Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Protein delivery into live cells by incubation with an endosomolytic agent

Abstract

We report that a tetramethylrhodamine-labeled dimer of the cell-penetrating peptide TAT, dfTAT, penetrates live cells by escaping from endosomes with high efficiency. By mediating endosomal leakage, dfTAT also delivers proteins into cultured cells after a simple co-incubation procedure. We achieved cytosolic delivery in several cell lines and primary cells and observed that only a relatively small amount of material remained trapped inside endosomes. Delivery did not require a binding interaction between dfTAT and a protein, multiple molecules could be delivered simultaneously, and delivery could be repeated. dfTAT-mediated delivery did not noticeably affect cell viability, cell proliferation or gene expression. dfTAT-based intracellular delivery should be useful for cell-based assays, cellular imaging applications and the ex vivo manipulation of cells.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Cytosolic delivery of dfTAT in live cells is efficient.
Figure 2: dfTAT penetrates the cytosol by escaping from the endocytic pathway.
Figure 3: dfTAT-mediated delivery does not substantially affect cell proliferation and transcription.
Figure 4: Delivery of intact and functional proteins using co-incubation with dfTAT.
Figure 5: dfTAT-mediated delivery improves the delivery and transcriptional output of a transcription factor.

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. Takeuchi, T. et al. Direct and rapid cytosolic delivery using cell-penetrating peptides mediated by pyrenebutyrate. ACS Chem. Biol. 1, 299–303 (2006).

    CAS  Article  Google Scholar 

  2. Sakakibara, D. et al. Protein structure determination in living cells by in-cell NMR spectroscopy. Nature 458, 102–105 (2009).

    CAS  Article  Google Scholar 

  3. Lee, Y.J., Datta, S. & Pellois, J.P. Real-time fluorescence detection of protein transduction into live cells. J. Am. Chem. Soc. 130, 2398–2399 (2008).

    CAS  Article  Google Scholar 

  4. Schwarze, S.R., Hruska, K.A. & Dowdy, S.F. Protein transduction: unrestricted delivery into all cells? Trends Cell Biol. 10, 290–295 (2000).

    CAS  Article  Google Scholar 

  5. Dietz, G.P.H. & Bähr, M. Delivery of bioactive molecules into the cell: the Trojan horse approach. Mol. Cell. Neurosci. 27, 85–131 (2004).

    CAS  Article  Google Scholar 

  6. Pan, C., Lu, B., Chen, H. & Bishop, C. Reprogramming human fibroblasts using HIV-1 TAT recombinant proteins OCT4, SOX2, KLF4 and c-MYC. Mol. Biol. Rep. 37, 2117–2124 (2010).

    CAS  Article  Google Scholar 

  7. Gratton, J.-P. et al. Cell-permeable peptides improve cellular uptake and therapeutic gene delivery of replication-deficient viruses in cells and in vivo. Nat. Med. 9, 357–362 (2003).

    CAS  Article  Google Scholar 

  8. Massignani, M. et al. Enhanced fluorescence imaging of live cells by effective cytosolic delivery of probes. PLoS ONE 5, e10459 (2010).

    Article  Google Scholar 

  9. Erazo-Oliveras, A., Muthukrishnan, N., Baker, R., Wang, T.Y. & Pellois, J.P. Improving the endosomal escape of cell-penetrating peptides and their cargos: strategies and challenges. Pharmaceuticals (Basel.) 5, 1177–1209 (2012).

    CAS  Article  Google Scholar 

  10. Hoyer, J., Schatzschneider, U., Schulz-Siegmund, M. & Neundorf, I. Dimerization of a cell-penetrating peptide leads to enhanced cellular uptake and drug delivery. Beilstein J. Org. Chem. 8, 1788–1797 (2012).

    CAS  Article  Google Scholar 

  11. Eguchi, A. et al. Efficient siRNA delivery into primary cells by a peptide transduction domain-dsRNA binding domain fusion protein. Nat. Biotechnol. 27, 567–571 (2009).

    CAS  Article  Google Scholar 

  12. Austin, C.D. et al. Oxidizing potential of endosomes and lysosomes limits intracellular cleavage of disulfide-based antibody–drug conjugates. Proc. Natl. Acad. Sci. USA 102, 17987–17992 (2005).

    CAS  Article  Google Scholar 

  13. Dominska, M. & Dykxhoorn, D.M. Breaking down the barriers: siRNA delivery and endosome escape. J. Cell Sci. 123, 1183–1189 (2010).

    CAS  Article  Google Scholar 

  14. Puri, V. et al. Cholesterol modulates membrane traffic along the endocytic pathway in sphingolipid-storage diseases. Nat. Cell Biol. 1, 386–388 (1999).

    CAS  Article  Google Scholar 

  15. Koivusalo, M. et al. Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling. J. Cell Biol. 188, 547–563 (2010).

    CAS  Article  Google Scholar 

  16. Johnson, L.S., Dunn, K.W., Pytowski, B. & McGraw, T.E. Endosome acidification and receptor trafficking: bafilomycin A1 slows receptor externalization by a mechanism involving the receptor's internalization motif. Mol. Biol. Cell 4, 1251–1266 (1993).

    CAS  Article  Google Scholar 

  17. Vercauteren, D. et al. The use of inhibitors to study endocytic pathways of gene carriers: optimization and pitfalls. Mol. Ther. 18, 561–569 (2010).

    CAS  Article  Google Scholar 

  18. Rejman, J., Bragonzi, A. & Conese, M. Role of clathrin- and caveolae-mediated endocytosis in gene transfer mediated by lipo- and polyplexes. Mol. Ther. 12, 468–474 (2005).

    CAS  Article  Google Scholar 

  19. Srinivasan, D. et al. Conjugation to the cell-penetrating peptide TAT potentiates the photodynamic effect of carboxytetramethylrhodamine. PLoS ONE 6, e17732 (2011).

    CAS  Article  Google Scholar 

  20. Sun, X. et al. Development of SNAP-tag fluorogenic probes for wash-free fluorescence imaging. ChemBioChem 12, 2217–2226 (2011).

    CAS  Article  Google Scholar 

  21. Johnson, J.R., Kocher, B., Barnett, E.M., Marasa, J. & Piwnica-Worms, D. Caspase-activated cell-penetrating peptides reveal temporal coupling between endosomal release and apoptosis in an RGC-5 cell model. Bioconjug. Chem. 23, 1783–1793 (2012).

    CAS  Article  Google Scholar 

  22. Wadia, J.S., Stan, R.V. & Dowdy, S.F. Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat. Med. 10, 310–315 (2004).

    CAS  Article  Google Scholar 

  23. Csaszar, E. et al. An automated system for delivery of an unstable transcription factor to hematopoietic stem cell cultures. Biotechnol. Bioeng. 103, 402–412 (2009).

    CAS  Article  Google Scholar 

  24. Amsellem, S. et al. Ex vivo expansion of human hematopoietic stem cells by direct delivery of the HOXB4 homeoprotein. Nat. Med. 9, 1423–1427 (2003).

    CAS  Article  Google Scholar 

  25. Krosl, J. et al. In vitro expansion of hematopoietic stem cells by recombinant TAT-HOXB4 protein. Nat. Med. 9, 1428–1432 (2003).

    CAS  Article  Google Scholar 

  26. Will, E. et al. HOXB4 inhibits cell growth in a dose-dependent manner and sensitizes cells towards extrinsic cues. Cell Cycle 5, 14–22 (2006).

    CAS  Article  Google Scholar 

  27. Muthukrishnan, N., Johnson, G.A., Erazo-Oliveras, A. & Pellois, J.P. Synergy between cell-penetrating peptides and singlet oxygen generators leads to efficient photolysis of membranes. Photochem. Photobiol. 89, 625–630 (2013).

    CAS  Article  Google Scholar 

  28. Muthukrishnan, N., Johnson, G.A., Lim, J., Simanek, E.E. & Pellois, J.P. TAT-mediated photochemical internalization results in cell killing by causing the release of calcium into the cytosol of cells. Biochim. Biophys. Acta 1820, 1734–1743 (2012).

    CAS  Article  Google Scholar 

  29. Scharf, B. et al. Annexin A2 binds to endosomes following organelle destabilization by particulate wear debris. Nat. Commun. 3, 755 (2012).

    Article  Google Scholar 

  30. Tung, C.-H., Mueller, S. & Weissleder, R. Novel branching membrane translocational peptide as gene delivery vector. Bioorg. Med. Chem. 10, 3609–3614 (2002).

    CAS  Article  Google Scholar 

  31. Jiang, T. et al. Tumor imaging by means of proteolytic activation of cell-penetrating peptides. Proc. Natl. Acad. Sci. USA 101, 17867–17872 (2004).

    CAS  Article  Google Scholar 

  32. Peitz, M., Pfannkuche, K., Rajewsky, K. & Edenhofer, F. Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: a tool for efficient genetic engineering of mammalian genomes. Proc. Natl. Acad. Sci. USA 99, 4489–4494 (2002).

    CAS  Article  Google Scholar 

  33. Pellois, J.-P. & Muir, T.W. A ligation and photorelease strategy for the temporal and spatial control of protein function in living cells. Angew. Chem. Int. Ed. Engl. 44, 5713–5717 (2005).

    CAS  Article  Google Scholar 

  34. Boukamp, P. et al. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J. Cell Biol. 106, 761–771 (1988).

    CAS  Article  Google Scholar 

  35. Woods, L.K. et al. Comparison of four new cell lines from patients with adenocarcinoma of the ovary. Cancer Res. 39, 4449–4459 (1979).

    CAS  PubMed  Google Scholar 

  36. Jiang, N., Bénard, C.Y., Kébir, H., Shoubridge, E.A. & Hekimi, S. Human CLK2 links cell cycle progression, apoptosis, and telomere length regulation. J. Biol. Chem. 278, 21678–21684 (2003).

    CAS  Article  Google Scholar 

  37. Gonzalez-Vallina, R. et al. Lipoprotein and apolipoprotein secretion by a newborn piglet intestinal cell line (IPEC-1). Am. J. Physiol. 271, 249–259 (1996).

    Google Scholar 

  38. Hunt, M.E., Scherrer, M.P., Ferrari, F.D. & Matz, M.V. Very bright green fluorescent proteins from the pontellid copepod Pontella mimocerami. PLoS ONE 5, e11517 (2010).

    Article  Google Scholar 

  39. Wessel, D. & Flügge, U.I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138, 141–143 (1984).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This article was supported by awards R01GM087227 and R01GM087981 from the US National Institute of General Medical Sciences, by the Norman Ackerman Advanced Research Program and by the Robert A. Welch foundation (grant A-1769). We are grateful to M. Lasagna for technical assistance with FRET assay as well as to R. Chapkin for access to the luminometer and flow cytometer in his laboratory. We thank L. Dangott for help with proteomic analysis; C. Cepko (Harvard Medical School) for pCALNL-GFP; K. Rajewsky (Max Delbrück Center for Molecular Medicine) for pTriEx-HTNC; G. Sauvageau (Montreal University) for pTAT-HA-HOXB4; P. Zandstra (University of Toronto) for the HOXB4 luciferase reporter, engineered 3T3 cells and β-gal vectors; and I.R. Correa (New England Biolabs) for pSNAP-H2B and Snap-Surface 488. We also thank R. Burghardt (Texas A&M University) for providing COLO 316 cells, J. Massagué (Memorial Sloan-Kettering Cancer Center) for HaCaT cells, J. Sacchettinni (Texas A&M University) for HDF and Neuro-2a cells, E. Shoubridge (Montreal Neurological Institute and Hospital) for MCH58 cells and G. Wu (Texas A&M University) for primary intestinal porcine epithelial cells.

Author information

Authors and Affiliations

Authors

Contributions

A.E.-O., K.N., L.D. and J.-P.P. designed experiments. A.E.-O., K.N. and L.D. generated and processed data. A.E.-O., K.N., L.D., T.-Y.W. and G.A.J. contributed reagents. A.E.-O., K.N. and J.-P.P. wrote, edited and approved the final manuscript.

Corresponding author

Correspondence to Jean-Philippe Pellois.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–21 (PDF 26679 kb)

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Erazo-Oliveras, A., Najjar, K., Dayani, L. et al. Protein delivery into live cells by incubation with an endosomolytic agent. Nat Methods 11, 861–867 (2014). https://doi.org/10.1038/nmeth.2998

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.2998

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing