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Originally published In Press as doi:10.1074/jbc.M401604200 on February 1, 2005

J. Biol. Chem., Vol. 280, Issue 15, 15300-15306, April 15, 2005
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Cellular Uptake of Unconjugated TAT Peptide Involves Clathrin-dependent Endocytosis and Heparan Sulfate Receptors*

Jean Philippe Richard{ddagger}§, Kamran Melikov¶§||, Hilary Brooks{ddagger}, Paul Prevot{ddagger}, Bernard Lebleu{ddagger}, and Leonid V. Chernomordik¶

From the {ddagger}UMR 5124 CNRS, Université Montpellier 2, place Eugène Bataillon, 34095 Montpellier cedex 5, France and Section on Membrane Biology, Laboratory of Cellular and Molecular Biophysics, NICHD, National Institutes of Health, Bethesda, Maryland 20892-1855

Received for publication, February 13, 2004 , and in revised form, December 20, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Delivery of macromolecules mediated by protein transduction domains (PTDs) attracts a lot of interest due to its therapeutic and biotechnological potential. A major reevaluation of the mechanism of PTD-mediated internalization and the role of endocytosis in this mechanism has been recently initiated. Here, we demonstrate that the entry of TAT peptide (one of the most widely used PTDs) into different primary cells is ATPand temperature-dependent, indicating the involvement of endocytosis. Specific inhibitors of clathrin-dependent endocytosis partially inhibit TAT peptide uptake, implicating this pathway in TAT peptide entry. In contrast, the caveolin-dependent pathway is not essential for the uptake of unconjugated TAT peptide as evidenced by the efficient internalization of TAT in the presence of the known inhibitors of raft/caveolin-dependent pathway and for cells lacking or deficient in caveolin-1 expression. Whereas a significant part of TAT peptide uptake involves heparan sulfate receptors, efficient internalization of peptide is observed even in their absence, indicating the involvement of other receptors. Our results suggest that unconjugated peptide might follow endocytic pathways different from those utilized by TAT peptide conjugated to different proteins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent advances in the identification of new molecular therapy targets and disease-relevant proteins, accelerated by the completion of the human genome project, emphasized an importance of high molecular weight information-rich biomolecules, such as peptides, proteins, antisense DNA, and small interfering RNA, for molecular therapy. However, the delivery of proteins and nucleic acids into cells is greatly hampered by the low permeability of the cell plasma membrane to polar molecules. Not surprisingly, the discovery that a number of cationic peptides known as protein transduction domains (PTDs)1 can facilitate cytoplasmic and nuclear delivery of a conjugated cargo has attracted a lot of interest (1-3). Up to date, a wide range of cargo molecules, including low molecular weight drugs (4), oligonucleotides (5), peptides (6) and even full-length proteins (7-10), have been successfully delivered into cells using PTDs and, most importantly, the functional activity of the delivered cargo has been observed (7-10).

Despite significant progress in the cytoplasmic and nuclear delivery of various cargo molecules using PTDs, the underlying mechanisms remain under active debate. Until recently, it was widely assumed that the internalization of cationic PTDs is an energyand receptor-independent process based on direct transport through the lipid bilayer (11-15). On the other hand, there have been indications that uptake of full-length TAT protein, from which one of the most commonly used PTDs referred to as TAT peptide is derived, occurs via endocytosis and depends on cell surface heparan sulfate receptors (16). Moreover, the validity of some of the important data, supporting a direct transport model for synthetic TAT peptide, has been questioned in several recent studies (17-19). It has been shown that apparent ATP and temperature independence of the cellular uptake of the TAT peptide as well as its fast nuclear accumulation results from experimental artifacts due to cell fixation and incomplete removal of cell-bound peptide (18, 19). Recently, the uptake of TAT and (Arg)9 peptides has been attributed to a vesiculation process inhibited by cellular ATP depletion and by low temperature (19). Although several very recent papers (20-23) provide additional evidence for the involvement of endocytic pathways in the transduction of PTDs alone as well as PTDs conjugated to a cargo, the alternative mechanism of direct translocation has been also argued for in recent literature (24, 25).

In this work, we further clarify the mechanisms by which TAT peptide enters living cells. As in the case of the stable cell lines used in our earlier work, TAT entry into several different primary cells is found to be ATPand temperature-dependent, indicating the involvement of endocytosis. Judging from the effects of specific inhibitors, unconjugated TAT peptide enters cells mainly by a clathrin-dependent endocytic pathway, whereas raft/caveolin-dependent pathway is not required for internalization. Although a significant part of TAT peptide uptake involves heparan sulfate receptors, these receptors are not a prerequisite for TAT entry.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Peptide Synthesis and Labeling—Synthesis of TAT peptide with sequence GRKKRRQRRRPP was carried out by solid phase on a Pioneer peptide synthesizer (Applied Biosystems) following the Fmoc (N-(9fluorenyl)methoxycarbonyl) chemistry protocol. A cysteine was added to the C-terminal end of the peptide to provide a sulfhydryl group for ligation to a fluorochrome. Peptides were purified by preparative high pressure liquid chromatography and characterized by analytical high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight analysis (data not shown). Labeling with the fluorochromes was performed by conjugation with a 10 molar excess of the fluorochrome-maleimide derivatives (Molecular Probes) in 50 mM Tris-HCl buffer, pH 7.2, for 4 h in the dark. Labeled peptides were purified by semi-preparative high pressure liquid chromatography, freeze-dried, and resuspended in deionized water. Peptides were stored frozen at -20 °C until further use.

Cells and Cell Cultures—HeLa and HepG2 cells were cultured as exponentially growing subconfluent monolayers on 90-mm plates in RPMI 1640 medium (Invitrogen) supplemented with 10% (v/v) fetal calf serum and 2 mM glutamine. Wild type CHO cells (CHO-K1 cell line) and mutants lacking all glycosaminoglycans (pgsA-745 cell line) or heparan sulfate (pgsD-677 cell line) were cultured in 75-cm2 flasks in Vitacell Ham's F12K medium (ATCC) supplemented with 10% (v/v) fetal bovine serum and 2 mM glutamine. Primary HUVEC (human umbilical vein endothelial cells) cells were cultured as exponentially growing subconfluent monolayers on 90-mm plates in endothelial cell basal medium (Clonetics) supplemented with 2% (v/v) fetal calf serum, 2 mM glutamine, 1 mg/ml hydrocortisone, 50 µg/ml gentamicin, 50 pg/ml amphotericin B, 0.75 µg/ml bovine brain extract, and 10 µg/ml recombinant human epidermal growth factor. Peripheral blood, obtained from healthy donors, was collected in heparinized tubes. The peripheral blood mononuclear cells were separated by Ficoll-Hypaque (Sigma) as described previously (26).

Flow Cytometry—In the case of HeLa, HepG2, and human macrophages, exponentially growing cells were dissociated with a nonenzymatic cell dissociation medium (Sigma). 5 x 105 cells were plated and cultured overnight on 30-mm dishes. The culture medium was discarded, and cells were washed with NaCl/Pi, pH 7.3. NaCl/Pi was discarded, and cell monolayers were preincubated with Opti-MEM for 30 min. Subsequently, the cell monolayers were incubated, as described below, with peptides and drugs dissolved in Opti-MEM. Following incubation, cells were washed with NaCl/Pi and incubated for 10 min with 0.1% trypsin (with the exception of HUVEC, which were treated with 0.05% trypsin) to detach them and to remove surface-bound material. In some experiments membrane-bound peptides were removed by a 15-min incubation at 4 °C with 0.1% Pronase and 1 mM EDTA. After the incubation, one volume of serum was added to stop the trypsin (or Pronase) treatment and 10 volumes of NaCl/Pi were added to detach the cells completely. The cell suspension was centrifuged at 800 x g, washed with NaCl/Pi, centrifuged again, and resuspended in 500 µl of NaCl/Pi.

In case of wild type and mutant CHO cells, exponentially growing cells were dissociated with a nonenzymatic cell dissociation medium and resuspended in serum-free Ham's F12K medium. Subsequently, 5 x 105 cells were incubated, as described in the figure legends, with 200 µl of TAT peptide dissolved in F12K medium. Following incubation, cell suspension was centrifuged at 800 x g, washed twice with 2 ml of cold NaCl/Pi, and incubated for 10 min with 200 µl of 0.1% trypsin to remove surface-bound peptide. Finally, cells were washed once with 2 ml of F12K medium supplemented with 10% fetal calf serum and once with 2 ml of cold NaCl/Pi and resuspended in 500 µl of NaCl/Pi containing 10 µM propidium iodide (Molecular Probes) to exclude dead cells.

Fluorescence analysis was performed with a FACScalibur fluorescence-activated cell sorter (BD Biosciences). A minimum of 30,000 events/sample was analyzed with the exception of HUVEC cells for which 10,000 events were analyzed. To quantify effects of various treatments on cellular uptake, the median of cell fluorescence distribution in experiment was normalized to the cell fluorescence distribution median in untreated control. Each experiment was performed at least twice in duplicate or triplicate. The means ± S.D. of a total number of analyzed samples (from 3 to 14) are indicated on the figures. The significance of the effects of various treatments as compared with untreated control was evaluated by paired Student's t test at the 95% confidence level.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Uptake of TAT Peptide by Primary Cells Is ATPand Temperature-dependent—Although established cell lines are convenient and useful experimental models, many important biotechnological and biomedical applications involve primary cells. We studied the effects of ATP depletion and low temperature on TAT peptide uptake by human peripheral blood mononuclear macrophages and HUVEC endothelial cells. The primary cells were incubated with Alexa Fluor 488-tagged TAT peptide for 30 min, and the amount of internalized peptide was evaluated using FACS. As shown on Fig. 1, TAT uptake was greatly inhibited at 4 °C in both of the tested cell types. When cells were preincubated with sodium azide and deoxyglucose to deplete the cellular ATP pool, a smaller but still significant inhibition of TAT uptake was observed (Fig. 1). Temperature and ATP dependence of TAT uptake by the primary cells suggest that entry by endocytosis is a general mechanism of TAT internalization valid for primary cells rather than a mechanism characteristic only for established cell lines.



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  		FIG. 1.
Cellular uptake of TAT is inhibited at 4 °C and by depletion of cellular ATP in primary cells. Panel A, HUVEC cells were incubated for 30 min in the presence of 500 nM Alexa Fluor 488-tagged TAT at 37 °C (red curve) or at 4 °C (green curve) or after depletion of the cellular ATP pool (blue curve). The black curve corresponds to cells incubated in the absence of TAT. Panel B, the effect of 4 °C (red) or depletion of the cellular ATP pool (green) on the Alexa Fluor 488-tagged TAT internalization in HUVEC and human macrophages. Uptake is expressed as the median of cell fluorescence distribution in an experiment normalized to the cell fluorescence distribution median in untreated control (internalization at 37 °C). Means ± S.D. are indicated. Values significantly (p < 0.05) different from control (internalization at 37 °C) are marked with asterisk. For depletion of cellular ATP pool, cells were preincubated for 1 h with 10 mM sodium azide and 6 mM 2-deoxy-D-glucose. a.u., arbitrary unit.

 
Heparan sulfate receptors involved in TAT peptide internalization—Earlier works indicate that the uptake of either full-length TAT protein (16) or TAT peptide conjugated to streptavidin (20) depends on the presence of heparan sulfate receptors. To evaluate the role of heparan sulfates on the uptake of nonconjugated TAT peptide, wild type CHO cells (CHO-K1 cell line) or mutant cells lacking surface heparan sulfate (pgs-A745 and pgs-D677 cell lines) were incubated with 10 µM fluorescein-labeled TAT peptide for 30 min. Peptide uptake was significantly inhibited in mutant cells lacking surface heparan sulfates compared with wild type CHO cells (Fig. 2, A and B). However, a significant amount of peptide was internalized even in the absence of heparan sulfate receptors (Fig. 2A). In control experiments, no decrease in the uptake of the lypophilic dye FM 4-64, a heparan sulfate-independent marker of endocytosis, was observed in both mutant cell lines compared with wild type cells (Fig. 2B). The involvement of heparan sulfate receptors in TAT peptide was confirmed with another experimental approach. The pretreatment of cells with heparinase III inhibited TAT uptake in wild type cells but not in heparan sulfate receptor-deficient cells (Fig. 2, C and D). Although these data indicate the importance of heparan sulfate receptors for the uptake of TAT peptide in wild type CHO cells, Fig. 2 also shows TAT internalization in the absence of heparan sulfate proteoglycans, indicating the existence of heparan sulfate-independent mechanisms of entry.



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  		FIG. 2.
Involvement of heparan sulfate receptors in the cellular uptake of TAT peptide. Panel A, CHO-K1 (red curve), pgsA-745 (green curve), or pgsD-677 (blue curve) cells were incubated for 30 min in the presence of 10 µM FITC-tagged TAT at 37 °C. Panel B, CHO-K1 (red), pgsA-745 (green) or pgsD-677 (blue) cells were incubated with 10 µM fluorescein isothiocyanate-tagged TAT or 10 µM FM4-64 for 30 min. Uptake is expressed as the median of cell fluorescence distribution in an experiment normalized to the cell fluorescence distribution median in CHO-K1 cells. Means ± S.D. are indicated. Values significantly (p < 0.05) different from control (uptake in CHO-K1 cells) are marked with asterisk. Panel C, CHO-K1 were preincubated for 40 min at 37 °C in Na/Pi containing 0.1% bovine serum albumin, 0.2% gelatin, 0.1% glucose with (in blue) or without (in red) 10 milliunits/ml heparinase III. Subsequently, cells were incubated for 30 min in the presence of 10 µM fluorescein isothiocyanate-tagged TAT at 37 °C. Panel D, effect of heparinase treatment on the uptake of fluorescein isothiocyanate-tagged TAT (in blue) in CHO-K1 and pgsA-745 cells. Untreated control is shown in red. Uptake is expressed as the median of cell fluorescence distribution in an experiment normalized to the cell fluorescence distribution median in untreated control. Means ± S.D. are indicated. Values significantly (p < 0.05) different from untreated control are marked with asterisk. a.u., arbitrary unit.

 
TAT Peptide Uptake Involves Caveolin-independent Pathways of Endocytosis—The uptake of TAT peptide fused to proteins has been recently attributed specifically to caveolin-dependent pathway (21, 22). In contrast, in our earlier work (19), efficient uptake of unconjugated TAT peptide was observed in a number of cell lines deficient in caveolin-1 expression, including Jurkat T cell line and HeLa cells (27). Likewise, we observed an ATP- and temperature-sensitive uptake of TAT in HepG2 hepatoma cells (data not shown), which have been reported to lack caveolin-1 (28). To specifically test whether internalization of free TAT peptide involves caveolin-dependent endocytosis, we explored the effects of nystatin and filipin III, both known inhibitors of caveolin-dependent uptake (29). Both inhibitors had little effect on the uptake of Alexa Fluor 488-tagged TAT peptide in HeLa cells (data not shown). That is not surprising considering the low level of caveolin-1 expression in these cells and the inefficient internalization of a well established marker of raft/caveolin-dependent endocytic pathway, BODIPY-lactosylceramide (BODIPY-LacCer) (27). Importantly, in CHO cells, which efficiently express caveolin-1 and actively internalize BODIPY-LacCer (27), the uptake of Alexa Fluor 488-tagged TAT peptide incubation also was not significantly affected in the presence of 50 µg/ml nystatin (Fig. 3, A and D), whereas in the same conditions, the uptake of BODIPY-LacCer was inhibited by ~30% (Fig. 3, B and D). As expected, nystatin had no effect on the uptake of fluorescein-tagged transferrin (Fig. 3, C and D). Taken together, these results indicate that cellular uptake of unconjugated TAT peptide does not require raft/caveolin-dependent endocytic pathway.



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  		FIG. 3.
Effects of inhibitors of caveolin-dependent endocytosis. Panel A, CHO cells were incubated for 30 min in the presence or in the absence of 50 µg/ml nystatin at 10 °C in a water bath. They were then incubated for 10 min at 37 °C in the presence (red curves) or in the absence (blue curves) of 50 µg/ml nystatin and 500 nM Alexa Fluor 488-tagged TAT. The black curve corresponds to cells incubated in the absence of TAT. Membrane-bound peptide was removed by a Pronase treatment before FACS analysis Panel B, CHO cells were incubated with 500 nM BODIPY-LacCer in the presence or absence of nystatin using the same protocol as in panel A. Membrane-bound material was removed according to the protocol described previously (27). Panel C, CHO cells were incubated with 25 µg/ml fluorescein-labeled transferrin and washed as described in panel A. Panel D, effect of nystatin treatment on the internalization of Alexa Fluor 488-tagged TAT (in blue), BODIPY-LacCer (in green), and fluorescein-tagged transferrin (in red). Uptake is expressed as the median of cell fluorescence distribution in experiment normalized to the cell fluorescence distribution median in untreated control. Means ± S.D. are indicated. Values significantly (p < 0.05) different from untreated control are marked with asterisk. a.u., arbitrary unit.

 
Cellular Uptake of TAT Peptide Involves Clathrin-dependent Endocytosis—To test whether TAT uptake involves clathrin-coated pit-mediated endocytosis, TAT uptake was measured in the presence of known inhibitors of this endocytic pathway, potassium depletion, and chlorpromazine. Treatment of HeLa cells with chlorpromazine (a 30-min preincubation in the presence of 30 µM chlorpromazine followed by a 30-min incubation with both 30 µM chlorpromazine and 1 µM Alexa Fluor 488-tagged TAT peptide) resulted in a 50% inhibition of peptide uptake (Fig. 4, A and C). Similarly, a 30-min preincubation of HeLa cells in potassium-free buffer followed by a 5-min hypotonic shock and a 30-min incubation with 1 µM Alexa Fluor 488-tagged TAT peptide in potassium-free buffer resulted in a 40% decrease in peptide uptake (Fig. 4, B and C). In control experiments, significant inhibition was observed for the uptake of fluorescently labeled transferrin, a well established marker of clathrin-dependent endocytosis (Fig. 4C). Inhibition of the uptake of Alexa Fluor 488-tagged TAT peptide and fluorescein-tagged transferrin in potassium-free buffer was completely reversible upon replenishment of potassium in the medium (Fig. 4, B and C). These data indicate that, in the absence of active caveolin-dependent endocytosis, TAT peptide is efficiently internalized by clathrin-dependent endocytic pathway.



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  		FIG. 4.
Effect of inhibitors of clathrin-dependent endocytosis on HeLa cells. Panel A, HeLa cells were preincubated for 30 min in the presence or in the absence of 30 µM chlorpromazine. They then were incubated for 30 min in the presence (blue curves) or in the absence (red curves) of 30 µM chlorpromazine and 1 µM Alexa Fluor 488-tagged TAT. The black curves correspond to cells incubated in the absence of TAT. Panel B, HeLa cells were preincubated for 60 min in regular buffer or in K+-free buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 1 mM MgCl2, 1 mg/ml D-glucose, pH 7.4). They then were incubated for 5 min in a hypotonic buffer (K+-free buffer diluted 1:1 with distilled water) to induce a hypotonic shock and finally incubated in K+-free buffer (red curves) or in regular buffer (blue curves) in the presence of 500 nM Alexa Fluor 488-tagged TAT for 60 min. The final incubation step was done in regular buffer (green curves) when testing for reversibility. The black curve corresponds to cells incubated in the absence of TAT. Panel C, effect of chlorpromazine (CPZ) (in blue), K+ depletion (in green), and K+ replenishment (in red) on the internalization of Alexa Fluor 488-tagged TAT and fluorescein-tagged transferrin. Uptake is expressed as the median of cell fluorescence distribution after treatment normalized to the cell fluorescence distribution median in untreated control. Means ± S.D. are indicated. Values significantly (p < 0.05) different from untreated control are marked with asterisk. a.u., arbitrary unit.

 
To test the involvement of clathrin-dependent pathway in cells efficiently expressing caveolin-1, we explored the effect of potassium depletion on the TAT peptide uptake in CHO cells, which efficiently express caveolin-1 (27). Cellular internalization of Alexa Fluor 488-tagged TAT peptide (Fig. 5, A and C) was inhibited by ~55% in potassium-free buffer as compared with ~85% inhibition of fluorescein-tagged transferrin uptake (Fig. 5, B and C). Again, as with HeLa cells, inhibition for both Alexa Fluor 488-tagged TAT peptide and fluorescein-tagged transferrin was completely reversible upon replenishment of potassium in the medium (Fig. 5). These data indicate that, even in cells efficiently expressing caveolin-1, the uptake of unconjugated TAT peptide involves clathrin-dependent pathway of endocytosis.



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  		FIG. 5.
Effect of K+ depletion on clathrin-dependent endocytosis in CHO cells. Panel A, CHO cells were preincubated for 60 min in regular buffer or in K+-free buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 1 mM MgCl2, 1 mg/ml D-glucose, pH 7.4). They were then incubated for 5 min in a hypotonic buffer (K+-free buffer diluted 1:1 with distilled water) to induce a hypotonic shock and finally incubated in K+-free buffer (red curves) or in regular buffer (blue curves) in the presence of 500 nM Alexa Fluor 488-tagged TAT for 60 min. When testing for reversibility, the final incubation step was done in regular buffer (green curves). The black curve corresponds to cells incubated in the absence of TAT. Panel B, same experiments in CHO cells with 25 µg/ml fluorescein-tagged transferrin. Panel C, effect of K+ depletion (in blue) and K+ replenishment (in red) on the internalization of Alexa Fluor 488-tagged TAT and fluorescein-tagged transferrin. Uptake is expressed as the median of cell fluorescence distribution after treatment normalized to the cell fluorescence distribution median in untreated control. Means ± S.D. are indicated. Values significantly (p < 0.05) different from untreated control are marked with asterisk. a.u., arbitrary unit.

 
Internalized TAT Peptide Is Targeted to Acidic Compartments—As endocytosed proteins pass from early to late endosomes and to lysosomes, the vesicular compartments undergo progressive acidification. Even early endosomes show a reduction in pH (from pH 5.9 to 6.0) to promote the release of ligands bound to receptors. Acidification of these luminal compartments is blocked by agents such as monensin (30). To test whether endocytosed TAT peptide is targeted into acidified compartments, we took advantage of the fact that the fluorescence of fluorescein significantly decreases at pH 5-6, typical for endosomes and lysosomes compared with fluorescence at pH 7.4 of cell culture medium. We incubated HeLa cells with 2 µM fluorescein-tagged TAT for 60 min followed by 30 min of incubation in the presence or in the absence of 50 µM monensin at 4 °C. As shown on Fig. 6, B and C, incubation in the presence of monensin resulted in a significant increase in the cell-associated fluorescence. To verify that the higher fluorescent signal was due to a pH-dependent increase in the fluorescence of TAT-fluorescein within acidified compartments, the fluorescein tag was replaced with an Alexa Fluor 488 probe, which does not alter with pH (in the pH 5-8 range). In contrast to cells with TAT-fluorescein, the fluorescence of cells incubated with TAT-Alexa Fluor 488 was not affected by monensin (Fig. 6, A and C). These experiments indicate that a significant fraction of the TAT peptide is delivered into acidic cellular compartments.



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  		FIG. 6.
Monensin treatment. HeLa cells were incubated for 60 min in the presence of 2 µM Alexa Fluor 488-tagged (panel A) or fluorescein-tagged (panel B) TAT peptide. After incubation, cells were washed with NaCl/Pi and treated with trypsin. Subsequently, they were incubated for 30 min in the presence (blue curves) or in the absence (red curves) of 50 µM monensin at 4 °C. Following this step, they were washed again with NaCl/Pi, and resuspended in 500 µl of NaCl/Pi for FACS analysis. The black curve corresponds to cells incubated in the absence of TAT. Panel C, effect of monensin treatment (in blue) on the internalization of Alexa Fluor 488-tagged TAT and fluorescein isothiocyanate (FITC)-tagged TAT. Untreated control is shown in red. Uptake is expressed as the median of cell fluorescence distribution in experiment normalized to the cell fluorescence distribution median in untreated control. Means ± S.D. are indicated. Values significantly (p < 0.05) different from untreated control are marked with asterisk. a.u., arbitrary unit.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although the number of publications confirming the great potential of PTDs for cellular delivery of many important compounds is increasing each year (1), the mechanism of PTD-mediated delivery remains elusive. For about a decade, it was commonly accepted that, despite their highly polar nature, PTDs are able to rapidly and efficiently translocate directly through the lipid bilayer, thus delivering their cargo into the cytoplasm and nucleus in a receptor- and energy-independent way (14, 15). This model was supported by a large number of indirect evidence including temperature, ATP, and receptor independence of cellular uptake (11-14), but attempts to directly validate this model on protein-free lipid bilayer systems gave contradictory results (31, 32). Moreover, it was recently shown that apparent ATP and temperature independence and fast kinetics of PTD uptake result from artifacts of cell fixation and incomplete removal of surface-bound peptide (18, 19). These recent findings have led to a major reevaluation of key characteristics of PTD internalization and the role of endocytosis in PTD-mediated uptake.

Our present results indicate that fluorescently labeled TAT peptide as full-length TAT protein (16) or TAT-streptavidin conjugate (20) uses heparan sulfate receptors for internalization. However, the uptake of small basic peptides such as the TAT PTD might be also mediated by other pathways as suggested by the fact that the internalization of TAT peptide is not completely inhibited in cells lacking surface heparan sulfates due to mutations or enzymatic treatment. The observed promiscuity of TAT peptide in terms of receptor use is not very surprising given the highly cationic nature of the peptide and the abundance of negatively charged moieties on the cell surface. Therefore, one might expect that, in addition to specific interactions with heparan sulfate proteoglycans, there may well be a high level of electrostatic interactions with other negatively charged cell surface determinants. The importance of cell surface receptors for TAT uptake, mentioned above and ATP and temperature dependence as shown in this and earlier work (18, 19) demonstrate that TAT peptide uptake involves endocytosis. In some of the recent reports arguing for the crucial role of endocytosis in the uptake of constructs in which the TAT peptide was fused to proteins, the uptake has been attributed specifically to caveolae-dependent pathway (21, 22). On the other hand, we found cells deficient in caveolin-1 expression or cells lacking caveolin-1 to be able to efficiently internalize unconjugated TAT peptide. This indicates that caveolin-dependent pathway is not required for TAT peptide uptake, and alternative pathways might be utilized for uptake of TAT in those cells. Therefore, we tested the effects of various inhibitors of clathrin- and caveolae-dependent endocytosis on the uptake of unconjugated TAT peptide. For both caveolin-1-expressing and caveolin-1-deficient cells, inhibitors of clathrin-dependent pathway (potassium depletion and chlorpromazine) significantly decrease TAT peptide uptake, whereas inhibitors of raft/ caveolin-dependent pathway (nystatin and filippin III) have no significant effect on the uptake. These findings support an involvement of endocytosis in the TAT internalization and show that clathrin-dependent pathway is involved in the cellular uptake of unconjugated TAT, whereas raft/caveolin-dependent pathway does not play a significant role. Along the same lines, the full-size TAT protein is internalized by a clathrin-dependent pathway (33). In contrast to our results, recent reports on the cellular uptake of TAT peptide conjugated to proteins in HeLa and CHO cells have attributed the uptake of these conjugates to caveolin-dependent pathway (21, 22). This apparent discrepancy suggests that free TAT peptide and TAT peptide conjugated to macromolecules utilize different pathways for internalization, possibly due to difference in receptor usage. Interestingly, a recent report (34) on the uptake of TAT conjugated to Cre recombinase in T cells, which are deficient in caveolin-1, implicates caveolin-independent lipid raft macropinocytosis in transduction of TAT-Cre recombinase into cells. Very recently, lipid raft macropinocytosis also has been implicated in the uptake of unconjugated TAT peptide (35). However, methyl-{beta}-cyclodextrin, which was used in this work to inhibit raft/caveolin-dependent pathway, has been shown to affect clathrin-dependent pathway (36), thus complicating interpretation of the data. Taken together, these data indicate that alternative pathways could be used for TAT uptake depending on cell type and nature (and presence) of conjugated cargo. It is possible that, whereas free fluorescently labeled TAT peptide is mainly directed into constitutive clathrin-dependent pathway, TAT peptide conjugated to macromolecular cargo upon binding to cellular receptors induces signaling cascades activating caveolin-dependent endocytosis or macropinocytosis.

Whatever the case, our results confirm that endocytosis is a major route of TAT peptide internalization. It is generally accepted that endocytosed material is targeted to late endosomes or lysosomes, compartments that contain active proteases and are involved in the degradation of internalized proteins and other macromolecules. In our experiments with monensin, an inhibitor of endosome acidification, we found that, within 1 h, at least a part of the TAT peptide is delivered into acidic compartments. Trapping and possible degradation of TAT peptides within these acidic vesicles is at odds with effective delivery of bioactive transported cargo within cytoplasm and/or nucleus. Nevertheless, the delivery of functionally active proteins into the cytoplasm and the nucleus (7, 9, 10) has been well documented. This apparent discrepancy could be explained by different sensitivities of the experimental approaches. Whereas in our experiments we measure the total amount of internalized peptide, the delivery of only a negligible fraction of total TAT conjugate to the cytoplasm or to the nucleus might be sufficient for functional detection of the transduction with very sensitive biological assays as an end-point. Therefore, it is possible that only a small fraction of all internalized conjugated cargo molecules is actually delivered into the cytoplasm or nucleus.

There are several hypothetical mechanisms by which PTDs can facilitate transduction of macromolecules into the cell. First, it is possible that, once inside, endosomes, TAT, and other cationic PTDs destabilize the endosomal membrane to leak a portion of the enclosed macromolecules into the cytoplasm as reported for polyethyleneimine (37). TAT-induced destabilization of endosomes might involve TAT interactions with proteins or lipids specific to endosomal compartments. These interactions can be promoted by acidic pH inside endosomes. It is also possible that a functionally relevant fraction of the TAT peptide utilizes the caveolin-dependent endocytic pathway that bypasses acidic cellular compartments. This pathway is used by some viruses as described recently (38) for SV40. Finally, one cannot exclude the possibility that a small but nevertheless all-important fraction of the TAT peptide directly crosses the lipid bilayer to deliver conjugated cargo into the cytoplasm. This latter mechanism was additionally supported by the recent study (39), demonstrating direct translocation of penetratin (another well studied PTD) through protein-free synthetic lipid bilayers. Translocation of penetratin was shown to be lipid composition-dependent, requiring the presence of anionic lipids and high (>100 mV) transmembrane potentials, and was observed in a much slower time frame than previously expected (several hours rather than minutes) (39). This slow direct translocation of PTD through membranes might be of importance for the release of PTD from endosomes and lysosomes into the cytosol.

In conclusion, our experiments further substantiate the key role of endocytosis in the PTD entry into living cells and provide new insights into utilized endocytic pathways and surface receptors. We believe that these results along with future work involving sensitive functional assays will bring a better understanding of the mechanisms by which relatively short PTDs deliver large cargos to the cytosol and nucleus.

Note Added in Proof—We note a recent study (40) on TAT peptide interaction with soluble heparan sulfate that supports our conclusion on the involvement of heparan sulfate receptors in the cellular uptake of unconjugated TAT peptide.


   FOOTNOTES
 
* This work has been supported by grants from the Association pour la Recherche contre le Cancer (5919) and from the European Commission (QLK3-CT-2002-01989) (to B. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Both authors contributed equally to this work. Back

|| To whom correspondence should be addressed: NICHD, National Institutes of Health, 10 Center Dr., Bldg. 10, Rm. 10D05, Bethesda, MD 20892. Tel.: 301-402-9010; Fax: 301-480-2412; E-mail: melikovk{at}mail.nih.gov.

1 The abbreviations used are: PTD, protein transduction domain; CHO, Chinese hamster ovary; HUVEC, high pressure liquid chromatography; FACS, fluorescence-activated cell sorter; LacCer, lactosylceramide. Back


   ACKNOWLEDGMENTS
 
We thank Dr. E. Vives for providing the fluorochrome-tagged TAT peptides used in this study and for valuable discussions. We also want to thank Dr. S. Kinet for providing lymphocytes, Dr. D. Marks for valuable advice on caveolin-dependent endocytosis, and Dr. J. C. Grivel for the kind and expert help in performing some of the FACS experiments.



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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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