Jahangir Md. Alam

Entry of cell-penetrating peptide transportan 10 into a single vesicle by translocating across lipid membrane and its induced pores.

The cell-penetrating peptide, transportan 10 (TP10), can translocate across the plasma membrane of living cells and thus can be used for the intracellular delivery of biological cargo such as proteins. However, the mechanisms underlying its translocation and the delivery of large cargo remain unclear. In this report we investigated the entry of TP10 into a single giant unilamellar vesicle (GUV) and the TP10-induced leakage of fluorescent probes using the single GUV method. GUVs of 20% dioleoylphosphatidylglycerol (DOPG)/80% dioleoylphosphatidylcholine (DOPC) were prepared, and they contained a water-soluble fluorescent dye, Alexa Fluor 647 hydrazide (AF647), and smaller vesicles composed of 20% DOPG/80% DOPC. The interaction of carboxyfluorescein (CF)-labeled TP10 (CF-TP10) with these loaded GUVs was investigated using confocal microscopy. The fluorescence intensity of the GUV membrane increased with time to a saturated value, then the fluorescence intensity due to the membranes of the smaller vesicles inside the GUV increased prior to leakage of AF647. This result indicates that CF-TP10 entered the GUV from the outside by translocating across the lipid membrane before CF-TP10-induced pore formation. The rate constant of TP10-induced pore formation in lipid membranes increased with an increase in TP10 concentration. Large molecules such as Texas Red Dextran 40,000, and vesicles with a diameter of 1-2 μm, permeated through the TP10-induced pores or local rupture in the lipid membrane. These results provide the first direct experimental evidence that TP10 can deliver large cargo through lipid membranes, without the need for special transport mechanisms such as those found in cells.

Stretch-Activated Pore of the Antimicrobial Peptide, Magainin 2

Antimicrobial peptide magainin 2 forms pores in lipid membranes and induces membrane permeation of the cellular contents. Although this permeation is likely the main cause of its bactericidal activity, the mechanism of pore formation remains poorly understood. We therefore investigated in detail the interaction of magainin 2 with lipid membranes using single giant unilamellar vesicles (GUVs). The binding of magainin 2 to the lipid membrane of GUVs increased the fractional change in the area of the membrane, δ, which was proportional to the surface concentration of magainin 2, X. This indicates that the rate constant of the magainin 2-induced two-state transition from the intact state to the pore state greatly increased with an increase in δ. The tension of a lipid membrane following aspiration of a GUV also activated magainin 2-induced pore formation. To reveal the location of magainin 2, the interaction of carboxyfluorescein (CF)-labeled magainin 2 (CF-magainin 2) with single GUVs containing a water-soluble fluorescent probe, AF647, was investigated using confocal microscopy. In the absence of tension due to aspiration, after the interaction of magainin 2 the fluorescence intensity of the GUV rim due to CF-magainin 2 increased rapidly to a steady value, which remained constant for a long time, and at 4-32 s before the start of leakage of AF647 the rim intensity began to increase rapidly to another steady value. In contrast, in the presence of the tension, no increase in rim intensity just before the start of leakage was observed. These results indicate that magainin 2 cannot translocate from the outer to the inner monolayer until just before pore formation. Based on these results, we conclude that a magainin 2-induced pore is a stretch-activated pore and the stretch of the inner monolayer is a main driving force of the pore formation.

The Single-Giant Unilamellar Vesicle Method Reveals Lysenin -Induced Pore Formation in Lipid Membranes Containing Sphingomyelin

Lysenin is a sphingomyelin (SM)-binding pore-forming toxin. To reveal the interaction of lysenin with lipid membranes, we investigated lysenin-induced membrane permeation of a fluorescent probe, calcein, through dioleoylphosphatidylcholine(DOPC)/SM, DOPC/SM/cholesterol(chol), and SM/chol membranes, using the single-giant unilamellar vesicle (GUV) method. The results clearly show that lysenin formed pores in all the membranes, through which membrane permeation of calcein occurred without disruption of GUVs. The membrane permeation began stochastically, and the membrane permeability coefficient increased over time to reach a maximum, steady value, Ps, which persisted for a long time(100--500 s), indicating that the pore concentration increases over time and finally reaches its steady value, NP s . The Ps values increased as the SM/lysenin ratio decreased, and at low concentrations of lysenin, the Ps values of SM/DOPC/chol (42/30/28)GUVs were much larger than those of SM/DOPC (58/42) GUVs. The dependence of Ps on the SM/lysenin ratio for these membranes was almost the same as that of the fraction of sodium dodecyl sulfate (SDS)-resistant lysenin oligomers, indicating that NP s increases as the SDS-resistant oligomer fraction increases. On the other hand, lysenin formed pores in GUVs of SM/chol(60/40) membrane, which is in a homogeneous liquid-ordered phase, indicating that the phase boundary is not necessary for pore formation. The Ps values of SM/chol (60/40) GUVs were smaller than those of SM/DOPC/chol (42/30/28) GUVs even though the SDS-resistant oligomer fractions were similar for both membranes, suggesting that not all of the oligomers can convert into a pore. On the basis of these results, we discuss the elementary processes of lysenin-induced pore formation.

Spontaneous insertion of lipopolysaccharide into lipid membranes from aqueous solution

Lipopolysaccharide (LPS), one of the main components of outer membranes of Gram-negative bacteria, consists of a hydrophobic lipid (lipid A) with six hydrocarbon chains and a large hydrophilic polysaccharide chain. LPS plays endotoxic roles and can stimulate macrophages and B cells. To elucidate the mechanism of the interaction of LPS with various cell membranes, it is important to investigate the interaction of wild type LPS in a buffer with lipid membranes. In this report we investigated the interaction of low concentrations of LPS in a buffer with giant unilamellar vesicles (GUVs) of dioleoylphosphatidylcholine (DOPC) membrane in the liquid-crystalline (L(α)) phase and sphingomyelin (SM)/cholesterol(chol) (molar ration; 6/4) membrane in the liquid-ordered (lo) phase. We found that low concentrations (less than critical micelle concentration) of LPS in aqueous solution induced the shape changes such as the transformation from a prolate to a two-spheres-connected by a very narrow neck in the DOPC-GUVs and also in the SM/chol (6/4)-GUVs above their threshold concentrations. The analysis of the shape changes of the GUVs indicates that the monomers of LPS can insert spontaneously into the external monolayer of the lipid membranes of these GUVs from the aqueous solution. Moreover, higher concentrations of LPS induced the vesicle fission of SM/chol(6/4)-GUVs above its higher threshold concentration. The vesicle fission of GUVs is similar to those induced by single long chain amphiphiles such as lysophosphatidylcholine. On the basis of these results, we discuss the interaction of wild type LPS with lipid membranes and cell membranes. These results suggest that LPS molecules can insert spontaneously into the external monolayer of the plasma membranes composed of the L(α) phase-membrane and the microdomain in the lo phase.

Initial Step of pH-Jump-Induced Lamellar to Bicontinuous Cubic Phase Transition in Dioleoylphosphatidylserine/Monoolein

Electrostatic interactions (EI) are an important factor for phase transitions between lamellar liquid-crystalline (L(α)) and inverse bicontinuous cubic (Q(II)) phases. We investigated the low pH-induced L(α) to double-diamond cubic (Q(II)(D)) phase transition in dioleoylphosphatidylserine (DOPS)/monoolein (MO) using time-resolved small-angle X-ray scattering. Using a stopped-flow apparatus, a suspension of liposomes (multilamellar vesicles (MLVs) or large unilamellar vesicles (LUVs)) of 20%-DOPS/80%-MO membrane at neutral pH was rapidly mixed with a low pH buffer, and then the structural change of the membranes in the resultant suspension was observed as a function of time (i.e., pH-jump experiment). At the initial step, the L(α) phase was directly transformed into the hexagonal II (H(II)) phase, and subsequently, the H(II) phase slowly converted into the Q(II)(D) phase. We obtained the rate constants of the initial step (i.e., the L(α) to H(II) phase transition) and of the second step (i.e., the H(II) to Q(II)(D) phase transition) using the non-negative matrix factorization method. The rate constant of the initial step was independent of the MLV concentration, indicating that single MLVs can convert into the HII phase without any interaction with other MLVs. On the other hand, the rate constant of the initial step increased with a decrease in pH, 0.041 s(-1) at pH 2.6 and 0.013 s(-1) at pH 2.8, and also exhibited a size dependence; for smaller vesicles such as LUVs and smaller MLVs with diameters of ~1 μm, the rate constant was smaller. They were reasonably explained by the classical nucleation theory. These results provide the first experimental evidence of the total kinetics of EI-induced L(α)/Q(II) phase transitions.

Activation Energy of the Low-pH-Induced Lamellar to Bicontinuous Cubic Phase Transition in Dioleoylphosphatidylserine/Monoolein

Electrostatic interaction is an important factor for phase transitions between lamellar liquid-crystalline (Lα) and inverse bicontinuous cubic (QII) phases. We investigated the effect of temperature on the low-pH-induced Lα to double-diamond cubic (QII(D)) phase transition in dioleoylphosphatidylserine (DOPS)/monoolein (MO) using time-resolved small-angle X-ray scattering with a stopped-flow apparatus. Under all conditions of temperature and pH, the Lα phase was directly transformed into an intermediate inverse hexagonal (HII) phase, and subsequently the HII phase slowly converted to the QII(D) phase. We obtained the rate constants of the initial step (i.e., the Lα to HII phase transition) and of the second step (i.e., the HII to QII(D) phase transition) using the non-negative matrix factorization method. The rate constant of the initial step increased with temperature. By analyzing this result, we obtained the values of its apparent activation energy, Ea (Lα → HII), which did not change with temperature but increased with an increase in pH. In contrast, the rate constant of the second step decreased with temperature at pH 2.6, although it increased with temperature at pH 2.7 and 2.8. These results indicate that the value of Ea (HII → QII(D)) at pH 2.6 increased with temperature, but the values of Ea (HII → QII(D)) at pH 2.7 and 2.8 were constant with temperature. The values of Ea (HII → QII(D)) were smaller than those of Ea (Lα → HII) at the same pH. We analyzed these results using a modified quantitative theory on the activation energy of phase transitions of lipid membranes proposed initially by Squires et al. (Squires, A. M.; Conn, C. E.; Seddon, J. M.; Templer, R. H. Soft Matter 2009, 5, 4773). On the basis of these results, we discuss the mechanism of this phase transition.

Elementary processes of antimicrobial peptide PGLa-induced pore formation in lipid bilayers

Antimicrobial peptide PGLa induces the leakage of intracellular content, leading to its bactericidal activity. However, the elementary process of PGLa-induced leakage remains poorly understood. Here, we examined the interaction of PGLa with lipid bilayers using the single giant unilamellar vesicle (GUV) method. We found that PGLa induced membrane permeation of calcein from GUVs comprised of dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylglycerol (DOPG) and its rate increased with time to reach a steady value, indicating that PGLa induced pores in the bilayer. The binding of PGLa to the GUV membrane raised its fractional area change, δ. At high PGLa concentrations, the time course of δ showed a two-step increase; δ increased to a value, δ1, which was constant for an extended period before increasing to another constant value, δ2, that persisted until aspiration of the GUV. To reveal the distribution of PGLa, we investigated the interaction of a mixture of PGLa and carboxyfluorescein (CF) -labeled PGLa (CF-PGLa) with single GUVs. The change of the fluorescence intensity of the GUV rim, I, over time showed a two-step increase from a steady value, I1, to another, I2, concomitant with the entering of CF-PGLa into the lumen of the GUV prior to AF647 leakage. The simultaneous measurement of δ and I indicated that their time courses were virtually the same and the ratios (δ2/δ1 and I2/I1) were almost 2. These results indicated that CF-PGLa translocated across the bilayer before membrane permeation. Based on these results, the elementary processes of the PGLa-induced pore formation were discussed.