Effects of Mechanical Properties of Lipid Bilayers on the Entry of Cell-Penetrating Peptides into Single Vesicles

Abstract

The translocation of cell-penetrating peptides (CPPs) through plasma membranes of living cells is an important physiological phenomenon in biomembranes. To reveal the mechanism underlying the translocation of a CPP, transportan 10 (TP10), through lipid bilayers, we examined the effects of the mechanical properties of lipid bilayers on the entry of carboxyfluorescein (CF)-labeled TP10 (CF-TP10) into a giant unilamellar vesicle (GUV) using the single GUV method. First, we examined the effect of lateral tension in membranes on the entry of CF-TP10 into single GUVs comprising a mixture of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) (2/8). CF-TP10 entered the GUV lumen before the membrane permeation of Alexa Fluor 647 hydrazide (AF647) from the GUV and thus before pore formation in the membrane. The fraction of entry of CF-TP10 before pore formation and the rate of membrane rupture increased with tension. The CF-TP10-induced fractional change in the membrane area increased continuously with time until membrane rupture, but it increased more slowly than did the CF-TP10 concentration in the GUV membrane. A high mole fraction of cholesterol inhibited the entry of CF-TP10 into single GUVs by suppressing the translocation of CF-TP10 from the external to the internal monolayer, although higher concentrations of CF-TP10 induced the formation of pores through which CF-TP10 rapidly translocated. Suppression of the translocation of CF-TP10 by cholesterol can be reasonably explained by the large line tension of a prepore. We discussed the role of mechanical properties in membranes on the entry of CF-TP10 into single GUVs and proposed a hypothesis of the mechanism that CF-TP10 translocates across a bilayer through transient hydrophilic prepores in the membrane.

To close or to collapse: the role of charges on membrane stability upon pore formation

“…Although it is not clear why charged membranes containing lipids with two unsaturated tails are more prone to disruption, this may be related to the increase in membrane fluctuations due to their lower bending rigidity. High-amplitude fluctuations were shown to increase the propensity to membrane rarefactions (prepores) that may eventually evolve into a large pore due to a lower activation energy of pore formation (75)(76)(77).…”

Action of antimicrobial peptides and cell-penetrating peptides on membrane potential revealed by the single GUV method

“…For the entry of CPPs into GUV lumen, the binding of fluorescent dye-labeled CPPs to the outer leaflet of the GUV membrane from aqueous solution, their translocation from the outer leaflet to the inner one of the membrane, and the unbinding of CPPs from the inner leaflet to the aqueous solution in the GUV lumen can be separated and their rates are estimated ( Fig. 1b) (Islam et al 2014a(Islam et al , 2017Sharmin et al 2016;Moghal et al 2018). Simultaneous measurement of entry of CPPs into the GUV lumen and the membrane permeation of internal contents using confocal laser scanning microscopy (CLSM) can reveal the relationship between the entry of CPPs and the CPPcaused pore formation (Islam et al 2014a(Islam et al , 2017Moghal et al 2018).…”

“…1b) (Islam et al 2014a(Islam et al , 2017Sharmin et al 2016;Moghal et al 2018). Simultaneous measurement of entry of CPPs into the GUV lumen and the membrane permeation of internal contents using confocal laser scanning microscopy (CLSM) can reveal the relationship between the entry of CPPs and the CPPcaused pore formation (Islam et al 2014a(Islam et al , 2017Moghal et al 2018). The time course of change in fluorescence intensity (FI) of a GUV membrane (i.e., rim intensity) due to fluorescent dye-labeled CPPs provides information on time course change in peptide concentration in the GUV membrane, which has revealed the time course of translocation of peptides across lipid bilayer (Islam et al 2014a(Islam et al , 2017Karal et al 2015).…”

“…Simultaneous measurement of entry of CPPs into the GUV lumen and the membrane permeation of internal contents using confocal laser scanning microscopy (CLSM) can reveal the relationship between the entry of CPPs and the CPPcaused pore formation (Islam et al 2014a(Islam et al , 2017Moghal et al 2018). The time course of change in fluorescence intensity (FI) of a GUV membrane (i.e., rim intensity) due to fluorescent dye-labeled CPPs provides information on time course change in peptide concentration in the GUV membrane, which has revealed the time course of translocation of peptides across lipid bilayer (Islam et al 2014a(Islam et al , 2017Karal et al 2015). On the other hand, the membrane tension in single GUVs can be accurately controlled by the micropipette aspiration method and the osmotic pressure, and thus, we can elucidate the effect of membrane tension on the action of AMPs and CPPs (Karal et al 2015;Shibly et al 2016;Islam et al 2017;Hasan et al 2018.…”

Recent Advances in Origins of Life Research by Biophysicists in Japan

“…The lab of Masahito Yamazaki in Shizuoka University has been researching cell penetrating peptides into vesicles and actual cells [96]. In certain cases, including when studying the 21-mer antimicrobial cationic peptide, PGLa [97], or the 21-mer cell penetrating peptide, TP10 [98], they discovered that in both cases, the peptide induces pore formation in mixed dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) phospholipid vesicle membranes, causing leakage of the internal components, which when applied to real bacterial cells, results in cell death. The result is translocation of the peptide into the interior of the vesicle.…”