Cytoplasmic and Nuclear Delivery of a TAT-derived Peptide and a β-Peptide after Endocytic Uptake into HeLa Cells
2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês
10.1074/jbc.m308719200
ISSN1083-351X
AutoresTerra Potocky, Anant K. Menon, Samuel H. Gellman,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoSeveral short, highly cationic peptides are able to enter the cytoplasm and nucleus of cells from the extracellular medium. The mechanism of entry is unknown. A number of fluorescence-based studies suggested that these molecules cross the plasma membrane by an energy-independent process, directly gaining access to the cytoplasm. Recent reports have questioned this conclusion, attributing the prior observations to artifacts resulting from fixation procedures used to prepare cells for fluorescence microscopy. These studies analyzed live cells and showed that the peptides entered through endocytosis and accumulated in endocytic vesicles, without necessarily entering the cytoplasm. To resolve this controversy and to extend the analyses to non-natural β-peptide sequences, we studied the cytoplasmic and nuclear delivery of a fluorescein-labeled 9-residue sequence derived from the human immunodeficiency virus transactivator of transcription (TAT) peptide, TAT-(47–57), as well as a similarly labeled 12-residue β-peptide, β-(VRR)4, in live cells. Using fluorescence confocal microscopy, we show that when added to cells, both peptides are found in endocytic vesicles containing the transferrin receptor as well as in the cytoplasm and nucleus (TAT-(47–57)) or nucleolus (β-(VRR)4). The cells were verified to be intact through all experimental procedures by demonstrating their ability to exclude propidium iodide. Endocytic entry of the peptides was blocked by the energy poisons sodium azide and 2-deoxyglucose, whereas staining of the nucleus (nucleolus), but not endocytic vesicles, was abrogated by treating the cells with ammonium chloride. Our observations are consistent with the proposal that TAT-(47–57) and β-(VRR)4 enter cells by endocytosis and then exit an endosomal compartment to enter the cytoplasm by means of a mechanism requiring endosome acidification. Several short, highly cationic peptides are able to enter the cytoplasm and nucleus of cells from the extracellular medium. The mechanism of entry is unknown. A number of fluorescence-based studies suggested that these molecules cross the plasma membrane by an energy-independent process, directly gaining access to the cytoplasm. Recent reports have questioned this conclusion, attributing the prior observations to artifacts resulting from fixation procedures used to prepare cells for fluorescence microscopy. These studies analyzed live cells and showed that the peptides entered through endocytosis and accumulated in endocytic vesicles, without necessarily entering the cytoplasm. To resolve this controversy and to extend the analyses to non-natural β-peptide sequences, we studied the cytoplasmic and nuclear delivery of a fluorescein-labeled 9-residue sequence derived from the human immunodeficiency virus transactivator of transcription (TAT) peptide, TAT-(47–57), as well as a similarly labeled 12-residue β-peptide, β-(VRR)4, in live cells. Using fluorescence confocal microscopy, we show that when added to cells, both peptides are found in endocytic vesicles containing the transferrin receptor as well as in the cytoplasm and nucleus (TAT-(47–57)) or nucleolus (β-(VRR)4). The cells were verified to be intact through all experimental procedures by demonstrating their ability to exclude propidium iodide. Endocytic entry of the peptides was blocked by the energy poisons sodium azide and 2-deoxyglucose, whereas staining of the nucleus (nucleolus), but not endocytic vesicles, was abrogated by treating the cells with ammonium chloride. Our observations are consistent with the proposal that TAT-(47–57) and β-(VRR)4 enter cells by endocytosis and then exit an endosomal compartment to enter the cytoplasm by means of a mechanism requiring endosome acidification. There is widespread interest in the use of protein-derived peptides (1.Derossi 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 (966) Google Scholar, 2.Vives E. Brodin P. Lebleu B. J. Biol. Chem. 1997; 272: 16010-16017Abstract Full Text Full Text PDF PubMed Scopus (2063) Google Scholar, 3.Pooga M. Hallbrink M. Zorko M. Langel U. FASEB J. 1998; 12: 67-77Crossref PubMed Google Scholar), designed peptides (4.Mitchell D.J. Kim D.T. Steinman L. Fathman C.G. Rothbard J.B. J. Peptide Res. 2000; 56: 318-325Crossref PubMed Scopus (907) Google Scholar, 5.Lindgren M. Hallbrink M. Prochiantz A. Langel U. Trends Pharmacol. 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Rev. 2003; 55: 281-294Crossref PubMed Scopus (152) Google Scholar). The prototypical sequences that constitute these so-called protein transduction domains are derived from the human immunodeficiency virus (HIV) 1The abbreviations used are: HIVhuman immunodeficiency virusTATtransactivator of transcriptionAntpantennapediaFlufluoresceinPBSphosphate buffered salinePIpropidium iodideTR-TfTexas Red-labeled transferrinHBSHepes-buffered salineTfRtransferrin receptor. transactivator of transcription (TAT) protein, the antennapedia (Antp) homeodomain, and the herpes simplex virus (HSV) type 1 DNA-binding protein VP22, all of which are rich in arginine residues (13.Derossi D. Chassaing G. Prochiantz A. Trends Cell Biol. 1998; 8: 84-87Abstract Full Text PDF PubMed Scopus (665) Google Scholar, 15.Prochiantz A. Curr. Opin. Cell Biol. 2000; 12: 400-406Crossref PubMed Scopus (272) Google Scholar, 16.Schwarze S.R. Hruska K.A. Dowdy S.F. Trends Cell Biol. 2000; 10: 290-295Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). The mechanism by which these highly charged peptides are able to translocate from the extracellular milieu into the cytoplasm and nucleus without breach of the plasma membrane permeability barrier is unknown. One possible translocation mechanism is direct transfer of the peptide across the plasma membrane by means of a reverse micelle, which would ensure that the peptide remains in a hydrophilic environment during membrane transit (1.Derossi 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 (966) Google Scholar, 13.Derossi D. Chassaing G. Prochiantz A. Trends Cell Biol. 1998; 8: 84-87Abstract Full Text PDF PubMed Scopus (665) Google Scholar, 15.Prochiantz A. Curr. Opin. Cell Biol. 2000; 12: 400-406Crossref PubMed Scopus (272) Google Scholar). Although functional studies leave little doubt that TAT and Antp peptides can carry cargo into cells, resulting in a biological effect (23.Tung C.H. Weissleder R. Adv. Drug Deliv. Rev. 2003; 55: 281-294Crossref PubMed Scopus (152) Google Scholar, 24.Leifert J.A. Whittion J.L. Mol. Ther. 2003; 8: 13-20Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), recent reports have called into question the fluorescence-based methodologies that are commonly used to assess peptide translocation (25.Lundberg M. Johansson M. Biochem. Biophys. Res. Commun. 2002; 291: 367-371Crossref PubMed Scopus (210) Google Scholar, 26.Richard 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 (1479) Google Scholar, 27.Thorén P.E.G. Persson D. Isakson P. Goksör M. Önfelt A. Nordén B. Biochem. Biophys. Res. Comm. 2003; 307: 100-107Crossref PubMed Scopus (269) Google Scholar). These concerns are highlighted in a study by Richard et al. (26.Richard 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 (1479) Google Scholar) that describes artifacts in analyses based on fluorescence microscopy of fixed cells and fluorescence-activated cell sorting; both of these techniques are widely used to characterize translocation of fluorescently labeled peptides. human immunodeficiency virus transactivator of transcription antennapedia fluorescein phosphate buffered saline propidium iodide Texas Red-labeled transferrin Hepes-buffered saline transferrin receptor. Fluorescence microscopy studies of peptide translocation into the cytoplasm and nucleus have commonly involved exposing cells to the fluorescently tagged peptide of interest, washing to remove residual peptide, fixing the cells, and examining them via confocal fluorescence microscopy to determine intracellular peptide distribution (1.Derossi 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 (966) Google Scholar, 2.Vives E. Brodin P. Lebleu B. J. Biol. Chem. 1997; 272: 16010-16017Abstract Full Text Full Text PDF PubMed Scopus (2063) Google Scholar, 4.Mitchell D.J. Kim D.T. Steinman L. Fathman C.G. Rothbard J.B. J. Peptide Res. 2000; 56: 318-325Crossref PubMed Scopus (907) Google Scholar). Richard et al. (26.Richard 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 (1479) Google Scholar) studied fluorescently labeled TAT-(48–60) (GRKKRRQRRRPP) and arginine nonamer (R9) by this method and found that the observed translocation of the peptides to the nucleus results from a fixation artifact. In live cells that were shown to be impermeable to propidium iodide, TAT-(48–60) and R9 accumulated in vesicles containing known endocytic markers such as transferrin receptor and the lipophilic dye FM 4–64 but showed no fluorescence in the cytoplasm or nucleus (26.Richard 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 (1479) Google Scholar). Studies conducted at low temperature or after depletion of cellular ATP with azide and 2-deoxy-d-glucose showed no evidence of uptake, implying that the entry process is energy-dependent, as expected for endocytosis. Similar conclusions of endocytic uptake have been reported for peptide cargo conjugates such as TAT-green fluorescent protein (28.Lundberg M. Wikström S. Johansson M. Mol. Ther. 2003; 8: 143-150Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar) and complexes such as fluorescein isothiocyanate-Avidin/TAT (29.Console S. Marty C. García-Echeverría C. Schwendener R. Ballmer-Hofer K. J. Biol. Chem. 2003; 278: 35109-35114Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). No cytoplasmic or nuclear staining was observed in either case. It has also been demonstrated that TAT-(44–57) and Antp-(43–58) are not able to translocate across liposomal membranes, providing further evidence for an energy-dependent pathway of internalization (30.Kramer S.D. Wunderli-Allenspach H. Biochim. Biophys. Acta. 2003; 1609: 161-169Crossref PubMed Scopus (86) Google Scholar, 31.Drin G. Cottin S. Blanc E. Rees A.R. Temsamani J. J. Biol. Chem. 2003; 278: 31192-31201Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar, 32.Thoren P.E.G. Persson D. Karlsson M. Norden B. FEBS Lett. 2000; 482: 265-268Crossref PubMed Scopus (214) Google Scholar). Endocytic uptake of Antp-(43–58) and SynB5, an analogue of protegrin 1, has also been reported (31.Drin G. Cottin S. Blanc E. Rees A.R. Temsamani J. J. Biol. Chem. 2003; 278: 31192-31201Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar). These peptides were also unable to enter liposomes. A recent critical review of the literature supports an endocytic mechanism of basic peptide uptake (24.Leifert J.A. Whittion J.L. Mol. Ther. 2003; 8: 13-20Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). In contrast, a report from Thorén et al. (27.Thorén P.E.G. Persson D. Isakson P. Goksör M. Önfelt A. Nordén B. Biochem. Biophys. Res. Comm. 2003; 307: 100-107Crossref PubMed Scopus (269) Google Scholar) contains somewhat different conclusions. This work showed that analogues of TAT-(48–60) and hepta-arginine containing one tryptophan residue (designated TAT P59W and R7W, respectively) were taken up into live PC-12 and Chinese hamster ovary cells. However, unlike other reports, (26.Richard 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 (1479) Google Scholar, 28.Lundberg M. Wikström S. Johansson M. Mol. Ther. 2003; 8: 143-150Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 29.Console S. Marty C. García-Echeverría C. Schwendener R. Ballmer-Hofer K. J. Biol. Chem. 2003; 278: 35109-35114Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar), Thorén et al. (27.Thorén P.E.G. Persson D. Isakson P. Goksör M. Önfelt A. Nordén B. Biochem. Biophys. Res. Comm. 2003; 307: 100-107Crossref PubMed Scopus (269) Google Scholar) observed weak fluorescence in the cytoplasm and nucleus of cells after incubation with these peptides. In addition, use of low temperature or depletion of cellular ATP with rotenone and 2-deoxy-d-glucose slowed but did not stop uptake, which led the authors to conclude that uptake occurs at least in part via an energy-independent process, i.e. a process other than endocytosis. Zaro and Shen (33.Zaro J.L. Shen W. Biochem. Biophys. Res. Comm. 2003; 307: 241-247Crossref PubMed Scopus (78) Google Scholar) have used a cell fractionation protocol to try to distinguish peptide entry via endocytosis from direct entry in the cytoplasm across the plasma membrane (the latter process is designated "transduction"). These authors concluded that the majority of TAT-(47–57) enters Chinese hamster ovary cells via transduction rather than endocytosis. Here we report fluorescence microscopy studies that extend recently reported conclusions (26.Richard 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 (1479) Google Scholar, 28.Lundberg M. Wikström S. Johansson M. Mol. Ther. 2003; 8: 143-150Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 29.Console S. Marty C. García-Echeverría C. Schwendener R. Ballmer-Hofer K. J. Biol. Chem. 2003; 278: 35109-35114Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 30.Kramer S.D. Wunderli-Allenspach H. Biochim. Biophys. Acta. 2003; 1609: 161-169Crossref PubMed Scopus (86) Google Scholar) in several important dimensions. Working with the TAT-(47–57) sequence, we show that fluorescently labeled peptide can gain access to the cytoplasm and ultimately the nucleus via endocytic uptake in live cells. The integrity of the plasma membrane permeability barrier was confirmed throughout our experimental procedures by demonstrating the ability of the cells to exclude propidium iodide. In addition, we show that a basic β-amino acid oligomer ("β-peptide") displays comparable or better uptake behavior than TAT itself. For both TAT-(47–57) and β-(VRR)4, experiments with ammonium chloride suggest that acidification plays an essential role in escape from endosomes to the cytoplasm and nucleus/nucleolus. Synthesis of Fmoc-β Amino Acid Monomers—Fmoc-protected β3hArg(PMC)-OH and β3hVal-OH were synthesized via Arndt-Eistert homologation (52.Guichard G. Abele S. Seebach D. Helv. Chim. Acta. 1998; 81: 187-206Crossref Scopus (178) Google Scholar) using a modified procedure described by Muller et al. (53.Muller A. Vogt C. Sewald N. Synthesis. 1998; 6: 837-841Crossref Scopus (98) Google Scholar). Fmoc-protected α-amino acids were obtained commercially. Peptide Synthesis and Labeling with Fluorescein (Flu)—Synthesis of Flu-α-TAT and Flu-β-(VRR)4 were performed on an Applied Biosystems Model 432A Synergy peptide synthesizer using the standard Fmoc solid-phase strategy with O-benzotriazol-1-yl-N-N-N′-N′-tetramethyluronium hexafluorophosphate (HBTU) activation and 1-hydroxybenzotriazol (HOBt) additive. Two-hour coupling times and extended deprotections were employed to synthesize β-(VRR)4. A β-homoglycine residue was incorporated at the N terminus of the peptide; the free N-terminal was then conjugated to 6-carboxyfluoresein manually using 3 equivalents of HBTU/HOBt in N,N-dimethylformamide. After a 12-h coupling, the resin was washed with 3× N,N-dimethylformamide, 3× methylene chloride, and 3× diethyl ether, and the peptide was then cleaved from resin using a solution of 95% trifluoroacetic acid, 5% thioanisole, and 2.5% ethanedithiol. Peptides were purified by preparative reverse-phase high pressure liquid chromatography and characterized by matrix-assisted laser desorption ionization time-of-flight analysis. The purified peptides were lyophilized and resuspended in deionized distilled water. Peptide concentrations were determined by UV-visible spectroscopy at 494 nm. Peptide stock solutions were stored frozen at –70 °C. Cell Culture—HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml) in a humidified incubator containing 5% CO2 (gas). Confocal Microscopy—HeLa cells that had been grown on 90-mm plates to sub-confluence were dissociated from the plates by treatment with Trypsin/EDTA for 15 min at 37 °C. An aliquot of 1 × 105 cells was plated on 30-mm glass-bottom culture dishes (MatTek Corp.) and cultured overnight in Dulbecco's modified Eagle's medium to allow the cells to adhere. The medium was removed and the cells were washed with phosphate buffered saline (PBS), pH 7.3. The cells were then incubated for 15 min at 37 °C with 1 ml Opti-MEM containing 7 μm peptide (Flu-TAT or Flu-β-(VRR)4) and 8 μg/ml propidium iodide (PI). The medium was again discarded, and the cells were washed 3 × 5 min with 2 ml PBS. Each wash solution contained 8 μg/ml PI. PBS (2 ml) containing 8 μg/ml PI was added to the cells, and the cells were then placed on ice. The cells were then viewed by confocal microscopy using a Bio-Rad MRC-1024 laser scanning confocal microscope. Co-localization Studies—Cells, grown and plated as described above, were incubated for 15 min at 37 °C with 1 ml of Opti-MEM containing 1.5 μm or 7 μm Flu-TAT or Flu-β-(VRR)4, 8 μg/ml PI, and either 25 μg/ml Texas Red-labeled transferrin (TR-Tf) or 1.25 μm Syto-60. The cells were washed after the incubation period and placed on ice as above. Fluorescence Bleed-through Controls—Control cells that had been incubated with only peptide or only the costain (TR-Tf or Syto-60) were used to determine appropriate microscope parameters that would prevent bleed-through among the far-red, red, and green fluorescence channels. These parameters were used to view cells that had been incubated with peptide, PI, and the desired costain. Energy Poison Studies—Cells were plated at a density of 1 × 105 cells/well in glass-bottom plates as described above, then incubated with 1 ml of glucose-free Hepes buffered saline (HBS) (pH 7.4) containing 50 mm 2-deoxy-d-glucose and 10 mm sodium azide (NaN3) for 30 min at 37 °C. Peptide (7 μm), 25 μg/ml TR-Tf, and 8 μg/ml PI were added to the HBS solution. The cells were incubated for an additional 15 min at 37 °C, washed with 3 × 2 ml of glucose-free HBS containing 2-deoxy-d-glucose and NaN3, and viewed by confocal microscopy. Treated cells were compared with mock-treated cells that had been incubated with HBS that did not contain NaN3 or 2-deoxy-d-glucose. Ammonium Chloride Studies—Cells plated to a density of 1 × 105 cells/well in glass-bottom plates were incubated with 1 ml of Opti-MEM containing 50 mm NH4Cl for 30 min at 37 °C (34.Nicola A.V. McEvoy A.M. Straus S.E. J. Virol. 2003; 77: 5324-5332Crossref PubMed Scopus (278) Google Scholar, 35.Poole B. Okhuma S. J. Cell Biol. 1981; 90: 665-669Crossref PubMed Scopus (567) Google Scholar). Peptide (7 μm) was added, and the cells were incubated for 15 min at 37 °C, washed with 3 × 2 ml of PBS containing 50 mm NH4Cl, and viewed by confocal fluorescence microscopy. Mock-treated cells that had been incubated with Opti-MEM that did not contain NH4Cl were viewed in parallel. The difference in uptake efficiencies in the presence and absence of NH4Cl was determined by cell counting of nuclear staining. The peptides used in this study are shown in Fig. 1. Flu-TAT is a fluorescein-labeled, 9-amino acid sequence corresponding to residues 47–57 (YGRKKRRQRRR) of HIV TAT; Flu-β-(VRR)4 is a fluorescein-labeled 12-residue β-peptide. HeLa cells were incubated with Flu-TAT and Flu-β-(VRR)4 for 15 min at 37 °C, washed, and examined by confocal fluorescence microscopy. All assays were conducted in small glass-bottom dishes, thus allowing the cells to be viewed in buffered solution instead of in mounted preparations. PI (8 μg/ml) was included during the incubation with peptide, as well as in all wash buffers, to establish cell viability. Because PI is membrane impermeant it cannot enter viable cells; non-viable cells with a compromised plasma membrane are readily detected because PI enters these cells and fluoresces strongly upon binding to nucleic acid. All of the data described in this paper are derived from an examination of live cells, i.e. those that excluded PI. Fig. 2A illustrates uptake of Flu-TAT into the cytoplasm and nucleus of a live cell; the fluorescence image also shows a non-viable cell displaying PI staining of the nucleus.Fig. 2Transport of Flu-TAT into endosomes, cytoplasm, and nucleus of live HeLa cells.A, confocal microscopy images of HeLa cells incubated for 15 min at 37 °C with 7 μm fluorescein-conjugated Tat peptide and 8 μg/ml propidium iodide in 1 ml of Opti-MEM. Cells were washed three times with PBS prior to viewing. Left, green fluorescent image of two cells stained with Flu-TAT-(47–57). Center, red fluorescent image of the same two cells, one of which is stained with propidium iodide. Right, transmission image of the two cells. Bar, 10 μm. B, HeLa cells were incubated with 7 μm Flu-TAT (47–57) and 8 μg/ml propidium iodide for 15 min at 37 °C. Left, image of punctate fluorescence, indicating endocytic uptake. Center, punctate and diffuse cytoplasmic and nuclear fluorescence. Right, cytoplasmic and nuclear fluorescence (note that the nucleolus appears to exclude fluorescence and can be seen as a dark spot in the brightly stained nucleus). Bar, 10 μm. C, HeLa cells were incubated with 7 μm Flu-TAT and 25 μg/ml TR-Tf for 15 min at 37 °C. Top row, images taken near the top of the cell; bottom row, same cell visualized at the equatorial plane. Left, green fluorescent image shows internalization of Flu-TAT. Center, red fluorescent image shows internalization of Texas-Red transferrin. Right, merged image of green and red fluorescent images showing co-localization of Flu-TAT with TR-Tf. Bar, 10 μm. D, bleed-through controls were performed to determine the appropriate microscope parameters for co-localization studies of Flu-TAT and TR-Tf. All images were taken using the same microscope parameters. Upper panels, left, green fluorescent image of a HeLa cell incubated with 7 μm TAT for 15 min at 37 °C; center, red fluorescent image of the same cell at levels that prevent bleed-through; right, transmission image of cell. Lower panels, left, green fluorescent image of a cell incubated with 25 μg/ml TR-Tf for 15 min at 37 °C; center, red fluorescent image of the cell, showing transferrin uptake; right, transmission image of cell. Bar, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Flu-TAT Is Distributed in Endocytic Vesicles as Well as in the Nucleus and Cytoplasm—When 7 μm Flu-TAT was incubated with HeLa cells for 15 min at 37 °C, the majority of the cells took up the peptide. A small fraction of cells (5–10%) showed no detectable fluorescence. Three distinct fluorescence patterns were observed (Fig. 2B). Some cells showed only punctate fluorescence (Fig. 2B, left panel), whereas others showed a combination of punctate fluorescence and more diffuse fluorescence in the cytoplasm and nucleus (Fig. 2B, center panel). A third group of cells showed only cytoplasmic and nuclear staining with little or no punctate fluorescence (Fig. 2B, right panel). The TAT peptide did not localize to nucleoli as reported in previous studies on fixed cells (3.Pooga M. Hallbrink M. Zorko M. Langel U. FASEB J. 1998; 12: 67-77Crossref PubMed Google Scholar), but instead showed a diffuse nuclear staining that tended to exclude nucleoli. Upon incubation with a lower concentration of Flu-TAT (1.5 μm), the majority of cells showed only punctate fluorescence as in Fig. 2B, left panel. At 7 μm peptide, ∼50% of the cells showed nuclear fluorescence (resembling the images in Fig. 2B, center and right panels), whereas the remaining cells showed only a punctate distribution with a faint, diffuse fluorescence background (as in Fig. 2B, left panel). To test whether the punctate distribution of Flu-TAT was due to endocytosis of the peptide, we performed a double-labeling experiment using a TR-Tf conjugate (Molecular Probes) and Flu-TAT (Fig. 2C). TR-Tf binds to the cell surface transferrin receptor (TfR) and is taken up by receptor-mediated endocytosis, thus providing a well characterized marker for the endocytic pathway (36.Dunn K.W. McGraw T.E. Maxfield F.R. J. Biol. Chem. 1989; 109: 3303-3314Google Scholar). Prior to imaging double-labeled cells, fluorescence images were taken of cells incubated with Flu-TAT or TR-Tf alone to determine the appropriate microscope parameters to minimize fluorescence bleed-through (Fig. 2D). Fig. 2C shows the top and equatorial views of cellular distribution of Flu-TAT (Fig. 2C, green, left panel), TR-Tf (Fig. 2C, red, center panel), and an electronically merged image (Fig. 2C, right panel) showing the overlap (yellow) between the Flu-TAT and TR-Tf distributions. The merged image clearly shows that there is considerable overlap between the punctate structures seen in the Flu-TAT image and the endocytic vesicles defined by TR-Tf, but that there are also TR-Tf-positive vesicles that do not contain Flu-TAT and a few Flu-TAT positive vesicles that do not contain TR-Tf. This result is consistent with other reports of endocytic uptake of the TAT peptide (26.Richard 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 (1479) Google Scholar, 28.Lundberg M. Wikström S. Johansson M. Mol. Ther. 2003; 8: 143-150Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). The TR-Tf-positive, Flu-TAT-negative structures may correspond to endosomes committed to the recycling pathway that is responsible for returning TfR to the cell surface (36.Dunn K.W. McGraw T.E. Maxfield F.R. J. Biol. Chem. 1989; 109: 3303-3314Google Scholar). Intracellular Localization of β-(VRR)4 to Endocytic Vesicles, Cytopl
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