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Positively Charged DNA-Binding Proteins Cause Apparent Cell Membrane Translocation
Biochemical and Biophysical Research Communications 291, 367–371 (2002) doi:10.1006/bbrc.2002.6450, available online at http://www.idealibrary.com on
Positively Charged DNA-Binding Proteins Cause Apparent Cell Membrane Translocation Mathias Lundberg* and Magnus Johansson† ,1 *Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-17177 Stockholm, Sweden; and †Division of Clinical Virology F68, Karolinska Institute, Huddinge University Hospital, S-14186 Stockholm, Sweden
Received January 8, 2002
Several positively charged DNA-binding proteins such as the human immunodeficiency virus Tat protein, the Antennapedia (Antp) homeobox protein, and the herpes simplex virus VP22 protein have been reported to translocate across cell membranes and accumulate in cell nuclei. The import occurs by a poorly understood mechanism that appears to be receptorand energy-independent. We showed that both VP22 and the positively charged histone H1 adhered to the cell membrane of living cells and were not removed by extensive washing. However, after fixation the proteins relocated to the cell nucleus. The nuclear accumulation of VP22 and histone H1 after fixation shows that positively charged proteins may appear to translocate across the cell membrane because of a fixation artifact. The majority of studies on “membrane permeable” proteins and peptides have been performed using fixation techniques, and our study shows that influx of these proteins may occur during fixation rather than in living cells. © 2002 Elsevier Science (USA) Key Words: protein transduction domain; protein delivery; gene therapy; membrane translocation; immunocytochemistry; protein therapeutics
Fusing macromolecules to “membrane-permeable” proteins or peptides is currently studied as a possible strategy for intracellular macromolecule delivery (1). Several proteins have been reported to efficiently translocate the membrane of mammalian cells. The family of “membrane-permeable” proteins includes the human immunodeficiency virus Tat protein (2–10), the Drosophila melanogaster homeobox protein Antennapedia (Antp) (11–13), and the herpes simplex virus VP22 protein (14 –21). These proteins, or peptides deAbbreviations used: Antp, Antennapedia homeobox protein; CHO, Chinese hamster ovary; FCS, fetal calf serum; GFP, green fluorescent protein. 1 To whom correspondence and reprint requests should be addressed. Fax: ⫹46-8-58587933. E-mail: [email protected].
rived from the proteins, are reported to rapidly translocate the cell membrane with high efficiency (1–21). Little is known about the molecular mechanism mediating the membrane translocation. The import occurs rapidly within minutes and is not dependent on energy, because the import occurs at both 37 and 4°C (4, 5, 11, 12, 14). The import mechanism appears also to be receptor-independent since peptides synthesized from either D- or L-amino acids are equally well imported, and even peptides with reversed amino acid sequences translocate the cell membrane (4, 12). We have recently studied the import of recombinant VP22 in cultured cells (22). When added to cells, VP22 was detected by immunocytochemistry in the nuclei of almost 100% of the cells when applied at either 37 or 4°C for as short as 10 s. The rapid import of VP22 to the nucleus prompted us to investigate alternative explanations to this phenomenon rather than import of the protein into living cells. In summary, we showed that VP22 adheres to the cell surface of living cells and relocates to the cell nucleus during fixation due to the affinity of VP22 to DNA (22). Antp and Tat are, similar to VP22, highly positively charged DNA-binding proteins. The artificial nuclear localization of VP22 raises concern that the influx of these “membrane permeable” proteins may occur during fixation rather than in living cells. To address this question, we have studied the localization of VP22 and the positively charged histone H1 before and after fixation when the proteins were applied to cultured cells. Histone H1 is unrelated to the “membrane permeable” proteins, but is similar to these proteins in regard to its positively charge and the ability to bind DNA. In summary, we showed that both VP22 and histone H1 adhered to the cell membrane of living cells and were relocated to the cell nucleus after fixation. The majority of studies on intracellular delivery of Tat, Antp, and VP22 have been performed on fixed cells (3–21), and our finding suggests that the membrane translocation observed occurs during fixation.
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0006-291X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
Vol. 291, No. 2, 2002
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS Expression of recombinant VP22-GFP. GFP cDNA was PCR amplified (5⬘-CCATGGTGAGCAAGGGCGAGGAGC and 5⬘-CTTGTACAGCTCGTCCATGCCGAG) from the pEGFP-N1 plasmid vector (Clontech) and cloned into the pCRT7/VP22-TOPO-1 plasmid (Invitrogen) to express GFP fused to the C-terminus of VP22 (aa 159 – 301) with a C-terminal polyhistidine affinity tag. VP22-GFP was expressed in BL21(DE3)pLysS Escherichia coli (Invitrogen) and the protein purified using Talon metal affinity resin column (Clontech) as described in the manufacturer’s protocol. Import of VP22 into CHO cells. Chinese hamster ovary (CHO) cells (Clontech) were cultured at 37°C with 5% CO 2 in McCoy 5A modified medium supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, and 0.1 mg/ml streptomycin. For experiments on adherent cells, the cells were plated in Lab-Tech II chamber slides (Nunc). For experiments on cells in suspension, the cells were detached by incubation with 0.1% EDTA in PBS. The cells were washed twice with PBS, and 1 g VP22-GFP or Alexa Fluor-488 fluorescentlabeled histone H1 (Molecular Probes) were added to the cells in 200 l cell culture medium without serum. The cells were incubated into at 4°C and washed three times in PBS. The cells in suspension were adhered to Bio-Rad Adhesion slides (Bio-Rad). The cells were fixed 15 min with 100% room-tempered methanol, rehydrated 2 min in PBS, stained 2 min with 300 nM DAPI, and washed 5 ⫻ 2 min in PBS. When indicated, the fixed cells were incubated 10 min with 10% fetal calf serum in PBS. The cells were mounted in 50% glycerol in PBS and fluorescence microscopy was performed on a Nikon Eclipse E600 microscope equipped with a SPOT RT digital camera.
RESULTS We used recombinant VP22-GFP and Alexa Fluor488 fluorescent-labeled histone H1 to study the localization of these proteins when added to cells. The proteins were first added to dead methanol fixed CHO cells (Fig. 1). Both VP22-GFP and histone H1 localized almost exclusively to the cell nuclei of the fixed cells. These results show that if the proteins are able to enter the cells, either before or after fixation, their affinity for DNA is sufficient to cause nuclear accumulation. We added VP22-GFP or fluorescent-labeled histone H1 for 2 min to CHO cells cultured adherent on chamber slides (Fig. 2). Live unfixed cells incubated with either protein showed distinct fluorescence at the cell surface, indicating adherence of the proteins to the plasma membrane. No nuclear fluorescence was observed for either protein. The proteins remained attached to the cell membrane even after extensive washing with PBS. In contrast, after methanol fixation and rehydration in PBS the cells exhibited intracellular fluorescence. VP22-GFP fluorescence was after fixation located distinctly to cell nuclei whereas histone H1 showed fluorescence in both the cytosol and nucleus. Accordingly, the proteins attached to the cell membrane were released during fixation and relocated intracellularly. Immunocytochemistry is one of the most commonly used methods for detection of intracellular proteins in studies on the membrane permeable proteins VP22, Antp and Tat. In immunohistochemistry, the samples are blocked after fixation with FCS or
FIG. 1. Methanol-fixed CHO cells incubated with VP22-GFP or Alexa Fluor fluorescent-labeled histone H1. The cells were cultured on chamber slides and fixed with methanol, the fluorescent proteins were added, and the cells were washed with PBS. The nuclei were contrastained with DAPI.
other protein-blocking reagent prior to addition of the antibodies to reduce unspecific antibody binding. Therefore, we incubated the fixed cells with 10% FCS to investigate if the presence of FCS affected the location of VP22-GFP or histone H1. VP22-GFP nuclear fluorescence remained similar in the presence or absence of FCS. However, incubation with FCS resulted in a change from a cytosolic/nuclear pattern to a distinct nuclear location for histone H1. One possible problem in these experiments is that the protein reservoir that becomes mobilized during fixation could be located on the chamber-slide glass or plastic walls. The fixation artifact could in this case be dependent on the material used for cell culture. To investigate this possibility, we repeated the experiment on cells in suspension, which allowed thorough washing and changing of the test tubes before fixation. However, cells in suspension incubated with histone H1 showed a similar pattern as the cells cultured on chamber slides (Fig. 3), indicating that the protein that relocated to the nucleus after fixation was located on the cell membrane of the living cells. DISCUSSION We have shown that the positively charged DNAbinding proteins VP22 and histone H1 adhered to the plasma membrane of living cells, remained attached during washing, and relocated and bound intracellular structures such as DNA when the cells were fixed. The relocation of the proteins during fixation results in an
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FIG. 2. CHO cells cultured on chamber slides incubated with VP22-GFP (A) or Alexa Fluor-labeled histone H1 (B) before (live) or after methanol (MeOH) fixation. The fixed cells were rehydrated with PBS in the presence or absence of fetal calf serum (FCS). The live cells were visualized by phase contrast (PC) microscopy and the nuclei of the fixed cells were contrastained with DAPI.
apparent, but not true, cell membrane translocation. The techniques most commonly used to visualize the influx of the positively charged “membrane permeable” proteins VP22, Tat and Antp all involve cell fixation (3–21), and our findings show that the influx of these proteins may occur during fixation rather than in living cells. The influx and nuclear accumulation of positively charged proteins during fixation, explains the independence of incubation time, incubation temperature, and receptors for protein internalization (4, 5, 11, 12, 14). Although alternative fixation techniques other than methanol fixation used in the present study may
diminish the membrane translocation artifact, all fixation techniques cause disruption of the cell membrane and cannot reliably be used for studies on positively charged membrane translocating proteins. Several investigators have used flow cytometry analysis of living cells to determine the uptake of the membrane permeable proteins (5–10, 18). Although this technique does not involve fixation, it may not discriminate between proteins adhered to the cell surface or internalized proteins. In the present study, we showed that positively charged proteins such as VP22 and histone H1 remained attached to the cell membrane even after
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FIG. 3. CHO cells in suspension incubated with Alexa Fluor-labeled histone-H1 before (live) or after methanol (MeOH) fixation. The fixed cells were rehydrated with PBS in the presence or absence of fetal calf serum (FCS). The live cells were visualized by phase contrast (PC) microscopy and the nuclei of the fixed cells were contrastained with DAPI.
extensive washing. Accordingly, it is likely that the flow-cytometry analysis reflect cell surface adherence rather than intracellular accumulation of the proteins. We conclude that imaging studies on cell membrane translocating proteins have to be performed on live unfixed cells with fluorescently labeled proteins to exclude artifacts. Several proteins fused to Tat, Antp and VP22 are reported to have physiological effects when added to cells (2, 7, 10, 13, 20, 21). These studies suggest that the positively charged proteins and their fusion partners have the ability to transverse the cell membrane. Although the rapid temperature independent cellular influx and nuclear localization of positively charged DNA-binding proteins is likely to be due to the fixation artifact reported in the present study, we cannot exclude that these protein have properties that may mediate delivery of macromolecules to cells. The positive charge of the proteins will cause adherence to the negatively charged cell membrane. Proteins adhered to the cell membrane will be undergo endocytosis and end up in into endosomes and lysosomes. A recent study shows that lysosomal -glucuronidase fused to Tat is internalized by endocytosis and the fusion protein restores enzyme activity in a mouse model of -glucuronidase deficiency (23). Accordingly, positively charged proteins such as Tat can be used to deliver proteins to lysosomes and endosomes. Whether Tat, Antp and VP22 have the ability to also enter the cytosol or nucleus of cells is less clear. Although cytosolic or nuclear delivery of fusion proteins is suggested by functional or phenotypic assays (2, 7, 10, 13, 20, 21), the concomitant use of fixation techniques in protein imaging or flow
cytometry analysis in these studies make the data difficult to evaluate. In a recent study, the diphtheria toxin A-fragment was fused to VP22 or Tat (24). Although the presence of only a few molecules of the diphtheria toxin in the cytosol is sufficient to cause cell death, no cytotoxic effects were observed when the fusion proteins were applied to cells. This study suggests that neither VP22 nor Tat have the ability to translocate the cell membrane. However, it cannot be excluded that the diphtheria toxin interfered with the delivery of VP22 and Tat to the cytosol, and further studies will be required to elucidate the possible use of positively charged proteins as vectors for protein delivery. These studies will have to be performed using functional assays and live cell imaging to avoid detection of artificial protein import. ACKNOWLEDGMENTS This work was supported by grants from the Swedish Medical Research Council, Swedish Society of Medicine, and the medical faculty of the Karolinska Institute.
REFERENCES
370
1. Schwarze, S. R., Hruska, K. A., and Dowdy, S. F. (2000) Protein transduction: Unrestricted delivery into all cells? Trends Cell Biol. 10, 290 –295. 2. Frankel, A. D., and Pabo, C. O. (1988) Cellular uptake of the tat protein from human immunodeficiency virus. Cell 55, 1189 – 1193. 3. Fawell, S., Seery, J., Daikh, Y., Moore, C., Chen, L. L. Pepinsky, B., and Barsoum, J. (1994) Tat-mediated delivery of heterologous proteins into cells. Proc. Natl. Acad. Sci. USA 91, 664 – 668.
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4. Futaki, S., Suzuki, T., Ohashi, W., Yagami, T., Tanaka, S., Ueda, K., and Sugiura, Y. (2001) Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery. J. Biol. Chem. 276, 5836 – 5840. 5. Vive`s, 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. 6. Schwarze, S. R., Ho, A., Vocero-Akbani, A., and Dowdy, S. F. (1999) In vivo protein transduction: Delivery of a biologically active protein into the mouse. Science 285, 1569 –1572. 7. Nagahara, H., Vocero-Akbani, A. M., Snyder, E. L., Ho, A., Latham, D. G., Lissy, N. A., Becker-Hapak, M., Ezhevsky, S. A., and Dowdy, S. F. (1998) Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration. Nat. Med. 4, 1449 –1452. 8. Ho, A., Schwarze, S. R., Mermelstein, S. J., Waksman, G., and Dowdy, S. F. (2001) Synthetic protein transduction domains: Enhanced transduction potential in vitro and in vivo. Cancer Res. 61, 474 – 477. 9. Lewin, M., Carlesso, N., Tung, C-H., Tang, X-W., Cory, D., Scadden, D. T., and Weissleder, R. (2000) Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nat. Biotechnol. 18, 410 – 414. 10. Vocero-Akbani, A. M., Heyden, N. V., Lissy, N. A., Ratner, L., and Dowdy, S. F. (1999) Killing HIV-infected cells by transduction with an HIV protease-activated caspase-3 protein. Nat. Med. 5, 29 –33. 11. 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 –10450. 12. 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.
13. Pietersz, G. A., Li, W., and Apostolopoulos, V. (2001) A 16-mer peptide (RQIKIWFQNRRMKWKK) from Antennapedia preferentially targets the class I pathway. Vaccine 19, 1397–1405. 14. Elliott, G., and O’Hare, P. (1997) Intercellular trafficking and protein delivery by a herpesvirus structural protein. Cell 88, 223–233. 15. Brewis, N., Phelan, A., Webb, J., Drew, J., Elliott, G., and O’Hare, P. (2000) Evaluation of VP22 spread in tissue culture. J. Virol. 74, 1051–1056. 16. Aints, A., Dilber, M. S., and Smith, C. I. E. (1999) Intercellular spread of GFP-VP22. J. Gene Med. 1, 275–279. 17. Elliott, G., and O’Hare, P. (1999) Intercellular trafficking of VP22-GFP fusion proteins. Gene Ther. 6, 149 –151. 18. Wybranietz, W. A., Prinz, F., Spiegel, M., Schenk, A., Bitzer, M., Gregor, M., and Lauer, U. M. (1999) Quantification of VP22-GFP spread by direct fluorescence in 15 commonly used cell lines. J. Gene Med. 1, 265–274. 19. Kueltzo, L. A., Normand, N., O’Hare, P., and Middaugh, C. R. (2000) Conformational lability of herpesvirus protein VP22. J. Biol. Chem. 275, 33213–33221. 20. Dilber, M. S., Phelan, A., Aints, A., Mohamed, A. J., Elliott, G., Smith, C. I. E., and O’Hare, P. (1999) Intercellular delivery of thymidine kinase prodrug activating enzyme by the herpes simplex virus protein, VP22. Gene Ther. 6, 12–21. 21. Phelan, A., Elliott, G., and O’Hare, P. (1998) Intercellular delivery of functional p53 by the herpesvirus protein VP22. Nat. Biotechnol. 16, 440 – 443. 22. Lundberg, M., and Johansson, M. (2001) Is VP22 nuclear homing an artifact? Nat. Biotechnol. 19, 713–714. 23. Xia, H., Mao, Q., and Davidson, B. L. (2001) The HIV Tat protein transduction domain improves the biodistribution of betaglucuronidase expressed from recombinant viral vectors. Nat. Biotechnol. 19, 640 – 644. 24. Falnes, P. O., Wesche, J., and Olsnes, S. (2001) Ability of the Tat basic domain and VP22 to mediate cell binding, but not membrane translocation of the diphtheria toxin A-fragment. Biochemistry 40, 4349 – 4358.
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Positively Charged DNA-Binding Proteins Cause Apparent Cell Membrane Translocation Mathias Lundberg* and Magnus Johansson† ,1 *Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-17177 Stockholm, Sweden; and †Division of Clinical Virology F68, Karolinska Institute, Huddinge University Hospital, S-14186 Stockholm, Sweden
Received January 8, 2002
Several positively charged DNA-binding proteins such as the human immunodeficiency virus Tat protein, the Antennapedia (Antp) homeobox protein, and the herpes simplex virus VP22 protein have been reported to translocate across cell membranes and accumulate in cell nuclei. The import occurs by a poorly understood mechanism that appears to be receptorand energy-independent. We showed that both VP22 and the positively charged histone H1 adhered to the cell membrane of living cells and were not removed by extensive washing. However, after fixation the proteins relocated to the cell nucleus. The nuclear accumulation of VP22 and histone H1 after fixation shows that positively charged proteins may appear to translocate across the cell membrane because of a fixation artifact. The majority of studies on “membrane permeable” proteins and peptides have been performed using fixation techniques, and our study shows that influx of these proteins may occur during fixation rather than in living cells. © 2002 Elsevier Science (USA) Key Words: protein transduction domain; protein delivery; gene therapy; membrane translocation; immunocytochemistry; protein therapeutics
Fusing macromolecules to “membrane-permeable” proteins or peptides is currently studied as a possible strategy for intracellular macromolecule delivery (1). Several proteins have been reported to efficiently translocate the membrane of mammalian cells. The family of “membrane-permeable” proteins includes the human immunodeficiency virus Tat protein (2–10), the Drosophila melanogaster homeobox protein Antennapedia (Antp) (11–13), and the herpes simplex virus VP22 protein (14 –21). These proteins, or peptides deAbbreviations used: Antp, Antennapedia homeobox protein; CHO, Chinese hamster ovary; FCS, fetal calf serum; GFP, green fluorescent protein. 1 To whom correspondence and reprint requests should be addressed. Fax: ⫹46-8-58587933. E-mail: [email protected].
rived from the proteins, are reported to rapidly translocate the cell membrane with high efficiency (1–21). Little is known about the molecular mechanism mediating the membrane translocation. The import occurs rapidly within minutes and is not dependent on energy, because the import occurs at both 37 and 4°C (4, 5, 11, 12, 14). The import mechanism appears also to be receptor-independent since peptides synthesized from either D- or L-amino acids are equally well imported, and even peptides with reversed amino acid sequences translocate the cell membrane (4, 12). We have recently studied the import of recombinant VP22 in cultured cells (22). When added to cells, VP22 was detected by immunocytochemistry in the nuclei of almost 100% of the cells when applied at either 37 or 4°C for as short as 10 s. The rapid import of VP22 to the nucleus prompted us to investigate alternative explanations to this phenomenon rather than import of the protein into living cells. In summary, we showed that VP22 adheres to the cell surface of living cells and relocates to the cell nucleus during fixation due to the affinity of VP22 to DNA (22). Antp and Tat are, similar to VP22, highly positively charged DNA-binding proteins. The artificial nuclear localization of VP22 raises concern that the influx of these “membrane permeable” proteins may occur during fixation rather than in living cells. To address this question, we have studied the localization of VP22 and the positively charged histone H1 before and after fixation when the proteins were applied to cultured cells. Histone H1 is unrelated to the “membrane permeable” proteins, but is similar to these proteins in regard to its positively charge and the ability to bind DNA. In summary, we showed that both VP22 and histone H1 adhered to the cell membrane of living cells and were relocated to the cell nucleus after fixation. The majority of studies on intracellular delivery of Tat, Antp, and VP22 have been performed on fixed cells (3–21), and our finding suggests that the membrane translocation observed occurs during fixation.
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0006-291X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.
Vol. 291, No. 2, 2002
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS Expression of recombinant VP22-GFP. GFP cDNA was PCR amplified (5⬘-CCATGGTGAGCAAGGGCGAGGAGC and 5⬘-CTTGTACAGCTCGTCCATGCCGAG) from the pEGFP-N1 plasmid vector (Clontech) and cloned into the pCRT7/VP22-TOPO-1 plasmid (Invitrogen) to express GFP fused to the C-terminus of VP22 (aa 159 – 301) with a C-terminal polyhistidine affinity tag. VP22-GFP was expressed in BL21(DE3)pLysS Escherichia coli (Invitrogen) and the protein purified using Talon metal affinity resin column (Clontech) as described in the manufacturer’s protocol. Import of VP22 into CHO cells. Chinese hamster ovary (CHO) cells (Clontech) were cultured at 37°C with 5% CO 2 in McCoy 5A modified medium supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, and 0.1 mg/ml streptomycin. For experiments on adherent cells, the cells were plated in Lab-Tech II chamber slides (Nunc). For experiments on cells in suspension, the cells were detached by incubation with 0.1% EDTA in PBS. The cells were washed twice with PBS, and 1 g VP22-GFP or Alexa Fluor-488 fluorescentlabeled histone H1 (Molecular Probes) were added to the cells in 200 l cell culture medium without serum. The cells were incubated into at 4°C and washed three times in PBS. The cells in suspension were adhered to Bio-Rad Adhesion slides (Bio-Rad). The cells were fixed 15 min with 100% room-tempered methanol, rehydrated 2 min in PBS, stained 2 min with 300 nM DAPI, and washed 5 ⫻ 2 min in PBS. When indicated, the fixed cells were incubated 10 min with 10% fetal calf serum in PBS. The cells were mounted in 50% glycerol in PBS and fluorescence microscopy was performed on a Nikon Eclipse E600 microscope equipped with a SPOT RT digital camera.
RESULTS We used recombinant VP22-GFP and Alexa Fluor488 fluorescent-labeled histone H1 to study the localization of these proteins when added to cells. The proteins were first added to dead methanol fixed CHO cells (Fig. 1). Both VP22-GFP and histone H1 localized almost exclusively to the cell nuclei of the fixed cells. These results show that if the proteins are able to enter the cells, either before or after fixation, their affinity for DNA is sufficient to cause nuclear accumulation. We added VP22-GFP or fluorescent-labeled histone H1 for 2 min to CHO cells cultured adherent on chamber slides (Fig. 2). Live unfixed cells incubated with either protein showed distinct fluorescence at the cell surface, indicating adherence of the proteins to the plasma membrane. No nuclear fluorescence was observed for either protein. The proteins remained attached to the cell membrane even after extensive washing with PBS. In contrast, after methanol fixation and rehydration in PBS the cells exhibited intracellular fluorescence. VP22-GFP fluorescence was after fixation located distinctly to cell nuclei whereas histone H1 showed fluorescence in both the cytosol and nucleus. Accordingly, the proteins attached to the cell membrane were released during fixation and relocated intracellularly. Immunocytochemistry is one of the most commonly used methods for detection of intracellular proteins in studies on the membrane permeable proteins VP22, Antp and Tat. In immunohistochemistry, the samples are blocked after fixation with FCS or
FIG. 1. Methanol-fixed CHO cells incubated with VP22-GFP or Alexa Fluor fluorescent-labeled histone H1. The cells were cultured on chamber slides and fixed with methanol, the fluorescent proteins were added, and the cells were washed with PBS. The nuclei were contrastained with DAPI.
other protein-blocking reagent prior to addition of the antibodies to reduce unspecific antibody binding. Therefore, we incubated the fixed cells with 10% FCS to investigate if the presence of FCS affected the location of VP22-GFP or histone H1. VP22-GFP nuclear fluorescence remained similar in the presence or absence of FCS. However, incubation with FCS resulted in a change from a cytosolic/nuclear pattern to a distinct nuclear location for histone H1. One possible problem in these experiments is that the protein reservoir that becomes mobilized during fixation could be located on the chamber-slide glass or plastic walls. The fixation artifact could in this case be dependent on the material used for cell culture. To investigate this possibility, we repeated the experiment on cells in suspension, which allowed thorough washing and changing of the test tubes before fixation. However, cells in suspension incubated with histone H1 showed a similar pattern as the cells cultured on chamber slides (Fig. 3), indicating that the protein that relocated to the nucleus after fixation was located on the cell membrane of the living cells. DISCUSSION We have shown that the positively charged DNAbinding proteins VP22 and histone H1 adhered to the plasma membrane of living cells, remained attached during washing, and relocated and bound intracellular structures such as DNA when the cells were fixed. The relocation of the proteins during fixation results in an
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FIG. 2. CHO cells cultured on chamber slides incubated with VP22-GFP (A) or Alexa Fluor-labeled histone H1 (B) before (live) or after methanol (MeOH) fixation. The fixed cells were rehydrated with PBS in the presence or absence of fetal calf serum (FCS). The live cells were visualized by phase contrast (PC) microscopy and the nuclei of the fixed cells were contrastained with DAPI.
apparent, but not true, cell membrane translocation. The techniques most commonly used to visualize the influx of the positively charged “membrane permeable” proteins VP22, Tat and Antp all involve cell fixation (3–21), and our findings show that the influx of these proteins may occur during fixation rather than in living cells. The influx and nuclear accumulation of positively charged proteins during fixation, explains the independence of incubation time, incubation temperature, and receptors for protein internalization (4, 5, 11, 12, 14). Although alternative fixation techniques other than methanol fixation used in the present study may
diminish the membrane translocation artifact, all fixation techniques cause disruption of the cell membrane and cannot reliably be used for studies on positively charged membrane translocating proteins. Several investigators have used flow cytometry analysis of living cells to determine the uptake of the membrane permeable proteins (5–10, 18). Although this technique does not involve fixation, it may not discriminate between proteins adhered to the cell surface or internalized proteins. In the present study, we showed that positively charged proteins such as VP22 and histone H1 remained attached to the cell membrane even after
369
Vol. 291, No. 2, 2002
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. CHO cells in suspension incubated with Alexa Fluor-labeled histone-H1 before (live) or after methanol (MeOH) fixation. The fixed cells were rehydrated with PBS in the presence or absence of fetal calf serum (FCS). The live cells were visualized by phase contrast (PC) microscopy and the nuclei of the fixed cells were contrastained with DAPI.
extensive washing. Accordingly, it is likely that the flow-cytometry analysis reflect cell surface adherence rather than intracellular accumulation of the proteins. We conclude that imaging studies on cell membrane translocating proteins have to be performed on live unfixed cells with fluorescently labeled proteins to exclude artifacts. Several proteins fused to Tat, Antp and VP22 are reported to have physiological effects when added to cells (2, 7, 10, 13, 20, 21). These studies suggest that the positively charged proteins and their fusion partners have the ability to transverse the cell membrane. Although the rapid temperature independent cellular influx and nuclear localization of positively charged DNA-binding proteins is likely to be due to the fixation artifact reported in the present study, we cannot exclude that these protein have properties that may mediate delivery of macromolecules to cells. The positive charge of the proteins will cause adherence to the negatively charged cell membrane. Proteins adhered to the cell membrane will be undergo endocytosis and end up in into endosomes and lysosomes. A recent study shows that lysosomal -glucuronidase fused to Tat is internalized by endocytosis and the fusion protein restores enzyme activity in a mouse model of -glucuronidase deficiency (23). Accordingly, positively charged proteins such as Tat can be used to deliver proteins to lysosomes and endosomes. Whether Tat, Antp and VP22 have the ability to also enter the cytosol or nucleus of cells is less clear. Although cytosolic or nuclear delivery of fusion proteins is suggested by functional or phenotypic assays (2, 7, 10, 13, 20, 21), the concomitant use of fixation techniques in protein imaging or flow
cytometry analysis in these studies make the data difficult to evaluate. In a recent study, the diphtheria toxin A-fragment was fused to VP22 or Tat (24). Although the presence of only a few molecules of the diphtheria toxin in the cytosol is sufficient to cause cell death, no cytotoxic effects were observed when the fusion proteins were applied to cells. This study suggests that neither VP22 nor Tat have the ability to translocate the cell membrane. However, it cannot be excluded that the diphtheria toxin interfered with the delivery of VP22 and Tat to the cytosol, and further studies will be required to elucidate the possible use of positively charged proteins as vectors for protein delivery. These studies will have to be performed using functional assays and live cell imaging to avoid detection of artificial protein import. ACKNOWLEDGMENTS This work was supported by grants from the Swedish Medical Research Council, Swedish Society of Medicine, and the medical faculty of the Karolinska Institute.
REFERENCES
370
1. Schwarze, S. R., Hruska, K. A., and Dowdy, S. F. (2000) Protein transduction: Unrestricted delivery into all cells? Trends Cell Biol. 10, 290 –295. 2. Frankel, A. D., and Pabo, C. O. (1988) Cellular uptake of the tat protein from human immunodeficiency virus. Cell 55, 1189 – 1193. 3. Fawell, S., Seery, J., Daikh, Y., Moore, C., Chen, L. L. Pepinsky, B., and Barsoum, J. (1994) Tat-mediated delivery of heterologous proteins into cells. Proc. Natl. Acad. Sci. USA 91, 664 – 668.
Vol. 291, No. 2, 2002
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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