Force Mapping Study of Actinoporin Effect in Membranes Presenting Phase Domains.

Katia Cosentino,Joseph D Unsay,Edward Hermann,Uris Ros,Ana J García-Sáez,Nicolai Von Kügelgen

doi:10.3390/toxins13090669

Abstract

Equinatoxin II (EqtII) and Fragaceatoxin C (FraC) are pore-forming toxins (PFTs) from the actinoporin family that have enhanced membrane affinity in the presence of sphingomyelin (SM) and phase coexistence in the membrane. However, little is known about the effect of these proteins on the nanoscopic properties of membrane domains. Here, we used combined confocal microscopy and force mapping by atomic force microscopy to study the effect of EqtII and FraC on the organization of phase-separated phosphatidylcholine/SM/cholesterol membranes. To this aim, we developed a fast, high-throughput processing tool to correlate structural and nano-mechanical information from force mapping. We found that both proteins changed the lipid domain shape. Strikingly, they induced a reduction in the domain area and circularity, suggesting a decrease in the line tension due to a lipid phase height mismatch, which correlated with proteins binding to the domain interfaces. Moreover, force mapping suggested that the proteins affected the mechanical properties at the edge, but not in the bulk, of the domains. This effect could not be revealed by ensemble force spectroscopy measurements supporting the suitability of force mapping to study local membrane topographical and mechanical alterations by membranotropic proteins.

Highlights

Pore-forming toxins (PFTs) are involved in the defence system of many organisms, ranging from bacteria to animals
It exploits the force spectroscopy tool of atomic force microscopy (AFM), which allows for getting information on both the force required to break the bilayer and its thickness [27]
We have developed the “Forcemap Analyser”, a fast, high-throughput and user-friendly analysis tool that generates a spatial map of the acquired force values

Summary

Introduction

Pore-forming toxins (PFTs) are involved in the defence system of many organisms, ranging from bacteria to animals. Equinatoxin II (EqtII) and Fragaceatoxin C (FraC) are PFTs from the family of actinoporins, isolated from the sea anemones Actina equine and Actinia fragacea, respectively [4] These toxins consist of a β-sandwich core skirted by two α-helices on opposite sides [5]. Evidence by single-molecule approaches have revealed that the number of monomeric units composing EqtII oligomers is variable [8], having a broad stoichiometry distribution [8,9] This variability is in agreement with a model in which these α-PFTs form toroidal pores where both proteins and lipids contribute to the channel architecture [7,10]. How membrane phase separation helps the action of actinoporins remains unclear [17,18,19,20], and even less is known about the changes that actinoporins induce in the membrane in order to permeabilize it

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Discussion

Conclusion