Nanoscale Visualization of Morphological Alteration of Live-Cell Membranes by the Interaction with Oligoarginine Cell-Penetrating Peptides.

The interactions between the cell membrane and biomolecules remain poorly understood. For example, arginine-rich cell-penetrating peptides (CPPs), including octaarginines (R8), are internalized by interactions with cell membranes. However, during the internalization process, the exact membrane dynamics introduced by these CPPs are still unknown. Here, we visualize arginine-rich CPPs and cell-membrane interaction-induced morphological changes using a system that combines scanning ion-conductance microscopy and spinning-disk confocal microscopy, using fluorescently labeled R8. This system allows time-dependent, nanoscale visualization of structural dynamics in live-cell membranes. Various types of membrane remodeling caused by arginine-rich CPPs are thus observed. The induction of membrane ruffling and the cup closure are observed as a process of endocytic uptake of the peptide. Alternatively suggested is the concave structural formation accompanied by direct peptide translocation through cell membranes. Studies using R8 without fluorescent labeling also demonstrate a non-negligible effect of the fluorescent moiety on membrane structural alteration.

An influenza-derived membrane tension-modulating peptide regulates cell movement and morphology via actin remodeling.

Tension in cell membranes is closely related to various cellular events, including cell movement and morphogenesis. Therefore, modulation of membrane tension can be a new approach for manipulating cellular events. Here, we show that an amphipathic peptide derived from the influenza M2 protein (M2[45-62]) yields lamellipodia at multiple sites in the cell. Effect of M2[45-62] on cell membrane tension was evaluated by optical tweezer. The membrane tension sensor protein FBP17 was involved in M2[45-62]-driven lamellipodium formation. Lysine-to-arginine substitution in M2[45-62] further enhanced its activity of lamellipodium formation. M2[45-62] had an ability to reduce cell motility, evaluated by scratch wound migration and transwell migration assays. An increase in neurite outgrowth was also observed after treatment with M2[45-62]. The above results suggest the potential of M2[45-62] to modulate cell movement and morphology by modulating cell membrane tension.

Loosening of Lipid Packing Promotes Oligoarginine Entry into Cells.

Despite extensive use of arginine-rich cell-penetrating peptides (CPPs)-including octaarginine (R8)-as intracellular delivery vectors, mechanisms for their internalization are still under debate. Lipid packing in live cell membranes was characterized using a polarity-sensitive dye (di-4-ANEPPDHQ), and evaluated in terms of generalized polarization. Treatment with membrane curvature-inducing peptides led to significant loosening of the lipid packing, resulting in an enhanced R8 penetration. Pyrenebutyrate (PyB) is known to facilitate R8 membrane translocation by working as a hydrophobic counteranion. Interestingly, PyB also actively induced membrane curvature and perturbed lipid packing. R8 is known to directly cross cell membranes at elevated concentrations. The sites of R8 influx were found to have looser lipid packing than surrounding areas. Lipid packing loosening is proposed as a key factor that governs the membrane translocation of CPPs.

Calmodulin EF-hand peptides as Ca2+ -switchable recognition tags.

Calmodulin is a representative calcium-binding protein comprised of four Ca2+ -binding motifs with a helix-loop-helix structure (EF-hands). In this study, we clarified the potential of peptide segments derived from the third and fourth EF-hands (EF3 and EF4) to act as recognition tags. Through an analysis of the mode of disulfide formation among cysteines inserted at the N- or C-terminus of these peptide segments, EF3 and EF4 peptides were suggested to form a heterodimer with a topology similar to that in the wild-type protein. Heterodimer formation was shown to be a function of the Ca2+ concentration, suggesting that these structures may be used as Ca2+ -switchable recognition tags. An example of an "EF-tag" system involving the membrane fusion of liposomes decorated with EF3 and EF4 peptides is presented. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2016.

Effect of amino acid substitution in the hydrophobic face of amphiphilic peptides on membrane curvature and perturbation: N-terminal helix derived from adenovirus internal protein VI as a model.

The N-terminal amphipathic helical segment of adenovirus internal protein VI (AdVpVI) plays a critical role in viral infection. Here, we report that the peptide segment corresponding to AdVpVI (positions 33-55) can induce positive membrane curvature together with membrane perturbation. The enhanced perturbation ability of the peptide was observed for membranes containing negatively charged phospholipids. Based on the liposome leakage assay, substitution of leucine at position 40 to other aliphatic (isoleucine) and aromatic (phenylalanine and tryptophan) residues yielded a similar degree of membrane perturbation by the peptides, which was considerably diminished by the substitution to glutamine. Further studies using the wild-type AdVpVI (33-55) (WT) and phenylalanine-substituted peptides (L40F) demonstrated that both peptides have positive membrane-curvature-inducing ability. These peptides showed higher binding affinity to 50-nm large unilamellar vesicles (LUVs) than to 200-nm LUVs. However, no enhanced perturbation by these peptides was observed for 50-nm LUVs compared to 200-nm LUVs, suggesting that both the original membrane curvature and the additional strain due to peptide insertion affect the membrane perturbation ability of these peptides. In the case of L40F, this peptide rather had a lower membrane perturbation ability for 50-nm LUVs than for 200-nm LUVs, which can be attributed to possible shallower binding of L40F on membranes. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 430-439, 2016.

Curvature engineering: positive membrane curvature induced by epsin N-terminal peptide boosts internalization of octaarginine.

Epsin-1 is a representative protein for inducing the positive curvature necessary for the formation of clathrin-coated pits. Here we demonstrate that the N-terminus 18-residue peptide of epsin-1 (EpN18) has this ability per se, as proved by differential scanning calorimetry (DSC) and solid-state NMR. Moreover, it is shown how this positive curvature promotion can be exploited for promoting the direct penetration of a representative cell-penetrating peptide (CPP), octaarginine (R8), through artificial and plasma membranes. This synergistic effect has been used for the efficient delivery of a proapoptotic domain peptide (PAD), which induced high level of apoptosis only when coadministered with R8 and EpN18, thus emphasizing the importance of positive curvature induction for achieving the desired ultimate cargo bioavailability.

Effects of pyrenebutyrate on the translocation of arginine-rich cell-penetrating peptides through artificial membranes: recruiting peptides to the membranes, dissipating liquid-ordered phases, and inducing curvature.

Arginine-rich cell-penetrating peptides, including octaarginine (R8) and HIV-1 TAT peptides, have the ability to translocate through cell membranes and transport exogenous bioactive molecules into cells. Hydrophobic counteranions such as pyrenebutyrate (PyB) have been reported to markedly promote the membrane translocation of these peptides. In this study, using model membranes having liquid-ordered (Lo) and liquid-disordered (Ld) phases, we explored the effects of PyB on the promotion of R8 translocation. Confocal microscopic observations of giant unilamellar vesicles (GUVs) showed that PyB significantly accelerated the accumulation of R8 on membranes containing negatively charged lipids, leading to the internalization of R8 without collapse of the GUV structures. PyB displayed an alternative activity, increasing the fluidity of the negatively charged membranes, which diminished the distinct Lo/Ld phase separation on GUVs. This was supported by the decrease in fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH). Additionally, PyB induced membrane curvature, which has been suggested as a possible mechanism of membrane translocation for R8. Taken together, our results indicate that PyB may have multiple effects that promote R8 translocation through cell membranes.