title: 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.
authors.affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Matej
authors.initials: M
authors.lastname: Skočaj
authors.affiliation: Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Nataša
authors.initials: N
authors.lastname: Resnik
authors.affiliation: Institute of Biophysics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Maja
authors.initials: M
authors.lastname: Grundner
authors.affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Katja
authors.initials: K
authors.lastname: Ota
authors.affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Nejc
authors.initials: N
authors.lastname: Rojko
authors.affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Vesna
authors.initials: V
authors.lastname: Hodnik
authors.affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia; National Institute of Chemistry, Ljubljana, Slovenia.
authors.forename: Gregor
authors.initials: G
authors.lastname: Anderluh
authors.affiliation: Nencki Institute of Experimental Biology, Warsaw, Poland.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Sobota
authors.affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Peter
authors.initials: P
authors.lastname: Maček
authors.affiliation: Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Peter
authors.initials: P
authors.lastname: Veranič
authors.affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Kristina
authors.initials: K
authors.lastname: Sepčić
mesh_terms: D000818:Animals; D002784:Cholesterol / Q000378:metabolism*; D004285:Dogs; D005656:Fungal Proteins / Q000494:pharmacology; D006460:Hemolysin Proteins / Q000494:pharmacology*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D061985:Madin Darby Canine Kidney Cells; D021962:Membrane Microdomains / Q000378:metabolism*; D013109:Sphingomyelins / Q000378:metabolism*; D000097573:Red Fluorescent Protein
chemical_list: D002784:Cholesterol; D005656:Fungal Proteins; D006460:Hemolysin Proteins; D008164:Luminescent Proteins; D013109:Sphingomyelins; D000097573:Red Fluorescent Protein; C461280:ostreolysin