title: S4(13)-PV cell-penetrating peptide forms nanoparticle-like structures to gain entry into cells.
abstract: Despite increasing interest in cell-penetrating peptides (CPPs) as carriers for drugs and in gene therapy, the current understanding of their exact internalization mechanism is still far from complete. The cellular translocation of CPPs and their payloads has been mostly described by fluorescence- and activity-based methods, leaving the more detailed characterization at the ultrastructural level almost out of attention. Herein, we used transmission electron microscopy to characterize the membrane interaction and internalization of a cell-penetrating peptide S4(13)-PV. We demonstrate that S4(13)-PV peptide forms spherical nanoparticle-like regular structures upon association with cell surface glycosaminoglycans on the plasma membrane. Insertion of S4(13)-PV particles into plasma membrane induces disturbances and leads to the vesicular uptake of peptides by cells. We propose that for efficient cellular translocation S4(13)-PV peptides have to assemble into particles of specific size and shape. The spherical peptide particles are not dissociated in intracellular vesicles but often retain their organization and remain associated with the membrane of vesicles, destabilizing them and promoting the escape of peptides into cytosol. Lowering the temperature and inhibition of dynamins' activity reduce the internalization of S4(13)-PV peptides, but do not block it completely. Our results provide an ultrastructural insight into the interaction mode of CPPs with the plasma membrane and the distribution in cells, which might help to better understand how CPPs cross the biological membranes and gain access into cells.
authors.affiliation: Department of Developmental Biology, Institute of Molecular and Cell Biology, University of Tartu, EE51010 Tartu, Estonia.
authors.forename: Kärt
authors.initials: K
authors.lastname: Padari
authors.forename: Kaida
authors.initials: K
authors.lastname: Koppel
authors.forename: Annely
authors.initials: A
authors.lastname: Lorents
authors.forename: Mattias
authors.initials: M
authors.lastname: Hällbrink
authors.forename: Miguel
authors.initials: M
authors.lastname: Mano
authors.forename: Maria C
authors.initials: MC
authors.lastname: Pedroso de Lima
authors.forename: Margus
authors.initials: M
authors.lastname: Pooga
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D046529:Microscopy, Electron, Transmission; D053758:Nanoparticles / Q000737:chemistry*; D010316:Particle Size; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D013696:Temperature; D014018:Tissue Distribution
chemical_list: D006025:Glycosaminoglycans; D010455:Peptides; C512366:S4(13)-PV peptide