Summary
Cell penetrating peptides (CPPs) are currently used to deliver various macromolecular cargos to intracellular sites of action both in vitro and in vivo on an experimental basis. During the last few years, even more evidence has accumulated indicating that the main route of entry for most CPPs is through endocytosis rather than direct membrane penetration, as initially proposed. The specific endocytosis pathway utilized by CPPs is, however, still ill-defined and potentially varies depending on what CPPs, cargos, and cell lines are being studied. In this chapter, we provide detailed protocols for an initial characterization of the uptake mechanism involved in CPP-mediated delivery of DNA. Methods to both quantitatively and qualitatively study the uptake using fluorescence-assisted cell sorting (FACS) and confocal microscopy, respectively, are provided. Furthermore, methods to study the intracellular fate of the internalized cargo by co-localization studies between internalized DNA and established endosomal markers, e.g., transferrin, dextran as well as caveolin-1, are described. Finally, we provide a protocol to determine the dependence on dynamin, i.e., a central mediator of vesicle fission at the cell membrane, for DNA–peptide complex uptake using a dominant-negative construct of dynamin-2.
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References
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, 16010–16017.
Derossi, D., Calvet, S., Trembleau, A., Brunissen, A., Chassaing, G., and Prochiantz, A. (1996) Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent. J Biol Chem 271, 18188–18193.
Lindgren, M., Hallbrink, M., Prochiantz, A., and Langel, U. (2000) Cell-penetrating peptides. Trends Pharmacol Sci 21, 99–103.
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 re-evaluation of the mechanism of cellular uptake. J Biol Chem 278, 585–590.
Richard, J. P., Melikov, K., Brooks, H., Prevot, P., Lebleu, B., and Chernomordik, L. V. (2005) Cellular uptake of unconjugated TAT peptide involves clathrin-dependent endocytosis and heparan sulfate receptors. J Biol Chem 280, 15300–15306.
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, 284–294.
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, 310–315.
Rejman, J., Oberle, V., Zuhorn, I. S., and Hoekstra, D. (2004) Size-dependent internalization of particles via the pathways of clathrin- and caveolae-mediated endocytosis. Biochem J 377, 159–169.
Sandgren, S., Cheng, F., and Belting, M. (2002) Nuclear targeting of macromolecular polyanions by an HIV-Tat derived peptide. Role for cell-surface proteoglycans. J Biol Chem 277, 38877–38883.
Suzuki, T., Futaki, S., Niwa, M., Tanaka, S., Ueda, K., and Sugiura, Y. (2002) Possible existence of common internalization mechanisms among arginine-rich peptides. J Biol Chem 277, 2437–2443.
Belting, M. (2003) Heparan sulfate proteoglycan as a plasma membrane carrier. Trends Biochem Sci 28, 145–151.
Mislick, K. A., and Baldeschwieler, J. D. (1996) Evidence for the role of proteoglycans in cation-mediated gene transfer. Proc Natl Acad Sci U S A 93, 12349–12354.
Shieh, M. T., WuDunn, D., Montgomery, R. I., Esko, J. D., and Spear, P. G. (1992) Cell surface receptors for herpes simplex virus are heparan sulfate proteoglycans. J Cell Biol 116, 1273–1281.
Patel, M., Yanagishita, M., Roderiquez, G., Bou-Habib, D. C., Oravecz, T., Hascall, V. C., and Norcross, M. A. (1993) Cell-surface heparan sulfate proteoglycan mediates HIV-1 infection of T-cell lines. AIDS Res Hum Retroviruses 9, 167–174.
Hopkins, C. R., Miller, K., and Beardmore, J. M. (1985) Receptor-mediated endocytosis of transferrin and epidermal growth factor receptors: a comparison of constitutive and ligand-induced uptake. J Cell Sci Suppl 3, 173–186.
Glebov, O. O., Bright, N. A., and Nichols, B. J. (2006) Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells. Nat Cell Biol 8, 46–54.
Pelkmans, L., Kartenbeck, J., and Helenius, A. (2001) Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER. Nat Cell Biol 3, 473–483.
Damke, H., Binns, D. D., Ueda, H., Schmid, S. L., and Baba, T. (2001) Dynamin GTPase domain mutants block endocytic vesicle formation at morphologically distinct stages. Mol Biol Cell 12, 2578–2589.
Lee, E., and De Camilli, P. (2002) Dynamin at actin tails. Proc Natl Acad Sci U S A 99, 161–166.
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Wittrup, A., Belting, M. (2009). Characterizing Peptide-Mediated DNA Internalization in Human Cancer Cells. In: Belting, M. (eds) Macromolecular Drug Delivery. Methods in Molecular Biology, vol 480. Humana Press. https://doi.org/10.1007/978-1-59745-429-2_7
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DOI: https://doi.org/10.1007/978-1-59745-429-2_7
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