title: Transient focal membrane deformation induced by arginine-rich peptides leads to their direct penetration into cells.
abstract: Endocytosis has been implicated in the cellular uptake of arginine-rich, cell-penetrating peptides (CPPs). However, accumulating evidence suggests that certain conditions allow the direct, non-endocytic penetration of arginine-rich peptides through the plasma membrane. We previously showed that Alexa Fluor 488-labeled dodeca-arginine (R12-Alexa488) directly enters cells at specific sites on the plasma membrane and subsequently diffuses throughout cells. In this study, we found that the peptide influx was accompanied by the formation of unique, "particle-like" multivesicular structures on the plasma membrane, together with topical inversion of the plasma membrane. Importantly, the conjugation of dodeca-arginine (R12) to Alexa Fluor 488 or a peptide tag derived from hemagglutinin (HAtag) significantly accelerated particle formation, suggesting that the chemical properties of the attached molecules (cargo molecules) may contribute to translocation of the R12 peptide. Coincubation with R12-HAtag allowed the membrane-impermeable R4-Alexa488 to permeate cells. These results suggest that R12 peptides attached to hydrophobic cargo molecules stimulate dynamic morphological alterations in the plasma membrane, and that these structural changes allow the peptides to permeate the plasma membrane. These findings may provide a novel mode of cell permeabilization by arginine-rich peptides as a means of drug delivery.
authors.affiliation: Institute for Chemical Research, Kyoto University, Kyoto, Japan.
authors.forename: Hisaaki
authors.initials: H
authors.lastname: Hirose
authors.forename: Toshihide
authors.initials: T
authors.lastname: Takeuchi
authors.forename: Hiroko
authors.initials: H
authors.lastname: Osakada
authors.forename: Sílvia
authors.initials: S
authors.lastname: Pujals
authors.forename: Sayaka
authors.initials: S
authors.lastname: Katayama
authors.forename: Ikuhiko
authors.initials: I
authors.lastname: Nakase
authors.forename: Shouhei
authors.initials: S
authors.lastname: Kobayashi
authors.forename: Tokuko
authors.initials: T
authors.lastname: Haraguchi
authors.forename: Shiroh
authors.initials: S
authors.lastname: Futaki
mesh_terms: D001120:Arginine / Q000737:chemistry* / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D002463:Cell Membrane Permeability / Q000187:drug effects; D057846:Cell-Penetrating Peptides / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D004058:Diffusion; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006388:Hemagglutinins / Q000737:chemistry; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008301:Maleimides; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D021381:Protein Transport
chemical_list: C504424:Alexa Fluor 488 C5-maleimide; D057846:Cell-Penetrating Peptides; D005456:Fluorescent Dyes; D006388:Hemagglutinins; D008301:Maleimides; D009842:Oligopeptides; D001120:Arginine