Single-Vesicle Detection and Analysis of Peptide-Induced Membrane Permeabilization

Abstract

The capability of membrane-active peptides to disrupt phospholipid membranes is often studied by investigating peptide-induced leakage of quenched fluorescent molecules from large unilamellar lipid vesicles. In this article, we explore two fluorescence microscopy-based single-vesicle detection methods as alternatives to the quenching-based assays for studying peptide-induced leakage from large unilamellar lipid vesicles. Specifically, we use fluorescence correlation spectroscopy (FCS) to study the leakage of fluorescent molecules of different sizes from large unilamellar lipid vesicles dispersed in aqueous solution, and we use confocal imaging of surface-immobilized large unilamellar lipid vesicles to investigate whether there are heterogeneities in leakage between individual vesicles. Of importance, we design an experimental protocol that allows us to quantitatively correlate the results of the two methods; accordingly, it can be assumed that the two methods provide complementary information about the same leakage process. We use the two methods to investigate the membrane-permeabilizing activities of three well-studied cationic membrane-active peptides: mastoparan X, melittin, and magainin 2. The FCS results show that leakage induced by magainin 2 is less dependent on the size of the encapsulated fluorescent molecules than leakage induced by mastoparan X and melittin. The confocal imaging results show that all three peptides induce leakage by a heterogeneous process in which one portion of the vesicles are completely emptied of their contents but another portion of the vesicles are only partially emptied. These pieces of information regarding leakage induced by mastoparan X, melittin, and magainin 2 could not readily have been obtained by the established assays for studying peptide-induced leakage from lipid vesicles.

Site-Specific Peptide Probes Detect Buried Water in a Lipid Membrane

“…Altogether, these site-specific peptide probes enabled us to view water penetration into the hydrophobic region of the bilayer, which may be otherwise thermodynamically unfavorable in peptide-free bilayers. Previous literature has observed negligible water presence beyond the ester carbonyl in pure lipid membranes (75)(76)(77) and has also suggested that embedded amino acids and peptides may facilitate water diffusion across lipid bilayers and into the alkyl bulk (6,(78)(79)(80)(81)(82). Our results are consistent with this literature; however, additional methods beyond the scope of this study are required to rule out potential peptide multimers in the membrane.…”

Lipid vesicle composition influences the incorporation and fluorescence properties of the lipophilic sulphonated carbocyanine dye SP-DiO

“…20 This dye, as well as other members of the carbocyanine dye family, have been widely used to label cell membranes, [21][22][23] viruses, 9,10,24 exosomes 12,25,26 and synthetic liposomes. [27][28][29][30][31] From a structural point of view, lipophilic carbocyanine dyes, are symmetric molecules consisting of two aromatic cyanine moieties joined by three methine groups, which together constitute the fluorophore part of the molecule. Furthermore, to introduce lipophilicity, alkyl chains are conjugated to each cyanine moiety as depicted in Fig.…”

Stopping Membrane-Enveloped Viruses with Nanotechnology Strategies: Toward Antiviral Drug Development and Pandemic Preparedness

“…An alternative strategy of pretreating dye-loaded vesicles with a pore-forming antimicrobial peptide before immobilizing the vesicles onto a surface for confocal imaging has also been utilized to characterize three peptides, mastoparan X, melittin, and magainin 2, while focusing on the percentage of fully dyeemptied vesicles and the average percentage of dye release per vesicle after the membrane−peptide interaction was completed. 146 To date, the reported tethering strategies have focused on synthetic lipid vesicles, while it would be advantageous to further develop labeling and tethering strategies to attach authentic, enveloped virus particles to surfaces. From a biosensing perspective, the transition from virus-mimicking model membrane systems to actual virus particles would represent a major advance to directly understand how membrane-disruptive compounds affect enveloped virus particles.…”

“…Using this approach, it was discovered that antiviral peptides can disrupt not only individually tethered vesicles but also clusters of tethered vesicles. An alternative strategy of pretreating dye-loaded vesicles with a pore-forming antimicrobial peptide before immobilizing the vesicles onto a surface for confocal imaging has also been utilized to characterize three peptides, mastoparan X, melittin, and magainin 2, while focusing on the percentage of fully dye-emptied vesicles and the average percentage of dye release per vesicle after the membrane–peptide interaction was completed …”