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      Cell Entry of Arginine-rich Peptides Is Independent of Endocytosis*S⃞

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          Abstract

          Arginine-rich peptides are a subclass of cell-penetrating peptides that are taken up by living cells and can be detected freely diffusing inside the cytoplasm and nucleoplasm. This phenomenon has been attributed to either an endocytic mode of uptake and a subsequent release from vesicles or to direct membrane penetration (transduction). To distinguish between both possibilities, we have blocked endocytic pathways suggested to be involved in uptake of cell-penetrating peptides. We have then monitored by confocal microscopy the uptake and distribution of the cell-penetrating transactivator of transcription (TAT) peptide into living mammalian cells over time. To prevent side effects of chemical inhibitors, we used genetically engineered cells as well as different temperature. We found that a knockdown of clathrin-mediated endocytosis and a knock-out of caveolin-mediated endocytosis did not affect the ability of TAT to enter cells. In addition, the TAT peptide showed the same intracellular distribution throughout the cytoplasm and nucleus as in control cells. Even incubation of cells at 4 °C did not abrogate TAT uptake nor change its intracellular distribution. We therefore conclude that this distribution results from TAT peptide that directly penetrated (transduced) the plasma membrane. The formation of nonselective pores is unlikely, because simultaneously added fluorophores were not taken up together with the TAT peptide. In summary, although the frequency and kinetics of TAT transduction varied between cell types, it was independent of endocytosis.

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          Most cited references48

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          Virus Entry: Open Sesame

          Detailed information about the replication cycle of viruses and their interactions with host organisms is required to develop strategies to stop them. Cell biology studies, live-cell imaging, and systems biology have started to illuminate the multiple and subtly different pathways that animal viruses use to enter host cells. These insights are revolutionizing our understanding of endocytosis and the movement of vesicles within cells. In addition, such insights reveal new targets for attacking viruses before they can usurp the host-cell machinery for replication.
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            Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis.

            The TAT protein transduction domain (PTD) has been used to deliver a wide variety of biologically active cargo for the treatment of multiple preclinical disease models, including cancer and stroke. However, the mechanism of transduction remains unknown. Because of the TAT PTD's strong cell-surface binding, early assumptions regarding cellular uptake suggested a direct penetration mechanism across the lipid bilayer by a temperature- and energy-independent process. Here we show, using a transducible TAT-Cre recombinase reporter assay on live cells, that after an initial ionic cell-surface interaction, TAT-fusion proteins are rapidly internalized by lipid raft-dependent macropinocytosis. Transduction was independent of interleukin-2 receptor/raft-, caveolar- and clathrin-mediated endocytosis and phagocytosis. Using this information, we developed a transducible, pH-sensitive, fusogenic dTAT-HA2 peptide that markedly enhanced TAT-Cre escape from macropinosomes. Taken together, these observations provide a scientific basis for the development of new, biologically active, transducible therapeutic molecules.
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              Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake.

              Cellular uptake of a family of cationic cell-penetrating peptides (examples include Tat peptides and penetratin) have been ascribed in the literature to a mechanism that does not involve endocytosis. In this work we reevaluate the mechanisms of cellular uptake of Tat 48-60 and (Arg)(9). We demonstrate here that cell fixation, even in mild conditions, leads to the artifactual uptake of these peptides. Moreover, we show that flow cytometry analysis cannot be used validly to evaluate cellular uptake unless a step of trypsin digestion of the cell membrane-adsorbed peptide is included in the protocol. Fluorescence microscopy on live unfixed cells shows characteristic endosomal distribution of peptides. Flow cytometry analysis indicates that the kinetics of uptake are similar to the kinetics of endocytosis. Peptide uptake is inhibited by incubation at low temperature and cellular ATP pool depletion. Similar data were obtained for Tat-conjugated peptide nucleic acids. These data are consistent with the involvement of endocytosis in the cellular internalization of cell-penetrating peptides and their conjugates to peptide nucleic acids.
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                Author and article information

                Journal
                J Biol Chem
                jbc
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology
                0021-9258
                1083-351X
                6 February 2009
                6 February 2009
                : 284
                : 6
                : 3370-3378
                Affiliations
                []Max Delbrueck Center for Molecular Medicine, D-13125 Berlin, the [§ ]Department of Biology/Biophysics, Humboldt University Berlin, D-10115 Berlin, the []Department of Molecular Biology, Max Planck Institute for Infection Biology, D-10117 Berlin, and the []Department of Biology, Technische Universität Darmstadt, D-64287 Darmstadt, Germany
                Author notes
                [1]

                To whom correspondence should be addressed. Tel.: 49-6151-16-2377; Fax: 49-6151-16-2375; E-mail: Cardoso@ 123456bio.tu-darmstadt.de .

                Article
                3370
                10.1074/jbc.M805550200
                2635027
                19047062
                f792f615-9a30-453e-a4f8-27f05ce8fdad
                Copyright © 2009, The American Society for Biochemistry and Molecular Biology, Inc.

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                Creative Commons Attribution Non-Commercial License applies to Author Choice Articles

                History
                : 21 July 2008
                : 11 November 2008
                Categories
                Membrane Transport, Structure, Function, and Biogenesis

                Biochemistry
                Biochemistry

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