Cell Entry of Arginine-rich Peptides Is Independent of Endocytosis

Abstract

Arginine-rich peptides are a subclass of cell-penetrating peptides that are taken up by living cells and can be detected freely diffusing inside the cytoplasm and nucleoplasm. This phenomenon has been attributed to either an endocytic mode of uptake and a subsequent release from vesicles or to direct membrane penetration (transduction). To distinguish between both possibilities, we have blocked endocytic pathways suggested to be involved in uptake of cell-penetrating peptides. We have then monitored by confocal microscopy the uptake and distribution of the cell-penetrating transactivator of transcription (TAT) peptide into living mammalian cells over time. To prevent side effects of chemical inhibitors, we used genetically engineered cells as well as different temperature. We found that a knockdown of clathrin-mediated endocytosis and a knock-out of caveolin-mediated endocytosis did not affect the ability of TAT to enter cells. In addition, the TAT peptide showed the same intracellular distribution throughout the cytoplasm and nucleus as in control cells. Even incubation of cells at 4°C did not abrogate TAT uptake nor change its intracellular distribution. We therefore conclude that this distribution results from TAT peptide that directly penetrated (transduced) the plasma membrane. The formation of nonselective pores is unlikely, because simultaneously added fluorophores were not taken up together with the TAT peptide. In summary, although the frequency and kinetics of TAT transduction varied between cell types, it was independent of endocytosis.

Nucleic acid delivery by cell penetrating peptides derived from dengue virus capsid protein: design and mechanism of action

“…S7). The kinetic profile also revealed an initial lag time for membrane adsorption and subsequent cellular internalization, which agrees with other studies showing that Tat enters cells using endocytic routes [32][33][34][35]. In Table 1.…”

110 Years of the Meyer–Overton Rule: Predicting Membrane Permeability of Gases and Other Small Compounds

“…In light of this, recent reports excluding endocytosis as an uptake mechanism do not appear convincing. [79] Since passive membrane diffusion of peptides has long been known as impossible, [80,81] what else can possibly be offered as a transport mechanism? Transient water wires across a lipid bilayer.…”

Arginine‐rich cell‐penetrating peptides

“…The diffuse staining was not due to the release of peptide from endosomes as it appeared first and the punctate staining later, and it continued to occur at 4°C when endocytosis is inhibited. Several other studies have also reported cellular uptake when the cells have been incubated at 4°C [26,[51][52][53]. It has also been observed that blocking specific endocytotic pathways does not affect the ability of TAT peptide to enter cells.…”

“…The TAT protein is an 86 amino acid long protein that is released by infected cells and is an essential regulatory gene for HIV replication [8]. In 1997, Vives et al [9] found that a 11-amino acid sequence, TAT (47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57), now known as the TAT peptide or TAT PTD, can not only enter cells but is more efficient than the full length protein. It was observed that the chirality of the peptide backbone has no effect on cellular uptake of TAT peptide; inverse and retro forms were able to enter cells as efficiently as the native peptide, suggesting uptake does not require a specific binding site.…”