Biological membranes have evolved in part to prevent xenobiotics from passively entering cells.1
Notwithstanding this barrier function, numerous organisms have developed proteins, many of which are transcription factors, that breach membranes through a variety of mechanisms.2
The protein HIV tat, for example, when used in vitro
, rapidly enters the cytosol (and nucleus) of a wide variety of cells by endocytosis.3
However, the nine-amino-acid peptide required for the uptake of HIV tat, residues 49–57 (RKKRRQRRR), appears to utilize an additional mechanism, as is evident from its uptake, even at 4 °C, by a route distinct from the intact protein,4
sensitive to the attached cargo size and composition and even to cell type. Our previous studies demonstrated that the guanidinium head groups of tat 49–57 are the key functionalities required for its entry into cells. Indeed, replacement of all non-arginine residues in tat 49–57 with arginines provides molecular transporters that exhibit superior rates of uptake. Charge itself is necessary, but not sufficient, as is evident from the comparatively poor uptake of lysine nonamers.5,6
Bidentate hydrogen bonding of the cationic guanidinium groups to anionic cell surface groups (phosphates, carboxylates, sulfates) is important, as indicated by the finding that mono- or dimethylation of the critical guanidinium groups, while preserving charge, reduces or eliminates uptake. The number of arginines and therefore guanidinium groups is also important, with optimal uptake for oligomers of 7–15 residues.6,7
Natural backbone chirality is not critical for uptake, as the unnatural peptides show increased uptake relative to the natural peptides. Even the position of attachment and length of the side chains can be altered, as shown, for example, with guanidinium-rich peptoids that exhibit highly efficient uptake.5
Changes in the backbone composition and in the side-chain spacing between residues also can increase uptake, with highly branched, guanidinium-rich oligosaccharides and dendrimers being efficient transporters.5,6,8–10
In contrast to receptor-mediated uptake, an increase in conformational flexibility of the guanidinium-rich transporters generally enhances uptake.
Several mechanisms could accommodate the above structure–function relationships for guanidinium-rich cell-penetrating peptides (CPPs) and, more generally, guanidinium-rich molecular transporters (GRTs).5,6,11
These mechanisms could even operate concurrently, depending on, for example, the properties of the CPPs and cargoes, CCP concentrations, the choice of selected cell lines, and assay conditions. A receptor-mediated process is inconsistent with the broad range of structural modifications that promote uptake and especially the observation that more-flexible systems show enhanced uptake. Endocytosis and macropinocytosis have been proposed and appear to be pertinent to entry involving high-molecular-weight CPP conjugates.11,18,19
Conventional passive diffusion across the nonpolar interior of the plasma membrane is seemingly difficult to reconcile with the polarity of the arginine oligomers (which are highly water soluble) and the dependency of uptake on the number of charges. However, our prior studies have suggested that an adaptive translocation mechanism might be operative for low-molecular-weight guanidinium-based conjugates.6,11,12
In this process, positively charged guanidinium oligomers, which alone are too polar to enter a membrane, form bidentate hydrogen-bonded ion-pair complexes with complementary charged cell surface functionalities of membrane-embedded groups (carboxylates, sulfates, phosphates) and are driven inward across the membrane under the influence of the membrane potential. It is especially noteworthy, with respect to this mechanism, that water-soluble arginine oligomers can be completely solubilized in octanol (a membrane dielectric mimic) by treatment with an equivalent of a fatty acid salt (sodium laurate).12
Various CPPs have been reported to carry exogenous molecules into cells.6,7,10,11
Understanding the internalization mechanism of peptide transporters is fundamental to their use as delivery vectors for drugs and probes, especially in view of the increasing interest in the delivery of biologicals (peptides, proteins, nucleic acids, etc). To address the recent controversy surrounding the uptake mechanism of arginine-rich CPP peptides,13–19
we decided to examine, by direct observation in real time, their membrane interactions on a single-molecule level in living cells.
Single-molecule imaging is a technique that has been used to probe the dynamics of molecules as well as their local environments in liquids, solids, surfaces, and living cell membranes.20–25
Unlike assays used in previous studies, this technique can be used to directly observe the motion of individual oligoarginines in real time rather than the motion inferred indirectly from the behavior of the ensemble average. Attempting to indirectly define the behavior on the basis of the average alone makes analysis extremely complex and open to a number of different interpretations, particularly when multiple mechanisms might be operating concurrently.15,17,18
This work is the first report describing the direct observation of the motion of CPPs on the plasma membrane in real time on a single-molecule level in living cells.
In this investigation, epifluorescence imaging is used to study the movement of single fluorophore-labeled octaarginine transporters using living Chinese hamster ovary (CHO) cells.26–29
A novel label, the DCDHF-V fluorophore, is used which belongs to a class of single-molecule fluorophores consisting of an amine donor and dicyanomethylenedihydrofuran (DCDHF) acceptor linked by a conjugated unit (e.g., benzene, naphthalene, styrene, etc.). Molecules in the DCDHF class exhibit useful properties for single-molecule studies, such as high quantum yields, photostability, and environmental reporter functions.26–29
DCDHF-V, with conjugation extended by an additional vinyl group, absorbs and emits at long wavelengths (λex
= 610 nm, λem
= 630 nm in water), thereby avoiding cellular autofluorescence. Here, a maleimide derivative of DCDHF-V is covalently attached to the octaarginine backbone through an N-terminal cysteine; as such, it reports on the mobility of the resultant octaarginine peptide conjugate (see ). This fluorophore is brighter when the molecule is interacting with the more constrained environment of the membrane (and the cell interior) compared to the aqueous buffer outside the cell; hence, contrast in single-molecule imaging is enhanced. Moreover, useful signal-to-background is obtained at physiological oxygen concentrations, and the contrast observed is superior to that of the Cy3 dye (Amersham) with or without oxygen scavengers. Our results indicate that the behavior of octaarginine on a cell surface is different from that of lipids, known to penetrate cellular membranes through passive diffusion, conventionally involving lateral diffusion followed by membrane flip-flop. Furthermore, while the octaarginine behavior shares some common features with protein uptake (endocytotic) processes, it also exhibits dissimilar diffusion properties at the single-molecule level. The mode by which octaarginine penetrates the cell membrane appears to be either a multimechanism uptake process or a mechanism different from passive diffusion and endocytosis. These results have relevance to the mechanism of cellular uptake of guanidinium-rich transporters conjugated to small molecules, drugs, and probes (MW ca. <3000).
Figure 1 (A) Structures of DCDHF-V labeled octaarginine (Arg8-D-V) and tetraarginine (Arg4-D-V). (B) Structure of DCDHF-V-labeled lipid analogue (D-V-12). (C) Schematic of the imaging arrangement. CHO cells were cultured in fibronectin-coated imaging chambers (more ...)