Cellular Uptake of Unconjugated TAT Peptide Involves Clathrin-dependent Endocytosis and Heparan Sulfate Receptors

Abstract

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. 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. 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) 1The abbreviations used are: PTD, protein transduction domain; CHO, Chinese hamster ovary; HUVEC, high pressure liquid chromatography; FACS, fluorescence-activated cell sorter; LacCer, lactosylceramide. can facilitate cytoplasmic and nuclear delivery of a conjugated cargo has attracted a lot of interest (1Lindsay M.A. Curr. Opin. Pharmacol. 2002; 2: 587-594Crossref PubMed Scopus (171) Google Scholar, 2Matsui H. Tomizawa K. Lu Y.F. Matsushita M. Curr. Protein Pept. Sci. 2003; 4: 151-157Crossref PubMed Scopus (66) Google Scholar, 3Wadia J.S. Dowdy S.F. Curr. Protein Pept. Sci. 2003; 4: 97-104Crossref PubMed Scopus (146) Google Scholar). Up to date, a wide range of cargo molecules, including low molecular weight drugs (4Rothbard J.B. Garlington S. Lin Q. Kirschberg T. Kreider E. McGrane P.L. Wender P.A. Khavari P.A. Nat. Med. 2000; 6: 1253-1257Crossref PubMed Scopus (576) Google Scholar), oligonucleotides (5Astriab-Fisher A. Sergueev D. Fisher M. Shaw B.R. Juliano R.L. Pharm. Res. (N. Y.). 2002; 19: 744-754Crossref PubMed Scopus (256) Google Scholar), peptides (6Shibagaki N. Udey M.C. J. Immunol. 2002; 168: 2393-2401Crossref PubMed Scopus (125) Google Scholar) and even full-length proteins (7Nagahara H. Vocero-Akbani A.M. Snyder E.L. Ho A. Latham D.G. Lissy N.A. Becker-Hapak M. Ezhevsky S.A. Dowdy S.F. Nat. Med. 1998; 4: 1449-1452Crossref PubMed Scopus (887) Google Scholar, 8Schwarze S.R. Ho A. Vocero-Akbani A. Dowdy S.F. Science. 1999; 285: 1569-1572Crossref PubMed Scopus (2210) Google Scholar, 9Joshi S.K. Hashimoto K. Koni P.A. Genesis. 2002; 33: 48-54Crossref PubMed Scopus (46) Google Scholar, 10Peitz M. Pfannkuche K. Rajewsky K. Edenhofer F. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4489-4494Crossref PubMed Scopus (270) Google Scholar), have been successfully delivered into cells using PTDs and, most importantly, the functional activity of the delivered cargo has been observed (7Nagahara H. Vocero-Akbani A.M. Snyder E.L. Ho A. Latham D.G. Lissy N.A. Becker-Hapak M. Ezhevsky S.A. Dowdy S.F. Nat. Med. 1998; 4: 1449-1452Crossref PubMed Scopus (887) Google Scholar, 8Schwarze S.R. Ho A. Vocero-Akbani A. Dowdy S.F. Science. 1999; 285: 1569-1572Crossref PubMed Scopus (2210) Google Scholar, 9Joshi S.K. Hashimoto K. Koni P.A. Genesis. 2002; 33: 48-54Crossref PubMed Scopus (46) Google Scholar, 10Peitz M. Pfannkuche K. Rajewsky K. Edenhofer F. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4489-4494Crossref PubMed Scopus (270) Google Scholar). 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 (11Futaki S. Suzuki T. Ohashi W. Yagami T. Tanaka S. Ueda K. Sugiura Y. J. Biol. Chem. 2001; 276: 5836-5840Abstract Full Text Full Text PDF PubMed Scopus (1449) Google Scholar, 12Derossi D. Calvet S. Trembleau A. Brunissen A. Chassaing G. Prochiantz A. J. Biol. Chem. 1996; 271: 18188-18193Abstract Full Text Full Text PDF PubMed Scopus (971) Google Scholar, 13Suzuki T. Futaki S. Niwa M. Tanaka S. Ueda K. Sugiura Y. J. Biol. Chem. 2002; 277: 2437-2443Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 14Vives E. Brodin P. Lebleu B. J. Biol. Chem. 1997; 272: 16010-16017Abstract Full Text Full Text PDF PubMed Scopus (2071) Google Scholar, 15Derossi D. Joliot A.H. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar). 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 (16Tyagi M. Rusnati M. Presta M. Giacca M. J. Biol. Chem. 2001; 276: 3254-3261Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar). 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 (17Lundberg M. Johansson M. Biochem. Biophys. Res. Commun. 2002; 291: 367-371Crossref PubMed Scopus (210) Google Scholar, 18Lundberg M. Wikstrom S. Johansson M. Mol. Ther. 2003; 8: 143-150Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 19Richard J.P. Melikov K. Vives E. Ramos C. Verbeure B. Gait M.J. Chernomordik L.V. Lebleu B. J. Biol. Chem. 2003; 278: 585-590Abstract Full Text Full Text PDF PubMed Scopus (1483) Google Scholar). 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 (18Lundberg M. Wikstrom S. Johansson M. Mol. Ther. 2003; 8: 143-150Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 19Richard J.P. Melikov K. Vives E. Ramos C. Verbeure B. Gait M.J. Chernomordik L.V. Lebleu B. J. Biol. Chem. 2003; 278: 585-590Abstract Full Text Full Text PDF PubMed Scopus (1483) Google Scholar). 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 (19Richard J.P. Melikov K. Vives E. Ramos C. Verbeure B. Gait M.J. Chernomordik L.V. Lebleu B. J. Biol. Chem. 2003; 278: 585-590Abstract Full Text Full Text PDF PubMed Scopus (1483) Google Scholar). Although several very recent papers (20Console S. Marty C. Garcia-Echeverria C. Schwendener R. Ballmer-Hofer K. J. Biol. Chem. 2003; 278: 35109-35114Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar, 21Ferrari A. Pellegrini V. Arcangeli C. Fittipaldi A. Giacca M. Beltram F. Mol. Ther. 2003; 8: 284-294Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 22Fittipaldi A. Ferrari A. Zoppe M. Arcangeli C. Pellegrini V. Beltram F. Giacca M. J. Biol. Chem. 2003; 278: 34141-34149Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 23Drin G. Cottin S. Blanc E. Rees A.R. Temsamani J. J. Biol. Chem. 2003; 278: 31192-31201Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar) 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 (24Sakai N. Matile S. J. Am. Chem. Soc. 2003; 125: 14348-14356Crossref PubMed Scopus (284) Google Scholar, 25Hariton-Gazal E. Rosenbluh J. Graessmann A. Gilon C. Loyter A. J. Cell Sci. 2003; 116: 4577-4586Crossref PubMed Scopus (99) Google Scholar). 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. 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, and in Peptides were further and Cell and cells were as on in with and mm cells cell and lacking cell or heparan sulfate cell were in in with and mm cells were as on in cell with mm 50 50 and 10 human from was in cells were by as V. S. S. B. M. C. D. C. P. P. N. N. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). the case of and human cells were with a cell cells were and on was and cells were with pH was and cell were with for the cell were as with peptides and drugs in cells were with and for 10 with the of HUVEC, which were with to and to In some peptides were by a 4 with and mm the one of was added to the and 10 of were added to the cells cell was with and in of In case of and cells were with a cell and in cells were as in the with of TAT peptide in cell was with of and for 10 with of to cells were with of with and with of and in of 10 (Molecular Probes) to cells. analysis was performed with a fluorescence-activated cell A of was with the of cells for which were effects of various on cellular uptake, the of cell in was to the cell in was performed in or of a number of to are on the of the effects of various as with was by the of TAT Peptide by ATPand cell lines are and experimental important biotechnological and primary cells. the effects of ATP depletion and low temperature on TAT peptide uptake by human and cells. primary cells were with TAT peptide for and the of peptide was using As shown on TAT uptake was greatly inhibited 4 in of the cell cells were with and to the cellular ATP a significant of TAT uptake was observed and ATP of TAT uptake by the primary cells suggest that entry by endocytosis is a mechanism of TAT internalization for primary cells a mechanism for cell sulfate receptors in TAT peptide that the uptake of full-length TAT protein (16Tyagi M. Rusnati M. Presta M. Giacca M. J. Biol. Chem. 2001; 276: 3254-3261Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar) or TAT peptide conjugated to (20Console S. Marty C. Garcia-Echeverria C. Schwendener R. Ballmer-Hofer K. J. Biol. Chem. 2003; 278: 35109-35114Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar) depends on the presence of heparan sulfate receptors. the role of heparan on the uptake of TAT peptide, cells cell or cells lacking surface heparan sulfate and cell were with 10 TAT peptide for Peptide uptake was inhibited in cells lacking surface heparan with cells A and However, a significant of peptide was even in the of heparan sulfate receptors In in the uptake of the a heparan of was observed in cell lines with cells involvement of heparan sulfate receptors in TAT peptide was with experimental of cells with inhibited TAT uptake in cells not in heparan sulfate cells and Although these the importance of heparan sulfate receptors for the uptake of TAT peptide in also TAT internalization in the of heparan sulfate indicating the of heparan mechanisms of entry. TAT Peptide of uptake of TAT peptide to proteins has been recently attributed to caveolin-dependent pathway A. Pellegrini V. Arcangeli C. Fittipaldi A. Giacca M. Beltram F. Mol. Ther. 2003; 8: 284-294Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 22Fittipaldi A. Ferrari A. Zoppe M. Arcangeli C. Pellegrini V. Beltram F. Giacca M. J. Biol. Chem. 2003; 278: 34141-34149Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar). In contrast, in our earlier (19Richard J.P. Melikov K. Vives E. Ramos C. Verbeure B. Gait M.J. Chernomordik L.V. Lebleu B. J. Biol. Chem. 2003; 278: 585-590Abstract Full Text Full Text PDF PubMed Scopus (1483) Google Scholar), efficient uptake of unconjugated TAT peptide was observed in a number of cell lines deficient in caveolin-1 including cell and cells V. R. Mol. Biol. 2003; PubMed Scopus (210) Google Scholar). we observed an and uptake of TAT in cells (data not which have been to caveolin-1 T. H. R. T. J. Cell Sci. 2000; PubMed Google Scholar). internalization of TAT peptide involves caveolin-dependent we the effects of and known inhibitors of caveolin-dependent uptake P. E. J. J. Cell Biol. 1994; PubMed Scopus Google Scholar). inhibitors on the uptake of TAT peptide in cells (data not shown). is not the low of caveolin-1 in these cells and the internalization of a well of raft/caveolin-dependent endocytic pathway, V. R. Mol. Biol. 2003; PubMed Scopus (210) Google Scholar). in which caveolin-1 and V. R. Mol. Biol. 2003; PubMed Scopus (210) Google Scholar), the uptake of TAT peptide also was not in the presence of 50 A and whereas in the the uptake of was inhibited by and As on the uptake of and these results that cellular uptake of unconjugated TAT peptide not raft/caveolin-dependent endocytic of TAT Peptide TAT uptake involves TAT uptake was in the presence of known inhibitors of this endocytic pathway, and of cells with in the presence of by a with and TAT in a of peptide uptake A and a of cells in by a and a with TAT peptide in in a in peptide uptake and In significant was observed for the uptake of a well of clathrin-dependent endocytosis of the uptake of TAT peptide and in was of in the and in the of active caveolin-dependent TAT peptide is by clathrin-dependent endocytic the involvement of clathrin-dependent pathway in cells we the of depletion on the TAT peptide uptake in which caveolin-1 V. R. Mol. Biol. 2003; PubMed Scopus (210) Google Scholar). internalization of TAT peptide A and was inhibited by in as with of uptake and as with for TAT peptide and was of in the even in cells the uptake of unconjugated TAT peptide involves clathrin-dependent pathway of endocytosis. TAT Peptide to proteins from to and to the a in pH pH to to the of to receptors. of these is by such as M. P. Biol. PubMed Scopus Google Scholar). TAT peptide is into we of the that the of pH for and with pH of cell cells with TAT for by of in the presence or in the of 50 4 As shown on and in the presence of in a significant in the that the was due to a in the of the was with an which not with pH the pH In to cells with the of cells with was not by A and that a significant of the TAT peptide is delivered into cellular Although the number of the of PTDs for cellular delivery of important is (1Lindsay M.A. Curr. Opin. Pharmacol. 2002; 2: 587-594Crossref PubMed Scopus (171) Google Scholar), the mechanism of PTD-mediated delivery a it was commonly their polar PTDs are to and through the lipid their cargo into the and in a and E. Brodin P. Lebleu B. J. Biol. Chem. 1997; 272: 16010-16017Abstract Full Text Full Text PDF PubMed Scopus (2071) Google Scholar, 15Derossi D. Joliot A.H. Chassaing G. Prochiantz A. J. Biol. Chem. 1994; 269: 10444-10450Abstract Full Text PDF PubMed Google Scholar). model was by a number of evidence including and independence of cellular uptake (11Futaki S. Suzuki T. Ohashi W. Yagami T. Tanaka S. Ueda K. Sugiura Y. J. Biol. Chem. 2001; 276: 5836-5840Abstract Full Text Full Text PDF PubMed Scopus (1449) Google Scholar, 12Derossi D. Calvet S. Trembleau A. Brunissen A. Chassaing G. Prochiantz A. J. Biol. Chem. 1996; 271: 18188-18193Abstract Full Text Full Text PDF PubMed Scopus (971) Google Scholar, 13Suzuki T. Futaki S. Niwa M. Tanaka S. Ueda K. Sugiura Y. J. Biol. Chem. 2002; 277: 2437-2443Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 14Vives E. Brodin P. Lebleu B. J. Biol. Chem. 1997; 272: 16010-16017Abstract Full Text Full Text PDF PubMed Scopus (2071) Google Scholar), to this model on lipid bilayer results D. M. B. 2000; PubMed Scopus Google Scholar, H. Biophys. 2003; PubMed Scopus Google Scholar). Moreover, it was recently shown that apparent ATP and temperature independence and fast of uptake from artifacts of cell fixation and incomplete removal of peptide (18Lundberg M. Wikstrom S. Johansson M. Mol. Ther. 2003; 8: 143-150Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 19Richard J.P. Melikov K. Vives E. Ramos C. Verbeure B. Gait M.J. Chernomordik L.V. Lebleu B. J. Biol. Chem. 2003; 278: 585-590Abstract Full Text Full Text PDF PubMed Scopus (1483) Google Scholar). recent have to a major reevaluation of of internalization and the role of endocytosis in PTD-mediated Our results that TAT peptide as full-length TAT protein (16Tyagi M. Rusnati M. Presta M. Giacca M. J. Biol. Chem. 2001; 276: 3254-3261Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar) or (20Console S. Marty C. Garcia-Echeverria C. Schwendener R. Ballmer-Hofer K. J. Biol. Chem. 2003; 278: 35109-35114Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar) heparan sulfate receptors for internalization. However, the uptake of small peptides such as the TAT might be also mediated by other pathways as by the that the internalization of TAT peptide is not inhibited in cells lacking surface heparan due to or observed of TAT peptide in of is not very the cationic of the peptide and the of on the cell one might in to specific with heparan sulfate there well be a high of with other cell surface importance of cell surface receptors for TAT uptake, and ATP and temperature as shown in this and earlier (18Lundberg M. Wikstrom S. Johansson M. Mol. Ther. 2003; 8: 143-150Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar, 19Richard J.P. Melikov K. Vives E. Ramos C. Verbeure B. Gait M.J. Chernomordik L.V. Lebleu B. J. Biol. Chem. 2003; 278: 585-590Abstract Full Text Full Text PDF PubMed Scopus (1483) Google Scholar) demonstrate that TAT peptide uptake involves endocytosis. In some of the recent for the role of endocytosis in the uptake of in which the TAT peptide was to proteins, the uptake has been attributed to pathway A. Pellegrini V. Arcangeli C. Fittipaldi A. Giacca M. Beltram F. Mol. Ther. 2003; 8: 284-294Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 22Fittipaldi A. Ferrari A. Zoppe M. Arcangeli C. Pellegrini V. Beltram F. Giacca M. J. Biol. Chem. 2003; 278: 34141-34149Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar). On the other hand, we found cells deficient in caveolin-1 or cells lacking caveolin-1 to be to unconjugated TAT that caveolin-dependent pathway is not required for TAT peptide uptake, and alternative pathways might be utilized for uptake of TAT in those cells. we the effects of various inhibitors of and endocytosis on the uptake of unconjugated TAT and inhibitors of clathrin-dependent pathway depletion and TAT peptide uptake, whereas inhibitors of caveolin-dependent pathway and have significant on the an involvement of endocytosis in the TAT internalization and that clathrin-dependent pathway is in the cellular uptake of unconjugated whereas raft/caveolin-dependent pathway not a significant the the TAT protein is by a clathrin-dependent pathway A. F. A. N. P. B. Mol. Biol. PubMed Scopus Google Scholar). In to our recent on the cellular uptake of TAT peptide conjugated to proteins in and cells have attributed the uptake of these to caveolin-dependent pathway A. Pellegrini V. Arcangeli C. Fittipaldi A. Giacca M. Beltram F. Mol. Ther. 2003; 8: 284-294Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 22Fittipaldi A. Ferrari A. Zoppe M. Arcangeli C. Pellegrini V. Beltram F. Giacca M. J. Biol. Chem. 2003; 278: 34141-34149Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar). apparent that TAT peptide and TAT peptide conjugated to macromolecules different pathways for due to in a recent J.S. Dowdy S.F. Nat. Med. PubMed Scopus Google Scholar) on the uptake of TAT conjugated to in which are deficient in lipid in transduction of into cells. recently, lipid also has been in the uptake of unconjugated TAT peptide J.S. Dowdy S.F. J. Scopus Google Scholar). However, which was used in this to inhibit raft/caveolin-dependent pathway, has been shown to clathrin-dependent pathway S.K. G. F. B. K. Mol. Biol. 1999; PubMed Scopus Google Scholar), of the these that alternative pathways be used for TAT uptake on cell and of conjugated It is whereas TAT peptide is mainly into clathrin-dependent pathway, TAT peptide conjugated to cargo to cellular receptors caveolin-dependent endocytosis or the our results that endocytosis is a major of TAT peptide internalization. It is that is to or that active and are in the of proteins and other In our with an of we found a part of the TAT peptide is delivered into and of TAT peptides these is with delivery of cargo the delivery of active proteins into the and the (7Nagahara H. Vocero-Akbani A.M. Snyder E.L. Ho A. Latham D.G. Lissy N.A. Becker-Hapak M. Ezhevsky S.A. Dowdy S.F. Nat. Med. 1998; 4: 1449-1452Crossref PubMed Scopus (887) Google Scholar, 9Joshi S.K. Hashimoto K. Koni P.A. Genesis. 2002; 33: 48-54Crossref PubMed Scopus (46) Google Scholar, 10Peitz M. Pfannkuche K. Rajewsky K. Edenhofer F. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4489-4494Crossref PubMed Scopus (270) Google Scholar) has been well apparent be by different of the experimental Whereas in our we the of peptide, the delivery of a of TAT to the or to the might be for functional of the transduction with very as an it is that a small of conjugated cargo molecules is delivered into the or are several mechanisms by which PTDs can facilitate transduction of macromolecules into the it is and other cationic PTDs the membrane to a of the macromolecules into the as for F. M.A. D. B. J.P. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of might TAT with proteins or specific to can be by pH It is also that a of the TAT peptide the caveolin-dependent endocytic pathway that cellular pathway is used by some as recently J. A. Nat. Cell Biol. 2001; PubMed Scopus Google Scholar) for one the that a small of the TAT peptide the lipid bilayer to conjugated cargo into the mechanism was by the recent D. 2003; PubMed Scopus Google Scholar), direct translocation of well through synthetic lipid of was shown to be lipid the presence of and high and was observed in a D. 2003; PubMed Scopus Google Scholar). direct translocation of through might be of importance for the of from and into the In our further the role of endocytosis in the entry into living cells and provide new into utilized endocytic pathways and surface receptors. that these results with functional a of the mechanisms by which PTDs to the and in a recent A. J. Biophys. J. Full Text Full Text PDF PubMed Scopus Google Scholar) on TAT peptide with heparan sulfate that our on the involvement of heparan sulfate receptors in the cellular uptake of unconjugated TAT E. Vives for the TAT peptides used in this and for also to S. for D. for on caveolin-dependent and J. C. for the and in some of the