DOI: 10.1002/cbic.201500045


Potential of Proapoptotic Peptides to Induce the Formation of Giant Plasma Membrane Vesicles with Lipid Domains Daniel Lauster,[a] Olalla Vazquez,[b] Roland Schwarzer,[a] Oliver Seitz,[b] and Andreas Herrmann*[a] We have established a method of preparing giant plasma membrane vesicles (GPMVs) by using cysteine mutants of the proapoptotic peptide (PAP) Ac-R7-GG-KLAKLAKKLAKLAK. A cysteine scan revealed that cytotoxicity and GPMV formation were dependent on the cysteine position within the PAP sequence. In comparison to GPMVs prepared by extensive treatment with paraformaldehyde (PFA) and dithiothreitol (DTT), our GPMVs were produced from HeLa cells at much lower concentrations of the blebbing agent. We found that only GPMVs derived from cysteine-containing PAP showed lipid phase separation. This membrane model was applied to investigate the phase partitioning of two relevant membrane proteins: influenza virus hemagglutinin (HA) and tetherin, which clamps budding HIV to infected cells. For tetherin, we show for the first time exclusion from cholesterol-rich domains in a GPMV model, thus documenting the potential of our approach for membrane-partitioning studies.

Since the discovery of lipid membrane domains with high cholesterol content, so-called “lipid rafts”, the relevance of lipid domains for membrane organization and functioning has become an extensively studied topic.[1] In biological membranes, the cholesterol-enriched domains are below the limit of optical resolution, and their characterization is rather difficult. To overcome this challenge, single-molecule techniques such as fluorescent correlation spectroscopy (FCS)[2] or total internal reflection fluorescence (TIRF) microscopy,[3] and superresolution microscopy have been introduced for studying living cells.[4] An alternative approach relies on micron-sized domains within membrane models; for a review see Czogalla et al.[5] Giant unilamellar vesicles (GUVs) of specific, defined lipid mixtures form large lipid domains that are visible by conventional fluorescence microscopy. The lipid mixtures can phase separate into a liquid-ordered phase (Lo), with saturated (sphingo-)lipids and cholesterol, and a liquid-disordered phase (Ld) with unsaturated phospholipids; both can condense to larger patches.[6, 7] [a] D. Lauster, Dr. R. Schwarzer, Prof. Dr. A. Herrmann Department of Biology, Molecular Biophysics Humboldt-Universit•t zu Berlin Invalidenstrasse 42, 10115 Berlin (Germany) E-mail: [email protected] [b] Dr. O. Vazquez, Prof. Dr. O. Seitz Department of Chemistry, Humboldt-Universit•t zu Berlin Brook-Taylor-Strasse 2, 12489 Berlin (Germany)

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To better mimic biological membranes, giant plasma membrane vesicles (GPMVs), or blebs, can be generated from living cells; this enables the investigation of protein–lipid or lipid– lipid interactions in a complex environment at the micron level.[8] Different protocols for generating GPMVs have been published. For example, GPMVs have been derived from rat basophilic leukemia (RBL) cells by treating cells with dithiothreitol (DTT) and paraformaldehyde (PFA).[9] In order to circumvent protein crosslinking, N-ethylmaleimide (NEM) has been proposed as an alternative chemical agent to induce GPMV formation.[10] Typically, lipid domains in GPMVs can be formed only upon cooling of the sample.[11] GPMV formation has been shown for different cell lines; however, the efficiency, that is, yield, of GPMVs varies significantly between cell lines. Usually, the bleb-triggering chemicals are added at a rather high concentration, typically in the millimolar range. Thus, it remains elusive, how well GPMVs reflect the organization of their origin membrane as, for example, extensive treatment with the electrophilic NEM or DTT leads to protein modifications.[12] A less-noticed blebbing agent was identified when HeLa cells were treated with the proapoptotic peptide [KLAKLAK]2 conjugated to the cell-penetrating peptide (CPP) octaarginine (R8).[13] [KLAKLAK]2 is an antimicrobial peptide that disrupts bacterial membranes.[13b] The formation of pores and destruction of the electrochemical gradient have been proposed as its mode of action; however, it does not affect the plasma membrane of eukaryotic cells. Therefore, Ellerby et al. linked this peptide to R8 to allow delivery of the peptide into eukaryotic cells.[14] They found that the peptide interacts with the mitochondrial membrane of HeLa cells and induces apoptosis.[13b, 15] Interestingly, this peptide conjugate was observed to cause blebbing even at micromolar concentrations.[14] As this concentration is much lower than that required to induce GPMV formation by NEM or DTT/PFA, proapoptotic peptides could offer a gentler way to generate GPMVs from native membranes. In our search for methods enabling the formation of giant blebs under conditions that do little harm to protein structure, we became aware of recent reports in which the cellular delivery of peptides was improved by incorporating thiol modifications.[16] We hypothesized that the “thiol effect” would provide an opportunity for the construction of improved blebbing agents. We surmised that the bleb-forming activity might depend on the position of the cysteine residue. Therefore, we envisioned a cysteine screen within the proapoptotic sequence [KLAKLAK]2 linked to the cell-penetrating heptaarginine (R7; Figure 1 A). We aimed to form GPMVs in which the separation


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Communications hour of exposure time. The less toxic BP2 and BP6 caused only a low amount of GPMVs, and BP9 did not induce any blebbing. This could suggest a correlation between toxicity and the potential for GPMV formation. However, this seems not to be a strict relationship. Despite similar toxicity, BP5 induced more blebbing than BP9. Note that, apart from the fact that millimolar concentrations were required, DTT/PFA-treated cells produced Figure 1. Sequence and toxicity of proapoptotic peptides linked to a cell-penetrating peptide. A) Sequences of the native proapoptotic peptide [KLAKLAK]2 and the cysteine mutants. R7 corresponds to the cell-penetrating pepmuch smaller membrane vesicles tide. B) Cell viability of HeLa cells 18 h post treatment with BP1 (*), BP4 (~), BP5 (&), BP9 (^), and BP24 (&) at varithan cells treated with BP (Figous concentrations. Error bars represent SD values (n Š 4). ure 2 A). Next, we studied lipid domain formation in GPMVs by using the lipid-like fluorophore R18, of lipid phases could be induced in order to study the domain which is known to partition preferentially into the Ld partition of two fluorescently tagged membrane proteins: the domain.[17] Having in mind that the purpose of our study was influenza virus spike protein hemagglutinin, and the cell-surto identify peptides that induce GPMV formation in large face protein tetherin. amounts, we selected BP4 and BP5 for a comparison with the We surmised that the GPMV-triggering potential of peptides original BP1. Although BP4 and BP8 showed similar cytotoxicimight correlate with their cytotoxicity. Therefore, we first charty, we excluded the latter as it was a highly aggressive “blebacterized the viability of HeLa cells upon exposure to R7-[KLAKLAK]2 and cysteine mutants. For the cysteine-lacking blebbing ber” that led to the GPMVs bursting early. The three studied peptide 1 (BP1), we found a half-maximal cytotoxicity concenGPMV isolates showed a homogenous R18 distribution at 21 8C tration, CC50, of 3.7 mm, which is similar to that determined by (Figure 2 B). Interestingly, we observed lipid phase separation Watkins and colleagues for R8-[KLAKLAK]2-treated KG1a with the cysteine-containing peptides BP4 and BP5 at 4 8C, but not with GPMVs derived from BP1 treatment. cells.[13a] Next, we studied lipid phase distributions of membrane proFrom the cysteine screen, we obtained candidates that were teins in lipid-domain-forming GPMVs. Two different membrane similar (BP3 and BP7) or more toxic (BP4 and BP8) than the proteins were tagged with the fluorescent protein YFP, and original sequence (Figure 1). Peptides BP2, -5, -6, and -9 their partitioning in lipid domains was studied. We transiently showed less cytotoxicity than BP1, and BP24 showed none. transfected HeLa cells with plasmids of the constructs; after The data thus show that the cytotoxicity is dependent on the 48 h, the cells were treated with BP4 or BP1. cysteine position within the peptide sequence. (Table 1) First, we studied the lipid domain preference of the influenNext, we characterized the potential of the various peptides za virus hemagglutinin (HA). The phase preference of this to induce GPMV formation. HeLa cells treated with some of membrane protein has been intensively investigated in biothe peptide variants are shown in Figure 2 A. By visual inspecchemical and biophysical assays.[18] The viral envelope origition, we qualitatively characterized the potential of the peptides to induce GPMV formation after one hour of treatment nates from the plasma membrane, the budding site of the inwith 4 mm peptide (Figure 2 A). Cells treated only with the cellfluenza virus. The lipid composition of the viral envelope repenetrating peptide BP24 did not show any bleb formation, sembles that of raft domains.[19] Important determinants of the and their morphology was similar to that of the untreated conHA structure for routing the protein to rafts have been implitrol. For the original peptide, BP1, we observed medium bleb cated;[10, 20] however, no clear preference of HA for Lo domains formation, whereas the most toxic compound, BP8, triggered could be observed, either for domain-forming model memthe formation of huge GPMVs, which were only stable for one branes (GUVs) or for cell-derived GPMVs.[21]

Table 1. Comparison of CC50 values with blebbing efficiencies of the individual blebbing peptides. Compound











CC50 [mm] blebbing

3.7 œ 0.3 +

8.2 œ 0.3 + /¢

4.0 œ 0.2 +

2.1 œ 0.2 ++

15.9 œ 0.1 ++

7.7 œ 0.1 + /¢

4.2 œ 0.2 ++

1.8 œ 0.3 +++

16.5 œ 0.1 ¢/¢

n.d. ¢

Half maximal cytotoxicity concentration (CC50), including the standard deviation (SD), was derived from a dose response fit of data from titration experiments. The blebbing potential was evaluated by visual inspection after 1 h exposure to 4 mm peptide. The blebbing efficiency was divided into groups: +++), strong (+ ++), medium (+ +), low (+ /¢), no (¢/¢) blebbing. very strong (+

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Figure 3. Fluorescently tagged influenza virus HA in GPMVs. HeLa cells were transfected with HA–YFP and treated with BP1 and BP4 (4 mm) 48 h post transfection. Cells treated with BP1 and BP4 showed intensive blebbing and HA–YFP incorporation into the GPMVs. Images were taken at 21 8C. Scale bars: 20 mm.

Figure 2. Peptide-induced GPMVs of HeLa cells and formation of lipid domains in GPMVs. A) HeLa cells treated with either DTT/PFA or 4 mm BP4, or BP24. Treatment with BP4 showed medium or strong bleb formation, whereas cells treated with BP24 did not form blebs. Cells treated with DTT (2 mm) and PFA (25 mm) formed small GPMVs. For a qualitative characterization of GPMV inducing potential, see Table 1. The cell membrane was stained with DiD (red fluorescence) to visualize GPMV formation (large spheres in the red channel). Black arrows indicate GPMVs. Scale bars: 20 mm. Images were taken by confocal microscopy. B) GPMV isolates from HeLa cells treated with BP1, BP4, or BP5. To visualize the Ld phase, cells were prelabeled with R18. GPMVs of both preparations showed a homogeneous R18 distribution at 21 8C. At 4 8C, GPMVs from BP4- or BP5-treated cells showed lipid phase separation, but not those obtained by BP1 treatment. Scale bars: 10 mm. The bright spot on the GPMV (BP1, 4 8C) corresponds to cell debris from its original cell. Images were taken by epifluorescence microscopy.

To study the lipid domain partition of HA in GPMVs, we expressed the fluorescent fusion protein HA–YFP in HeLa cells. Upon treatment of HA–YFP-expressing cells with BP1 or BP4, we observed efficient incorporation of the HA construct into GPMVs (Figure 3). As expected, at 21 8C both R18 and HA–YFP showed a rather homogenous membrane distribution (Figure 4). Upon cooling to 4 8C, a clear domain separation was achieved, as indicated by the segregation of the Ld marker R18 (Figure 4). However, we found two GPMV populations that differed in their pattern of lateral arrangement of HA–YFP. For the majority of GPMVs, ChemBioChem 2015, 16, 1288 – 1292

HA–YFP accumulated essentially in the Ld domain, whereas in other GPMVs of the same batch HA was also present in the Lo domain, even though it still preferentially enriched in the Ld domain. Obviously, as deduced from this different lipid domain partition behavior of HA–YFP, the GPMVs generated are heterogeneous with respect to the properties of their lipid domains. Our results suggest that there are at least two different types of Lo domain in blebbing-peptide-induced GPMVs: one type from which HA–YFP is excluded and one type to which HA– YFP has access. This observation of heterogeneous Lo domains supports the view that subtypes of Lo and/or Ld domains exist in biological membranes.[22] Notably, in our previous study on the lipid domain partition of HA in GUVs made from synthetic lipids, we never observed HA in Lo domains.[21] Evidently, the order difference between Lo and Ld domains in those GUVs is much more pronounced than in GPMVs, excluding HA–YFP from the Lo domain. This indicates that the order difference between lipid domains in biological membranes is smaller than for model systems. Similar observations and conclusions have been made previously upon comparison of the partition of various fluorescent lipid analogues between Lo and Ld domains in GUVs made from synthetic lipids and in GPMVs.[23] Finally, we emphasize that the redistribution of HA–YFP to Lo domains in GPMVs, even though partial, is basically in agreement with the lipid domain partition behavior of HA found for plasma membranes of intact cells,[10, 19–20] and recommend GPMVs as a system that mimics biological membranes much better than do GUVs in respect to lipid domains and their properties. However, the system is not perfect, as we found that GPMV formation does not result in a homogenous population of vesicles with respect to lipid domains. It might be that, in addition to the plasma membrane, intracellular membranes might also be involved to different extent in modulating the composition of GPMVs. Tetherin, also known as bone marrow stromal antigen (Bst2/ CD317), has been shown to tether budding HIV to the surface of infected cells.[24] Tetherin has membrane anchors at both protein termini: a transmembrane domain and the lipid-like membrane anchor glycosylphosphatidylinositol (GPI).[24–25] Both termini have competing properties in terms of lipid domain partition. As GPI is known to recruit to lipid rafts,[22, 26] a colocali-


Ó 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Communications Experimental Section Peptide synthesis, purification, and analysis: The peptides were prepared by automated solidphase peptide synthesis by using N-Fmoc-protected building blocks. We have recently published the synthesis, purification, and characterization of these compounds.[29] The concentrations of the samples were determined by HPLC and UV/ Vis. Cell culture: HeLa cells (Cell Culture Service) were maintained in Dulbecco’s modified eagle medium (DMEM) at 37 8C supplemented with glutamine (2 mm) and 10 % (v/v) fetal calf serum under 5 % CO2. Cell-viability assay: HeLa cells were seeded in a black 96-well plate to a density of 10 000 cells per well. The next day, the cells Figure 4. Lipid phase preference of I) influenza HA–YFP and II) tetherin-YFP in GPMVs after BP4 treatment at 21 were treated for 17 h at 37 8C with and 4 8C. GPMVs were prelabeled with R18 to visualize Ld domains. I) Upon cooling, GPMVs containing HA–YFP copeptides in a twofold serial dilulocalized with the Ld domain (first row), whereas other GPMVs show only a slight preference of HA–YFP for the Ld tion starting at 32 mm. Afterwards, domain if at all (second row). II) Tetherin-YFP colocalizes with the Ld domains at 4 8C. Scale bars: 10 mm. The bright Cell Titer-Blue reagent (20 mL per spots inside the GPMVs correspond to cell debris from their original cells. Images were taken after incubation at well, Promega) was added, and the the indicated temperature for 15–20 min. plates were incubated for 1 h at 37 8C. Fluorescence was detected with a plate reader (Fluostar zation of tetherin with lipid rafts has been suggested. In conoptima, BMG; lex = 520 nm, lem = 590 nm). Cell viability was calcutrast, the transmembrane domain of tetherin has been suglated from fluorescence intensity F according to Equation (1):

gested to act as a motif that excludes the protein from lipid rafts.[27] We found a clear preference of tetherin–YFP for the Ld domain in BP4-derived GPMVs at 4 8C (Figure 4); this supports the view that the transmembrane domain dominates the recruitment behavior of tetherin in lipid-domain-forming membranes but not the GPI anchor of the protein. Our results are in agreement with super-resolution microscopy investigations on intact cells for which tetherin was tagged with the fluorescent protein mEos.[28] In this study no colocalization of tetherin with other lipid raft proteins was found. In summary, this study shows that cysteine mutants of the R7-[KLAKKLAK]2 peptide are powerful inducers of GPMVs from HeLa cells. The unmodified peptide, which is delivered into cells through a heptaarginine repeat, furnished giant blebs. For most compounds, the results suggest at least a qualitative correlation between cell toxicity and the extent of GPMV formation. Of note is that the incorporation of cysteine residues at certain positions proved detrimental to the construction of GPMVs that allow separation of the lipid phases Lo and Ld. GPMVs induced by peptides such as R7-KLACKLAKKLAKKLAK might provide an alternative membrane model to study lipid domain partitioning of proteins, as demonstrated here for HA– YFP and tetherin–YFP fusion proteins.

ChemBioChem 2015, 16, 1288 – 1292

Cell viability ½%¤ ¼

F peptide ¢F medium   100 F untreated cells ¢F medium


Experiments were performed with two peptide synthesis batches, each at least twice in triplicate dilution series. Data were fitted with a dose–response curve by using GraphPad Prism 5 to obtain CC50 values. Plasmid constructs: The plasmid coding for HA–YFP is described in ref. [30]. Details for primers and cloning can be provided upon request. Plasmid encoding fluorescently labeled tetherin was provided by Martin Lehmann (FMP, Berlin Buch).[28] Cell transfection: One day before transfection HeLa cells were seeded at 200 000 per well in a 6-well plate format. On the next day, plasmid (4 mg) was transfected with TurboFect (8 mL, Thermo Fisher Scientific). For strong protein expression, cells were incubated for 48 h at 37 8C before GPMV preparation. Blebbing visualization: One day before the experiment, a m-Slide 8-well glass bottom dish (ibidi, Martinsried, Germany) was seeded at 100 000 HeLa cells per well. The next day, the cells were labeled with dialkylcarbocyanine D (DiD, 20 mm) at 4 8C and washed in PBS + + (137 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, 2 mm KH2PO4, 1 mm CaCl2, 0.5 mm MgCl2, pH 7.4). The wells were then treated independently for 1 h at 37 8C with BP1–BP9 and the control BP24 (4 mm) or with paraformaldehyde (PFA, 25 mm) and DTT (2 mm). In order to visualize blebbing, confocal images were taken with the focus set on the apical cell membrane.


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Communications Giant plasma membrane vesicle preparation: Cells transfected for 48 h at an approximate confluency of 80 % were washed three times with PBS + + . GPMV formation was either performed with PFA (25 mm) and DTT (2 mm) according to ref. [31] or with proapoptotic peptides (4 mm) at 37 8C for 1 h. GPMVs were labeled with either octadecyl rhodamine B chloride (R18, 1 mm) or DiD (20 mm). Cell nuclei were stained with 4’,6-diamidin-2-phenylindol (DAPI, 1 mg mL¢1). Afterwards, cells were again washed twice with PBS + + , and then twice with GPMV buffer (150 mm NaCl, 25 mm HEPES, 0.901 mm CaCl2, pH 7.4). The supernatant was transferred to 1.5 mL centrifugation tubes and stored at 4 8C for at least 1 h to allow the GPMVs to settle down. Twenty objects from each of two independent GPMV preparations with BP1, BP4, and BP5 were recorded and analyzed at either 21 or 4 8C. Phase separation was detected by inspecting lateral R18 distribution. Fluorescence microscopy and lipid phase separation: Epifluorescence microscopy was conducted on an inverted Olympus IX-81 microscope equipped with a cooled monochrome CCD camera. Images were obtained with a 20 Õ UPlanFL air objective (numerical aperture 0.75) by using U-MWNiba filters (BP470–495, BA520IF, DM510–550) for green fluorescence such as YFP, and a U-MWG2 filter set (BP510–550, BA590, DM570) for red fluorescence like DiD or R18. Depending on the individual protein concentration and labeling efficiency, images were obtained with exposure times from a half to several seconds for the green channel and < 0.5 s for the red channel. Lipid phase separation was studied in an air-conditioned room at 21 8C, and cooling was achieved with a peltier element.

Acknowledgements O.V. is grateful for a Marie Curie Fellowship. We thank Martin Lehmann (FMP, Berlin Buch) for the tetherin–YFP plasmid. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) through SFB 765. Keywords: cysteine · giant vesicles · hemagglutinin · influenza virus · phase separation · tetherin [1] K. Simons, E. Ikonen, Nature 1997, 387, 569 – 572. [2] C. Marquer, S. L¦vÞque-Fort, M.-C. Potier, J. Visualized Exp. 2012, e3513. [3] a) A. Asanov, A. Zepeda, L. Vaca, Biochim. Biophys. Acta Mol. Cell Biol. Lipids 2010, 1801, 147 – 155; b) H. Schneckenburger, Curr. Opin. Biotechnol. 2005, 16, 13 – 18. [4] D. M. Owen, A. Magenau, D. Williamson, K. Gaus, Bioessays 2012, 34, 739 – 747. [5] A. Czogalla, M. Grzybek, W. Jones, U. Coskun, Biochim. Biophys. Acta Mol. Cell Biol. Lipids 2014, 1841, 1049 – 1059. [6] H. J. Kaiser, D. Lingwood, I. Levental, J. L. Sampaio, L. Kalvodova, L. Rajendran, K. Simons, Proc. Natl. Acad. Sci. USA 2009, 106, 16645 – 16650. [7] V. Betaneli, R. Worch, P. Schwille, Chem. Phys. Lipids 2012, 165, 630 – 637.

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Potential of Proapoptotic Peptides to Induce the Formation of Giant Plasma Membrane Vesicles with Lipid Domains.

We have established a method of preparing giant plasma membrane vesicles (GPMVs) by using cysteine mutants of the proapoptotic peptide (PAP) Ac-R7-GG-...
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