title: Characterisation of plasmalemmal shedding of vesicles induced by the cholesterol/sphingomyelin binding protein, ostreolysin A-mCherry.
abstract: Ostreolysin A (OlyA) is a 15-kDa protein that binds selectively to cholesterol/sphingomyelin membrane nanodomains. This binding induces the production of extracellular vesicles (EVs) that comprise both microvesicles with diameters between 100nm and 1μm, and larger vesicles of around 10-μm diameter in Madin-Darby canine kidney cells. In this study, we show that vesiculation of these cells by the fluorescent fusion protein OlyA-mCherry is not affected by temperature, is not mediated via intracellular Ca2+signalling, and does not compromise cell viability and ultrastructure. Seventy-one proteins that are mostly of cytosolic and nuclear origin were detected in these shed EVs using mass spectroscopy. In the cells and EVs, 218 and 84 lipid species were identified, respectively, and the EVs were significantly enriched in lysophosphatidylcholines and cholesterol. Our collected data suggest that OlyA-mCherry binding to cholesterol/sphingomyelin membrane nanodomains induces specific lipid sorting into discrete patches, which promotes plasmalemmal blebbing and EV shedding from the cells. We hypothesize that these effects are accounted for by changes of local membrane curvature upon the OlyA-mCherry-plasmalemma interaction. We suggest that the shed EVs are a potentially interesting model for biophysical and biochemical studies of cell membranes, and larger vesicles could represent tools for non-invasive sampling of cytosolic proteins from cells and thus metabolic fingerprinting.
authors: Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, Ljubljana, Slovenia; Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, Ljubljana, Slovenia. Electronic address: matej.skocaj@mf.uni-lj.si.
authors: Matej
authors: M
authors: Skočaj
authors: Bioorganic Chemistry Laboratory, Department of Physics, Via Sommarive 14, University of Trento, Povo (TN), Italy. Electronic address: bx07yy8@gmail.com.
authors: Yang
authors: Y
authors: Yu
authors: Institute of Biophysics, Faculty of Medicine, Vrazov trg 2, University of Ljubljana, Ljubljana, Slovenia. Electronic address: majagrund@yahoo.com.
authors: Maja
authors: M
authors: Grundner
authors: Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, Ljubljana, Slovenia. Electronic address: natasa.resnik@mf.uni-lj.si.
authors: Nataša
authors: N
authors: Resnik
authors: Laboratory of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, Ljubljana, Slovenia. Electronic address: polona.bedina@ki.si.
authors: Apolonija
authors: A
authors: Bedina Zavec
authors: Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Jamova 39, Ljubljana, Slovenia. Electronic address: adrijana.leonardi@ijs.si.
authors: Adrijana
authors: A
authors: Leonardi
authors: Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Jamova 39, Ljubljana, Slovenia; Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, Večna pot 113, University of Ljubljana, Ljubljana, Slovenia. Electronic address: igor.krizaj@ijs.si.
authors: Igor
authors: I
authors: Križaj
authors: Bioorganic Chemistry Laboratory, Department of Physics, Via Sommarive 14, University of Trento, Povo (TN), Italy. Electronic address: guella@science.unitn.it.
authors: Graziano
authors: G
authors: Guella
authors: Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, Ljubljana, Slovenia. Electronic address: peter.macek@bf.uni-lj.si.
authors: Peter
authors: P
authors: Maček
authors: Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, Ljubljana, Slovenia. Electronic address: mateja.erdani@mf.uni-lj.si.
authors: Mateja Erdani
authors: ME
authors: Kreft
authors: Institute of Physiology, Pharmacology and Toxicology, Veterinary Faculty, Gerbičeva 60, University of Ljubljana, Ljubljana, Slovenia. Electronic address: robert.frangez@vf.uni-lj.si.
authors: Robert
authors: R
authors: Frangež
authors: Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, Ljubljana, Slovenia. Electronic address: peter.veranic@mf.uni-lj.si.
authors: Peter
authors: P
authors: Veranič
authors: Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, Ljubljana, Slovenia. Electronic address: kristina.sepcic@bf.uni-lj.si.
authors: Kristina
authors: K
authors: Sepčić
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D055252:Cell-Derived Microparticles / Q000737:chemistry* / Q000187:drug effects; D002784:Cholesterol / Q000737:chemistry / Q000302:isolation & purification; D004285:Dogs; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D017136:Ion Transport; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D008244:Lysophosphatidylcholines / Q000737:chemistry / Q000302:isolation & purification; D061985:Madin Darby Canine Kidney Cells; D055432:Metabolomics; D010196:Pancreatic Elastase / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D013109:Sphingomyelins / Q000737:chemistry / Q000302:isolation & purification; D000097573:Red Fluorescent Protein
chemical_list: D002118:Calcium; D002352:Carrier Proteins; D002784:Cholesterol; D005656:Fungal Proteins; D006460:Hemolysin Proteins; D008164:Luminescent Proteins; D008244:Lysophosphatidylcholines; D010196:Pancreatic Elastase; D011993:Recombinant Fusion Proteins; D013109:Sphingomyelins; D000097573:Red Fluorescent Protein; C461280:ostreolysin; C017130:cholesterol-binding protein
keywords: Aegerolysin proteins; Extracellular vesicles; Ostreolysin A; Vesiculation