Delineating the mechanisms by which cell-penetrating peptides, such as HIV-Tat peptide, oligoarginines and penetratin, gain access to cells has recently received intense scrutiny. Heightened interest in these entities stems from their ability to enhance cellular delivery of associated macromolecules, such as genes and proteins, suggesting that they may have widespread applications as drug-delivery vectors. Proposed uptake mechanisms include energy-independent plasma membrane translocation and energy-dependent vesicular uptake and internalization through endocytic pathways. In the present study, we investigated the effects of temperature, peptide concentration and plasma membrane cholesterol levels on the uptake of a model cell-penetrating peptide, L-octa-arginine (L-R8) and its D-enantiomer (D-R8) in CD34+ leukaemia cells. We found that, at 4–12 °C, L-R8 uniformly labels the cytoplasm and nucleus, but in cells incubated with D-R8 there is additional labelling of the nucleolus which is still prominent at 30 °C incubations. At temperatures between 12 and 30 °C, the peptides are also localized to endocytic vesicles which consequently appear as the only labelled structures in cells incubated at 37 °C. Small increases in the extracellular peptide concentration in 37 °C incubations result in a dramatic increase in the fraction of the peptide that is localized to the cytosol and promoted the binding of D-R8 to the nucleolus. Enhanced labelling of the cytosol, nucleus and nucleolus was also achieved by extraction of plasma membrane cholesterol with methyl-β-cyclodextrin. The data argue for two, temperature-dependent, uptake mechanism for these peptides and for the existence of a threshold concentration for endocytic uptake that when exceeded promotes direct translocation across the plasma membrane.
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Research Article|
March 26 2007
Temperature-, concentration- and cholesterol-dependent translocation of L- and D-octa-arginine across the plasma and nuclear membrane of CD34+ leukaemia cells
Marjan M. Fretz;
Marjan M. Fretz
*Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3XF, Wales, U.K.
†Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, PO Box 80082, 3508 TB Utrecht, The Netherlands
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Neal A. Penning;
Neal A. Penning
*Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3XF, Wales, U.K.
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Saly Al-Taei;
Saly Al-Taei
*Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3XF, Wales, U.K.
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Shiroh Futaki;
Shiroh Futaki
‡Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
§PRESTO, Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan
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Toshihide Takeuchi;
Toshihide Takeuchi
‡Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
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Ikuhiko Nakase;
Ikuhiko Nakase
‡Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
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Gert Storm;
Gert Storm
†Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, PO Box 80082, 3508 TB Utrecht, The Netherlands
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Arwyn T. Jones
Arwyn T. Jones
1
*Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3XF, Wales, U.K.
1To whom correspondence should be addressed (email jonesat@cardiff.ac.uk).
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Publisher: Portland Press Ltd
Received:
December 05 2006
Revision Received:
January 09 2007
Accepted:
January 12 2007
Accepted Manuscript online:
January 12 2007
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Authors Journal compilation, The Biochemical Society, London
2007
Biochem J (2007) 403 (2): 335–342.
Article history
Received:
December 05 2006
Revision Received:
January 09 2007
Accepted:
January 12 2007
Accepted Manuscript online:
January 12 2007
Citation
Marjan M. Fretz, Neal A. Penning, Saly Al-Taei, Shiroh Futaki, Toshihide Takeuchi, Ikuhiko Nakase, Gert Storm, Arwyn T. Jones; Temperature-, concentration- and cholesterol-dependent translocation of L- and D-octa-arginine across the plasma and nuclear membrane of CD34+ leukaemia cells. Biochem J 15 April 2007; 403 (2): 335–342. doi: https://doi.org/10.1042/BJ20061808
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