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.
Highlights
Biological membranes are composed of thousands of species of proteins and lipids [1]
We investigated whether pre-treatment of living Madin-Darby canine kidney (MDCK) cells for 60 min with a cholesterol-scavenging agent, as 5 mM methyl-b-cyclodextrin (Sigma-Aldrich, USA), or for 30 min and 60 min with 0.5 U/mL sphingomyelinase from Staphylococcus aureus (Sigma-Aldrich USA) influenced the staining of these cells with Ostreolysin A (OlyA)-mCherry
Internalisation of OlyA-mCherry in MDCK cells As the presence of OlyA-mCherry inside MDCK cells was observed at prolonged incubation times (Fig. 6C, D), we further investigated the pathways of OlyA-mCherry internalisation
Summary
Biological membranes are composed of thousands of species of proteins and lipids [1]. As one of the major lipids of the vertebrate plasma membrane, SM is mainly located in the plasmalemma outer leaflet, and it can be recognised by lysenin [19,20], a protein that is secreted through the dorsal pores of the earthworm Eisenia foetida [21], and by equinatoxin II (EqTII), a cytolysin from the sea anemone Actinia equina [22] Both of these toxins have been shown to bind to SM, they interact with distinct membrane pools of this lipid [23]. Binding of OlyA to cholesterol/SM is essential for recruitment of the membrane attack complex/perforin-domaincontaining 59-kDa protein pleurotolysin B (PlyB) onto cholesterol/ SM-rich model lipid membranes and onto cell membranes to form the binary pore-complex that is permeable to solutes [27,31] This specific recognition of cholesterol/SM-enriched membrane domains means that the non-cytolytic OlyA and similar mushroom proteins are potential tools for the detection of cellular raft-like membrane domains. Our data indicate that OlyA can be used to direct a fused protein of choice as a cargo via caveolae into the cell endosomal recycling system
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