Tracking Cholesterol/Sphingomyelin-Rich Membrane Domains with the Ostreolysin A-mCherry Protein

Abstract

Ostreolysin A (OlyA) is an ∼15-kDa protein that has been shown to bind selectively to membranes rich in cholesterol and sphingomyelin. In this study, we investigated whether OlyA fluorescently tagged at the C-terminal with mCherry (OlyA-mCherry) labels cholesterol/sphingomyelin domains in artificial membrane systems and in membranes of Madin-Darby canine kidney (MDCK) epithelial cells. OlyA-mCherry showed similar lipid binding characteristics to non-tagged OlyA. OlyA-mCherry also stained cholesterol/sphingomyelin domains in the plasma membranes of both fixed and living MDCK cells, and in the living cells, this staining was abolished by pretreatment with either methyl-β-cyclodextrin or sphingomyelinase. Double labelling of MDCK cells with OlyA-mCherry and the sphingomyelin-specific markers equinatoxin II–Alexa488 and GST-lysenin, the cholera toxin B subunit as a probe that binds to the ganglioside GM1, or the cholesterol-specific D4 domain of perfringolysin O fused with EGFP, showed different patterns of binding and distribution of OlyA-mCherry in comparison with these other proteins. Furthermore, we show that OlyA-mCherry is internalised in living MDCK cells, and within 90 min it reaches the juxtanuclear region via caveolin-1–positive structures. No binding to membranes could be seen when OlyA-mCherry was expressed in MDCK cells. Altogether, these data clearly indicate that OlyA-mCherry is a promising tool for labelling a distinct pool of cholesterol/sphingomyelin membrane domains in living and fixed cells, and for following these domains when they are apparently internalised by the cell.

Fungal aegerolysin-like proteins: distribution, activities, and applications

“…PlyA2 co-localized with the raft marker CD59 and with a late endosomal marker, but not with the transferrin receptor or with markers of early endosomes or the Golgi apparatus. Similarly, the mode of OlyA-mCherry binding to the plasma membrane was compared to the binding patterns of natural toxin analogs that specifically target raftassociated lipids (Skočaj et al , 2014. These data clearly showed that OlyA-mCherry labels different pools of lipids compared to other toxins that bind to components of membrane rafts.…”

“…3) (Skočaj et al 2014). Similarly, another almost identical aegerolysin, pleurotolysin A2 (PlyA2), which has also been shown to bind to sphingomyelin-and cholesterolrich membranes, was fused with enhanced green fluorescent protein (EGFP) and used to label fixed HeLa cells (Bhat et al 2013).…”

“…These data clearly showed that OlyA-mCherry labels different pools of lipids compared to other toxins that bind to components of membrane rafts. OlyA-mCherry was also endocytosed when incubated with living Madin-Darby canine kidney cells, and the internalization pathway was followed (Skočaj et al 2014). The protein entered the cell mainly by the raft-dependent caveolin-1 pathway.…”

“…OlyA-mCherry and PlyA2-EGFP have demonstrated lipid rafts in cells, and therefore these nontoxic proteins show good potential to be used as versatile tools to track membrane rafts in living cells, as well as providing tools to investigate their overall biological roles in healthy and abnormal tissues or cells. Moreover, OlyA was suggested to be used as a vehicle to direct other proteins of choice into the endosomal recycling route upon fusion with OlyA (Skočaj et al 2014). …”

Ostreolysin A/Pleurotolysin B and Equinatoxins: Structure, Function and Pathophysiological Effects of These Pore-Forming Proteins

“…Selectivity of OlyA/PlyB is based on the cholesterol and cholesterol/sphingomyelin-rich membrane domains in urothelial cancer cells, in contrast to normal urothelial cells. Fluorescent-labelled OlyA-mCherry has been utilised as a highly specific probe to visualise cholesterol/sphingomyelin rich membrane microdomains in living and fixed cells, and to study membrane trafficking [31].…”