Biochimica et Biophysica Acta 1858 (2016) 2882–2893

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Biochimica et Biophysica Acta

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Characterisation of plasmalemmal shedding of vesicles induced by thecholesterol/sphingomyelin binding protein, ostreolysin A-mCherry

Matej Skočaj a,b, Yang Yu c, Maja Grundner d, Nataša Resnik b, Apolonija Bedina Zavec e, Adrijana Leonardi f,Igor Križaj f,g, Graziano Guella c, Peter Maček a, Mateja Erdani Kreft b, Robert Frangež h,Peter Veranič b,⁎, Kristina Sepčić a,⁎⁎a Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, Ljubljana, Sloveniab Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, Ljubljana, Sloveniac Bioorganic Chemistry Laboratory, Department of Physics, Via Sommarive 14, University of Trento, Povo (TN), Italyd Institute of Biophysics, Faculty of Medicine, Vrazov trg 2, University of Ljubljana, Ljubljana, Sloveniae Laboratory of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, Ljubljana, Sloveniaf Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Jamova 39, Ljubljana, Sloveniag Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, Večna pot 113, University of Ljubljana, Ljubljana, Sloveniah Institute of Physiology, Pharmacology and Toxicology, Veterinary Faculty, Gerbičeva 60, University of Ljubljana, Ljubljana, Slovenia

Abbreviations: [Ca2+]i, intracellular Ca2+ concentratOlyA, ostreolysin A;MS, mass spectroscopy; PC, phosphati⁎ Correspondence to: P. Veranič, Institute of Cell Biology

of Ljubljana, Vrazov trg 2, Ljubljana, Slovenia.⁎⁎ Correspondence to: K. Sepčić, Department of Biology,of Ljubljana, Večna pot 111, Ljubljana, Slovenia.

E-mail addresses: matej.skocaj@mf.uni-lj.si (M. Skočajmajagrund@yahoo.com (M. Grundner), natasa.resnik@mfpolona.bedina@ki.si (A. Bedina Zavec), adrijana.leonardi@igor.krizaj@ijs.si (I. Križaj), guella@science.unitn.it (G. Gue(P. Maček), mateja.erdani@mf.uni-lj.si (M.E. Kreft), robert(R. Frangež), peter.veranic@mf.uni-lj.si (P. Veranič), kristi

http://dx.doi.org/10.1016/j.bbamem.2016.08.0150005-2736/© 2016 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 3 March 2016Received in revised form 10 August 2016Accepted 30 August 2016Available online 31 August 2016

Ostreolysin A (OlyA) is a 15-kDa protein that binds selectively to cholesterol/sphingomyelin membranenanodomains. This binding induces the production of extracellular vesicles (EVs) that comprise bothmicrovesicles with diameters between 100 nm and 1 μm, and larger vesicles of around 10-μm diameter inMadin-Darby canine kidney cells. In this study, we show that vesiculation of these cells by the fluorescent fusionprotein OlyA-mCherry is not affected by temperature, is not mediated via intracellular Ca2+ signalling, and doesnot compromise cell viability and ultrastructure. Seventy-one proteins that are mostly of cytosolic and nuclearorigin were detected in these shed EVs using mass spectroscopy. In the cells and EVs, 218 and 84 lipid specieswere identified, respectively, and the EVs were significantly enriched in lysophosphatidylcholines and cholester-ol. Our collected data suggest that OlyA-mCherry binding to cholesterol/sphingomyelinmembrane nanodomainsinduces specific lipid sorting into discrete patches, which promotes plasmalemmal blebbing and EV sheddingfrom the cells. We hypothesize that these effects are accounted for by changes of local membrane curvatureupon the OlyA-mCherry-plasmalemma interaction. We suggest that the shed EVs are a potentially interestingmodel for biophysical and biochemical studies of cell membranes, and larger vesicles could represent tools fornon-invasive sampling of cytosolic proteins from cells and thus metabolic fingerprinting.

© 2016 Elsevier B.V. All rights reserved.

Keywords:Aegerolysin proteinsOstreolysin AExtracellular vesiclesVesiculation

1. Introduction

Aegerolysins (Pfam PF06355) are ~15-kDa proteins that are pro-duced by some evolutionarily distant organisms, including bacteria,fungi and plants. Their exact functions and biological roles are not

ion; EVs, extracellular vesicles;dylcholine; SM, sphingomyelin., Faculty ofMedicine, University

Biotechnical Faculty, University

), bx07yy8@gmail.com (Y. Yu),.uni-lj.si (N. Resnik),ijs.si (A. Leonardi),lla), peter.macek@bf.uni-lj.si.frangez@vf.uni-lj.sina.sepcic@bf.uni-lj.si (K. Sepčić).

clear [1,2]. Some of the fungal aegerolysins have been reported to behaemolytic [3,4], while those from mushrooms of the genus Pleurotuspromote cytolysis as binding components in transmembrane binarypore-forming complexes [5–7]. Binding of these Pleurotus aegerolysinshas been reported to be specific for membranes composed ofsphingomyelin (SM)/sterol and/or ceramide phosphoethanolamine/sterol, and also for ceramide phosphoethanolamine alone [8–11].

Ostreolysin A (OlyA), an aegerolysin from the oyster mushroomPleurotus ostreatus (Jacq.) P. Kumm. (1871), as well as its fluorescentfusion variant OlyA-mCherry, have been shown to bind specific mem-brane domains enriched in cholesterol and SM, which fulfil the criteriafor so-called “membrane rafts” [8,9,12–15]. In concert with pleurotolysinB (PlyB), a 59-kDa protein with a membrane-attack complex/perforindomain, OlyA promotes the formation of transmembrane pores,through recruitment of PlyB to membranes that are rich in cholesteroland SM [6,7,9,16–18]. OlyA alone can bind these membranes, although

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permeabilisation only occurs when OlyA is combined with PlyB. How-ever, OlyA binding to artificial cholesterol/SM (molar ratio, 1:1) largeunilamellar vesicles causes membrane budding and fission, and out-ward vesiculation [9].

When bound to living Madin-Darby kidney (MDCK) cells, OlyA-mCherry is partially internalised via caveolae after ~30 min, and within90min it accumulates in the juxtanuclear region, in caveolin-1–positivestructures. However, when applied to MDCK cells at ≥1 μM, OlyA-mCherry immediately triggers the outward plasmalemmal vesicula-tion [15]. Taken together, these previous studies suggest that in-creased concentrations of OlyA and OlyA-mCherry can induceblebbing and vesiculation of artificial membranes that are rich incholesterol and SM, and of cell membranes containing cholesterol/SMmembrane nanodomains.

Vesiculation of cellular membranes is a constitutive process,although it can also be induced by external physico-chemical factors.Submicrometre-sized, anuclear extracellular vesicles (EVs) are ubiqui-tously produced by prokaryotic and eukaryotic cells, and these are in-volved in a broad range of physiological and pathological processes.Eukaryotic EVs have been classified as: (i) microvesicles/microparticlesthat are produced by outward budding and fission of the plasmalemma;(ii) exosomes that are formed within the endosomal network; and (iii)apoptotic bodies released from cells undergoing apoptosis [19].Microvesicles that are shed from the plasmalemma are 100 nm to~1 μm in diameter, while exosomes of endocytotic origin are 40 nm to100 nm in diameter, and apoptotic bodies that are released as blebsfrom cells undergoing apoptosis can be larger (diameter, 1–5 μm)[19–21].

Eukaryotic microvesicles that are shed from the cell surface areformed from the plasmalemma, and can contain lipids, cytokines,prions, infective particles, proteins, mRNAs, and miRNAs. As these EVscan freely move through the body fluids and can reach distant cells,they can spread inflammation and infection, modulate immune re-sponses, and contribute to progression of several diseases, includingcancers [20–24]. In addition, blebbing and vesiculation of the plasma-lemma can be induced by a variety of exogenous factors, such as expo-sure of cells to oxidising and reducing agents, as well as in situationswhen the cortical cytoskeleton of the cell is constitutively weakened,such as in filamin-deficient melanoma cells [25–30]. Finally, membraneblebbing that includes outward vesiculation was proposed as an impor-tant cell defencemechanism against the complementmembrane-attackcomplex [31], and for removal of sublytic concentrations of differentbacterial toxins [32–35].

In this study, we explored the previously observed process ofblebbing and vesiculation of MDCK cells induced by OlyA-mCherry inmore detail. We analysed the sizes, and protein and lipid compositionsof EVs induced by OlyA-mCherry in living MDCK cells. Furthermore,we examined whether this production of EVs is temperature-dependent, related to increases in intracellular Ca2+ concentration([Ca2+]i), and affects cell viability and ultrastructure. We show herethat vesiculation of MDCK cells induced by OlyA-mCherry is not de-creased by low temperatures and does not lead to increased [Ca2+]i.Our proteomic analysis revealed that these shed EVs contained 71proteins. These were mostly of cytosolic and nuclear origin, andonly a fewwere assigned asmembrane-associated or typicalmembraneraft residents. Furthermore, we identified 218 different lipid species inthese cells, and 84 different lipid species in these EVs. Compared tothe cellular lipids, a reduced number of lipid species was observedin the EVs in terms of: negatively charged glycerophospholipids(e.g., phosphatidylserine, phosphatidylinositol); phosphatidylethanol-amine; ether glycerophospholipids (ether phosphatidylcholine [ether-PC] and ether phosphatidylethanolamine); ceramides; and diglycer-ides. However, glycosphingolipids were not detected in the EVs. In con-trast, there was significant increase in the relative ratio of lysoPC andcholesterol in the EVs. As cell viability and ultrastructure were not af-fected by OlyA-mCherry, and the vesiculation was not temperature-

dependent, we assume that EVs formation is a result of a direct physicaleffect of OlyA-mCherry on the plasmalemma.

2. Materials and methods

2.1. Reagents and materials

2.1.1. ProteinsOlyA, OlyA-mCherry and Δ48PlyB were produced as described by

Ota et al. [9] and Skočaj et al. [15]. GeneBank accession numbers forOlyA and PlyB are KC012711.1 and AB177870.1, respectively. Proteinconcentrations were determined using a microvolume spectrophotom-eter (Nanodrop2000; Thermo Scientific, Hudson, NH, USA). Proteinsizes and purities were determined using SDS-PAGE electrophoresison homogenous 12% acrylamide gels, with the proteins stained withCoomassie blue.

2.1.2. Cell cultureMDCK cells were originally derived from a kidney of a normal cocker

spaniel (NBL-2, ATCC-CCL-34), and theywere obtained fromATCC (Ma-nassas, VA, USA). These cells were recently authenticated and tested forcontamination. They were maintained at 37 °C and 5% CO2, in plasticdishes (TPP Techno Plastic Products, Trasadingen, Switzerland). Theywere subcultured using TrypLE Select (Gibco, Paisley, UK) when 90%to 100% confluence. The growth medium was Advanced Dulbecco'sModified Eagle's Medium (A-DMEM)/Nutrient Mixture F-12 (1:1),10% fetal calf serum, 50 U mL−1 crystacylin (Pliva, Zagreb, Croatia),and 50 U mL−1 streptofatol (Fatol, Griefswald, Germany). Culturemedia and supplements were purchased from Invitrogen (Life Technol-ogies, Grand Island, NY, USA), unless otherwise stated.

2.2. Methods

2.2.1. Cell viability assayCell viability was measured using the colorimetric CellTiter 96 AQue-

ous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA),which uses 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). This is based on the conversionof the tetrazolium salt into a coloured,water-soluble formazanproduct bythe mitochondrial activity of viable cells at 37 °C, where the amount offormazan produced by the dehydrogenase enzymes is proportional tothe number of living cells. MDCK cells were plated in 96-well microtitreplates (Sigma-Aldrich, St. Louis, MO, USA) at 5.0 × 104 cells cm−2

in growth medium. After 48 h, the cells were washed twice withpre-warmed PBS and treated for 1 h with 1 μM or 5 μM OlyA-mCherry (dissolved in pre-warmed A-DMEM). Control cells weretreated with A-DMEM only. After 1 h, the cells were washed. The cellsin one plate were incubated in growth media for an additional 23 h,while the other cells were immediately prepared for the MTS test. Ab-sorbance was measured at 490 nm using a microplate reader (EL-800;Bio-Tek, Winooski, VT, USA). The viability index was expressed as theratio between the absorbance at 490 nm of the treated and controlcells (×100). Before performing the MTS assay, the cells were washedthree times with growth medium. For each well, 20 μL MTS reagent(CellTiter 96 Aqueous Reagent; Promega, Madison, WI, USA) wasadded directly to the cell culture.

2.2.2. Intracellular Ca2+ measurementsThe [Ca2+]i was measured using Fura-2 (applied as Fura-2-AM;

Molecular Probes Inc., Eugene, OR, USA). The cells were incubated for30min at 37 °C in PBS containing 4 μMFura-2-AM (or in PBS for the con-trol). The loading solutionwas thenwashed out and replaced by growthmedium for an additional 30 min. After 30 min, 1 μM or 5 μM OlyA-mCherry or OlyA was added for 10 min. The kinetics of fluorescencechanges of the Fura-2 was measured using a multi-mode microplatereader (Cytation™ 3; Bio-Tek,Winooski, VT, USA). Ratio measurements

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were performed by exciting the cells at 340 nmand 380 nm, respective-ly and measuring emission spectra at 510 nm.

Additionally, the [Ca2+]i was measured in individual MDCK cells,treated with either 5 μMOlyA or 5 μM/0.2 μMOlyA/Δ48PlyB (dissolvedin pre-warmed growth medium with no FCS or antibiotics) on aninverted Leica multispectral laser scanning confocal microscope(Leica Microsystems, Heidelberg, Germany). The cells were plated(5.0 × 104 cm−2) on borosilicate cover glasses at the bottom of poly-styrene chambers (Lab-Tek 8-well), andwere loadedwith Fluo-4 AM(Molecular probes Inc., Eugene, OR, USA; final concentration 8 μM) inthe dark at 37 °C for 40 min. The fluorescence imaging was performedafter washing the cells 3 times with pre-warmed growth medium con-taining no fetal calf serum or antibiotics. To obtain the fluorescence sig-nal of almost an entire cell, a large pinhole diameter (10 Airy disc) andan oil immersion objective lens (Leica, Planapo ×40, NA = 1.25) wereused. The excitation of the calcium indicator Fluo-4 stain was achievedwith the use of an argon laser excitation line at 488 nm. Time series(10 min, 1 frame/1.67 s) were collected using a scanning format of512 × 512 pixels. Five independent experiments were performed ineach experimental condition. Leica digital image software was usedfor fluorescence intensity analysis.

2.2.3. Light microscopyThe cells were plated in plastic dishes (5.0 × 104 cm−2; TPP,

Trasadingen, Switzerland) and grown for 48 h. After 48 h, the cellswere washed twice with pre-warmed PBS, and incubated with 5 μMOlyA-mCherry (diluted in pre-warmed A-DMEM) or with A-DMEM incontrol experiment. Phase-contrast imaging was done within 1 h afteradding OlyA-mCherry at room temperature using water-immersionobjective (63× W/NA 0.95).

Additionally, we examined the presence of detached EVs from thelivingMDCK cells. The cells were washed with pre-warmed PBS and in-cubated for 10 min with 5 μM OlyA-mCherry or with 5 μM OlyA. At theend of the incubation, the detached EVs, suspended in growth mediumwere collected and incubated for 3 h on glass slides, coveredwith APTES(Thermo Scientific, USA) in order to capture free EVs to the glass slidesurface. Nuclear stain DAPI (Vector) was used to determine DNA in ex-tracellular vesicles. Fluorescence images of the vesicles were acquiredusing oil-immersion objective (63× oil/NA 1.4) while phase-contrastimages were acquired using water-immersion objective (63× W/NA0.95). All images were acquired using the Axio-Vision programme(Carl Zeiss, Oberkochen, Germany).

2.2.4. Transmission electron microscopyTheMDCK cells were plated at 5.0 × 104 cm−2 and grown for 48 h in

plastic Petri dishes in growth medium. The cells were then washedtwice with pre-warmed PBS, and treated with 1 μM or 5 μM OlyA-mCherry (in PBS) for 10 min at 37 °C. Control cells were treated withPBS only. The cells were then washed with PBS and fixed in 2.5% glutar-aldehyde in 0.2 M cacodylate buffer, for 2 h at 4 °C. After rinsing in0.33 M sucrose solution in 0.1 M cacodylate buffer at 4 °C, the cellswere post-fixed in 1% osmium tetroxide (Serva, Heidelberg, Germany)in 0.1M cacodylate buffer, for 1 h at 4 °C. The cellswere then dehydratedthrough graded ethanol concentrations (30%–100%) and embedded inEpon (Serva, Heidelberg, Germany). Ultrathin sections were stainedwith uranyl acetate and lead citrate (both from Merck, Darmstadt,Germany). Sections were examined in a transmission Philips CM100electron microscope (Philips/FEI Corporation, Eindhoven, Holland).

For negative staining, the MDCK cells were plated at 5.0 × 104 cm−2

and grown for 48 h in growth medium. The cells were then washedtwice with pre-warmed PBS and treated for 10 min with 5 μM OlyA-mCherry (in A-DMEM) at 37 °C. After this treatment, the cell mediumwas collected. As a control, the medium was collected also from theuntreated cells. Fifteen microlitres of the vesicle samples was put onformvar-carbon coated grids and allowed to adsorb for 20 min at37 °C. The grids with the adherent vesicles were pre-washed with PBS,

fixed with 1% glutaraldehyde, and then washed in distilled water. Thegrids were stained with 1% uranyl acetate and air dried.

2.2.5. Flow cytometryThe MDCK cells were plated in plastic dishes (TPP, Trasadingen,

Switzerland) at 5.0 × 104 cm−2 and grown for 48 h in growth medium.The cellswere thenwashed twicewith pre-warmed PBS, and treated for10, 30 or 60 min with 1 μM or 5 μM OlyA-mCherry (in PBS), at 4 °C or37 °C. After these treatments, 1 mL of the EV suspensions was carefullycollected and centrifuged for 5min at 1000 ×g, to remove any detachedcells. The flow cytometry data acquisition and analysis were performedusing a flow cytometer (Altra; Beckman Coulter, Brea, CA, USA) with ahigh-power 488-nm laser (200 mW, water-cooled). The presence ofparticles was determined by the forward and side scatter (FS/SS)parameters, set at logarithmic gain. The EXPO32 Beckman Coulter soft-ware was used for analysis of the data. A minimum of 1.0 × 104 eventswere recorded for each sample. For measurement of the EV concentra-tion, 20 μL of calibrating beads (Flow Count Fluorospheres, BeckmanCoulter, Brea, CA, USA) of 10 μm diameter and known concentration(1.0 × 106 beads mL−1) was added to the samples (final volume500 μL). The concentration of EVs was calculated using the formula:EVs (μL−1) = Events in EVs gate × concentration of Flow Countbeads ∕ events in the gate of the bead population.

2.2.6. Proteomic analysisThe MDCK cells were plated in plastic dishes (T75; TPP Techno

Plastic Products, Trasadingen, Switzerland) at 5.0 × 104 cm−2 andgrown for 48 h in growth medium. The cells were then washed twicewith pre-warmed PBS and treated with 5 μM OlyA-mCherry (in PBS)for 10 min at room temperature. After the treatment, 3 mL EVs suspen-sion was carefully collected and subjected to centrifugation for 5 min at1000 ×g, to remove any detached cells. The EV suspensionswere storedat 4 °C until their use the next day.

2.2.6.1. Isolation of proteins. As observed from preliminary studies, it wasnecessary to remove the unbound OlyA-mCherry from the EVs, as it in-terfered with the MS analysis. For the isolation of the proteins and re-moval of the unbound OlyA-mCherry from the EVs, two differentstrategies were used. The EV suspensionwas either: (i) first centrifugedat 15,000 ×g for 30 min at 4 °C, with the sedimented EVs treated with12.5% (w/v) trichloroacetic acid (final concentration); or (ii) first centri-fuged in 100-kDa cut-off tubes (Amicon; Sigma-Aldrich, St. Louis, MO,USA) for 10 min at 1000 ×g, with the retentate treated with 12.5%(w/v) trichloroacetic acid (final concentration). After the trichloroaceticacid had been added to the samples, the mixtures were incubated for10 min on ice, and then centrifuged at 16,100 ×g at 4 °C. The superna-tants were carefully removed, and the sediments were washed withcooled acetone. These washed samples were vacuum-dried and storedat−20 °C until the proteomic analysis.

2.2.6.2. Preparation of the protein samples for SDS-PAGE. The proteinsamples were dissolved overnight in 50 μL buffer containing30 mM Tris, 7 M urea, 2 M thiourea, 2.5% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 0.25%(w/v) ABS-14 (alkylamidesulfobetain-14), and 0.002% (w/v),bromophenol blue. In-house prepared 10% polyacrylamide gels [36]were used for the separation of proteins. After silver staining, thegels were imaged with Image Scanner, using the LabScan 5 software(GE Healthcare, Amersham Biosciences, Amersham, UK). The proteinbands were cut out of the gels and stored at −20 °C.

2.2.6.3. In-gel digestion and identification of proteins. Immediately prior toMS analysis, the gel pieces containing protein spots were thawed anddestained in a solution containing 30 mM potassium ferricyanide and100 mM sodium thiosulfate (1:1; v/v) for 15 min, with shaking atroom temperature. The gel pieces were washed twice with water for

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15min with shaking, then for 20min in 200 mM ammonium hydrogencarbonate without shaking, and finally twice again with water for15 min. Then the pieces of gel were dehydrated with acetonitrile andvacuum-dried. In-gel reduction and alkylation of the proteins followed.Reduction preceded by incubation of the gel pieces in 10 mM dithio-threitol and 25 mM ammonium hydrogen carbonate at 56 °C. After45 min, the reducing solution was removed and the proteins werealkylated with 55 mM iodoacetamide in 25 mM ammonium hydrogencarbonate at room temperature in the dark for 30 min. The supernatantwas removed, and the gel pieceswerewashed by shaking in 25mMam-monium hydrogen carbonate for 15 min. After dehydration with aceto-nitrile, the gel pieces were vacuum dried. The proteins inside the gelwere fragmented usingMS-grade modified trypsin (Promega, Madison,WI, USA; 12.5 ng μL−1 in 25 mM ammonium hydrogen carbonate),overnight at 37 °C. The resulting peptides were extracted from the gelwith 50% (v/v) acetonitrile and 5% (v/v) formic acid, and concentratedunder a vacuum to 15 μL. The extracted peptides were purified usingC-18 Stage Tips (Fisher Scientific, Waltham, MA, USA) according to themanufacturer protocol, and stored at −20 °C.

2.2.6.4. Mass spectrometry. LC-MS/MS analyses were performed usingan ion-trap mass spectrometer (1200 series HPLC-Chip-LC/MSD TrapXCT Ultra; Agilent Technologies, Waldbronn, Germany) with theelectrospray ionisation source operating in the positive mode andcontrolled by ChemStation LC 3D system Rev. B.01.03 SR1 (AgilentTechnologies, Santa Clara, CA, USA), and LC/MSD Trap Control softwareversion 6.0 (Bruker Daltonik GmbH, Bremen, Germany). An HPLC-Chip(Agilent Technologies, Waldbronn, Germany) was used to separatemixtures of tryptic peptides, which comprised a 40 nL enrichmentcolumn and 43 mm × 75 μm analytical columns packed with Zorbax300SB-C18:5 particles. The peptides were loaded on the enrichmentcolumn in 97% (v/v) solvent A (0.1% [v/v] formic acid in water) and 3%(v/v) B (0.1% [v/v] formic acid in acetonitrile) at 4 μL min−1. Thepeptides were then eluted from the column with a gradient from 3%(v/v) B to 50% (v/v) B in 41 min, followed by a steep gradient to 90%(v/v) B in 1 min, at a flow rate of 0.35 μL min−1. Mass spectra were re-corded in the range from 400m/z to 2200m/z. The five most abundantions in each MS spectra were MS/MS scanned. The mass spectra wereanalysed using the Spectrum Mill software Rev. A.03.03.084 (AgilentTechnologies, USA) and the non-redundant NCBI (National Center forBiotechnology Information) protein data bank species Lupus canis Lin-naeus. The following restrictions were applied: two miss cleavageswere allowed, precursor and product mass tolerance of ±2.5 Da and±0.7 Da, respectively, carboxyamidomethylcysteine (C) as fixed modi-fication, and oxidised methionine (Mox) as variable. These results werefurther validated using the Scaffold 2 Software (Proteome Software,Portland, OR, US) with parameters set as: at least two distinct peptidesdetermined, protein and peptide probability of 0.95 or above.

2.2.7. Lipidomic analysisTheMDCK cells were plated in plastic flasks (T75; TPP Techno Plastic

Products, Trasadingen, Switzerland) at 5.0 × 104 cm−2 and grown for48 h in growth medium. The cells were then washed twice with pre-warmed PBS and treated with 5 μM OlyA-mCherry (in PBS) for 10 minat room temperature. After the treatment, 3 mL EV suspension wascarefully collected and centrifuged for 5 min at 1000 ×g, to removeany detached cells. Meanwhile, the control untreated cells were scrapedfrom the plastic dishes. The EV suspensions and cell pellets were storedunder a N2 atmosphere at −20 °C until the next day.

2.2.7.1. Extraction of the lipids from the MDCK cells and extracellularvesicles. The lipid extraction process was performed as described inour previous study [37]withmodifications to provide a two-step proto-col to expand the lipid recovery [38]. First, the cell pellets were resus-pended in 1 mL Milli-Q water by gentle pipetting, and transferred intoa glass bottle, while the EV suspensions were transferred straight into

a glass bottle. Before lipid extraction, 60 μL and 10 μL of 1,2-dilauroyl-sn-glycero-3-phosphocholine (100 ng μL−1), a lipid standard that isdifferent from any natural cellular lipid, were added to the cell and EVsuspensions, respectively. Then three volumes of chloroform:methanol(10:1, v/v) was added to each suspension, followed by sonication andincubation for 2 h at room temperature while shaking. After centrifu-gation at 2500 ×g for 5min, the organic (lower) phases were careful-ly collected in glass vials, and three volumes of chloroform:methanol(2:1, v/v) was added to the aqueous (upper) phase, followed by 1 hshaking at room temperature, and repeated centrifugation. The sec-ond collected lower organic extracts were combined with the previ-ous ones, evaporated using a rotary evaporator (Rotavapor R-134,V700; Büchi, Flawil, Switzerland), dried under N2, and stored at−20 °C. The lipid extracts were initially dissolved in perdeuteratedmethanol (CD3OD) for the NMR measurements. After NMR analysis,the samples were fluxed with N2 to reconstitute them, in 300 μLMeOH (for the cell lipid samples) and in 50 μL (for the EV lipid samples)for theMS analyses. In our study, NMRwas used initially to estimate theinter-class distributions, while theMS analysis was performed to deter-mine the intra-class distributions of the lipid molecular species.

2.2.7.2. NMR measurements. 1H NMR (400 MHz) and 31P NMR(162 MHz) spectra of the raw organic lipid extracts were recorded ind4-methanol (99.90% CD3OD) at 300 K on a NMR spectrometer(Bruker-Avance 400 MHz; Bruker, Billerica, MA, USA) using a 5 mmbroadband inverse probe equipped with pulsed-gradient field utility.The following analyseswere carried out: 1HNMR (proton chemical shifts,scalar couplings J); 1H\\1H COSY (proton-proton scalar correlations);1H\\13C HSQC (proton-carbon one-bond correlations); 1H\\13C HMBC(proton-carbon multiple-bond correlations); fully 1H-decoupled 31PNMR. One-dimensional NMR spectra (1H, 31P) of the lipid extracts werefitted and integrated using the MestreNova 10.1 software (MestrelabResearch S.L., Escondido, CA, USA).

2.2.7.3. HPLC-electrospray ionisationMS/MSmeasurements.Our previous-ly developed LC-MS/MS method [37] was used to separate and analysethe lipidmolecular species in theMDCK cell and EV extracts. This meth-od combines a ShimadzuHigh Performance LC™ (CBM-20A; Shimadzu,Kyoto, Japan) equipped with a binary pump (LC-20AB; Shimadzu,Kyoto, Japan) with electrospray ionisation triple-quadrupole MS/MS(API 3000; Applied Biosystems, Hudson, NH, USA). In short, a KinetexC18 column (pore size, 100 Å; column length, 100 mm; inner diameter,4.6 mm; particle size 2.6 μm; Phenomenex, Castel Maggiore, Italy) wasused to separate the lipids. The mobile phase was composed of metha-nol/water (7:3, v/v) with 10 mM ammonium acetate (phase A), andmethanol with 10 mM ammonium acetate (phase B). The gradient elu-tion programme went from 70% B to 100% B in 45 min, and was thenmaintained at 100% B for 20 min, with the next re-equilibration at 70%B for 15 min. The flow rate was 1 mL min−1 and 10 μL of sample wasinjected. The MS analysis was performed in positive ionisation modewith the MS/MS class-specific scans including precursor ion scanningand neutral loss scanning, using the optimised parameters of: nebulisergas, 9; curtain gas, 10; temperature, 300 °C; ion spray voltage, 5 kV; col-lision gas, 4; declustering potential, 65; focusing potential, 250; en-trance potential, 5; collision cell exit potential, 18; collision energy, 40or 50 for different classes of lipids; Q1/Q3 with unit resolution andstep size of 0.1 amu. The MS/MS class-specific scan of precursor ionscanning for m/z 184 was used to characterise PC, lysoPC, ether-PCand SM, and precursor ion scanning for m/z 264 for sphingosine-based(d18:1) glycosphingolipids and ceramide with the precursor ion formof [M + H]+, while neutral loss scanning for 185 Da and 141 Da wereapplied to analyse the phosphatidylserine and phosphatidylethanol-amine lipids with precursor ion [M+ H]+, as well as neutral loss scan-ning for 115 Da, 189 Da, 277 Da and 35 Da for phosphatidic acids,phosphatidylglycerols, phosphatidylinositols and diglycerides with[M+ NH4]+ as precursor ion forms, respectively.

Fig. 1. Effects of OlyA-mCherry on survival of MDCK cells. The viability indices of MDCKcells treated with 1 μM and 5 μM OlyA-mCherry are expressed as the optical density ofthe treated cells/optical density of control cells ×100 (%), after 1 h and 24 h of exposure.Data are means ± s.d. from three independent experiments. The differences wereanalysed using Student's t-tests on the two populations, and one-way ANOVA. Treatedcell viability was not significantly different from control cell viability (i.e., without OlyA-mCherry).

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2.2.7.4. Data processing. One-dimensional NMR spectra (1H and 31P)were processed using the MestreNova 10.1 software (Mestrelab Re-search S.L., Escondido, CA) and compared with our NMRmeasurementsof commercial lipid standards, to initially identify lipid classes. The spec-tra for the LC-MS/MS class-specific scans (i.e., precursor ion scanning,neutral loss scanning) were preprocessed using Analyst, version 1.42(Applied Biosystems, Hudson, NH, USA) by extracting ions from thetotal ion chromatograms, which helped to identify lipid molecular spe-cies considering the retention times of the LC, as well as by the ‘LipidMass Spec. Prediction’ (LIPID MAPS) enquiry with the specified param-eters of ‘Mass’ (m/z of a lipid species), ‘Mass tolerance’ (±0.5 m/z),‘Head group’ (e.g., PC, phosphatidylethanolamine, phosphatidylinositol,phosphatidylserine), and ‘Precursor ion’ ([M+H]+, [M+NH4]+). Peakareas were integrated from the extracted ion chromatograms to repre-sent the quantities of individual lipid species. The intra-class relativeratio of individual lipid species was estimated by normalising its peakarea to the total peak area of all of the lipids in the same class. For exam-ple, the relative molar fraction of each PC species in a given sample wasobtained by the ratio of its area to the total area of PC lipids, and thesame procedure was performed for all other lipid classes. Based onthis, each lipid species was considered a variable and hence a data ma-trixwith rows that indicated different samples and columns of variablesshowing the relative ratio of each lipid was subjected to multivariatedata analysis using SIMCA-P 13.0 (Umetrics, Umeå, Sweden). Pareto-scalingwas used to pre-process the data, and principal component anal-ysis was first applied to visualise the preprocessed MS/MS data to seewhether and how the EV lipid samples differed from the cellular lipidsamples, and to reveal such differences as well as any systematic pat-terns and trends of variation. Finally, the distribution/variation of lipidfatty acyl carbon chain lengths and double bonds (db) were evaluated.Univariate unpaired t-tests were performed using the OriginPro soft-ware (OriginPro 8, OriginLab Corporation, Northampton, MA, USA) toestimate variation of each lipid, as well as the variation of the acylchain lengths and double bonds.

2.2.7.5. Determination of cholesterol content. The total cholesterol in thecontrol cell samples and the EVs (induced by 5 μM OlyA-mCherry)was determined by the enzymatic fluorometric method using Amplex®Red Cholesterol Assay kits (Thermo Fisher Scientific, Waltham, MA,USA), and a multi-mode microplate reader (Cytation™ 3; Bio-Tek,Winooski, VT, USA). To determine the relative amounts of cholesterolin the EV samples, we normalised its concentration against the totalcholine lipids, quantitatively determined by the colorimetric Phospho-lipid LabAssay™ (Wako, Richmond, VA, USA).

3. Results

3.1. OlyA-mCherry is not toxic to MDCK cells

In agreement with our previous study [15], cell viability assays with1 μM or 5 μM OlyA-mCherry showed no acute toxicity towards MDCKcells after 1 h of incubation. Moreover, if the cells were grown for anadditional 23 h after washing out the OlyA-mCherry, their survivalwas comparable to control cells (Fig. 1). Furthermore, no increase in[Ca2+]i was detected after treatments with 1 μM or 5 μM OlyA orOlyA-mCherry. In contrast, when MDCK cells were treated with com-bined OlyA and PlyB that assemble into a transmembrane pore complex[9], a time-dependent increase in the [Ca2+]i was triggered (Fig. 2),followed by a rapid decrease of the fluorescence which is suggestive ofcell lysis, as reported previously in other mammalian cell types [17,18].

3.2. OlyA-mCherry induces outward vesiculation of the plasmalemma inliving MDCK cells

The formation of EVswas firstmonitored under thefluorescence andphase contrast microscope. In contrast to control MDCK cells without

OlyA-mCherry (Fig. 3A), EVs were observed after treatment of thesecells with 5 μMOlyA-mCherry (Fig. 3B), and some of these EVs also de-tached from themembranes of mother cells andwere found inmediumcollected from cell cultures (Fig. 3C,D). Most of these EVs had diametersbetween 1 and 5 μm, with the largest diameters observed between 10and 20 μm that were strongly positive for OlyA-mCherry and negativefor DAPI nuclear stain (Fig. 3D). The same phenomenon was observedalso when MDCK cells were treated with OlyA (Fig. S1). OlyA-mCherry(5 μM) also induced ruffling, budding, and vesiculation of blood cells,as demonstrated for erythrocytes, thrombocytes and leukocytes(Supplementary File 1, Fig. S2).

Further analyses of the cellular ultrastructure after the application ofOlyA-mCherry to livingMDCK cells were carried out using transmissionelectron microscopy (Fig. 4). In contrast to untreated MDCK cells(Fig. 4A), in those treatedwith 1 μMor5 μMOlyA-mCherry, the buddingof a large number of small EVs was detected (diameter ~ 100 nm) thathad not been detected with fluorescence microscope because of alower resolution power. These EVs were mainly localised close to orattached to filopodial cell protrusions (Fig. 4B, C) and they wereelectronically more transparent than the cytosol of the cells. The ultra-structure of the cells treated with 1 μM or 5 μM OlyA-mCherry wasnot changed.

Since by fluorescence and phase-contrast microscopy it was impos-sible to distinguish vesicles with diameters below 500 nm from smallmembrane protrusions on the cell surface, electron microscopy ofnegatively stained vesicles isolated from cell culture medium was per-formed (Fig. 4D, E). This method confirmed that the majority of thefree floating EVs had a diameter around 100 nm (Fig. 4D) and thattheir number was considerably larger in OlyA-mCherry – treated cellscompared to the control (Fig. 4D and E).

3.3. Quantification of extracellular vesicles induced by OlyA-mCherry usingflow cytometry

To determine the influence of temperature and OlyA-mCherry con-centration and time of incubation on the budding of EVs from livingMDCK cells, the concentrations of the EVs produced were determinedusing flow cytometry. The cells were treated at 4 °C and 37 °C, and theEVs were harvested after 10, 30, or 60 min of incubation of the cellswith 1 μMor 5 μMOlyA-mCherry (with growthmediumas the control).Fig. 5 shows that the higher OlyA-mCherry concentration (5 μM) pro-duced greater numbers of EVs in the growth medium. Furthermore,the extent of blebbing and EV shedding was not dependent on

Fig. 2.OlyAdoesnot induce the increase in intracellular Ca2+ activity. Representative images of the Fluo-4AMfluorescence inMDCKcells before (A) and after (B) a 3-min exposure toOlyA(5 μM), or before (D) and after (E) addition of OlyA/PlyB (5 μM/0.2 μM). C, F: The time-course traces of the fluorescence intensity changes in selected MDCK cells corresponding to theregions of interest (ROIs) in panels A and B, or D and E respectively. Arrows indicate the time of application of OlyA-mCherry. Note that after the addition of OlyA, no changes influorescence were observed in the nuclear region and in the cytosol of the MDCK cells, while after the addition of OlyA/PlyB, a strong increase in fluorescence was obvious. Scale bars:30 μm.

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temperature; notably, lowered temperature did not prevent cellsfrom vesiculation. Generally, the highest number of EVs was detect-ed after the 10-min incubation of OlyA-mCherry with the MDCKcells, and the number of these EVs progressively dropped for the

Fig. 3. Vesiculation of livingMDCK cells treated with 5 μmOlyA-mCherry. Representative phasephase-contrast image of MDCK cells treated with 5 μM OlyA-mCherry (B); representative phmCherry that were captured on glass slides (C); representative fluorescent images of isolatedglass slides (D). Scale bars: 20 μm. Arrows indicate vesicles of 1–5 μm while arrowheads indica

longer incubation times (i.e., 30, 60 min). The concentration of EVswas calculated according to the known concentration of calibrationbeads that were used in all of the experiments. We were able to de-tect 1.0 × 105 EVs mL−1 that were released into the medium when

-contrast image of MDCK cells where no formation of EVs is observed (A); representativease-contrast images of isolated detached EVs from MDCK cells treated with 5 μM OlyA-detached EVs from MDCK cells treated with 5 μM OlyA-mCherry that were captured onte larger vesicles.

Fig. 4. EVs induced by OlyA-mCherry and revealed by transmission electronmicroscopy.MDCK cells were cultured for 48 h and treatedwith growthmedium (A, D, control), or with 1 μM(B) or 5 μM (C, E) OlyA-mCherry for 10min, and prepared for Epon embedding (A–C) or negative staining (D, E). Arrowheads, EVs. Scale bars: 1 μm (A–C); 200 nm (D, E). Twenty imageswere analysed per each treatment conditions.

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5 μM OlyA-mCherry was applied to the cells for 10 min. Treatmentwith the higher concentration of OlyA-mCherry caused more exten-sive plasmalemma vesiculation and resulted in vesicles with in-creased amounts of bound OlyA-mCherry. The highest amount ofOlyA-mCherry bound to the EV s was observed after the 10-mintreatment of the MDCK cells with 5 μM OlyA-mCherry (Fig. S3).

3.4. Proteomic analysis of extracellular vesicles induced by OlyA-mCherry

Proteomic analysis was carried out only for the EVs induced with5 μM OlyA-mCherry in living MDCK cells, as the numbers of vesiclesin the EVs induced with 1 μM OlyA-mCherry, and consequently theprotein levels, were under the limit for reliable quantification. Ashigh levels of OlyA-mCherry in these samples exacerbate the detec-tion of other proteins on two-dimensional electrophoresis, the

proteins in these EV preparations were resolved in only one dimen-sion, by SDS-PAGE. The profile of the proteins associated with theEVs induced by OlyA-mCherry is shown in Fig. S4. Using mass spec-trometry (MS), we identified 71 different proteins in these EVs (seeTable S1, where the proteins identified are grouped according totheir most typical cellular localisation, as specified by gene ontologyanalysis). The majority of these proteins were either cytoplasmic(e.g., cytosolic, ribosomal, endoplasmic reticulum, endosomal, mito-chondrial) or nuclear. Many of them were connected with the struc-ture or function of the cytoskeleton. Moreover, different heat-shockproteins involved in cell stress responses were identified. A few ofthese proteins were membrane-associated proteins, among whichsome were associated with the plasmalemma e.g., annexin A1, vas-cular endothelial growth factor receptor, and V-type ATPase. Againstexpectations, only two of these 71 proteins were described as

Fig. 5. Influence of temperature andOlyA-mCherry concentration and time of incubation on the number of extracellular vesicles released fromMDCK cells. (A) Representative forward andside scatter (FS/SS) plots showing beads and EVs in the control (untreated) and after treatment of the cells with 5 μMOlyA-mCherry for 10min at 37 °C. (B)MDCK cells were incubated at4 °C (left) and 37 °C (right) with 1 μM and 5 μM OlyA-mCherry for increasing periods of time. The concentrations of the extracellular vesicles (EVs) were determined using the knownconcentrations of the 10-μm calibration beads. Each data point represents the concentration of EVs obtained from the cells treated with OlyA-mCherry after subtracting thebackground from the untreated cells. Data are means ± s.d. of two independent determinations.

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components of lipid rafts: insulin-like growth factor 2mRNA bindingprotein 1, and annexin A2.

3.5. Lipidomic analysis of extracellular vesicles induced by OlyA-mCherry

The initial lipidomic analyses showed that the number of EVs pro-duced in living MDCK cells with 1 μM OlyA-mCherry was below thelimit for reliable quantification of lipids. Therefore, the lipidomic analy-siswas only carried out on the EVs inducedwith 5 μMOlyA-mCherry. Toevaluate the lipidomic profile of the EVs produced, and to have an over-view of the differences in the lipid profiles between the MDCK cells andthe EVs, nuclear magnetic resonance (NMR) was initially used as a non-destructive tool to determine the distribution of the different lipid clas-ses in each sample. Then liquid chromatography–tandem MS (LC-MS/MS) was used to characterise the individual lipids in each of the lipidclasses determined using NMR.

The NMRmeasurements demonstrated that the total amount of thelipids in these EVs induced by 5 μMOlyA-mCherry represented approx-imately 1% of the total lipid in the MDCK cells. The LC-MS/MS analysesrevealed 218 different lipid species in MDCK cells, and 84 lipid speciesin the EVs, although it was not possible to determine the amount of cho-lesterol (Table S2). This analysis showed that the numbers and types ofspecies of choline-containing lipids that represented themain lipid classof the MDCK cell membranes (i.e., PC, ether-PC, SM, lysoPC) were simi-lar to those in the EVs, except for the large reduction in ether-PC in theEVs. In addition, as shown by the total ion chromatogram overlay inFig. 6A, there were significant differences compared with the MDCK

cells for the relative peak intensities of the lysoPC lipids with respectto the other choline-containing lipids in the EVs. Further inter-class dis-tribution analysis of all of the choline-containing lipids (Fig. 6B) showedthat the relative ratio of lysoPC in the EVs (~18.3%) was significantlyhigher than the relative ratio of lysoPC in the MDCK cells (~0.5%). Thisincrease was compensated for by reductions in PC and ether-PC in theEVs. There were no significant changes in the proportions of SM. Anoth-er interesting observation (see Table S2) was that while 36 species ofphosphatidylethanolamine and ether-phosphatidylethanolamine wereseen for the MDCK cells, these were almost depleted (or below the de-tection limits) in the EVs, where there were only three species of phos-phatidylethanolamine. Similarly, the EVs lacked phosphatidylserinespecies, with the exception of one single phosphatidylserine with a sat-urated short chain (28 carbons; phosphatidylserine 28:0), and saturat-ed diglyceride lipids.

To define the variation pattern of the lipid species between theMDCK cells and the EVs, multivariate data analysis was performed bylooking systematically at the individual lipid species. As an unsuper-vised multivariate method, principal component analysis was per-formed first on the pre-processed LC-MS/MS data for the PC lipidspecies. Pareto scalingwas applied for all of these analyses. The principalcomponent analysis scores plot shows distinctive separation betweentheMDCK cells and the EVs for the first principal component dimension,indicating that the PC lipid profile had prominent variations betweenthe MDCK cells and the EVs (Fig. S5A). Moreover, the first two compo-nents of this principal component analysis model cumulatively de-scribed 94.6% of the total variability, which showed as a significant

Fig. 6. Comparison of the choline-containing lipids in MDCK cells and in the extracellular vesicles induced by OlyA-mCherry. Analysis of samples from untreated MDCK cells and EVsinduced by 5 μM OlyA-mCherry for 10 min. (A) Ion chromatogram overlay obtained by LC-MS/MS precursor ion scanning for m/z 184 for phosphatidylcholine (PC), ether-PC (e-PC),lysoPC and SM from MDCK cells (blue) and from the EVs (red). (B) Relative ratios of PC, ether-PC, lysoPC and sphingomyelin (SM) of the MDCK cells and the EVs, as estimated bynormalisation of the peak area to the total peak area of all of the lipids in the same class. Data are means ± s.e.m. of three independent determinations. The differences were analysedusing univariate unpaired t-tests. **, 0.001 b p b 0.05; ***, p b 0.001.

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difference between the MDCK cells and the EVs. Furthermore, the con-tribution of individual PC lipid species to the differences between theMDCK cells and the EVs was analysed according to a correspondingloadings column plot (Fig. S5B), from which it can be seen that the PCspecies with relatively longer chain length and higher unsaturationwere much more represented in the EVs. Multivariate data analysis ofthe molecular species from other classes was carried out according toa similar workflow.

According to principal component analysis, to highlight the generaltrends in the MDCK cells and the EVs, we compared the distributionsand the variations of the fatty acyl carbon chain lengths (i.e., carbonnumber; Figs. S6, S7), as well as their level of unsaturation (doublebonds; Figs. S8–S11). These data show that compared to the wholeMDCK cell lipid extract, the EVswere enriched in PC species with longeracyl chains (Fig. S6), and SM lipid species with shorter acyl chains(Fig. S7). Furthermore, the EVs were relatively enriched in choline-containing lipids with higher numbers of double bonds for lysoPC, PCand ether-PC (Figs. S8–S10), as well as in SM lipids with one doublebond (Fig. S11).

The NMR measurements provided the ratios of cholesterol to totallipids (see Fig. S12), which in the MDCK cells was around 25 mol%.This is in line with previous analyses of the MDCK cell lipidome usinga shotgun lipidomic approach [39], where it was shown that MDCKcells contain 28 mol% cholesterol. However, the amount of cholesterolin the EVs in the present studywas too low to be accurately determinedby NMR. Therefore, an enzymaticmethodwas used tomeasure the cho-lesterol levels of these EVs. The cholesterol contents were normalisedfor the control MDCK cells and the EVs using enzymatic measurementsof choline-containing lipids in both of these samples. These data provid-ed the ratios of cholesterol to the total choline-containing lipids in theMDCK cells and the EVs as 0.57 and 1.0, respectively (Fig. S13).

4. Discussion

The data obtained in the present study and in our previousstudy [15] have shown that: (i) when applied at ≥1 μM, the cholesterol/SM - binding protein OlyA-mCherry induces the formation of EVs with alarge spectrum of sizes in different cells in a concentration-dependent

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manner; (ii) this vesiculation is an intrinsic property of OlyA, since itcan be induced also by the untagged protein; (iii) this vesiculation isnot temperature-dependent or associated with changes in [Ca2+]i,and it does not compromise the viability of the MDCK cells; (iv) theEVs that are shed from the plasmalemma of the MDCK cells arepoor in membrane proteins, including typical raft-associated mem-brane proteins; and (v) these EVs are relatively enriched in cholesteroland lysophospholipids. The formation of these EVs seems to be the spe-cific consequence of the binding of OlyA-mCherry to cholesterol/SMmembrane nanodomains in MDCK cell membranes [8,9,12,15]. In fact,in our previous work no binding of OlyA-mCherry, and consequentlyno formation of EVs were observed after the treatment of these cellswith either methyl-β-cyclodextrin or sphingomyelinase [15].

Regarding the size, these MDCK-derived EVs that are labelled byOlyA-mCherry were shown to be composed of two main populations.One population had diameter between 100 nm and 1 μm and probablycorresponded to the microvesicles, while the other comprised of largervesicles of around 10-μm diameter. The number of EVs released in-creased with increased concentrations of OlyA-mCherry. Furthermore,the rate of vesiculation was not dependent on temperature, and itstarted immediately upon OlyA-mCherry application, thus precedingthe OlyA-mCherry internalisation via caveolae that we observed previ-ously [15]. Altogether, these observations support the conclusion thatthese EVs induced by OlyA-mCherry are not related to exosomes or ap-optotic bodies, which indicates that this vesiculation process is notexocytosis-related or apoptosis-related, respectively.

Studies of membrane repair mechanisms have revealed that atmoderate and sublytic concentrations, pore-forming toxins can inducecell blebbing and shedding of plasmalemma [32,40–42]. In addition tothe toxin endocytosis, plasmalemmal blebbing and vesiculation havebeen proposed as a defence mechanism used by eukaryotic cells to re-move membrane-perturbing molecules [32,42–46]. Studies with strep-tolysin O and pneumolysin have revealed the pivotal role of a transientrise in [Ca2+]i for membrane repair, including plasmalemmal blebbingand vesiculation [35,45–47]. Thus, we also examined the possibilitythat the OlyA-mCherry treatment triggers transient rises in [Ca2+]ifrom intracellular stores, as observed for the combined OlyA/PlyB cyto-lytic complex [17,18, this work].With OlyA-mCherry or OlyA alone, thiswas not the case.

On the basis that this outward vesiculation induced by OlyA-mCherry is not dependent on temperature or [Ca2+]i, and consideringthe fact that OlyA inducesmembrane budding andoutward vesiculationupon its binding to artificial cholesterol/SM (molar ratio, 1:1) largeunilamellar vesicles [9], it would appear that this vesiculation arises asa consequence of the direct physical effects of OlyA-mCherry bindingto the extracellular leaflet of the cell membrane, which would result inlocal membrane bending. Similarly, a recent study of an ectocytosis-like process evoked by streptolysin O has shown that this toxin can in-duce blebbing and vesiculation of living cells in a Ca2+-free medium,and also in cells that are chemically fixed [32]. Suchmembrane bendingeffects have been extensively studied with intracellular proteins in-volved in budding of the plasmalemma and intracellular membranes[48,49].

We furthermore took advantage of the proteomic and lipidomicapproaches here to specify the composition and origin of these EVsinduced by OlyA-mCherry. Considering that OlyA and fused OlyA-mCherry are proteins targeting cholesterol/SMmembrane nanodomainsthat correspond to the membrane rafts [8,9,15], we speculated thatthese EVs that are labelled with OlyA-mCherry might be enriched inproteins and lipids typical of membrane rafts. Interestingly, however,in the present study, the proteomic and lipidomic profiles of these EVsshed from MDCK cell membranes by OlyA-mCherry treatment werein contrast with our hypothesis. The EVs collected in the presentstudy were poor in membrane proteins and contained only two raft-associated proteins. Annexin A1 detected in the EVs proteome is awell-known protein involved in plasmalemma repair processes that

may include vesiculation, too [50]. Recruitment of cytosolic annexinsto plasmalemma is known to be triggered by the increased [Ca2+]i[51], thereforewe suggest that the detected annexin A1was incorporat-ed into the larger detached EVs as a cytosol-soluble protein. Thus over-all, this proteomic analysis supports the view that these EVs induced byOlyA-mCherry contain only part of the plasmalemmal proteome, alongwith some constituents of the cytoplasm that are entrapped by the EVsupon their formation. Some of these proteins might also originate fromcontamination of the EVs during their isolation from the MDCK cells, asthe contamination of microvesicle preparations with cells is a commonproblem [20,52].

Notably also, no caveolins were identified in these EV preparations.These proteins are typical constituents of caveolar rafts, and have beenshown to be highly enriched also in detergent-resistant membranes de-rived from MDCK cells [53]. The concept of these detergent-resistantdomains has been an operational term for membrane rafts in severalstudies [54], as an assumption based on their liquid-ordered physicalstate that is more resistant to solubilisation by detergents [55]. How-ever, as detergents themselves can be responsible for the formationof ordered domains, detergent-resistant domains should not be as-sumed to describe biological rafts in terms of their extent, structure,composition, or even existence [55,56].

Despite these open questions, very recent lipidomic analyses ofdetergent-resistantmembranes derived fromMDCK cells have revealedtheir increased cholesterol [57] and SM [57,58] content, alongwith theirenrichment in PC species with more saturated fatty acyl chains [58].Similarly, a quantitative shotgun MS analysis of the lipidome ofpolarised MDCK cells infected with a raft-interacting influenza virusshowed that the viral envelopes that budded from the apical cell mem-braneswere significantly enriched in cholesterol and SM, at the expenseof glycerophospholipids [39]. In the present study, the SM content inthese EVs induced by OlyA-mCherry was comparable to that of theMDCK cells; however, cholesterol and lysophospholipids were relative-ly more abundant in the EVs than in the cell lipids extract. At the sametime, the EVs lacked glycosphingolipids and had a significantly lowernumber of different lipid species, including ceramides, negativelycharged phospholipids, diglycerides, phosphatidylethanolamines,and ether glycerophospholipids. Furthermore, for the EV choline-containing lipids (i.e., lysoPC, PC, ether-PC) there was a higher degreeof unsaturation than in the total MDCK cell lipids extract. These differ-ences in the lipid composition of theseMDCK-derivedmembrane struc-tures most probably reflect the different mechanisms that lead to theirformation, in terms of detergent-resistant membranes [57,58], theMDCK-cell-derived influenza virus envelope [39], and these EVs in-duced by OlyA-mCherry (present study).

Membrane bending can lead to the reshaping of flat cellular and ar-tificial lipid membranes into vesicles, tubules, and other shapes, whichwill be induced by the interplay of several factors. In cells, these includespecific proteins that bind tomembranes as scaffolds, or that can changethe local membrane curvature by insertion of their amphipathic regionsinto the lipid matrix, or that can penetrate into only one lipid leaflet,thereby developing curvature via the bilayer-coupled mechanism[59–63]. Another way to change membrane curvature via bilayer-coupled mechanisms is through the redistribution of lipids betweenthe inner and outer membrane leaflet, as caused by flippases,scramblases, and lipid-modifying enzymes [64–66]. Moreover, differ-ences in local spontaneous curvature of laterally segregated lipid mem-brane domains, such as between liquid ordered and liquid disordereddomains, and the line tension between them,might additionally inducelocal membrane bending or modify the membrane shape, as has beenshown in artificial membrane systems [67,68].

Our collected data suggest that OlyA or OlyA-mCherry bindingto cholesterol/SM membrane nanodomains causes local outwardplasmalemmal bending due to a direct physical action, which is mostlikely to be through the development of locally positive membrane cur-vature. This results in formation of the spherical EVs of two types:

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microvesicles that are shed from the cell surface, as well as larger blebs.The latter can be connected to tubular structures that can be seen totether the blebs to the mother membrane of cells, but can also detachfrom the cell membrane. The lipid composition of these EVs, and in par-ticular their increased fraction of cholesterol and lysoPC species, and therelatively high proportion of unsaturation of their choline-containinglipids, suggest that this vesiculation induced by OlyA-mCherry isaccompanied by specific lipid sorting into membrane patches thatbud outwards from the plasmalemma to create vesicles and tubules,which is a process governed by a mechanism reported for giantunilamellar vesicles [67,69]. These studies have shown that imposedmembrane curvature can result in segregation of lipids and also fissionof tubular structures, which can be facilitated by a lipid-bound protein[70].

In this way, we speculate that this imposed membrane curvature,and in particular in tubular structures, promotes the sorting of mem-brane proteins into these EVs, as also implied by our proteomic anal-ysis. This budding induced by OlyA-mCherry produces sphericalvesicles and tubules that are shapes that both contain high positiveand negative curvatures. This coincides with the observed relativelyhigher EV content of lysophospholipids and cholesterol, which rep-resent lipids with positive and negative spontaneous curvature, re-spectively [60].

Finally, as at least a portion of these EVs induced by OlyA-mCherrycontains soluble cytoplasmic proteins, and as OlyA-mCherry is notcytotoxic, this makes such EVs potentially interesting tools for rela-tively non-invasive sampling of cytosolic proteins from differentcells, and for lipidomic and proteomic analyses, as have been reportedfor other EVs [21]. Furthermore, as these EVs induced by OlyA andOlyA-mCherry are relatively large, have a non-permeabilised mem-brane, and are easy to prepare in relatively large amounts, we suggestthat they can be used asmodel EVs in biophysical and biochemical stud-ies of cell membranes as well as a tool for sampling of cytosolic mole-cules from cells and thus metabolic fingerprinting.

Competing interests

No competing interests declared.

Author contributions

Conceived and designed the experiments: KS, PV, GG, PM, IK, RF.Performed the experiments: MS, YY, MG, NR, ABZ, AL, RF.Analysed the data: MS, YY, MG, NR, ABZ, AL, IK, GG, PV, KS, RF.Wrote the paper: KS, PV, PM, MEK, GG, IK, MS, YY, NR, ABZ, AL, RF.

Transparency document

The Transparency document associated with this article can befound, in online version.

Acknowledgements

The authors gratefully acknowledge Dr. Christopher Berrie for criti-cal reading and appraisal of the manuscript, the Slovenian ResearchAgency for financial support (grants J4-7162, P1-0207, and P4-0053),and Mrs. Nina Orehar and Mrs. Sanja Čabraja for excellent technicalhelp. GG thanks CIBIO-UNITN for financing the Doctoral Scholarshipfor YY.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.bbamem.2016.08.015.

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