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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Interactions of Cell-Penetrating Peptides in the Model of GiantPlasma Membrane VesiclesPille Säälik 1,2 , Aira Niinep 1 , Mats Hansen 3 , Ülo Langel 3,4 , andMargus Pooga 1,21 Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia; 2 EstonianBiocentre, Tartu, Estonia; 3 Department of Neurochemistry, Stockholm University, Sweden;4 Institute of Technology, University of Tartu, EstoniaIntroductionThe cellular uptake mechanism of cell-penetrating peptides (CPP) is still a matter of debatein spite of the remarkable development in the area. Both endocytosis and direct membranetranslocation are shown to be involved in the process. However, the exact mechanism anddeterminants of the translocation process have remained elusive.We have exploited the model of giant plasma membrane vesicles (GPMV) [1] from ratbasophilic leukaemia cells (RBL) to study the membrane interactions of six mostwell-known cell-penetrating peptides. Since GPMVs are derived from the cell’s plasmamembrane, their composition is very close to this organelle, being thus more native modelsystem for CPP studies compared to artificial vesicles consisting of just a small number ofvarying lipids. The membrane of GPMVs segregates into liquid-ordered (L o ) andliquid-disordered (L d ) lipid phases domains at low temperature, which can be visualizedwith fluorescently labeled cholera toxin B subunit and annexin V, respectively. Therefore,GPMVs have been used earlier to study the lipid phase dynamics and the phase preferenceof different plasma membrane proteins [2,3]. We aimed to use these vesicles to elucidatethe involvement of membrane domains with different organization in the cellular uptake ofsix CPPs - Tat peptide (pTat), nonaarginine (R 9 ), model amphipathic peptide (MAP),Penetratin (pAntp), Transportan (TP), and its shorter analogue Transportan10 (TP10).Results and discussionIn parallel with the results that the primary and secondary amphipathic CPPs [4] - TP,TP10, pAntp and MAP, clearly preferred the L d phase when interacting with plasmamembrane components, we also detected the translocation and accumulation of all thetested CPPs into GPMVs at two temperatures below physiological (Figure 1). Thiscorroborates the ability of CPPs to cross the biological membrane without using endocyticprocesses [5,6]. The result that the control peptide, a slightly negative (general charge -1)18-mer peptide from the C-terminal part of rat adrenergic receptor 1 either didn’tinternalize or entered the GPMVs in a very low extent suggested that the property ofaccumulation into GPMVs is characteristic only to peptides with penetration ability.The preference of all the membrane-affine CPPs, TP, TP10, pAntp and MAP, for theL d phase in GPMVs suggests that in live cell, these peptides might also interact favorablywith the membrane areas of more dynamic and less densely packed nature. Our suggestionthat the L d phase is a preferred portal for CPPs when translocating into GPMVs is alsosupported by the fact that the vesicles showing preferentially only CtxB-staining, i.e.containing mainly L o phase, were sometimes devoid of CPPs. Furthermore, GPMVsdepleted of cholesterol by methyl- -cyclodextrin treatment revealed more uniform uptakeof all tested CPPs, confirming that the presence of densely packed membrane areas actsrather as a barrier than a contributor of CPP uptake.The accumulation of all the tested CPPs into the giant plasma membrane vesicles wasthe highest for pTat and R 9 , as detected by flow cytometry analysis. The continuousaccumulation of these CPPs into vesicles took place during one hour. On the contrary topTat and R 9 , Transportan rapidly interacted with GPMVs and reached a plateau, revealingthe fast and stable interaction with the membrane components.410