Temporary Membrane Permeabilization via the Pore-Forming Toxin Lysenin.

Nisha Shrestha,Devon Richtsmeier,Malyk Walker,Gamid Abatchev,Andrew Bogard,Rebecca Hermann,Raquel J Brown,Daniel Fologea,Christopher A Thomas

doi:10.3390/toxins12050343

Abstract

Pore-forming toxins are alluring tools for delivering biologically-active, impermeable cargoes to intracellular environments by introducing large conductance pathways into cell membranes. However, the lack of regulation often leads to the dissipation of electrical and chemical gradients, which might significantly affect the viability of cells under scrutiny. To mitigate these problems, we explored the use of lysenin channels to reversibly control the barrier function of natural and artificial lipid membrane systems by controlling the lysenin’s transport properties. We employed artificial membranes and electrophysiology measurements in order to identify the influence of labels and media on the lysenin channel’s conductance. Two cell culture models: Jurkat cells in suspension and adherent ATDC5 cells were utilized to demonstrate that lysenin channels may provide temporary cytosol access to membrane non-permeant propidium iodide and phalloidin. Permeability and cell viability were assessed by fluorescence spectroscopy and microscopy. Membrane resealing by chitosan or specific media addition proved to be an effective way of maintaining cellular viability. In addition, we loaded non-permeant dyes into liposomes via lysenin channels by controlling their conducting state with multivalent metal cations. The improved control over membrane permeability might prove fruitful for a large variety of biological or biomedical applications that require only temporary, non-destructive access to the inner environment enclosed by natural and artificial membranes.

Highlights

IntroductionFor well over a century, investigations focused on introducing non-permeant cargoes into cells while preventing irreversible damage or unintentional perturbations of cellular functionalities led to numerous strategies for mediating intracellular transport [1,2,3,4,5,6,7,8,9,10,11,12,13]
Our explorations on the temporary permeabilization of membranes via lysenin channels Our explorations on the temporary permeabilization of membranes via lysenin channels inserted inserted into the membrane of live cells rely on dyes and media more complex than the simple ionic into the membrane of live cells rely on dyes and media more complex than the simple ionic solutions that are traditionally used for assessments performed in artificial membrane systems
Multivalent cations significantly inhibit the macroscopic conductance of lysenin channels [39,40], which affect their capability to create transmembrane conductive pathways that provide access to which affect their capability to create transmembrane conductive pathways that provide access to the the cytosol

Summary

Introduction

For well over a century, investigations focused on introducing non-permeant cargoes into cells while preventing irreversible damage or unintentional perturbations of cellular functionalities led to numerous strategies for mediating intracellular transport [1,2,3,4,5,6,7,8,9,10,11,12,13] With this goal in mind, gaining access to the cytosol by using pore-forming proteins (PFPs) that disrupt the membrane barrier function and introduce conductive pathways that facilitate a diffusion-driven transport of solutes across membranes is a promising new mechanism [8,9,10,14,15]. Ion channels present high transport rate, selectivity, and regulation [17,18,19]; with a few notable exceptions [2], their narrow opening and high selectivity limit their applicability to intracellular delivery of specific, small-sized ions and compounds

Methods

Results

Conclusion