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Preferential invasion of mitotic cells by Salmonellareveals that cell surface cholesterol is maximalduring metaphase

António J. M. Santos1,2, Michael Meinecke3, Michael B. Fessler4, David W. Holden1 and Emmanuel Boucrot5,*1Section of Microbiology, MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, UK2Graduate Program in Areas of Basic and Applied Biology (GABBA), University of Porto, Portugal3Department for Biochemistry II, Georg-August-University Göttingen, 37073 Göttingen, Germany4Laboratory of Respiratory Biology, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA5Institute of Structural and Molecular Biology, Division of Biosciences, University College London, London WC1E 6BT, UK

*Author for correspondence (e.boucrot@ucl.ac.uk)

Accepted 28 April 2013Journal of Cell Science 126, 2990–2996� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.115253

SummaryCell surface-exposed cholesterol is crucial for cell attachment and invasion of many viruses and bacteria, including the bacteriumSalmonella, which causes typhoid fever and gastroenteritis. Using flow cytometry and 3D confocal fluorescence microscopy, wefound that mitotic cells, although representing only 1–4% of an exponentially growing population, were much more efficientlytargeted for invasion by Salmonella. This targeting was not dependent on the spherical shape of mitotic cells, but was instead SipB

and cholesterol dependent. Thus, we measured the levels of plasma membrane and cell surface cholesterol throughout the cell cycleusing, respectively, brief staining with filipin and a fluorescent ester of polyethylene glycol-cholesterol that cannot flip through theplasma membrane, and found that both were maximal during mitosis. This increase was due not only to the rise in global cell

cholesterol levels along the cell cycle but also to a transient loss in cholesterol asymmetry at the plasma membrane during mitosis.We measured that cholesterol, but not phosphatidylserine, changed from a ,20:80 outer:inner leaflet repartition during interphase to,50:50 during metaphase, suggesting this was specific to cholesterol and not due to a broad change of lipid asymmetry duringmetaphase. This explains the increase in outer surface levels that make dividing cells more susceptible to Salmonella invasion andperhaps to other viruses and bacteria entering cells in a cholesterol-dependent manner. The change in cholesterol partitioning alsofavoured the recruitment of activated ERM (Ezrin, Radixin, Moesin) proteins at the plasma membrane and thus supported mitoticcell rounding.

Key words: Mitosis, Cholesterol, Plasma membrane, Bacterial pathogenesis, Salmonella

IntroductionCholesterol is a key metabolic precursor and a determinant of

membrane fluidity and lipid lateral clustering (Maxfield and

van Meer, 2010). Cell surface-exposed cholesterol is crucial

for the cell binding and invasion of many viruses and

pathogenic bacteria, including Salmonella (Garner et al.,

2002; Lafont et al., 2002; Rawat et al., 2003). Salmonella

causes typhoid fever and a large proportion of gastroenteritis

cases in humans. To invade cells, Salmonella assembles a type

3 secretion system (T3SS), encoded in its pathogenicity island-

1 (SPI-1). T3SS acts as a ‘molecular needle’ that translocates

virulence proteins into the host cell to trigger entry of the

bacterium (Galán, 2001). The binding of Salmonella to its host

depends on the presence of cholesterol in the targeted

membrane (Garner et al., 2002). As the total cholesterol

levels double between G1 and G2 (Fielding et al., 1999),

we investigated whether Salmonella enterica serovar

Typhimurium (Salmonella) preferentially invades cells at

specific stages of their cell cycle. We found that Salmonella

invades mitotic cells preferentially because cell surface

cholesterol is maximal when cells divide.

Results and DiscussionSalmonella invades mitotic cells preferentially

Using flow cytometry and confocal microscopy, we showed that

Salmonella targeted mitotic cells more efficiently than cells in

other phases of their cell cycle (Fig. 1A–E; supplementary

material Fig. S1A–F and Movies 1, 2), consistent with a recent

observation (Misselwitz et al., 2011). This was true for the

different wild-type strains (12023, SL1344 and LT2) and cell

lines tested (supplementary material Fig. S1A). HeLa cells have

been widely used to study Salmonella entry but, as tumour cells

can have a perturbed cholesterol homeostasis (Gerlier et al.,

1982), we also used the diploid epithelial cell line RPE1. After an

infection of 10 minutes at a multiplicity of infection of 100, 60%

of G2 cells but 93% of mitotic cells (although representing only

1% of the total population) were infected by SL1344 (Fig. 1D,E;

supplementary material Fig. S1E). Less than 20% of interphase

but 60% of mitotic cells contained more than two bacteria per

cell (Fig. 1F; supplementary material Fig. S1G). As a

consequence, 26% of all intracellular bacteria in the sample

were inside mitotic cells (supplementary material Fig. S1H).

Mitotic cell preference was not due to the different shape

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(spherical) as it remained when the infection was performed on

cells rounded after detachment (Fig. 1G).

Surface cholesterol mediates targeting of mitotic cells bySalmonella

The translocon protein SipB binds directly to cholesterol and

mediates the cholesterol-dependent attachment of Salmonella to

targeted cells (Hayward et al., 2005). A mutant with a functional

T3SS but lacking effectors required for invasion (DsopE/E2/B)retained preferential binding to mitotic compared with interphase

cells (Fig. 2A–D), but, as expected, was unable to invade.

However, a mutant lacking a T3SS altogether (DprgH) or onelacking SipB did not preferentially bind to mitotic cells (Fig. 2A–

D). To test whether other bacterial cell surface proteins could

mediate the targeting to mitotic cells, we induced the binding and

internalization of the DsipB mutant by expressing Yersiniainvasin, which allows cell entry by a different mechanism than

the one used by Salmonella (Aiastui et al., 2010). Expression of

the invasin did not restore the preference of DsipB for mitoticcells (Fig. 2E), confirming that SipB mediates the targeting.

Cholesterol depletion abolished the preferential targeting to

mitotic cells (Fig. 2F–H; supplementary material Fig. S2). By

contrast, cholesterol enrichment, resulting in comparable levels

in interphase and control mitotic cells (Fig. 2F), significantly

reduced the preference of Salmonella for mitotic cells (Fig. 2H).

Thus, cholesterol and SipB mediate the preferential targeting

of mitotic cells by Salmonella. Interestingly, Salmonella

invades cycling but not quiescent stem cells in a SipB- and

Fig. 1. Salmonella invades mitotic cells preferentially. (A) Representative FACS profiles of RPE1 cells exposed to EGFP-expressing S. Typhimurium SL1344

(MOI 100) for 10 minutes, fixed and stained with propidium iodide (DNA). Gating for EGFPpositive cells identified uninfected and infected cells (left). DNA

profiles of total, uninfected and infected cells are shown (middle and right). Arrow shows the enrichment in the infected sample and corresponding depletion in the

uninfected population. (B) Interphase and mitotic cells (phospho-Histone H3-negative and -positive, respectively) were gated, and infected and uninfected cells

were identified as in A. (C) Representative images of RPE1 cells treated as in A, stained for DNA (blue) and a-tubulin (red). Arrow indicates a mitotic cell. Scale

bar: 10 mm. (D) RPE1 cells infected with 12023, SL1344 and LT2, stained and gated as in A and B. (E) Experiments carried out as in D. Ratios of uninfected toinfected cells at each stage of the cell cycle. A ratio of 1 (horizontal line) represents no preference. (F) Percentage of interphase cells (left) or mitotic cells (right)

infected by one or more than two bacteria [LT2 (light grey), 12023 (grey) or SL1344 (dark grey)] after 10 minutes, scored by immunofluorescence. (G) Ratios of

infected cells in mitosis to infected cells in interphase (identified as in B using MPM-2), in adhered or detached (trypsinized) cells. Data are mean 6 s.e.m. ns, not

significant. *P,0.05; ***P,0.001.

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cholesterol-dependent manner (Kolb-Mäurer et al., 2002; Yu

et al., 2009), giving a potential relevance for our findings to

infections in vivo.

Cell surface cholesterol is maximal during mitosis

The preferential targeting of mitotic cells suggested they could

have more cholesterol at their surface. Short (1 minute)

incubation of live cells with the fluorescent cholesterol-binding

compound filipin stained the plasma membrane without being

significantly internalized (Fig. 3A–C; supplementary material

Fig. S3). Because cholesterol (and thus filipin) can flip across the

bilayer (Garg et al., 2011), we also used a fluorescent ester of

polyethylene glycol-cholesterol (fPEG-cholesterol; Fig. 3D–F),

which partitions in cholesterol-rich domains. Because of its size,

PEG cannot flip across the plasma membrane (Madenspacher

et al., 2010; Sato et al., 2004) and thus quantitatively accounts

for the cell surface-exposed, endogenous cholesterol. In

addition, fPEG-cholesterol can be fixed and thus coupled with

phospho-Histone H3 (pH 3) labelling to identify mitotic cells by

flow cytometry. However, pH 3 labels all phases within mitosis

(prophase to cytokinesis) (Li et al., 2005), and because we

observed two populations (Fig. 3F), we also used 3D confocal

microscopy. It showed that the strongest signals corresponded to

metaphase cells and, thus, that plasma membrane and cell

surface exposed cholesterol were maximal during this stage

(Fig. 3G–J).

The large differences between G2 and metaphase are unlikely

to be due to changes in cholesterol synthesis; therefore, we

investigated its transbilayer repartition. Studies in neurons, red

blood cells and epithelial cells have determined that plasma

Fig. 2. Cell-surface cholesterol mediates the targeting of Salmonella to mitotic cells. (A) Scheme depicting the mutants used in the study. (B) RPE1 cells

incubated for 10 minutes with SL1344 wild-type or DsopE/E2/B, DprgH or DsipB. EGFP-expressing bacteria (green) stained for DNA (blue), a-tubulin (red) and

extracellular bacteria [CSA-1, yellow (green+red double-stained)]. Scale bars: 10 mm. (C) Ratios of bacteria adhered to mitotic cells to bacteria adhered interphasecells, determined by immunofluorescence. (D) Average number of bacteria in interphase cells (grey) or mitotic cells (red) infected as in B scored by

immunofluorescence. (E) Percentage of interphase (grey) or mitotic cells (red) infected by SL1344 (WT), or WT and DsipB mutant expressing Yersinia invasin

protein (‘+ invasin’), scored by flow cytometry. (F) Total cellular cholesterol levels upon cholesterol depletion (‘2cholesterol’) or loading (‘+cholesterol’) in

interphase or metaphase cells (‘Mitosis’). (G,H) Percentage of infected cells (G) and ratios of infected cells in mitosis to infected cells in interphase (H) in control,

‘2cholesterol’ or ‘+cholesterol’ samples, determined by flow cytometry. Data are mean 6 s.e.m. ns, not significant. *P,0.05; **P,0.01; ***P,0.001.

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Fig. 3. Cell surface cholesterol is maximal during mitosis. (A) Representative FACS profile of live RPE1 cells double-stained for DNA (DRAQ5) and plasma

membrane cholesterol (filipin). (B) Quantification of experiments carried out as in A in HeLa or RPE1 cells (N.4 experiments). (C) Representative image of a cell

sample used in A,B. Plasma membrane cholesterol (filipin, false-coloured green), DNA (blue). (D) Calibration of fPEG-cholesterol concentrations in living cells. A

concentration of 1 mg/ml was chosen for subsequent experiments as it represented the smallest saturating concentration. (E) Representative FACS profile of RPE1cells from control, cholesterol depleted (‘2cholesterol’) or cholesterol loading (‘+cholesterol’) stained with fPEG-cholesterol. (F) Representative FACS profile of

RPE1 cells stained with fPEG-cholesterol, DRAQ5 and phospho-Histone H3. G1, S and G2 cells were identified by DNA gating as in A and mitotic cells were

phospho-Histone H3-positive. (G) Scheme of imaging strategy. (H) Single-cell measurements of plasma membrane cholesterol. Representative image of a cell

sample used. Plasma membrane cholesterol (filipin, false-coloured green), DNA (blue). The values are integrals of the filipin signals from whole 3D stacks of images.

Stages of interphase (G1, S and G2) were determined by gating the integral DNA signals (2n, intermediate and 4n) of each cell, metaphase cells were identified by

morphology. (I) Single-cell measurements of cell surface cholesterol. Representative images of a cell sample used, fPEG-cholesterol (green), DNA (blue). The values

are integrals of fPEG-cholesterol signals from whole stacks of images. (J) Single-cell measurements of cell surface phosphatidylserine (FITC-Annexin V). Apoptosis

was induced by staurosposine (1 mM, 6 hours). (K) DHE fluorescence before (non-permeabilized) and after (permeabilized) TNBS in mitotic cells (rounded) orinterphase. The values were normalized to that of ‘interphase before’. (L) Remaining DHE fluorescence after TNBS corresponding to the fraction of inaccessible

(cytoplasmic) fraction of cholesterol at the plasma membrane. (M) Fluorescence from various concentrations of DHE loaded (1 hour, 37 C̊) on giant unilamellar

vesicles (GUVs). TNBS was applied to GUVs loaded with the highest concentration. (N) Model proposing the changes in cholesterol distribution at the plasma

membrane of mitotic cells. Scale bars: 10 mm. Data are mean 6 s.e.m. ns, not significant. *P,0.05; **P,0.01; ***P,0.001.

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membrane cholesterol is mainly (70–80%) located at the

cytoplasmic leaflet (Mondal et al., 2009; Schroeder et al.,

1991; Wood et al., 2011). Interestingly, phosphatidylserine and

phosphatidylethanolamine are mainly located at the cytoplasmic

leaflet during interphase but are transiently exposed at the

cleavage furrow during cytokinesis (Emoto et al., 2005). We

assessed cholesterol asymmetry using an established assay that

takes advantage of the fluorescence of the natural derivative of

cholesterol DHE and of its fast quenching by TNBS (Mondal

et al., 2009). As TNBS is cell impermeable, it only quenches

DHE located at the outer leaflet of the plasma membrane. Fast

3D stacks of images of DHE located at the plasma membrane

(,1 minute after loading) were acquired before and after TNBSapplication. In interphase cells, TNBS quenched ,20% of DHEsignals (Fig. 3K). As .90% signals were quenched inpermeabilized cells, we concluded that ,80% of DHEmolecules were protected and thus located in the inner leaflet

of the plasma membrane, in agreement with the literature

(Mondal et al., 2009). In mitotic cells, however, TNBS quenched

,45% of the DHE signals, indicating a partitioning closer to50:50 between both leaflets (Fig. 3K,L). This suggests that the

increase of cell surface-exposed cholesterol we observed with

fPEG-cholesterol on metaphase cells was due to a loss of

transbilayer asymmetry. The decreased quenching in mitotic cells

was not due to the differences in starting intensities as on giant

unilamellar vesicles (GUV) DHE signals were linear across a

range of concentrations encompassing those used in cells

(Fig. 3M). TNBS quenched ,50% of signals at the highestDHE concentration, as expected for an equal partitioning of the

probe between the two leaflets. Thus, we concluded that the

asymmetrical distribution of plasma membrane cholesterol ceases

during mitosis, exposing more at the outer surface, thereby

explaining why dividing cells are more susceptible to Salmonella

invasion. Interestingly, this was not observed for

phosphatidylserine during metaphase, which remained mostly atthe inner leaflet (Fig. 3J), indicative of a cholesterol-specific

property.

Changes in cholesterol asymmetry support ERM proteinrecruitment during mitosis

Loss of cholesterol asymmetry induces a concomitant decrease at

the inner leaflet (Fig. 3N), consistent with the decrease in the

cholesterol-binding protein caveolin-1 at the plasma membrane

during metaphase (Boucrot et al., 2011). To mitigate the decrease in

inner leaflet levels of cholesterol during mitosis, we enriched cells

with cholesterol complexes. Addition of cholesterol perturbed cell

compaction and rounding causing metaphase cells to become

significantly bigger (Fig. 4A; supplementary material Movies 3, 4).

The formation of the rigid cortical actin network that drives mitotic

compaction is mediated by RhoA (Maddox and Burridge, 2003) and

activated ERM (Ezrin, Radixin, Moesin) proteins that bridge actin

filaments to the plasma membrane (Kunda et al., 2008). Cholesterol

addition did not affect recruitment of Rho GTPases to the plasma

membrane during mitosis, but strongly decreased the recruitment of

activated ERM proteins (Fig. 4B,C; supplementary material Fig.

S4A,B and Movies 5, 6), potentially explaining the defect in mitotic

cell rounding caused by cholesterol addition. As ERM proteins are

not known to bind directly to cholesterol but do bind to

phosphatidylinositol-4,5-bis-phosphate [PtdIns(4,5)P2] (Roch et al.,

2010), a decrease in cholesterol in the inner leaflet during mitosis

might favour ERM proteins recruitment by supporting higher levels

of PtdIns(4,5)P2. Indeed, cholesterol enrichment led to a decrease in

PtdIns(4,5)P2 levels during metaphase (Fig. 4D), in agreement with

a previous report (Chun et al., 2010). Clarification of the

mechanisms involved will help our understanding of mitotic cell

rounding as well as the propensity of dividing cells to be

preferentially targeted by viruses and bacteria.

Materials and MethodsCell culture and cholesterol level manipulation

HeLa (ECACC 93021013) and hTERT-RPE1 (ATCC CRL-4000) cells weregrown at 37 C̊ in 5% CO2 in DMEM (Gibco) or DMEM:F12 HAM 0.25% (w/v)sodium bicarbonate (Sigma), respectively, supplemented with 1 mM glutamine(Sigma) and 10% fetal calf serum. Cholesterol levels were decreased and increasedusing 15 mM methyl-beta-cyclodextrin (MbCD) and 16 mg/ml water-solublecholesterol, respectively (both Sigma).

Antibodies and reagents

Antibodies used were: rabbit anti-phospho-HistoneH3 and anti-phospho-ERM,FITC-Annexin V (both Cell Signaling Technology), mouse anti-MPM-2

Fig. 4. Changes in cholesterol levels support ERM protein recruitment

during mitosis. (A) 3D rendering (grid side 13.5 mm) of metaphase cellsstained for cholesterol (filipin, false-coloured green), actin (red) and DNA

(blue) from control or cholesterol loading (‘+cholesterol’) samples. Bar graph

shows the metaphase cells volume in control or ‘+cholesterol’ samples.

(B) Immunofluorescence of pERM (green), actin (red) and DNA (blue) in

control or ‘+cholesterol’ samples. (C) Recruitment at the plasma membrane

of endogenous Rho GTPases and pERM in metaphase cells from control or

‘+cholesterol’ samples. (D) Immunofluorescence of PtdIns(4,5)P2 (green),

actin (red) and DNA (blue) in control or ‘+ cholesterol’ samples. Bar graph

shows the quantification. Scale bars: 10 mm. Data are mean 6 s.e.m. ns, notsignificant. *P,0.05; **P,0.01.

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(Millipore) and anti-a-tubulin (Sigma), goat anti-Salmonella (CSA-1) (KPL),mouse anti-RhoA 26C4, rabbit anti-Cdc42 P1 (both Santa Cruz Technologies),mouse anti-Rac1 (BD Biosciences), mouse anti-PIP2 2C1 (AbCam). Antibodyspecificity was verified on cells overexpressing the targeted proteins. Secondaryantibodies (Alexa488-, Alexa555- or Alexa647-conjugated), phalloidin-rhodamineand wheat germ agglutinin Texas-Red-x conjugate were from Invitrogen. DRAQ5(Biostatus), filipin III, dehydroergosterol (DHE), (2,4,6-trinitrobenzene sulfonicacid) TNBS, staurosporine, 1-butanol and propidium iodide were from Sigma.Synthesis and characterization of fPEG-cholesterol was described earlier(Madenspacher et al., 2010).

Bacterial strains and infectionsWild-type S. Typhimurium strains were 12023, SL1344 and LT2. SL1344 mutantswere: DsopE/sopE2/sopB/sptP (called DsopE/E2/B), DprgH (both from DrUnsworth; Imperial College London) and DsipB (generated using l redrecombinase method; 59-CGGAGACAGAGCAGCACAGTGAACAAGAAAA-GGAATAATTGTGTAGGCTGGAGCTGCTTCG-39 and 59-GCGGCGGGATT-TATTCCCACATTACTAATTAACATATTTTCATATGAATATCCTCCTTAG-39). Strains harboured pFPV25.1 plasmid carrying gfpmut3A (rpsM constitutivepromoter), or pRI203 plasmid, encoding for the Yersinia pseudotuberculosisinvasion region, as indicated.

Infection of adhered and detached cellsCells grown overnight were incubated with sub-cultured bacteria (OD600 1.8) atMOI 100, incubated in Earle’s Balanced Salt Solution (Gibco), at 37 C̊ for10 minutes, washed and fixed [3% paraformaldehyde (PFA) for 20 minutes]. Insome experiments, cells were detached using 0.025% Trypsin-EDTA (Sigma) justbefore infection.

Microscopy of fixed cellsCells infected or treated as described above were permeabilized (0.1% saponin),stained with primary and secondary antibodies, mounted using Aqua polymount(Polysciences Inc.) and imaged using a confocal laser-scanning microscope (LSM510 or 710, Zeiss). In some experiments, cells infected with EGFP-expressingSalmonella were labelled without permeabilization with CSA-1 to labelextracellular bacteria. Total fluorescence (sum of the Integrated Density of eachimages in 3D stacks) of cholesterol, Rho GTPases and pERM was quantified usingImageJ. Volumes of metaphase cells were measured using Huygens software(Scientific Volume Imaging) and visualized (3D rendering) using Volocity 5.0(PerkinElmer).

Flow cytometryCells on dishes infected as described above were permeabilized (0.08% Triton X-100 for 10 minutes) and stained with primary and secondary antibodies. Data werecollected on FACS CaliburTM (BD Biosciences) and analysed with FlowJo 7.6(TreeStar).

Cell surface cholesterol and asymmetry measurementsPlasma membrane cholesterol staining (filipin)Detached cells (flow cytometry) or cells grown on glass-bottom dishes(microscopy) were successively incubated in imaging buffer (DMEM withoutPhenol Red, 5% FBS, 15 mM HEPES) containing 5 mM DRAQ5 (10 minutes,37 C̊) and 5 mg/ml filipin for 1 minute (to limit the staining to the plasmamembrane) before measuring each samples live.

Cell surface cholesterol staining (fPEG-cholesterol)Cells grown on dishes or coverslips were stained successively with 5 mM DRAQ5(10 minutes, 37 C̊) and with 1 mg/ml fPEG-cholesterol for 20 seconds beforefixation with 4% PFA. Cells were then immunostained and prepared for flowcytometry or confocal microscopy.

Live-cell 3D stack imaging was performed on a spinning-disk confocalmicroscope (Eclipse TE-2000, Nikon; UltraVIEW VoX, Perkin-Elmer)controlled by Volocity 5.0. Flow cytometry and fixed-cell microscopy wereperformed on instruments described above.

Cholesterol asymmetry measurementsQuenching of DHE by TNBS was performed as described by Mondal et al.(Mondal et al., 2009). Each MatTek dishes were placed onto the microscope stage(37 C̊ chamber) and a field of view containing mitotic cells selected (brightfield).Cells were incubated for 1 minute in imaging medium containing 0.5 mM DHE-MbCD complexes (DHE in ethanol dried under argon and dissolved in buffercontaining MbCD and DHE at 8:1 mol:mol ratio, sonicated and incubatedovernight at 37 C̊ under gentle agitation), washed once and rapidly imaged (3Dstacks) using an epifluorescence microscope equipped with 350/50 and 420LPfilters (Chroma). TNBS (10 mM) was added and another set of images taken after1 minute. In some experiments, the cells were permeabilized using 40 mg/mldigitonin (Sigma).

GUVs were produced as described previously (Meinecke et al., 2013) usinga lipid mixture (25% phosphatidylcholine, 15% phosphatidylethanolamine,2% phosphatidylinositol, 8% phosphatidylserine, 10% sphingomyelin, 40%cholesterol) and imaged as described above.

Statistical analysis

Results shown are mean 6 standard error of the mean (s.e.m.). Statistical testingwas performed using Student’s t-test (continuous data, two groups), chi-square test(binomial data) or one-way ANOVA and Dunnett’s test (continuous data, at leastthree groups), as appropriate. ns, not significant; *P,0.05; **P,0.01;***P,0.001. n, number of cells, was .30,000 and .50 cells per FACS andmicroscopy experiment, respectively; N, number of experiments, was greater thanthree for each quantification.

AcknowledgementsWe thank Francisco Garcı́a-del Portillo (Centro Nacional deBiotecnologia) and Kate Unsworth for providing reagents; wethank Richard Hayward, Mair Thomas and António Ferreira forcomments on the manuscript; and the members of the Holden andBoucrot labs, especially Kieran McGourty, for discussions. E.B.thanks Harvey McMahon (Cambridge) for kind access to reagentsand equipment.

Author contributionsE.B. and D.W.H. designed the research, A.J.M.S. performed andanalyzed all the experiments with the exception of the filipin andDHE experiments that were performed and analyzed by E.B., M.M.produced the GUVs, M.B.F. provided fPEG-cholesterol and adviceon its use and E.B. supervised the project. The manuscript waswritten by E.B. with input of all the other authors.

FundingThis work was supported in part by Fundação para a Ciência eTecnologia (to A.J.M.S.); the Human Frontier Science Program;Deutsche Forschungsgemeinschaft [grant number SFB 803 to M.M.];the Intramural Research Program of the National Institutes of HealthNational Institute of Environmental Health Sciences [grant numberZ01 ES102005 to M.B.F.]; the Medical Research Council and theWellcome Trust [to D.W.H.]; and a Biotechnology and BiologicalSciences Research Council David Phillips Research Fellowship [toE.B.]. Deposited in PMC for release after 6 months.

Supplementary material available online at

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