Cell Transduction Pathways of Transportans - Bioconjugate Chemistry

Bioconjugate Chem. 2005, 16, 1399−1410

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Cell Transduction Pathways of Transportans Ka¨rt Padari,†,‡ Pille Saä¨lik,† Mats Hansen,§ Kaida Koppel,† Raivo Raid,‡ U ¨ lo Langel,§ and Margus Pooga*,†,‡ Estonian Biocenter, 23 Riia Street, 51010 Tartu, Estonia, Institute of Zoology and Hydrobiology, University of Tartu, 46 Vanemuise Street, 51014 Tartu, Estonia, and Department of Neurochemistry and Neurotoxicology, Arrhenius Laboratories, Stockholm University, S-10691 Stockholm, Sweden. Received April 25, 2005; Revised Manuscript Received September 8, 2005

Attempts to unravel the cell translocation mechanism of a growing number of cell-penetrating peptides (CPP) have revealed molecular determinants essential for internalization ability. The peptide sequence and the charge have been proposed to be the major factors in determining the membrane interaction mode and subsequent internalization pathway. Recent research in this field has shifted to search and design of novel CPPs with predefined vectorial properties and elucidation of the mechanism of cell entry of CPPs with high cargo delivery efficiency. Here we present a map of interaction modes with cell surface and intracellular traffic of transportan and its analogue TP10 complexed with fluorescently labeled avidin or streptavidin-gold conjugates. The protein cargo complexed with either peptide is transduced into HeLa and Bowes cells mostly in the endocytic vesicles with heterogeneous morphology and size as demonstrated by transmission electron microscopy (TEM) and confocal laser scanning fluorescence microscopy. Most of the induced vesicles are large, with 0.5-2 µm diameter, probably macropinosomes, but the complexes are present also in smaller vesicles, suggesting involvement of different pathways. Later the majority of complexes are translocated from the cell periphery into vesicles of perinuclear region and partly to lysosomes. A fraction of transportanstreptavidin complexes is present also freely in cytoplasm, both in the close vicinity of plasma membrane and more centrally, suggesting the escape from endosomal vesicles, since vesicles with discontinuous membrane were also detected by TEM. The cell-translocation process of transportanprotein complexes is temperature dependent and strongly inhibited at 8-10 °C and blocked at 4 °C when only interaction with the plasma membrane takes place.

INTRODUCTION

One of the major bottlenecks in introduction of oligonucleotide and peptide or protein-based drugs is the poor uptake of hydrophilic compounds by cells. For a decade, great expectations to overcome this obstacle were connected with cell-penetrating peptides (CPP) (also called protein transduction domains, membrane translocating sequences, or Trojan peptides), a class of 10-30 amino acid long peptides. Cationic and hydrophilic CPPs of natural and synthetic origin were first suggested to cross cellular membrane in a receptor- and temperatureindependent mode (1-3). The model of direct passage of CPPs across the plasma membrane, however, has been displaced by the more cell-centered concept of mainly endocytic uptake. Evidence that the cellular uptake of CPPs and their cargoes is highly dependent on physical parameters such as temperature (4-7), charge of the peptide (8-10), and experimental conditions (4, 11, 12) has been accumulated rapidly during the past years. Taking into consideration that more than 200 peptide sequences have shown cellular delivery properties and the number is still growing, one has to assume that the details of internalization mechanisms might vary. The rapidly growing number of examples of successful CPPmediated cargo delivery (13) demonstrates the potential * To whom correspondence should be addressed. Tel: (3727) 375 023, Fax: (372-7) 420 286. E-mail: [email protected]. † Estonian Biocenter. ‡ University of Tartu. § Stockholm University.

and development in the field of application of peptidic delivery vectors for biotechnological applications. The detailed knowledge about the internalization mechanism and intracellular trafficking of CPPs and their cargoes is still of fundamental significance. Efforts to define the intracellular pathway used by different CPP-protein complexes and fusion proteins have provided controversial results. It has been shown that Tat-GFP fusion proteins are taken up by HeLa and Jurkat cell lines via lipid raft-dependent caveolar endocytosis (14, 15). On the other hand, rapid internalization by lipid raft-dependent macropinocytosis without the involvement of caveolar marker protein caveolin has been demonstrated for TatCre fusion protein in mouse lymphocytes (16). Recently, Nakase and co-workers demonstrated that the uptake of penetratin is not mediated by macropinocytosis in HeLa cells (7). On the other hand, slightly improved oligoarginine uptake was observed when coapplied to HeLa cells with epidermal growth factor (7) suggesting a macropinocytic mechanism. Macropinocytosis is an actin-mediated endocytic process initiated usually by growth factors and characterized by the membrane ruffles and outgrowth of protrusions, which collapse and fuse with the plasma membrane generating large endocytic vesicles (17). Macropinosomes might be dissimilar, it has been reported about the different translocation speed of Adenovirus type 2 virus (Ad2)-containing and EGFinduced macropinosomes and the shorter lifetime of former (18). The same group also observed the increase of Ad2 uptake by clathrin-mediated endocytosis and the reduction of viral escape from endosomes as a response

10.1021/bc050125z CCC: $30.25 © 2005 American Chemical Society Published on Web 10/22/2005

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to the inhibition of macropinocytosis (18). It is not clear yet, whether the CPP-containing vesicular structures are of the same character/type as adenovirus- or EGFinduced macropinosomes and if the CPPs switch between the endocytic routes. The CPP-containing vesicular structures could be disrupted leading to the initiation of escape and the increased functional activity of delivered proteins as demonstrated by including membrane destabilizing sequences into fusion protein or exposing cells to multifunctional peptides containing both a CPP and the membrane disrupting motif (16, 19). The polyargininepeptide conjugates labeled with the fluorescent dye molecule are released from vesicular structures upon irradiation at an appropriate wavelength (20). Although it might provide a potentially useful technique for increasing the efficiency of CPP-mediated delivery of active cargoes, the mechanism of photo-destabilization of vesicular membranes is not well understood and the putative toxic effects of this method are not evaluated yet. Therefore, application of CPPs with intrinsic membrane-destabilizing properties is still of interest. One of the potential membrane-interacting peptides is transportan, a 27 amino acid long chimeric peptide (21), the C-terminal part of which has helical structure both in the aqueous environment and in the presence of lipid vesicles (10) and interacts with phospholipids using the hydrophobic face of the R-helix (22). Transportan has been demonstrated to be an efficient carrier for PNA (23), fluorophore-labeled peptide (24), and protein (12). One possibility to explain the high delivery efficiency of MAP (24) and transportan is the more efficient escape from vesicular structures as compared to penetratin and Tat peptide due to stronger interaction with membrane lipids. Therefore, the more detailed analysis of internalization and cellular trafficking of transportan are of interest. Since transportan modulates the activity of membranous G-proteins at high concentration (21), an analogue TP10, with no effect on the activity of GTPases, was designed (25). The cellular uptake and fate of CPPs and their cargoes has been mainly studied by using fluorescence or activity-based methods. The vesicular structures mediating the cell entry of CPPs are specified either by looking for colocalization with the respective marker proteins or by specifically inhibiting a certain type of endocytic process. Visualization on ultrastructural level might be helpful in characterizing the sites of initial interaction of CPPs with the plasma membrane or membrane-bound extracellular structures, changes of membrane morphology, size and shape of vesicular structures hiding CPPs, etc. Therefore, we used a preembedding labeling procedure and transmission electron microscopy (TEM) to visualize protein cellular delivery by transportan and TP10. The cellular uptake and trafficking of biotin-CPP complexes with colloidal-goldlabeled streptavidin was characterized after fixation and embedding of cells by TEM. The results obtained by electron microscopy in fixed cells were compared with confocal laser scanning microscopy (CLSM) data in living cells using analogous experimental setup, i.e., by using the complexes of biotin-CPP with fluorescently labeled avidin. MATERIALS AND METHODS

Peptide Synthesis. Transportan (GWTLNSAGYLLGKINLKALAALAKKIL-amide) and TP10 (AGYLLGKINLKALAALAKKIL-amide) were synthesized stepwise on a 0.1 mmol scale on an automated peptide synthesizer (Applied Biosystems Model 431A) using the t-Boc solidphase peptide synthesis strategy. tert-Butyloxycarbonyl

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amino acids were coupled as hydroxybenzotriazole esters to a p-methylbenzylhydrylamine resin (Neosystem, Strasbourg, France) to obtain C-terminally amidated peptides. Biotin was coupled to the side-chain of Lys13 or Lys7 in transportan and transportan 10, respectively, as previously described (12). The final cleavage and purification of peptides was performed, as described (12). The molecular weights of biotinylated peptides were determined by MALDI-TOF mass spectroscopy (Voyager STR). Cell Cultures. The human breast cancer cell line HeLa (ATCC CCL-2) and the human Bowes melanoma cells (ATCC CRL-9607) were cultivated in a humidified atmosphere containing 5% CO2 at 37 °C in Iscove’s Modified Dulbecco’s Medium (IMDM) or in Dulbecco’s Modified Eagle’s Medium (DMEM), respectively. Culture medium was supplemented with 10% fetal calf serum (FCS), 100 IU/mL penicillin, and 100 µg/mL streptomycin. Delivery of Streptavidin-Gold into Cultured Cells by Biotinyl-Transportan and Biotin-TP10. The cells used for experiments were grown for 2 days on round Thermanox tissue culture coverslips (diam 13 mm, Electron Microscopy Science, Fort Washington, PA) in 24well plates to 80-100% confluence. The stock solution of biotin-transportan (1 mM) was treated with 10 mg/ mL L-leucine in a minimal volume for 5 min at room temperature in order to dissociate the multimers of peptide. The biotinylated transportan or TP10 was complexed with streptavidin-gold (d 10 nm, Amersham Pharmacia Biotech, UK) for 3 min, diluted with the respective culture medium and applied to cells. Cells were incubated with the complexes of 25 or 5 µM biotintransportan or -TP10 and streptavidin-gold (1:50 dilution) at 10, 20, or 37 °C for 20 min to 72 h depending on the experiment. Labeling of Transferrin with Colloidal Gold. The colloidal gold solution of particles with 20 nm diameter was prepared, and absorption of transferrin was performed essentially as described earlier by Bendayan (26). Briefly, the gold sol was prepared by adding 1.5 mL of 1% sodium citrate solution to 100 mL of boiling solution of 0.01% HAuCl4 dropwise over 5 min and boiling the mixture for 5 min. The resulting gold colloid was coated with 2.5 mg transferrin (Sigma, dissolved in 2 mL of water) at room temperature for 30 min. Transferringold was stabilized with 1 mg/mL PVP40, 0.5 mg/mL PEG20000, and 0.5 mg/mL BSA and harvested by centrifugation at 10000g for 1 h at 4 °C. The residual free protein and aggregated material was removed by centrifugation at 5000 rpm for 45 min in discontinuous gradient of 10, 30, and 50% glycerol in PBS in a SW41 rotor. The most intensely colored fraction of transferringold with homogeneous particle size was collected and stabilized for storage with 2% PVP (poly(vinylpyrrolidone)), 0.2 mg/mL PEG and 0.05% NaN3. Comparative Uptake of Transferrin and Transportan-Streptavidin Complexes by HeLa Cells. HeLa cells were grown on Thermanox coverslips in 24well plates as described above. The nearly confluent cell layer was washed twice with serum-free IMDM and incubated in serum-free culture medium for 1 h in order to upregulate the transferrin receptors on plasma membrane (27). The starved cells were incubated with complexes of 25 or 5 µM biotin-TP and 1:50 dilution of streptavidin-gold (10 nm) along with the transferringold complex (20 nm) at 1:50 dilution in serum-free IMDM in CO2 incubator at 37 °C for 30 min. In some experiments cells were first incubated with TP-streptavidin complexes for 30 min, and then the solution

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containing complexes was removed and replaced with the culture medium with gold-labeled transferrin and incubated for an additional 30 min. An analogous experiment with reversed order, first transferrin followed by TPstreptavidin complexes, was also carried out. Electron Microscopy. Cells were fixed for transmission electron microscopy with 3% glutaraldehyde in 0.1 M sodium cacodylate-HCl buffer (pH 7.3) containing 0.05 M sucrose for 1-2 h. After being rinsed with cacodylate buffer, the cells were postfixed with 1.5% osmium tetraoxide in the same buffer containing 0.7% potassium ferrocyanate at 4 °C for 30 min and dehydrated in ethanol series from 50% up to 96%. Cells on Thermanox coverslips were embedded in epoxy resin, and the material was cut into ultrathin sections of 40-60 nm by using the Reichert Om U2 ultramicrotome. The sections were collected on grids and stained with 2% uranyl acetate in 50% ethanol and lead citrate according to instruction (28). Microtomed sections on the grids were examined and the images recorded by using a JEM-100S (JEOL, Japan) electron microscope. The scanned electron microphotos were analyzed and processed with Adobe Photoshop version 7.0 software. Fluorescence Microscopy Experiments with Live Cells. HeLa cells (8 × 103) were seeded onto eight-well chambered coverglasses (Nalge Nunc International, Rochester, NY) 2 days before uptake experiments. The glasses were coated with collagen-fibronectin solution (10 µg/ mL fibronectin, 30 µg/mL collagen, 0.01% bovine serum albumin, 100 IU/mL penicillin, and 100 µg/mL streptomycin in DMEM) for 30 min before seeding the cells. Cells were incubated with the complexes of 0.15 µM Alexa Fluor (AF) 488 labeled avidin (Molecular Probes, Leiden, Netherlands) and 0.5 or 1 µM biotin-CPP in serum free IMDM for 1 h at 37 °C. The incubation solution was supplemented with 25 µg/mL transferrin (labeled with AF 594, Molecular Probes) or 150 nM Lyso Tracker Red DND-99 (Molecular Probes) depending on experiment. For visualizing the Golgi apparatus, the cells were incubated with 0.5 µM BODIPY-TR-C5 ceramide complex with BSA (prepared according to manufacturer’s instructions, Molecular Probes) in serum free IMDM for 30 min before applying peptide-avidin complexes into the same medium. After 1 h incubation, the medium with CPPavidin complexes and the markers of cellular organelles was removed and the cells were rinsed with PBS (containing 1 mM of CaCl2 and 0.5 mM MgCl2) followed by a brief rinse with 0.05% trypan blue in PBS and two additional washing steps with PBS. Cells were covered with calcium and magnesium-supplemented PBS, and the serial images from live cells were recorded using the laser confocal scanning microscope MRC-1024 with excitation at 488 and 568 nm (Bio-Rad Laboratories, Hercules, CA). To avoid cross-talk between the channels, emission signals were collected independently. Obtained images were processed with Adobe Photoshop version 6.0 software. Fluorescence Microscopy Experiments with Fixed Cells. HeLa cells (2 × 104) were seeded 2 days before experiment on round glass coverslips into 24-well plate. Incubation with peptide-protein complexes was performed in serum-containing (10% FCS) medium. For the pulse-chase study, the medium with complexes was replaced after 1 h by media containing no complexes for 4 or 24 h according to the experiment. Before applying fresh media, the cells were rinsed twice in PBS to completely remove the nonassociated complexes. Cells were fixed for 20 min in 4% paraformaldehyde on ice, followed by 5 min permeabilization in 0.1% Triton X-100

in PBS. Nonspecific binding sites were blocked with 10% (w/v) nonfat dry milk solution in PBS for 30 min. Lysosomes were visualized with 1:50 (hybridoma supernatant) solution of LAMP2 primary antibody and 10 µg/ mL AF 594-labeled goat anti-mouse secondary antibody. For revealing the actin fibers, cells were stained with 30 nM Texas Red-labeled phalloidin. Images were recorded using the laser confocal scanning microscope or Olympus BX61 microscope equipped with CCD camera DX70. RESULTS

Association of Transportan-Streptavidin Complexes with the Cell Surface. To characterize the cellular delivery of proteins by transportan peptides, we followed the uptake of noncovalent complexes of biotintransportan (or biotin-TP10) with streptavidin-gold by electron microscopy. The complexes between the biotinCPP and gold-labeled streptavidin form quickly and were assembled by coincubating the respective components before applying to cells. The association of transportanand TP10-protein complexes with the cell surface was examined in HeLa and human Bowes melanoma cells, which slightly differ in morphology. The surface of HeLa cells was usually more structured, possessing protrusions, long filopodia, and smaller microvillar extensions, whereas Bowes cells had a rather smooth plasma membrane with fewer protrusions and long extensions spread only between adjacent cells. The peptide-protein complexes associated quickly with the surface of the cells, already upon a 5-10 min incubation and were detectable by electron microscopy between the cells mostly as elongated structures of 0.4-2 µm on the electron dense background (Figure 1A-C). The initial interaction of transportan-streptavidin complexes induced formation of a small pit in the plasma membrane (Figure 1A). The aggregates were more typical for the complexes with transportan rather than with TP10. Formation of big peptide-protein aggregates was reduced by hindering the multimerization of transportan during formation of complexes with gold-labeled streptavidin by using leucine. The cells incubated with such “dissociated” complexes at 3 µM peptide concentration resembled the complexes of TP10 and showed significantly lower degree of aggregation (Figure 1D). The smaller aggregates but not the extensive agglomerates still formed, and the complexes were distributed more diffusely on the protrusions of cells. The peptide-protein complexes preferentially associated with filopodia and extensions of plasma membrane in HeLa cells. A smaller fraction of complexes associated with the flat areas, and most of these seemed to be in close contact with the lipids of the plasma membrane. Transportan-Avidin Complexes Associate with Membrane Protrusions and Actin Cytoskeleton. The nature of membrane protrusions involved in the transportan-mediated protein internalization was further assessed by confocal microscopy in cells with visualized actin. Staining of actin cytoskeleton with Texas Redlabeled phalloidin revealed highly structured morphology of HeLa cells’ plasma membrane with numerous protrusions. The protrusions were most abundant between the neighboring cells and oriented parallel bridging the cells, whereas thinner and more heterogeneous microspikes were observed in other regions of cellular membrane (Figure 2A). Transportan-avidin complexes docked to the intercellular membrane protrusions starting from 5 min incubation (data not shown). After 1 h incubation, the complexes were detected as punctate structures abundantly decorating the actin-filament-containing mem-


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Figure 1. Interaction of CPP-protein agglomerates at the plasma membrane of cells. Human Bowes melanoma cells incubated with the complexes of gold-labeled streptavidin (10 nm, dilution 1:50) and 5 µM biotin-transportan for 30 min (A, B) or 2 h (C). HeLa cells (D) incubated for 1 h with the complexes of streptavidin-gold (10 nm, 1:50) and 2.5 µM biotin-transportan, treated with leucine (10 mg/mL) before complexing with protein-gold.

brane protrusions. The colocalization of peptide-protein complexes with actin fibers was detected also in the cortical cytoplasm of cells where actin filaments were packed less densely (Figure 2A). This suggests that intracellular translocation of peptide-avidin complexes might at least partially be mediated by actin cytoskeleton. Morphology of the Plasma Membrane Changes upon Internalization of Transportan-Streptavidin Complexes. Streptavidin-gold complexes with transportan or TP10 entered the cells by using at least two morphologically clearly distinguishable mechanisms. First, docking of larger CPP-protein aggregates induced morphological changes in the plasma membrane leading to the formation of membrane invaginations (Figure 1BD). Later the complexes shifted deeper into the cytoplasm, being finally fully engulfed by the cell into endocytic vacuoles (arrows in Figure 1C and enlarged sections of Figure 1B and C). The membrane bound aggregates seemed to repulse or force the indentation of plasma membrane. Various cell surface extensions were frequently involved in the translocation process as demonstrated in Figure 1B and D. The second mode of internalization was characteristic for smaller complexes containing few or a single gold particle, which did not induce detectable changes in the membrane morphology upon interaction with the plasma membrane (arrows in Figure 3A and the enlarged section of Figure 3A and B). Later the complexes with a single gold particle were also detectable in cortical cytoplasm, and changes in morphol-

ogy of plasma membrane were not observed (Figure 3C). The second mode was not the major cell entry mechanism of streptavidin-gold complexes and was more characteristic for TP10 than transportan complexes and at the lower concentrations of peptide. It has to be mentioned that despite the differences in HeLa and Bowes cells morphology, both internalization modes were observed in both cell lines. Distribution of Protein Delivered into Cells by Transportans. Streptavidin-gold complexes with transportan or TP10 were detectable in the cortical cytoplasm of Bowes and HeLa cells already after 10 min of incubation with complexes at 37 °C. Later in 1-2 h, transportan-streptavidin complexes spread over the cytoplasm, including the perinuclear region. The cell-transduced peptide-protein aggregates resided mainly in vesicular structures of irregular shape and varying diameter of 0.5-2.0 µm, which primarily seemed to depend on the size of internalized aggregate (Figure 4). The gold particles localized mostly in large vacuoles of 0.5-1.0 µm and in the multivesicular relatively spherical structures of 200-300 nm in diameter (large arrows in Figure 4A and B), the latter resembling the structures of endosomal-lysosomal pathway. Some complexes were present in long tubulovesicular structures, which were more typical for Bowes cells (Figure 4C and small arrow in Figure 4A). The complexes containing vesicles in HeLa cells were more homogeneous and spherical with maximal diameter of 0.3-0.5 µm. A small fraction of peptideprotein complexes was also present in small caveosome-



Figure 2. Localization of peptide-avidin complexes in relation to the actin cytoskeleton and organelles. Complexes of 1 µM (A) or 0.5 µM (B-D) biotinylated transportan with 0.15 µM AF 488-labeled avidin in HeLa cells after 1 h incubation (green). Actin fibers were stained with 30 nM phalloidin-Texas Red (A). Coincubation of cells with TP-avidin complexes with 0.5 µM BODIPY-TR-C5 ceramide (B) or 50 µg/mL AF 594-labeled transferrin (D) was performed to reveal Golgi apparatus, and endosomes, respectively. Pulse-chase study: cells were incubated for 1 h with TP-avidin complexes, followed by a 24 h chase period. Lysosomes were visualized by staining with LAMP2 antibody and AF 594-labeled anti mouse antibody (red) (C). Colocalized structures are visible as yellow. Fluorescence microphoto of HeLa cells (A) and confocal sections at the equatorial level of nuclei (B-D).

Figure 3. Nonaggregated CPP-protein complexes associate directly with the plasma membrane. HeLa cells incubated for 1 h with streptavidin-gold (10 nm, 1:50) complexed with 2.5 µM biotin-transportan (A, B) or biotin-TP10 (C) in the presence of leucine.

like vesicles of about 100 nm diameter or less (arrowhead in Figure 4A). Most of the complexes containing vesicles in HeLa cells were intact and surrounded by a continuous membrane. However, some of the vesicles that contained peptide-protein complexes had a discontinuous membrane, and the electron density of the vesicle content was undistinguishable from that of cytosol (arrows in Figure 4C). Moreover, considerable part of gold particles was spread apparently free in cytosol, often in the close proximity of vesicles with discontinuous membrane as indicated by the arrowheads in the enlarged section of Figure 4C. One might assume that the membrane was

destabilized by transportan or TP10, and some vesicles were broken enabling the escape of complexes into cytosol. Data obtained by electron microscopy were complemented by analogous experiments using the confocal laser scanning microscopy for analyzing the localization of biotinylated transportan complexed with AF 488labeled avidin. After 1 h incubation, the transportanavidin complexes localized in living cells in a punctate manner mostly at the cellular membrane or in the cortical cytoplasm but also in the perinuclear area (Figure 2B-D). However, fluorescent punctate structures


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Figure 4. Localization of CPP-protein complexes in the cells after incubation at 37 °C. HeLa cells (A, B) incubated for 1 h with streptavidin-gold (10 nm, 1:50) complexed with 3 µM biotin-transportan (A) or 2.5 µM biotin-TP10 (B) in the presence of leucine. Bowes cells (C, D) incubated with the complexes of streptavidin-gold (10 nm, 1:50) and 5 µM biotin-transportan for 2 h (C) and 72 h (D). p ) plasma membrane; n ) nucleus.

were differentsspherical aggregates with the higher diameter concentrated to the perinuclear area into one or two centers, whereas smaller dots were distributed in the cortical cytoplasm (Figure 2A-D). Some big fluorescent aggregates were attached to the cellular membrane, and very few were occasionally moving freely in the extracellular media. Large aggregates probably resulted from the multimerization of the peptide and the assembly of the formed complexes into bigger agglomerates, which was observed also at higher peptide concentrations in electron microscopy experiments (Figure 1A-C). Some agglomerates were not in contact with the cells, and their presence in the media after many washing steps suggests that initially membrane-bound aggregates could detach during treatment/visualization. CPP-Protein Complexes Accumulated in the Perinuclear Area of Cells upon Extended Incubation at 37 °C. To find out whether the cell-transduced protein is later mainly directed to the cellular degradative pathway or remains in the cytoplasm, we extended the incubation of Bowes cells with transportan-streptavidin complexes to 2 days or longer. After 20-72 h, peptideprotein complexes were concentrated in structures resembling late endosomes and lysosomes as observed by electron microscopy (enlarged section and arrows in Figure 4D). However, some gold particles were still scattered diffusely in the cytoplasm. Upon extended incubation of the cells with the complexes, the amount

of gold particles was significantly increased, especially in the perinuclear region, confirming the continuous uptake for more than 4 h (12). Our data indicated that some complexes of CPP-protein retained intactness for at least several hours before accumulation to the degradative organelles after 20 h. The intracellular trafficking of peptide-protein complexes in relation to structures of the degradative pathway was studied by visualizing the plasma membrane and Golgi complex with BODIPY TR C5-labeled ceramide and acidic intracellular vesicles, including lysosomes, with Lyso Tracker Red DND-99. Fluorescent ceramide derivative inserts into the plasma membrane of live cells and is preferentially targeted to the Golgi apparatus. After incubating HeLa cells with TR-ceramide, the whole cell cytoplasm exhibited reticular staining reminiscent with the endoplasmic reticulum. Labeled ceramide concentrated also into one or two perinuclear centers probably in Golgi apparatus (Figure 2B). The transportan-avidin complexes showed, after translocation into the HeLa cells, a partial colocalization with TR-ceramide in perinuclear areas (Figure 2B). Specific accumulation of transportan-avidin complexes in the reticular structures of cytoplasm was not observed. Lyso Tracker intensely stained the perinuclear area of the HeLa cells and some cortically localized vesicles, but the entire cell area fluoresced weakly red probably due to nonspecific interaction with other membranous structures. Localization of transportan-avidin complexes



Figure 5. Interaction of CPP-protein complexes with plasma membrane of cells and internalization at 10 °C. Bowes (A) or HeLa (B, C) cells were incubated with complexes of 5 µM biotin-transportan (A, B) or 3 µM biotin-TP10 (C) with 5 nm (A) or 10 nm (B, C) streptavidin-gold (1:50) for 1 h at 10 °C.

taken up by the cells and Lyso Tracker overlapped partially only in large vesicles of the perinuclear area (data not shown). A more specific staining of lysosomes in HeLa cells was achieved with antibody specific to lysosome-associated membrane protein-2 (LAMP2). For enlightening the intracellular targeting of peptideprotein complexes in time, we performed pulse-chase experiments by growing the cells for 4 or 24 h in culture media after a 1 h pulse with complexes of transportan and avidin. After a 4-h chase, the fraction of complexes in lysosomes was not substantial (data not shown). However, after a 24-h chase, the amount of complexes inside the HeLa cells had markedly increased, suggesting the continuing accumulation of internalized complexes (Figure 2C) corroborating the data obtained by electron microscopy. Colocalization of transportan-avidin complexes with lysosomes was detected mostly in the perinuclear region including some very big vesicular structures. However, even after a 24 h chase, a fraction of complexes-containing vesicles retained their localization distinct from the degradative organelles marked by LAMP2 antibody (Figure 2C). Translocation of CPP-Streptavidin Complexes into HeLa and Bowes Cells Is Not Blocked at 10 °C. The pinocytosis and receptor-mediated endocytosis are the potential cellular uptake mechanisms of large molecular structures. Endocytosis occurs usually within minutes to several hours and is cellular-energy-dependent. In general, if the temperature is lowered below 18 °C, endocytosis is considered to be blocked, and also the flexibility and the fluidity of plasma membrane is drastically reduced (29) and the membrane traffic is slowed (30). We evaluated whether peptide-protein complexes could enter the cultured cells at the endocytosis abolishing temperatures, e.g. 8-10 °C. After incubation of Bowes or HeLa cells with complexes of biotin-transportan and streptavidin-gold for 1 h at 10 °C, the gold particles were clearly detectable attached at the plasma membrane but also in the vesicular structures of cortical cytoplasm. The amount of internalized complexes at 10 °C was markedly lower compared to the complexes visible at the physiological conditions but still clearly detectable by electron microscopy. As demonstrated in Figure 5, transportan-

streptavidin complexes resided mainly in rather small spherical vesicles of 0.2-0.3 µm diameter (arrows in Figure 5A and B) and localized at the plasma membrane of cells (enlarged section of Figure 5B). TP10-protein complexes were mostly localized randomly as single particles near the plasma membrane or very rarely in vesicles in the peripheral cytoplasm (enlarged section of Figure 5C). In addition, no big agglomerates of peptideprotein complexes formed nor were large cell surface invaginations observed at 8-10 °C. Thus, under the conditions of cellular energy deficiency, the cellular uptake of biotin-transportan complexes with streptavidin-gold was not completely abolished, but the formation of very big vesicular structures and their further translocation toward the cell center were strongly inhibited, as the complexes were detected only in the cortical cytoplasm. On the contrary, at 20 °C, the transportanstreptavidin complexes were distributed in the cells in vesicular structures analogously as observed at the physiological temperature. Still, the amount of complexes in cells was lower, and the large membrane invaginations so typical to physiological temperature were almost missing (data not shown). Cellular Uptake of Transportan-Streptavidin Complexes Is Abolished at 4 °C and Lower Temperature. Although the internalization of CPP-protein complexes was diminished at 8 °C, we assessed whether transportan or TP10 could transduce streptavidin-gold into cells at 0-4 °C also. After incubation of the cells with transportan-streptavidin complexes on ice for 1 h, only a negligible amount of gold particles had associated with the plasma membrane. Association of transportanstreptavidin complexes with the plasma membrane was rather rare, and no complexes were detected in the cells. The absence of uptake at 4 °C suggests the prevalence of energy-dependent processes in the internalization of peptide-protein complexes. However, some complexes of streptavidin-gold with transportan or TP10 translocated into cells even at 10 °C support the ideas about uptake pathways that are active at low temperatures (31) also. Cellular Uptake of Transportan-Streptavidin Complexes in Parallel with Transferrin. Our results indicate that CPP-protein complexes are taken up by


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Figure 6. Colocalization of transferrin and CPP-protein complexes in HeLa cells. Cells were incubated in serum free culture medium containing the complexes of streptavidin-gold (10 nm, 1:50) with 5 µM (A, B) or 2.5 µM (C) biotin-transportan, and 20 nm gold-labeled transferrin (1:50) at 37 °C for 1 h.

the cells mainly via temperature-dependent endocytic pathways that originate from the cell membrane invaginations and result in the formation of cellular vesicles of different shape and size. Transferrin, an iron-transport protein, is known to be internalized via clathrin-coated pits and vesicles (32). To assess whether this endocytic pathway is involved in the internalization of transportan-streptavidin complexes, we tested the putative colocalization of internalized complexes with transferrin. Prior to the addition of the peptide-protein complexes, HeLa cells were first incubated with serum-free culture medium for 30 min to induce the targeting of transferrin receptors to the plasma membrane (27). Transferrin, tagged with colloidal gold, induced the formation of clathrin-coated pits and the respective vesicles and in addition associated with the terminals of microvilli and filopodia. Cells incubated with the peptide-streptavidin complexes labeled with a 10 nm gold particle along with a 20 nm gold particle-tagged transferrin in serum-free medium for 30 min revealed no specific colocalization of these proteins. Still, in some areas at the plasma membrane and cellular vesicles, location of transferrin and transportan-streptavidin complexes overlapped. Complexes of streptavidin with TP10 showed less colocalization with transferrin than transportan complexes. The ultrastructural analysis, however, revealed that if transportan-protein complexes and transferrin were used at high concentrations (respectively 5 µM biotintransportan and 1:25 transferrin-gold), colocalization on the plasma membrane was prevailing. As shown in Figure 6A and B, transferrin associated mainly with the regions of plasma membrane where peptide-protein complexes had concentratedspreferentially at the protrusions and also on smooth areas. Inside the cell, both the peptide-protein complexes and transferrin molecules were usually confined to the same big vacuoles of 0.5-1 µm diameter in the cortical and perinuclear cytoplasm (enlarged section of Figure 6B and arrows in Figure 6C). Some peptide-protein complexes localized in cytoplasm/ cells without transferrin as indicated by arrowheads on the enlarged section of Figure 6C. Distribution of peptide-protein complexes in the cells coincubated with the gold-labeled transferrin was very similar to that observed in cells treated with the complexes only, whereas the localization of transferrin at the plasma membrane and

inside the cells was radically altered by coincubation with transportan-streptavidin complexes. The colocalization of CPP-protein complexes with transferrin was observed mainly at higher concentrations. Surprisingly, a considerably lower number of transferrin-gold particles associated with the cells incubated with transferrin only as compared to the cells coincubated with transportanstreptavidin complexes (data not shown). In addition to peptide-streptavidin complexes, transportan also induced increased association of transferrin with the plasma membrane and subsequent uptake. In principle the cationic transportan might associate at high concentration with anionic transferrin and increase association with the plasma membrane and the uptake of transferrin by cells. To exclude the putative association of transportan with transferrin in solution or at the plasma membrane, we incubated cells with these consecutively. Cells were first incubated with TP-streptavidin complexes for 30 min, washed several times, and then incubated with transferrin-gold complexes for an additional 30 min. An analogous experiment with reversed order of incubations was also performed. Under these conditions, no noticeable colocalization was detected between transferrin and transportan-protein complexes (data not shown). In the cells, transferrin and peptide-protein complexes localized mainly to different vesicular compartments, whereas a fraction of both was colocalizing in a certain type of vesicular structures, probably in late endosomes. This suggests that transportan-streptavidin complexes are taken up to large vesicular structures and that this pathway does not overlap with the well-characterized clathrin-mediated endocytic pathway of the transferrin receptor. However, during later targeting, the structures containing peptide-protein complexes may fuse with multivesicular bodies on their way to lysosomes. The involvement of receptor-mediated endocytosis in the internalization of peptide-avidin complexes was proven by confocal microscopy as well, following their cellular uptake in the culture medium containing AF 594labeled transferrin. After 1-h incubation, transferrin had concentrated in a punctate manner in the cytoplasm of cells with more intense signal in the vesicular structures of perinuclear area (Figure 2D). Structures containing transportan-avidin complexes rarely overlapped with


the transferrin-positive vesicles in the cortical cytoplasm and were usually less abundant, whereas in larger perinuclear vesicles the colocalization with transferrin was more common but still not prevailing (Figure 2D). DISCUSSION

The power of cell-penetrating peptides to transduce various payloads of very different nature and size into living cells in culture and in animal models has found rapidly growing application (13). Despite intense studies during the past decade, the cell entry mechanism of “Trojan peptides” has remained elusive (33), though several cellular pathways have been demonstrated to be involved in the uptake of CPPs (4, 14-16, 31, 34-37). We characterized the uptake and cellular whereabouts of proteins complexed with delivery peptides transportan and TP10 by transmission electron microscopy and confocal fluorescence microscopy. Electron microscopy reveals that the complexes of gold-labeled streptavidin with biotin-transportans are mostly detectable as elongated agglomerates on the electron-dense background associating with the plasma membrane of the cells frequently at the cell surface extensions, microspikes and filopodia. Since large aggregates of peptide-protein complexes formed only at higher peptide concentration (5 µM) and were more typical for transportan rather than TP10-protein complexes, the aggregation might be caused by multimerization in solution (21). Complexation of biotin-transportan at 0.5-1 µM concentration with avidin did not induce formation of aggregates as observed by fluorescence microscopy, whereas by increasing the peptide concentration the size of cell-associating assemblages also increased. Peptide-protein complexes associated with the cellular surface were mostly on the electron dense background, which was not present when the complexes were assembled in solution in the absence of the cells. Therefore, the electron dense material is obviously of cellular origin and may represent exoplasmic membrane proteins or cell surface proteoglycans. Considering the role of proteoglycans in cell-cell and cellmatrix interactions (38, 39), in peptide-mediated cellular transduction (35, 40, 41) and strong interaction of cationic CPPs with heparan sulfates (42), the interaction of transportan-protein complexes with the cell surface proteoglycans is highly probable. The mechanisms used by CPPs to translocate into mammalian cells may vary in different cells and between peptides; so far endocytosis via clathrin-coated pits (34, 43) and via lipid rafts (14, 40) have been suggested. In parallel, macropinocytosis has been proposed as a route for CPPs translocation (7, 16). Macropinocytosis is a lipid raft-mediated, clathrin- or caveolae-independent endocytic process (44), which starts with the induction of actin-dependent lamellipodia and membrane ruffling leading internalization of extracellular material into the macropinosomes of 1 µm or greater. The results obtained by electron microscopy reveal that transportan-streptavidin complexes associated with the extensions of cell surface and induced invaginations of the plasma membrane are reminiscent of the changes preceding the macropinocytosis of some pathogenic bacteria (45, 46). The vacuoles formed in the cytoplasm could fuse with lysosomes or release their content into the cytosol (17). Our results demonstrate that the cell-transduced goldtagged streptavidin localized mainly in intracellular vesicular structures with different size (0.5-2 µm) and in Bowes cells the vesicular membrane was not always intact (47). On the basis of these considerations, it seems probable that transportan-mediated cellular uptake of


protein molecules proceeds at least partially by macropinocytosis and transportan is able to destabilize the macropinosomes. The peptide-protein complexes marked by gold particles were also found in vesicles characteristic to receptor-mediated endocytic pathway, e.g. multivesicular bodies and endosomal-lysosomal structures suggesting the involvement of clathrin-dependent endocytosis. In most animal cells, the clathrin-dependent pathway guarantees the uptake of specific macromolecules, nutrients, and ions from the extracellular fluid (48). Transferrin, a typical marker of clathrin-dependent endocytosis, and transportan-protein complexes colocalized partially on the plasma membrane of the HeLa cells and later in the endocytic vacuoles. Colocalization of transportan-streptavidin complexes and transferrin was more characteristic at higher concentrations of substances and less obvious at lower concentration. The results of confocal laser scanning microscopy confirmed that vesicular structures containing transportan-avidin complexes rarely overlapped with transferrin-AF 594 positive vesicles, suggesting that a small fraction of transportan-protein complexes was cointernalized with transferrin or targeted into the same vesicles intracellularly. At high concentrations weak electrostatic interaction of cationic transportan complexes with anionic transferrin might be envisaged, leading to the increase of transferrin uptake and altering its intracellular distribution. Different endocytic pathways target the ingested material to specific cellular destinations: degradative organelles, the plasma membrane, ER, Golgi apparatus. The destination of the protein molecules translocated into cells by CPPs is of primary importance both from the view of mechanisms and application. Neither electronnor fluorescence microscopy revealed accumulation of transportan-protein complexes to endoplasmic reticulum or Golgi apparatus, though some colocalization with the respective markers was detected by fluorescence microscopy in perinuclear area. On the contrary, part of transportan-protein complexes was targeted to late endosomes and lysosomal structures, especially after 20 h or later. However, a substantial amount of labeled complexes was detectable in the cytoplasm or vesicles of nondegradative character after 24 or even 48 h of incubation. Caveolae-mediated endocytosis which starts from cholesterol and sphingolipid rich membrane rafts has been proposed for the cellular translocation of Tat fusion proteins (14, 15). Electron microscopy revealed that streptavidin complexed with transportans was occasionally present also in caveosome-like structures, mainly in the cortical cytoplasm. This internalization mechanism is not prevailing since by confocal fluorescence microscopy we did not observe the accumulation of peptide-protein complexes to the Golgi apparatus, but it suggests that complexes can use different endocytic pathways simultaneously. Apparently all endocytic pathways have been shown to be used to gain access into eukaryotic cells by viruses (49). Cell-entry of peptide-protein complexes has several features similar to internalization of Simian Virus 40 (SV40). We detected peptide-protein complexes in vesicles of different size and morphology with little overlap with clathrin-coated vesicles, indicating other transduction modes; for example, SV40 is known to enter cells dynaminII-independently (50) and the recruitment of caveolae seems not to be obligatory (51). Moreover, like the infectivity of SV40, internalization of transportan/TP10(strept)avidin complexes can be blocked by actin depo-


lymerization and depletion of cholesterol from the plasma membrane (Saä¨lik and Padari, unpublished data). It has to be assessed if the same or different inducible actinand cholesterol-dependent endocytic processes lead to internalization of some viruses, bacteria, and peptidecargo complexes and which receptors are involved, if any. In parallel to translocation into cells in membranesurrounded vesicles, transportan-streptavidin complexes with a single gold particle were detectable in the cortical cytoplasm of cells in the close proximity of the intact plasma membrane, suggesting direct passage across the membrane. The amount of complexes that seemingly directly crossed the plasma membrane was very small compared to that in vesicles and was higher for TP10 rather than transportan complexes. The presence of transportan-streptavidin complexes with a single (or few) gold particle in the cortical cytoplasm could be caused by redistribution of complexes from the cell surface into cells upon fixation (4, 11) or an embedding procedure. Neither can we exclude that those complexes are encompassed by the membrane, which remains undetectable by electron microscopy, in analogy with SV40 particles internalization within the vesicles devoid of the visible coat (51), which were found when studying a novel clathrin-, caveolae-, and dynaminII-independent cholesterol-sensitive endocytic pathway (51, 52). On the other hand, we cannot completely ignore the possibility that a small fraction of transportan-protein complexes translocates into cells by inducing a pore-like structure in the plasma membrane hypothesized for complexes of carrier peptides MPG and Pep-1 with cargo molecules (36) or the nondefined mechanism observed in studies of Seelig and collaborators (31). Presence of oligoarginine both in cytoplasmic and vesicular fraction after fractionation of CHO cell material (53, 54) and the activity of biological cargo delivered into cells by transportan and/ or TP10 (55, 56) supports the cytoplasmic localization of peptide-protein complexes observed in our experiments. The translocation mode has been also suggested to depend on the size of the particle as proposed by Morris and co-workers (57) which could be the case for transportan/TP10-streptavidin complexes of different size. However, until now this hypothesis has not found unambiguous affirmation. The contribution of endocytosis in the cellular uptake process of CPPs is often estimated after blocking this pathway by lowering temperature, which has sometimes led to the contradicting results. Cellular translocation of gold-tagged streptavidin-transportan complexes was highly temperature/energy-dependent. Lowering temperature to endocytosis-permissive 20 °C markedly reduced amount of internalized complexes. Minimal uptake of transportan-streptavidin complexes was detected also at endocytosis nonpermissive 8-10 °C but not at 0-4 °C. Unexpectedly, the internalization detected at 8-10 °C was mainly vesicular and cortical; the vesicles had not translocated markedly toward perinuclear region in 1 h. It has been demonstrated earlier that FITC-labeled avidin complexes with biotin-Tat and biotin-penetratin cannot enter CHO cells at 0-4 °C (41). Very recently, transduction of Cy3-tagged streptavidin into HEK293 cells after complexing with biotin-maurocalcine, a 33 amino acid long scorpion toxin (58), was shown, but only after fixation of cells. Our results are in line with the suggestion of mainly endocytic uptake of CPP-protein complexes (41). Reduction of uptake at 20 °C and ceasing at 0-4 °C suggests that the decrease in fluidity of the membrane has also considerable effect on the internalization of CPPs and their constructs as proposed earlier

Padari et al.

Figure 7. Internalization and intracellular routing of CPPprotein complexes. Internalization via macropinosomes (1), clathrin-coated vesicles (2) and caveosomes (3) leading to different cellular compartments: escape from macropinosomes (4), targeting to lysosomes (5) and partial direction to Golgi complex (6). Putative translocation of peptide-protein complexes across plasma membrane without induction of vesicular structures (7).

for penetratin peptide (29). The reduced association of CPP-avidin/streptavidin complexes with cell surface structures at 0-4 °C observed by Console and collaborators (41) and by us might be also partly ascribed to the increased rigidity of the plasma membrane. Summing up, as we schematically represent in Figure 7, complexes of transportan and TP10 with protein can translocate into cells by different mechanisms in parallel, mainly by endocytic processes using diverse vesicles and probably also by translocation without inducing the membrane invagination and vesicle formation. The latter mode could be more common for CPPs alone than detected here for constructs with proteins. The morphology and size of formed vesicles suggests that macropincytosis is the main mechanism of transportan-protein complexes’ uptake by Bowes and HeLa cells. It seems also that the size (degree of concentration) of complexes on the cell surface influences the size of the forming vesicle, the mode of internalization and further cellular targeting. ACKNOWLEDGMENT

This work was supported by grants from European Community (QLK3-CT-2002-01989), Estonian Science Foundation (ESF 5588 and 5995), the Swedish Institute Visby Program, Swedish Royal Academy of Sciences, and Swedish Research Councils NT and Med. LITERATURE CITED (1) Derossi, D., Joliot, A. H., Chassaing, G., and Prochiantz, A. (1994) The third helix of the Antennapedia homeodomain translocates through biological membranes. J. Biol. Chem. 269, 10444-50. (2) Futaki, S., Goto, S., and Sugiura, Y. (2003) Membrane permeability commonly shared among arginine-rich peptides. J. Mol. Recognit. 16, 260-4. (3) Vives, E., Brodin, P., and Lebleu, B. (1997) A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J. Biol. Chem. 272, 16010-7. (4) Richard, J. P., Melikov, K., Vives, E., Ramos, C., Verbeure, B., Gait, M. J., Chernomordik, L. V., and Lebleu, B. (2003) Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake. J. Biol. Chem. 278, 585-90.

Cell Transduction Pathways of Transportans (5) Thoreń, P. E., Persson, D., Esbjorner, E. K., Gokso¨r, M., Lincoln, P., and Nordeń, B. (2004) Membrane binding and translocation of cell-penetrating peptides. Biochemistry 43, 3471-89. (6) Christiaens, B., Grooten, J., Reusens, M., Joliot, A., Goethals, M., Vandekerckhove, J., Prochiantz, A., and Rosseneu, M. (2004) Membrane interaction and cellular internalization of penetratin peptides. Eur. J. Biochem. 271, 1187-97. (7) Nakase, I., Niwa, M., Takeuchi, T., Sonomura, K., Kawabata, N., Koike, Y., Takehashi, M., Tanaka, S., Ueda, K., Simpson, J. C., Jones, A. T., Sugiura, Y., and Futaki, S. (2004) Cellular uptake of arginine-rich peptides: roles for macropinocytosis and actin rearrangement. Mol. Ther. 10, 101122. (8) Persson, D., Thoreń, P. E., Herner, M., Lincoln, P., and Nordeń, B. (2003) Application of a novel analysis to measure the binding of the membrane-translocating peptide penetratin to negatively charged liposomes. Biochemistry 42, 421-9. (9) Terrone, D., Sang, S. L., Roudaia, L., and Silvius, J. R. (2003) Penetratin and related cell-penetrating cationic peptides can translocate across lipid bilayers in the presence of a transbilayer potential. Biochemistry 42, 13787-99. (10) Magzoub, M., Kilk, K., Eriksson, L. E., Langel, U ¨ ., and Gra¨slund, A. (2001) Interaction and structure induction of cell-penetrating peptides in the presence of phospholipid vesicles. Biochim. Biophys. Acta 1512, 77-89. (11) Lundberg, M., and Johansson, M. (2002) Positively charged DNA-binding proteins cause apparent cell membrane translocation. Biochem. Biophys. Res. Commun. 291, 367-71. (12) Saä¨lik, P., Elmquist, A., Hansen, M., Padari, K., Saar, K., Viht, K., Langel, U ¨ ., and Pooga, M. (2004) Protein cargo delivery properties of cell-penetrating peptides. A comparative study. Bioconjugate Chem. 15, 1246-53. (13) Dietz, G. P., and Ba¨hr, M. (2004) Delivery of bioactive molecules into the cell: the Trojan horse approach. Mol. Cell Neurosci. 27, 85-131. (14) Fittipaldi, A., Ferrari, A., Zoppe, M., Arcangeli, C., Pellegrini, V., Beltram, F., and Giacca, M. (2003) Cell membrane lipid rafts mediate caveolar endocytosis of HIV-1 Tat fusion proteins. J. Biol. Chem. 278, 34141-9. (15) Ferrari, A., Pellegrini, V., Arcangeli, C., Fittipaldi, A., Giacca, M., and Beltram, F. (2003) Caveolae-mediated internalization of extracellular HIV-1 tat fusion proteins visualized in real time. Mol Ther 8, 284-94. (16) Wadia, J. S., Stan, R. V., and Dowdy, S. F. (2004) Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat. Med. 10, 310-5. (17) Conner, S. D.. and Schmid, S. L. (2003) Regulated portals of entry into the cell. Nature 422, 37-44. (18) Meier, O., Boucke, K., Hammer, S. V., Keller, S., Stidwill, R. P., Hemmi, S., and Greber, U. F. (2002) Adenovirus triggers macropinocytosis and endosomal leakage together with its clathrin-mediated uptake. J. Cell Biol. 158, 111931. (19) Michiue, H., Tomizawa, K., Wei, F. Y., Matsushita, M., Lu, Y. F., Ichikawa, T., Tamiya, T., Date, I., and Matsui, H. (2005) The NH2 Terminus of Influenza Virus Hemagglutinin-2 Subunit Peptides Enhances the Antitumor Potency of Polyarginine-mediated p53 Protein Transduction. J. Biol. Chem. 280, 8285-8289. (20) Maiolo, J. R.r., Ottinger, E. A., and Ferrer, M. (2004) Specific redistribution of cell-penetrating peptides from endosomes to the cytoplasm and nucleus upon laser illumination. J. Am. Chem. Soc. 126, 15376-7. (21) Pooga, M., Ha¨llbrink, M., Zorko, M., and Langel, U ¨ . (1998) Cell penetration by transportan. FASEB J. 12, 67-77. (22) Barany-Wallje, E., Andersson, A., Gra¨slund, A., and Ma¨ler, L. (2004) NMR solution structure and position of transportan in neutral phospholipid bicelles. FEBS Lett. 567, 265-9. (23) Pooga, M., Soomets, U., Ha¨llbrink, M., Valkna, A., Saar, K., Rezaei, K., Kahl, U., Hao, J. X., Xu, X. J., WiesenfeldHallin, Z., Ho¨kfelt, T., Bartfai, T., and Langel, U ¨ . (1998) Cell

Bioconjugate Chem., Vol. 16, No. 6, 2005 1409 penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo. Nat. Biotechnol. 16, 857-61. (24) Ha¨llbrink, M., Floreń, A., Elmquist, A., Pooga, M., Bartfai, T., and Langel, U ¨ . (2001) Cargo delivery kinetics of cellpenetrating peptides. Biochim. Biophys. Acta 1515, 101-9. (25) Soomets, U., Lindgren, M., Gallet, X., Ha¨llbrink, M., Elmquist, A., Balaspiri, L., Zorko, M., Pooga, M., Brasseur, R., Langel, U ¨ ., Prochiantz, A., and Brakenhielm, E. (2000) Deletion analogues of transportan. Biochim. Biophys. Acta 1467, 165-76. (26) Bendayan, M. (1984) Protein A-Gold Electron Microscopic Immunocytochemistry, Applications and Limitations. J. Electron. Microsc. Technique 1, 243-270. (27) Chitambar, C. R., and Zivkovic-Gilgenbach, Z. (1990) Influence of cellular iron status on the release of soluble transferrin receptor from human promyelocytic leukemic HL60 cells. J. Lab. Clin. Med. 116, 345-53. (28) Venable, J. H., and Coggeshall, R. (1965) A Simplified Lead Citrate Stain for Use in Electron Microscopy. J. Cell Biol. 25. (29) Letoha, T., Gaal, S., Somlai, C., Czajlik, A., Perczel, A., and Penke, B. (2003) Membrane translocation of penetratin and its derivatives in different cell lines. J. Mol. Recognit. 16, 272-9. (30) Haylett, T., and Thilo, L. (1991) Endosome-lysosome fusion at low temperature. J. Biol. Chem. 266, 8322-7. (31) Ziegler, A., Nervi, P., Du¨rrenberger, M., and Seelig, J. (2005) The cationic cell-penetrating peptide CPP(TAT) derived from the HIV-1 protein TAT is rapidly transported into living fibroblasts: optical, biophysical, and metabolic evidence. Biochemistry 44, 138-48. (32) Schmid, S. L., McNiven, M. A., and De Camilli, P. (1998) Dynamin and its partners: a progress report. Curr. Opin. Cell Biol. 10, 504-12. (33) Torchilin, V. P. (2005) Fluorescence microscopy to follow the targeting of liposomes and micelles to cells and their intracellular fate. Adv Drug Delivery Rev. 57, 95-109. (34) Richard, J. P. M., K. Brooks, H. Prevot, P. Lebleu, B. Chernomordik, L. V. (2005) Cellular uptake of unconjugated TAT peptide involves clathrin-dependent endocytosis and heparan sulfate receptors. J. Biol. Chem. 280, 15300-6. (35) Tyagi, M., Rusnati, M., Presta, M., and Giacca, M. (2001) Internalization of HIV-1 tat requires cell surface heparan sulfate proteoglycans. J. Biol. Chem. 276, 3254-61. (36) Deshayes, S., Heitz, A., Morris, M. C., Charnet, P., Divita, G., and Heitz, F. (2004) Insight into the mechanism of internalization of the cell-penetrating carrier peptide Pep-1 through conformational analysis. Biochemistry 43, 1449-57. (37) Potocky, T. B., Menon, A. K., and Gellman, S. H. (2003) Cytoplasmic and nuclear delivery of a TAT-derived peptide and a beta-peptide after endocytic uptake into HeLa cells. J. Biol. Chem. 278, 50188-94. (38) Gallo, R. L., Ono, M., Povsic, T., Page, C., Eriksson, E., Klagsbrun, M., and Bernfield, M. (1994) Syndecans, cell surface heparan sulfate proteoglycans, are induced by a proline-rich antimicrobial peptide from wounds. Proc. Natl. Acad. Sci. U.S.A. 91, 11035-9. (39) Belting, M. (2003) Heparan sulfate proteoglycan as a plasma membrane carrier. Trends Biochem. Sci. 28, 14551. (40) Sandgren, S., Wittrup, A., Cheng, F., Jonsson, M., Eklund, E., Busch, S., and Belting, M. (2004) The human antimicrobial peptide LL-37 transfers extracellular DNA plasmid to the nuclear compartment of mammalian cells via lipid rafts and proteoglycan-dependent endocytosis. J. Biol. Chem. 279, 17951-6. (41) Console, S., Marty, C., Garcia-Echeverria, C., Schwendener, R., and Ballmer-Hofer, K. (2003) Antennapedia and HIV transactivator of transcription (TAT) “protein transduction domains” promote endocytosis of high molecular weight cargo upon binding to cell surface glycosaminoglycans. J. Biol. Chem. 278, 35109-14. (42) Gonç alves, E., Kitas, E., and Seelig, J. (2005) Binding of oligoarginine to membrane lipids and heparan sulfate: struc-

1410 Bioconjugate Chem., Vol. 16, No. 6, 2005 tural and thermodynamic characterization of a cell-penetrating Peptide. Biochemistry 44, 2692-702. (43) Vendeville, A., Rayne, F., Bonhoure, A., Bettache, N., Montcourrier, P., and Beaumelle, B. (2004) HIV-1 Tat enters T cells using coated pits before translocating from acidified endosomes and eliciting biological responses. Mol. Biol. Cell 15, 2347-60. (44) Swanson, J. A. and Watts, C. (1995) Macropinocytosis. Trends Cell Biol. 5, 424-8. (45) Ammendolia, M. G., Bertuccini, L., Minelli, F., Meschini, S., and Baldassarri, L. (2004) A Sphingomonas bacterium interacting with epithelial cells. Res. Microb. 155, 636-46. (46) Garcia-Perez, B. E., Mondragon-Flores, R., and LunaHerrera, J. (2003) Internalization of Mycobacterium tuberculosis by macropinocytosis in non-phagocytic cells. Microb. Pathog. 35, 49-55. (47) Pooga, M., Kut, C., Kihlmark, M., Ha¨llbrink, M., Fernaeus, S., Raid, R., Land, T., Hallberg, E., Bartfai, T., and Langel, U ¨ . (2001) Cellular translocation of proteins by transportan. FASEB J. 15, 1451-3. (48) Lamaze, C. and Schmid, S. L. (1995) The emergence of clathrin-independent pinocytic pathways. Curr. Opin. Cell Biol. 7, 573-80. (49) Pelkmans, L. and Helenius, A. (2003) Insider information: what viruses tell us about endocytosis. Curr. Opin. Cell Biol. 15, 414-22. (50) Pelkmans, L., Puntener, D., Helenius, A., and Kartenbeck, J. (2002) Local actin polymerization and dynamin recruitment in SV40-induced internalization of caveolae. Science 296, 535-9. (51) Damm, E. M., Pelkmans, L., Kartenbeck, J., Mezzacasa, A., Kurzchalia, T., and Helenius, A. (2005) Clathrin- and caveolin-1-independent endocytosis: entry of simian virus 40 into cells devoid of caveolae. J. Cell Biol. 168, 477-88.

Padari et al. (52) Kirkham, M., Fujita, A., Chadda, R., Nixon, S. J., Kurzchalia, T. V., Sharma, D. K., Pagano, R. E., Hancock, J. F., Mayor, S., and Parton, R. G. (2005) Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles. J. Cell Biol. 168, 465-76. (53) Zaro, J. L. and Shen, W. C. (2003) Quantitative comparison of membrane transduction and endocytosis of oligopeptides. Biochem. Biophys. Res. Commun. 307, 241-7. (54) Zaro, J. L. and Shen, W. C. (2005) Evidence that membrane transduction of oligoarginine does not require vesicle formation. Exp. Cell Res. 307, 164-73. (55) Kaushik, N., Basu, A., Palumbo, P., Myers, R. L., and Pandey, V. N. (2002) Anti-TAR polyamide nucleotide analog conjugated with a membrane-permeating peptide inhibits human immunodeficiency virus type 1 production. J. Virol. 76, 3881-91. (56) Fisher, L., Soomets, U., Cortes Toro, V., Chilton, L., Jiang, Y., Langel, U ¨ ., and Iverfeldt, K. (2004) Cellular delivery of a double-stranded oligonucleotide NFkappaB decoy by hybridization to complementary PNA linked to a cell-penetrating peptide. Gene Ther. 11, 1264-72. (57) Morris, M. C., Depollier, J., Mery, J., Heitz, F., and Divita, G. (2001) A peptide carrier for the delivery of biologically active proteins into mammalian cells. Nat. Biotechnol. 19, 1173-6. (58) Esteve, E., Mabrouk, K., Dupuis, A., Smida-Rezgui, S., Altafaj, X., Grunwald, D., Platel, J. C., Andreotti, N., Marty, I., Sabatier, J. M., Ronjat, M., and De Waard, M. (2005) Transduction of the scorpion toxin Maurocalcine into cellsEvidence that the toxin crosses the plasma membrane. J. Biol. Chem. 280, 12833-9.

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