Single-Molecule Motions of Oligoarginine Transporter Conjugates on the Plasma Membrane of Chinese Hamster Ovary Cells

Department of Chemistry, Stanford University, Stanford, California 94305, USA.
Journal of the American Chemical Society (Impact Factor: 12.11). 08/2008; 130(29):9364-70. DOI: 10.1021/ja710798b
Source: PubMed


To explore the real-time dynamic behavior of molecular transporters of the cell-penetrating-peptide (CPP) type on a biological membrane, single fluorescently labeled oligoarginine conjugates were imaged interacting with the plasma membrane of Chinese hamster ovary (CHO) cells. The diffusional motion on the membrane, characterized by single-molecule diffusion coefficient and residence time (tau R), defined as the time from the initial appearance of a single-molecule spot on the membrane (from the solution) to the time the single molecule disappears from the imaging focal plane, was observed for a fluorophore-labeled octaarginine (a model guanidinium-rich CPP) and compared with the corresponding values observed for a tetraarginine conjugate (negative control), a lipid analogue, and a fluorescently labeled protein conjugate (transferrin-Alexa594) known to enter the cell through endocytosis. Imaging of the oligoarginine conjugates was enabled by the use of a new high-contrast fluorophore in the dicyanomethylenedihydrofuran family, which brightens upon interaction with the membrane at normal oxygen concentrations. Taken as a whole, the motions of the octaarginine conjugate single molecules are highly heterogeneous and cannot be described as Brownian motion with a single diffusion coefficient. The observed behavior is also different from that of lipids, known to penetrate cellular membranes through passive diffusion, conventionally involving lateral diffusion followed by membrane bilayer flip-flop. Furthermore, while the octaarginine conjugate behavior shares some common features with transferrin uptake (endocytotic) processes, the two systems also exhibit dissimilar traits when diffusional motions and residence times of single constructs are compared. Additionally, pretreatment of cells with cytochalasin D, a known actin filament disruptor, produces no significant effect, which further rules out unimodal endocytosis as the mechanism of uptake. Also, the involvement of membrane potential in octaarginine-membrane interaction is supported by significant changes in the motion with high [K(+)] treatment. In sum, this first study of single transporter motion on the membrane of a living cell indicates that the mode by which the octaarginine transporter penetrates the cell membrane appears to either be a multimechanism uptake process or a mechanism different from unimodal passive diffusion or endocytosis.

1 Follower
16 Reads
  • Source
    • "The availability of STX-NH 3 + through chemical synthesis offers a convenient starting material from which to append functional groups through mild and selective conjugation strategies (Figure 1B; Figure S1 available online). For our purposes, Cy5 and DCDHF (Lord et al., 2009) fluorophores serve as bright, photostable, long wavelength emitting probes that, when appropriately derivatized, are soluble and chemically stable in aqueous cell media (Lee et al., 2008). Both Cy5 and DCDHF exhibit high total numbers of emitted photons and large fluorescence quantum yields, and possess absorbance and emission wavelengths that avoid cellular autofluorescence. "
    [Show abstract] [Hide abstract]
    ABSTRACT: A desire to better understand the role of voltage-gated sodium channels (Na(V)s) in signal conduction and their dysregulation in specific disease states motivates the development of high precision tools for their study. Nature has evolved a collection of small molecule agents, including the shellfish poison (+)-saxitoxin, that bind to the extracellular pore of select Na(V) isoforms. As described in this report, de novo chemical synthesis has enabled the preparation of fluorescently labeled derivatives of (+)-saxitoxin, STX-Cy5, and STX-DCDHF, which display reversible binding to Na(V)s in live cells. Electrophysiology and confocal fluorescence microscopy studies confirm that these STX-based dyes function as potent and selective Na(V) labels. The utility of these probes is underscored in single-molecule and super-resolution imaging experiments, which reveal Na(V) distributions well beyond the optical diffraction limit in subcellular features such as neuritic spines and filopodia.
    Chemistry & biology 07/2012; 19(7):902-12. DOI:10.1016/j.chembiol.2012.05.021 · 6.65 Impact Factor
  • Source
    • "Understanding the modes of CPP transduction is fundamental to their use as delivery vectors for biologicals (e.g., peptides, proteins, and nucleic acids) and probes, especially in view of the increasing interest in the delivery of anticancer drugs (Lee et al., 2008). It is especially noteworthy that the cell-penetrating mechanism is dependent upon the properties of the CPPs, the selected cell lines, the concentrations of CPPs and the assay conditions (temperature or serum). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The application of cell-penetrating peptides (CPPs) for delivering various cargo molecules with biological functions into cells has gained much attention in recent years. However, the internalization mechanisms and delivery properties of CPP-cargo remains controversial. In this study, low- and high-molecular-weight cargoes attached to arginine-rich CPPs were employed: the former was the fluorescein isothiocyanate-labeled nona-arginine (CPP-FITC), and the latter was the fluorescently labeled nona-arginine-avidin complex (CPP-avidin). We measured the intracellular trafficking of CPP-FITC and CPP-avidin in four cancer cell lines in a series of microenvironments altered by the presence or absence of serum, different temperatures and different incubation times. The results revealed that CPP-cargo delivery exhibited no specificity toward any cell line, but the levels were found to be related to cell type and cargo. Furthermore, their endocytic mechanisms were investigated via incubation with related endocytic inhibitors. Two different types of CPP-cargo were required to cross the plasma membrane to bind to cell surface-associated heparan sulfate proteoglycans in a time-dependent manner. CPPs and small cargoes attached to CPP may enter cells rapidly via direct translocation in addition to the endocytic route. Translocation of large components linked to CPP tended to be mediated by macropinocytosis in an energy-dependent manner with slower rates for larger compounds. In contrast, the clathrin-dependent pathway is not essential to the translocation of either type of CPP-cargo.
    International Journal of Pharmaceutics 08/2011; 419(1-2):200-8. DOI:10.1016/j.ijpharm.2011.08.001 · 3.65 Impact Factor
  • Source
    • "Cationic CPPs have multiple arginine and/or lysine residues while amphipathic CPPs exhibit amphipathic structures (e.g., α-helix in model amphipathic peptide, MAP) (Patel et al., 2007). e mechanism of internalization has yet to be established, and has been shown to occur through endocytosis-dependent mechanism (Lundberg & Johansson, 2001; Nakase et al., 2004; Richard et al., 2005) and independent ((orén et al., 2003; Rothbard et al., 2004; Cahill, 2010) mechanisms, or a combination of both (Zaro & Shen, 2003; Zaro & Shen, 2005a; Zaro et al., 2006; Lee et al., 2008; Wender et al., 2008; Zhang et al., 2009; Sawant & Torchilin, 2010; Schmidt et al., 2010). Not only do these peptides accumulate in cells, but they are also able to ferry cargo molecules into the cells, including small molecule drugs, peptides, proteins, RNAs or DNAs, and even large particles, such as liposomes (Vives et al., 2008). is property draws lots of attention, because many new potential drugs, in particular proteins and peptides, have targets in the cytosol or nucleus, but they cannot access these sites eeciently. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Properties of different arginine-rich peptides, including net charge and charge distribution, were evaluated for their influence on surface binding, internalization, and intracellular localization. The peptides were radiolabeled and subsequently tested for surface binding and internalization in CHO-K1 cells. Subcellular fractionation assays were performed to separate the amount of peptides associated within vesicles from those inside the cytosol. Net neutral charged peptides, YGR(6)E(6) and YG(RE)(6), showed large decreases in both surface binding and cell uptake compared to their net positive charged counterparts, YGR(6)G(6) and YG(RG)(6). The peptides with clustered arginine residues, YGR(6)G(6) and YGR(6)E(6), exhibited significantly higher binding and uptake than those with alternating arginine and glycine/glutamate residues, YG(RG)(6) and YG(RE)(6). The intracellular distribution analysis for all of the peptides tested showed that, regardless of the net uptake, the arginine-rich peptides were preferentially localized in the cytosolic compartment of the cells. Both net positive charge and a clustered arginine sequence enhance the surface binding and internalization of peptides; however, the cytosolic versus vesicular intracellular distribution does not change. The results presented in this report provide important information regarding the specificity of binding and internalization of arginine-rich peptides, which is necessary for the future design of targeted drug delivery systems.
    Journal of Drug Targeting 12/2010; 19(8):675-80. DOI:10.3109/1061186X.2010.531729 · 2.74 Impact Factor
Show more

Similar Publications

Preview (2 Sources)

16 Reads
Available from