

488-nm line of an argon ion laser.

856 Traffic 2007; 8: 848–866

Duchardt et al.

uptake and the failure to detect vesicles questioned

whether the interference of CPZ with this import pathway

was in fact a result of the inhibition of clathrin-dependent

endocytosis. To address this point by an independent

experimental approach, HeLa cells were used in which

endocytosis by clathrin-coated pits and caveolae/lipid rafts

could be disrupted through the inducible expression of

a DN mutant dynamin-2. Dynamin is a guanosine triphos-

phatase, which is critically involved in the scission of

vesicles in these endocytic pathways (38,39). As ex-

pected, the expression of DN dynamin inhibited the

internalization of transferrin, a classical marker for CME

(Figure 8B, lower panels), leading to the enrichment of

receptor-bound transferrin at the plasma membrane.

Figure 8: Uptake through NZ is sen-

sitive to CPZ and is dynamin and

heparan sulfate dependent. A) Inhibi-

tion of NZ formation by CPZ. Cells were

incubated with the compound 30 min

before addition of 20 mM peptide. B)

HeLa dynK44A cells were incubated for

24 h without or with tetracycline (1 mg/

mL). Then, the cells were incubated

with 20 mM R9 or with a transferrin

Alexa Fluor 633 conjugate for 30 min

at 378C. White arrows indicate NZs. C)

Cells were treated for 30 min with

either ammonium chloride (50 mM) or

chloroquine (100 mM) or remained

untreated. The images show the uptake

of R9 after 30 min. Scale bars represent

20 mm. D) HeLa cells were treated for

6 h with heparinases I, II and III (10, 5,

and 2 U/mL, respectively) or remained

untreated. Then, the cells were washed

and incubated with peptide-containing

medium. Images were taken 20 min

after addition of peptide. Removal of

HSPG from the cell surface was con-

firmed by immunofluorescent staining

of treated and untreated cells. Scale

bars correspond to 20 mm.

Traffic 2007; 8: 848–866 857

Uptake of Cationic Cell-Penetrating Peptides

In addition, the induction of DN dynamin abolished the

development of NZs and consequently the delivery of

peptide into the cytoplasm (Figure 8B, upper panels).

For the full-length Tat protein and CPPs, it had been shown

that acidification of the endosomal content is required for

the release of CPPs into the cytoplasm (12,20,21). We

therefore probed for a role of acidification in the NZ-

mediated rapid peptide import into the cytoplasm. Contra-

dictory results were obtained for the two well-established

inhibitors of endosomal acidification, ammonium chloride

and chloroquine (Figure 8C). Treatment of cells with

ammonium chloride completely abolished the develop-

ment of NZs. In contrast, a clear vesicular staining could

be detected. In contrast, chloroquine did not interfere with

the rapid cellular uptake through NZ.

Moreover, we probed for an involvement of heparan sulfate

in peptide uptake. For the uptake of the Tat protein, heparan

sulfate proteoglycans were shown to be involved in uptake

(40–42). In addition, the interaction of R9 with these

molecules was directly shown by biochemical experiments

(43). Heparan sulfate was selectively removed from the cell

surface by treatment of cells with heparinases. Interest-

ingly, different effects on uptake were observed at high and

low concentrations of R9 (Figure 8D). At a low peptide

concentration, heparinase treatment was without effect.

Neither the number of peptide-loaded vesicles nor their

cellular localization was altered. In contrast, at high peptide

concentration, the number of cells showing the develop-

ment of NZs as well as the rapid delivery of R9 into the

cytoplasm and nucleus was reduced. Therefore, the contri-

bution of heparan sulfate to peptide uptake is restricted to

the highly efficient uptake through NZs.

By electron microscopy, NZs are indistinguishable

from the rest of the cell

Light microscopy had failed to provide evidence for an

involvement of vesicular structures in the NZ-mediated

cellular uptake of nona-arginine and Tat. However, the

abolition of NZ formation in cells expressing DN dynamin

and the inhibitory effect of CPZ suggested a clathrin-

dependent endocytic process. To address the presence

of densely packed small vesicles that could not be resolved

by fluorescence light microscopy, a fine structural analysis

of NZ was performed by electron microscopy. Growth of

cells on coverslips structured with co-ordinates enabled

a matching of fluorescence and electron microscopy

images (Figure 9). Paraformaldehyde fixation required for

sample preparation for electron microscopy maintained

the distribution of fluorescence typical for NZ. By electron

microscopy, the NZs were indistinguishable from the

remainder of the cell. Individual clathrin-coated vesicles

could be identified within the cells (not shown). However,

there was no indication of an enrichment of clathrin-

containing structures either inside the cytoplasm or at

the plasma membrane.

The internalization of R9 at high peptide

concentrations is sensitive to the

PKC inhibitor rottlerin

Electron microscopy strongly indicated that uptake

through NZ occurs by an endocytosis-independent mech-

anism. In this case, the activity of CPZ should be because

of interference with processes other than assembly of the

AP-2 coat on nascent clathrin-coated vesicles (44). As CPZ

was shown to interfere with calcium-dependent signaling,

we therefore tested a panel of inhibitors reported to also

interfere with calcium-dependent and calcium-independent

signaling processes. Ro-318220, which inhibits the calcium-

dependent PKCa (45), was without effect on the inter-

nalization of 20 mM R9 through NZ. The PI-3 kinase inhibitor

wortmannin, which had been shown to interfere with

several steps in endosomal trafficking including clathrin-

dependent processes (46,47), was also without effect. In

contrast, preincubation of cells with rottlerin, a compound

that had been reported to be a preferential inhibitor of

PKCd (48), albeit with specificity that was questioned later

(45), abolished the development of the NZ (Figure 10A). R9

uptake at low peptide concentrations remained unaffected

(Figure 10B). Rottlerin had no effect on the endocytosis of

transferrin, providing further evidence that the activity of

CPZ on NZ-dependent import was not a result of interfer-

ence with clathrin-dependent endocytosis. In cells not

treated with rottlerin, the formation of NZ also had no

effect on the endocytosis of transferrin, showing that both

clathrin-dependent processes were spatially and function-

ally distinct (Figure 10A, top panels). Even though these

further inhibitor-based analyses do not provide a conclusive

picture on the molecular mechanism involved in the NZ-

mediated uptake, the inhibition by both CPZ and rottlerin

constitutes a distinct pharmacological profile of this pro-

cess, which may guide the further elucidation of this

uptake mechanism.

The formation of NZs is a characteristic of different

cell types

Having shown distinct characteristics of the uptake of Tat

and R9 through NZ, we next wanted to learn whether this

uptake mechanism is a particular characteristic of HeLa

cells. For this reason, we selected a panel of cells derived

from different species, different tissues and with different

growth characteristics: MC57, mouse fibrosarcoma cells;

Chinese hamster ovary cells; primary human dendritic cells

and human Jurkat T-cell leukemia cells. In all cell lines, we

observed a concentration-dependent formation of NZs

(Figure 11). In addition, in all cases, the cell population

was heterogeneous with respect to the formation of NZ.

These results show that the formation of NZ is not only

a mechanism present in cancer cells and furthermore that

cell attachment is not a prerequisite for this uptake

mechanism. For Jurkat cells, we tested whether incuba-

tion with MbCD could also induce cytoplasmic delivery of

Antp. In correspondence to the observation with HeLa

cells, Jurkat cells showed a homogeneous cytoplasmic

fluorescence, albeit for a higher fraction of cells.

858 Traffic 2007; 8: 848–866

Duchardt et al.

Relevance of the import mechanism for the cellular

activity of cargo peptides

Antennapedia, R9 and Tat have been used as vectors for

the cellular delivery of bioactive cargos. Having shown

that the cellular uptake and cytoplasmic delivery of Antp

are different from the one of the other two peptides, we

finally investigated the relevance of these import charac-

teristics for the bioactivity of a peptide delivered through

Figure 9: Electron microscopy of NZs. A and B) NZs in two different cells within one field of view. HeLa cells were grown on surface-

structured coverslips that enabled a matching of cells in fluorescence and electron microscopy images. Confocal laser scanning

microscopy was performed on paraformaldehyde-fixed cells. For each magnification, a pair of fluorescence and electron microscopy

images is shown. The fluorescence images in the center and bottom panels weremagnified beyond the resolution limit and are intended to

show the correspondence of the electron micrographs with NZ based on larger morphological features, such as the dark spots (left). Scale

bars are 30 mm (top), 6 mm (center) and 3 mm (bottom).

Figure 10: Sensitivity of R9 up-

take at high concentrations to

rottlerin. Cells were either treated

with rottlerin (20 mM) or remained

untreated and then medium contain-

ing either A) 20 mM R9 and transferrin

Alexa Fluor 633 conjugate (10mg/mL)

or B) 5 mM R9 was added to the cells.

Scale bars represent 20 mm. Note

that instrument settings for fluo-

rescein detection at 5 and 20 mM

were different because of the 10-

fold difference in intracellular signal

intensity.

Traffic 2007; 8: 848–866 859

Uptake of Cationic Cell-Penetrating Peptides

conjugation either to Antp or to R9. We have shown

previously that after endocytosis, a large fraction of

imported peptides is broken down by endolysosomal

proteases (49). One should therefore expect that an

uptake mechanism that bypasses the endolysosomal

compartment should lead to an increased activity of the

cargo peptide. As bioactive cargo, we selected a peptide

corresponding to the N-terminal seven amino acids of the

proapoptotic protein Smac (23) (Table 1). Cytoplasmic

delivery of the Smac peptide enhances apoptosis by

promotion of caspase-3 activation (25). Because the free

N-terminus of this peptide is required for bioactivity, the

peptide was C-terminally elongated by the respective

CPP, carrying a fluorescein moiety at its C-terminus

(50). Analysis of the uptake of both conjugates confirmed

that the internalization mechanisms observed for the

CPPs alone remained valid for the CPP–cargo conjugates

(Figure 12A). Over the tested concentration range, inter-

nalized Smac–Antp exhibited a concentration-independent

vesicular staining. In contrast, Smac–R9 at 5 mM showed

an exclusive vesicular staining. At 10 mM, the cell popu-

lation was heterogeneous, with some cells showing

bright cytoplasmic and nuclear fluorescence. At 20 mM,

the peptide was internalized through NZ and delivered

efficiently into the cytoplasm and nucleus. Surprisingly,

for this peptide, we observed an enrichment of fluores-

cence within the nucleoli, an observation that we cannot

explain in the moment, however, which has been reported

before (19).

To compare the bioactivity of the Smac peptide delivered

through either Antp or R9, caspase-3 activation and cell

viability after stimulation of the death receptor Fas were

selected as readouts. We had shown previously that in

contrast to the tumour necrosis factor (TNF) receptors, Fas

was not subject to CPP-dependent downregulation from

the plasma membrane (50). Stimulation of Fas with an

agonistic antibody in the presence of cycloheximide resulted

in a 10-fold induction of caspase-3 activity. Pretreatment of

cells with the Smac–R9 conjugate potentiated the induction

of caspase-3 activation in a concentration-dependent man-

ner byup to 100% (Figure 12B). In contrast,when theSmac-

derived peptidewasdelivered into the cells through theAntp

peptide, the enhancement of Fas-mediated caspase-3 acti-

vation was less efficient (only up to 30%). A similar differ-

ence in efficiencies exerted by Smac–R9 and Smac–Antp

was observed when cell viability after 24 h was analyzed

as a parameter for the efficiency of apoptosis induction

(Figure 12C). While the treatment of cells with the

Smac–R9 peptide reduced the cell viability by up to 50%,

Smac–Antp had no detectable effect on viability. This experi-

ment supports the intracellular integrity of the peptides

after rapid cytoplasmic delivery, which is a requirement for

the expected biological effect.

Discussion

Concentration dependence of the uptake of Antp,

R9 and Tat

This work shows that Antp on one hand and R9 and Tat on

the other hand differ with respect to the contributions of

individual endocytic processes to the uptake of these

peptides. Moreover, the mechanism of entry depends on

peptide concentration and the availability of alternative

endocytic routes. Above a critical threshold, internalization

Figure 11: Existence of the uptake mechanism in other cell

types. Mouse MC57 fibrosarcoma cells, Chinese hamster ovary

cells, human primary dendritic cells and human Jurkat leukemia

T cells were incubated withmedium containing R9 (2 or 20 mM). The

images were taken either 30 min (for 2 mM R9) or 15 min (for 20 mM

R9) after peptide addition. The scale bars represent 10mm.Note the

differences in the cell morphology between cell types. The nuclei of

dendritic cells are smaller than those of MC57 cells, and the

perinuclear area exhibits less intensive peptide staining.

860 Traffic 2007; 8: 848–866

Duchardt et al.

occurs through a highly efficient nonendocytic pathway.

This pathway originates from spatially confined areas of the

plasma membrane and leads to a rapid release of peptides

into the cytoplasm. The integrity of the plasmamembrane is

fully maintained. Antennapedia differs from the arginine-rich

CPPs nona-arginine and Tat in that much higher concen-

trations and at least partial blockade of endocytic uptake are

required for the induction of this pathway.

In the concentration range up to 40 mM, for the Antp

peptide, there is strong evidence that at least three

endocytic pathways, that is macropinocytosis, CME and

caveolae/lipid-raft-mediated endocytosis contribute to

uptake. Neither of these three pathways dominates.

Pharmacological interference with any one pathway yields

a distinct phenotype with respect to the subcellular

distribution of fluorescence and is without effect on the

other two pathways. The simultaneous inhibition of two

endocytic pathways leads to the addition of the respective

inhibitory effects.

For R9 and Tat at concentrations below 10 mM, entry of

these peptides is sensitive to MbCD and EIPA, strongly

indicative of entry through caveolae/lipid-raft-mediated

endocytosis and macropinocytosis. Remarkably, there

was no evidence for an involvement of CME at this

concentration. At a concentration of 5 mM, inhibition of

caveolae/lipid-raft-mediated endocytosis or macropinocy-

tosis induces the rapid NZ-dependent uptake mechanism.

The induction of this mechanism at a concentration

of peptide at which, in the absence of inhibitors, up-

take occurs through endocytosis indicates that a critical

concentration of peptide associated with the plasma

membrane is required. Because of the redirection of pepti-

des toward this pathway, the effect of EIPA and MbCD is

somewhat of a paradox: instead of inhibiting the uptake of

Figure 12: Relevance of the uptake mechanism for the bioactivity of a cargo peptide. A) HeLa cells were incubated with either

Smac–Antp or Smac–R9 at the indicated concentrations for 30 min at 378C. The scale bars represent 20 mm. B and C) After incubation of

cells with peptides for 30 min, the cells were washed and apoptosis was induced by stimulation with an agonistic Fas-specific antibody

(100 ng/mL) and CHX (2 mg/mL). B) For measurements of caspase-3 activity, cells were harvested 4 h after the induction of apoptosis. The

bars represent the results of three independent experiments. C) Determination of cell viability was performed using crystal violet staining

24 h after stimulation of Fas.

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Uptake of Cationic Cell-Penetrating Peptides

peptides into the cells, at this concentration, these inhibi-

tors actually promote the uptake. This pathway mediates

a more rapid peptide uptake than the other two pathways,

one indication for a nonendocytic nature of this import

mechanism. When the rapid uptake mechanism is blocked

by CPZ, the phenotypes observed for cells treated with

MbCD or EIPA are recovered.

At higher concentrations, uptake occurs primarily by a

mechanism leading to rapid entry of the peptides into the

cytoplasm and nucleus. The EIPA- and MbCD-sensitive

mechanisms still contribute to peptide uptake as shown

by the fact that the inhibition of one of these pathways

still increases the cellular fluorescence even more. Con-

sidering our previous observations on the CPP-induced

endocytosis of TNF receptors by these three cationic CPPs

(50), one should note that Antp was more effective in

inducing the internalization of the receptors than R9 and

the Tat peptide. At low peptide concentrations, Antp was

in fact the only peptide inducing receptor internalization.

The internalization of EGF receptors suggested that this

internalization occurred through a clathrin-dependent mech-

anism. Consistently, at low concentrations, only entry of

Antp was CPZ sensitive. The internalization induced by Tat

and R9 at higher concentrations suggests that a fraction of

these peptides, in addition to the rapid NZ-mediated

uptake, was also internalized by clathrin-dependent endo-

cytosis, albeit with a much slower kinetics. For Antp, this

rapid cytoplasmic uptake was only observed at sig-

nificantly higher peptide concentrations for cells treated

with MbCD.

Uptake through NZs

The highly efficient uptake of R9 through NZs was heparin

sulfate and dynamin dependent; sensitive to ammonium

chloride, CPZ and rottlerin and insensitive to chloroquine.

The dynamin dependence and sensitivity to CPZ, when

considered in isolation, are strongly indicative of clathrin-

dependent endocytosis. However, the failure to detect

vesicles by electron microscopy; the rapid, stepwise

and spatially confined uptake kinetics and finally the con-

comitant formation of membrane blebs all point toward a

nonendocytic mechanism.

The ability to abolish this uptake by pharmacological

interventions that inhibit molecular processes inside the

cell indicates that association of peptide with the plasma

membrane alone is insufficient to initiate this process.

Instead, accumulation of peptide at the plasma membrane

induces an active process inside the cell. Apparently, this

process requires a certain minimum concentration of

membrane-associated peptide. At medium peptide con-

centrations, peptides are steadily removed from the

plasma membrane by endocytosis. Different import rates

of Antp and the other two peptides by the various

endocytic processes may therefore be one factor contri-

buting to the lower propensity of Antp to enter along this

pathway – a hypothesis that is supported by the ability to

induce this uptake by MbCD in HeLa and Jurkat cells.

Once endocytosis is blocked, peptides accumulate and the

threshold is reached. The initial formation of the NZ, bleb

formation and rapid release into the cytoplasm are tightly

coupled processes. All interventions that blocked this

uptake also blocked the formation of a NZ. It will be

interesting to seewhether an intervention can be identified

that only blocks the second step.

Considering the absence of vesicles in the electron

microscopy images, the blockade of the uptake by expres-

sion of DN dynamin is somewhat surprising. However, the

formation of blebs is a process involving rapid changes of

membrane morphology. Dynamins couple to a large num-

ber of cellular signaling processes (51), and even though it

is not clear why the peptides induce the formation of

outward protrusions, dynamins could well be involved in

such a process.

Interestingly, for any given cell for which uptake occurred

by distinctive steps, these steps frequently were similar

in size. One may hypothesize that the peptides are

enriched on specific domains of the plasma membrane

and that these domains have a certain capacity to bind

peptide that varies from cell to cell. At this point, neither

the nature of these domains nor the details of the uptake

mechanism is clear. At least, our observations favor an

‘accumulate and discharge’ mechanism over a mecha-

nism involving a transient formation of pores or continu-

ous transfer of peptides across the lipid bilayer. The FLIP

experiments also strongly support a transient association

of the peptides with molecules inside and on the surface

of the cell.

Heparan sulfates are candidate molecules for mediating

this association. Cell surface heparan sulfates have been

considered to play a role as multivalent, low-affinity

receptors of cationic CPPs. Remarkably, however, at low

peptide concentrations, removal of cell surface heparan

sulfates was without effect on uptake, at least as detect-

able by fluorescence microscopy. Consistent with our

results, for the Tat peptide at 100 mM, a requirement for

cell surface heparan sulfate for peptide was shown (14),

while at 1 mM, heparan sulfates were not required (52). It is

difficult to understand why these molecules should func-

tionally act as receptors only at high concentrations. One

possible mechanism may be through cross-linking by

polyvalent binding of peptides. Guanidinium groups have

been proposed to engage in bidentate interactions, pos-

sibly with heparan sulfates on the cell surface (53,54). The

higher fraction of arginine side chains in the R9 and Tat

oligopeptides in comparison with Antp may explain the

higher propensity of the former to induce this uptake

mechanism. Moreover, such a mechanism would be

consistent with ammonium chloride acting as an agent

that shields off negative charges on the cell surface.

862 Traffic 2007; 8: 848–866

Duchardt et al.

Reassessment of previous results

To this point, a number of publications have addressed the

mechanisms contributing to the cellular uptake of CPPs,

with sometimes conflicting results. Observations of a rapid

endocytosis-independent mechanism have been the latest

addition in this field (18,19). Our model provides a frame-

work to accommodate these previous observations. First

of all, our data show that the individual CPPs, albeit all

cationic in nature, possess remarkable differences in their

import pathways. Moreover, it is now clear that peptide

concentration is a key experimental condition when ana-

lyzing peptide uptake. Potocky et al. investigated the

cellular delivery of the Tat peptide at a concentration of

7 mM. Three different phenotypes with respect to the

distribution of the peptide in HeLa cells were observed

(21). While some cells exclusively exhibited a vesicular

staining, some cells showed a combination of vesicular

and cytoplasmic fluorescence, and finally in some cells,

fluorescence was only present in the cytoplasm and

nucleus. We show that this heterogeneity in the distribu-

tion of the peptide is likely related to the fact that

a concentration higher than 5 mM had been used. When

cells were incubated with the Tat peptide at concentra-

tions of 1–5 mM, the peptide was located only in vesicles

(20). The partial colocalization of the Tat peptide with

transferrin (11,21) as well as with dextran (20) may now

readily be explained by the finding that the peptides

simultaneously use several endocytic pathways.

To this point, we restricted ourselves to CPPs, two ofwhich

correspond to PTDs of the respective full-length proteins. It

needs to be resolved to which degree the import mecha-

nisms of the PTDs correspond to the ones of the full-length

proteins. Still observations reported for the Antp and Tat

peptide conjugated to protein cargos as well as for the full-

length Tat protein are also fully consistent with our model.

Antennapedia and Tat peptides differed substantially in

their capacity to deliver avidin into HeLa cells (29). For

a Tat-derived peptide (aa 11) and the Tat protein, conjugated

to green fluorescent protein (GFP), evidence was pre-

sented that both conjugates are internalized through the

same lipid-raft-dependent mechanism (17). The concentra-

tion of the peptide–GFP conjugate was less than 1 mM. At

this concentration, lipid-raft-mediated endocytosis as a key

uptake mechanism is fully consistent with our model.

Moreover, for a Tat–Cre conjugate used at a concentration

of less than 1 mM, both lipid-raft-mediated endocytosis and

macropinocytosis were identified as mechanisms contrib-

uting to internalization (15). Finally, both the Tat peptide and

the full-length Tat protein induce the internalization of TNF

receptors from the plasma membrane (50), indicating the

same mechanism of uptake.

The relevance of our findings for the application of CPPs is

supported by the fact that the uptake mechanisms identi-

fied for CPPs alone retain their validity also for cargo

peptides that are introduced into the cells through coupling

to CPPs. Moreover, the demonstration that the uptake

mechanism determines the cargo bioactivity stresses the

importance of understanding in detail the mode of inter-

nalization of CPPs for their application as delivery vectors.

In conclusion, our results provide a comprehensive frame-

work to encompass most previous observation on the

cellular uptake of cationic CPPs. One given CPP may not

only use different endocytic pathways for cellular entry but

may also use nonendocytic entry routes. Considering the

differences between Antp and the other two peptides, the

fraction of arginine side chains may be a relevant structural

characteristic, influencing the contribution of individual

entry pathways to uptake, possibly by induction of cross-

linking of cell surface heparan sulfates. Concerning the

rapid cytoplasmic uptake, a distinctive pharmacological

profile was defined. The data indicate that the uptake is

a specific response of the cell to a local enrichment of

cationic molecules at the plasma membrane. It is not clear

so far whether the CPPs initiate the enrichment of the

cellular molecules to which they associate or whether the

NZ represent preformedmembrane domains. Finally, it will

be interesting to investigate whether a physiological role

for this pathway exists.

Materials and Methods

Cells and reagentsThe human cervical carcinoma cell line HeLa was obtained from the

American Type Culture Collection (Manassas, VA, USA). HeLadynK44A

expressing a DN form of dynamin-2 under the control of a tet on/off

promoter, cultured in medium containing 1 mg/mL tetracycline, was a kind

gift from Bo van Deurs (University of Copenhagen, Copenhagen, Denmark).

The expression of mutant dynamin was induced by tetracycline deprivation

for at least 24 h. Transferrin Alexa Fluor 633 conjugate, dextran Alexa Fluor

647 conjugate and CTB Alexa Fluor 555 conjugate and Zenon mouse IgG1

labeling kit (specific for the Fc part of immunoglobulin G1 antibodies) were

purchased from Mobitech (Gottingen, Germany). Fluorogenic caspase-3

substrate (Ac-DEVD-AMC), wortmannin, Ro-318220, rottlerin and CPZ

were from Calbiochem (Bad Soden, Germany); ammonium chloride, EIPA,

MbCD, MTT and heparinase I, II and III were obtained from Sigma

(Deisenhofen, Germany). The anti-Fas-activating antibody was obtained

from Upstate (Hamburg, Germany). BSA was obtained from SIGMA

(Steinheim, Germany). The anti-heparan sulfate proteoglycan (HSPG)

vesicular stomatitis virus (VSV)-tagged single-chain antibody HS4C3 (55)

and the mouse anti-VSV (clone P5D4) antibody were a kind gift of Toin van

Kuppevelt (Radboud University Nijmegen Medical Centre, Nijmegen, The

Netherlands).

Peptide synthesisAutomated peptide synthesis was performed by solid-phase Fmoc/tBu-

chemistry using an automated peptide synthesizer for multiple peptide

synthesis (RSP5032; Tecan, Hombrechtlikon, Switzerland) in 2-mL syringes

according to a protocol described elsewhere (20). Smac–Antp and Smac–

R9 (Table 1) were synthesized using a Na-carboxyfluorescein-labeled lysyl-

Rink amide resin (56). The purity of all peptides was determined by

analytical high-performance liquid chromatography (HPLC). The identity of

the peptides was confirmed by MALDI-TOF mass spectrometry. Peptides

with a purity of less than 95% were purified by preparative HPLC.

Flow cytometryHeLa cells were incubated with medium containing peptides at the

indicated concentrations for 30 min at 378C. After incubation, the cells

Traffic 2007; 8: 848–866 863

Uptake of Cationic Cell-Penetrating Peptides

were washed with medium, detached by trypsinization for 5 min, washed,

suspended in PBS and measured immediately by flow cytometry (BD

FACSCalibur System; Becton Dickinson, Heidelberg, Germany). In each

case, the fluorescence of 10 000 vital cells was acquired. Vital cells were

gated based on sideward and forward scatter.

Confocal laser scanning microscopyConfocal laser scanning microscopy was performed on an inverted LSM510

laser scanning microscope (Carl Zeiss, Gottingen, Germany) using a Plan-

Apochromat 63� 1.4 N.A. lens. All measurements of peptide uptake were

performed with living, nonfixed cells grown in eight-well chambered cover

glasses (Nunc, Wiesbaden, Germany). Cells were seeded at a density of

4 � 104/well 1 day before the experiment and cultured in RPMI-1640

supplemented with 10% fetal calf serum. For detection of fluorescein-

labeled peptides, the 488-nm line of an argon ion laser was directed over an

HFT UV/488 beam splitter, and fluorescence was detected with a BP 505–

550 band pass filter. For the simultaneous detection of fluorescein-labeled

peptides and Alexa Fluor 633 or -647 conjugates, the 488-nm line of an

argon ion laser and the light of a 633-nm helium neon laser were directed

over an HFT UV/488/633 beam splitter, and fluorescence was detected

using an NFT 545 beam splitter in combination with a BP 505–530 band

pass filter for fluorescein detection and an LP 650 long pass filter for Alexa

Fluor 633 and Alexa Fluor 647 detection. Life cell microscopy was

performed at room temperature.

Quantitative analysis of colocalizationFirst, color channels containing the signal of fluorescein-labeled Antp and

the respective tracer were extracted from the multichannel confocal

images. After low-pass filtering to reduce image noise, for both channels,

binary masks corresponding to vesicle-associated fluorescence were

generated (MaskAntp and MaskTracer). Threshold levels were selected by

visual inspection. A third mask containing those pixels in which vesicle-

associated fluorescence was present in both channels was generated by

applying an AND-operation to the masks for peptide- and tracer-associated

fluorescence (MaskColoc). Because of the presence of peptide and tracer in

the medium, all three masks also included all pixels outside the cells. For

this reason, individual cells were selected as ROI and the number of objects

(NAntp, NTracer and NColoc) within each cell corresponding to individual

vesicles or small groups of vesicles in all three binary masks was counted

using the object analysis routines within Image Pro Plus 4.5 (Media

Cybernetics Inc., Silver Spring, MD, USA). The fraction of tracer-positive

vesicles colocalizing with Antp was calculated by dividing NColoc by NTracer,

and the fraction of Antp-positive vesicles colocalizing with tracer was

calculated by dividing NColoc by NAntp.

Electron microscopyHeLa cells were grown on CELLocate 5245, with a square size of 55 mm

(Eppendorf, Hamburg, Germany). After addition of medium containing R9 in

a concentration of 20 mM, formation of NZ was followed by confocal laser

scanning microscopy as described previously. Directly after appearance of

NZ, cells were washed with ice-cold PBS and subsequently fixed with 4%

formaldehyde in PBS for 30 min at room temperature. For electron

microscopy, cells were stored for 1 h at 48C, postfixed with 1% osmium

tetroxide in 100 mM phosphate buffer at pH 7.2 for 1 h on ice, washed with

H2O, treated with 1% aqueous uranyl acetate for 1 h at 48C, dehydratedthrough a graded series of ethanol and embedded in Epon. Ultrathin

sections were stained with uranyl acetate and lead citrate and viewed in

a Philips CM10 electron microscope. The imprint of the cellocate coverslip

in the Epon resin was used as an orientation for the matching of

fluorescence and electron microscopy images.

Incubation with inhibitorsCells were treated with the indicated inhibitors for 30 min at 378C. Then,the medium was removed and medium containing peptide as well as the

corresponding inhibitor was added. After 30 min of incubation at 378C,the cells were washed twice with medium and analyzed by fluorescence

microscopy or flow cytometry.

ImmunofluorescenceHeLa cells were seeded at a density of 4 � 104/well in eight-well

chambered covered glasses and incubated with medium containing 10 U/

mL of heparinase I, 5 U/mL heparinase II and 2 U/mL heparinase III for 6 h at

378C. Then, the cells were washed with ice-cold PBS containing 0.1% (w/v)

BSA and incubated with the anti-HSPG antibody HS4C3 on ice. After 1.5 h

of incubation, cells were washed with ice-cold PBS/BSA. To visualize bound

antibodies, the cells were incubated for 1 h with an anti-VSV antibody

(P5D4)/Zenon Alexa Fluor 647 conjugate on ice. The antibody staining was

analyzed by confocal laser scanning microscopy.

Caspase-3 activity assayHeLa cells were incubated with medium containing peptides for 30 min at

378C. After one washing step, cells were treated as indicated with agonistic

Fas antibody and cycloheximide (CHX) for the induction of apoptosis,

followed by incubation for further 3 h. Cells were harvested by scraping,

washed with ice-cold PBS and lysed in lysis buffer [1% Triton, 150 mM

NaCl, pH 7.7, supplemented with protease inhibitor cocktail tablets (Roche

Diagnostics, Mannheim, Germany)] for 30min on ice. The protein content in

lysates was determined using a commercially available Bradford protein

assay kit (Bio Rad Laboratories, Munchen, Germany). Equivalents of 30 mg

protein for each sample were diluted in caspase activity buffer (20 mM

HEPES, 10 mM dithiothreitrol, 10% glycerol, 100 mM NaCl, pH 7.5).

Caspase-3 substrate was added to the samples to a final concentration of

2 mM. The efficiency of the substrate cleavage by active caspase-3 was

analyzed immediately after substrate addition and after 1 h of incubation at

378C using a luminescence spectrometer LS50B (PerkinElmer, Norwalk,

CT, USA).

MTT assayHeLa cells were seeded in 96-well microtiter plates (1.5 � 104/well) and

cultivated over night. The next day, cells were incubated with peptides for

6 h at 378C. Cell viability was measured using the colorimetric MTT dye.

Cells were incubated with MTT at a concentration of 1 mg/mL for 4 h. The

formazan product was solubilized with SDS [10% (w/v) in 10 mM HCl]. Cell

viability was determined by measuring the absorbance of each sample at

570 nm using a microplate reader (Molecular Devices SpectraMax 340;

GMI, Ramsey, Minnesota, USA).

Cytotoxicity assayHeLa cells were treated with the indicated peptides for 30 min at 378C,washed and stimulated with the agonistic Fas antibody (100 ng/mL) and

CHX (2 mg/mL) for the induction of apoptosis. After an additional 24-h

incubation at 378C, cells in both groups were washed with PBS, followed by

crystal violet staining [20% (v/v) methanol, 0.5% (w/v) crystal violet] for

15 min. The wells were washed with H2O and air-dried. The dye was dis-

solved in methanol and the optical density at 550 nm measured with an

enzyme-linked immunosorbent assay plate reader.

Acknowledgments

We thank Viktoria Wolf for the isolation and cultivation of human dendritic

cells, Brigitte Sailer for oriented cutting of ultrathin sections and Anne

Spang and Mark Trautwein for helpful discussions. F. D. is a scholar of the

Graduiertenkolleg 794. R. B. gratefully acknowledges financial support

from the Volkswagen Foundation (‘‘Nachwuchsgruppen an Universitaten’’

I/77 472).

Supplementary Materials

Figure S1: The effect of inhibitors of endocytosis on the internaliza-

tion of tracer molecules. HeLa cells were incubated for 30 min at 378C

with the indicated inhibitors of endocytosis (CPZ 10 mg/mL, MbCD 5 mM

864 Traffic 2007; 8: 848–866

Duchardt et al.

and EIPA 100 mM) or remained untreated (control groups).Then, the

medium was removed and cells were incubated for further 30 min with

a transferrin Alexa Fluor 633 conjugate (10 mg/mL), a dextran Alexa Fluor

647 conjugate (10 mM) or CTB Alexa Fluor 555 (10 mg/mL) in the presence or

absence of the corresponding inhibitor. The scale bars represent 20 mm.

Figure S2: The effect of endocytic inhibitors on cell viability. HeLa cells

were incubated with inhibitors of endocytosis, either alone or in combina-

tion, at the indicated concentrations for 2 h at 378C. Cell viability was

determined using crystal violet staining. The frames indicate the concen-

trations of inhibitors applied in the experiments.

Video S1: Endocytosis of R9 through NZs. Medium containing R9 at

a concentration of 20 mM was added to HeLa cells and uptake of

fluorescence was followed by time-lapse confocal microscopy. The time

series was started 1 min after addition of peptide. The video comprises 50

images, with a time interval of 10 seconds between each image.

Supplemental materials are available as part of the online article at http://

www.blackwell-synergy.com

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