Temperature-, concentration- and cholesterol-dependent translocation of L- and D-octa-arginine across the plasma and nuclear membrane of CD34+ leukaemia cells
Résumé
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 abilities to enhance cellular delivery of associated macromolecules such as genes and proteins, suggesting they may have widespread applications as drug-delivery vectors. Proposed uptake mechanisms include energy-independent plasma membrane translocation and energy-dependent vesicular uptake and internalisation through endocytic pathways. Here we investigated the effects of temperature, peptide concentration and plasma membrane cholesterol levels on the uptake of a model cell penetrating peptide, octaarginine (L-R8) and its D-enantiomer (D-R8) in CD34 +} leukaemia cells. We find that at 4-12 °C, L-R8 uniformly labels the cytoplasm and nucleus but in cells incubated with D-R8 there is additional labeling of the nucleolus, that is still prominent at 30 °C incubations. At temperatures between 12-30 °C, the peptides are also localised to endocytic vesicles, that 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 localised to the cytosol and promoted the binding of D-R8 to the nucleolus. Enhanced labeling of the cytosol, nucleus and nucleolus was also achieved by extraction of plasma membrane cholesterol with methyl-{beta}-cyclodextrin. The data argues 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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