Directly Observing the Lipid-Dependent Self-Assembly and Pore

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Directly observing the lipid-dependent self-assembly and pore-forming mechanism of the cytolytic toxin listeriolysin O Estefania Mulvihill, Katharina van Pee, Stefania A Mari, Daniel J. Müller, and Özkan Yildiz Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.5b02963 • Publication Date (Web): 24 Aug 2015 Downloaded from http://pubs.acs.org on August 26, 2015

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Directly observing the lipid-dependent self-assembly and pore-forming mechanism of the cytolytic toxin listeriolysin O

Estefania Mulvihill‡,#, Katharina van Pee§,#, Stefania A. Mari‡, Daniel J. Müller‡,*, and Özkan Yildiz§,* ‡

Department

of

Biosystems

Science

and

Engineering,

Eidgenössische

Technische Hochschule (ETH) Zurich, Mattenstrasse 26, 4058 Basel, Switzerland §

Department of Structural Biology, Max-Planck-Institute of Biophysics, Max von

Laue Str. 3, 60438 Frankfurt am Main, Germany

Running

title:

Lipid-dependent

dynamic

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of

LLO

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ABSTRACT Listeriolysin O (LLO) is the major virulence factor of Listeria monocytogenes and a member of the cholesterol-dependent cytolysin (CDC) family. Grampositive pathogenic bacteria produce water-soluble CDC monomers that bind cholesterol-dependent to the lipid membrane of the attacked cell or of the phagosome, oligomerize into prepores and insert into the membrane to form transmembrane pores. However, the mechanisms guiding LLO towards pore formation are poorly understood. Using electron microscopy and time-lapse atomic force microscopy, we show that wild-type LLO binds to membranes, depending on the presence of cholesterol and other lipids. LLO oligomerizes into arc- or slit-shaped assemblies, which merge into complete rings. All three oligomeric assemblies can form transmembrane pores and their efficiency to form pores depends on the cholesterol and the phospholipid composition of the membrane. Furthermore, the dynamic fusion of arcs, slits and rings into larger rings and their formation of transmembrane pores does not involve a height difference between prepore and pore. Our results reveal new insights into the pore-forming mechanism and introduce a dynamic model of pore formation by LLO and other CDC pore-forming toxins.

Keywords: transmembrane pore formation; cholesterol-dependent cytolysins (CDC);

pore-forming

toxins

(PFT);

erythrocyte

ghosts;

brain

lipids;

phospholipids


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A large number of bacterial pathogens and some higher organisms have developed pore-forming toxins (PFT) to perforate the membranes of host cells or of intracellular organelles for the purpose of attacking or defending.1-4 PFTs can be small molecules,5-8 peptides,9-11 or proteins and are classified based on the secondary structure elements they insert into the membrane.12 α-PFTs fold hydrophobic α-helical stretches that span cellular membranes to form transmembrane channels.13 β-PFTs, after having been secreted by the bacteria as soluble proteins, bind receptors of the host cell membrane and insert amphipathic β-hairpins into the lipid membrane.14-17 Once inserted these βhairpins are thought to assemble into transmembrane β-barrels, which form transmembrane pores with diameters varying from 0.5 nm to 100 nm.18,19 The largest family within the β-PFTs are the cholesterol-dependent cytolysins (CDC).20 CDC monomers range from 47 to 60 kDa21 and share 40–70% sequence identity, suggesting that they all adopt very similar tertiary structures and have similar modes of action.22 CDCs are secreted as water-soluble monomers and bind to the target membrane, where 35–50 monomers oligomerize into ring-shaped structures to form transmembrane pores.23 The lipid composition of the target membrane influences the binding and activity of CDC, which is mainly attributed to the accessibility of cholesterol to CDC in the membrane.24

The sequence and structural similarity within the bacterial CDCs suggests a common pore-forming mechanism. Studies on perfringolysin25 and pneumolysin18 indicate that some CDCs form circular prepores before they insert into the membrane to form the pore, while other CDCs like streptolysin O26,27 and suilysin28 insert into the membrane and form pores before ring formation is complete. Even though pore formation intermediates were stabilized by low temperature25 or locked by disulfide bonds,25,29 the "preporeto-pore" model was for a long time the most accepted mechanism for CDC pore formation. The "prepore-to-pore" model does not explain the step-wise increase of the CDC ion-conductivity observed by electrophysiology30,31 or the presence of arc-shaped transmembrane pores shown by atomic force microscopy (AFM)32,33 and electron microscopy (EM).28,34,35 Therefore, the question whether the CDCs conform to the "prepore-to-pore" or the "growing pore" mechanism29 ACS Paragon Plus Environment

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remains unsolved. Recently, it was shown that listeriolysin O (LLO), a member of the CDC family, which had been mutated near a pH-sensitive cluster of charged residues, assembles into arcs and that these arcs fuse into ringshaped structures, forming transmembrane pores at room temperature.30 However, how wild-type LLO (LLOWT) assembles transmembrane pores at physiological temperature (37°C) and how the lipid composition of the membrane affects transmembrane pore formation has not yet been addressed.

We recently reported the X-ray structure of LLO and showed the interactions between monomers in oligomeric structures.35 Nevertheless, additional data are required to understand the mechanisms of membrane insertion and pore formation by LLO. Here we explore the pore-forming mechanism of LLO in different membrane systems by high-resolution EM and time-lapse AFM at physiological temperature (37°C). We observe several distinct steps of pore formation, in which LLO monomers bind to lipid membranes in a cholesterol-dependent manner and assemble into larger arc-, slit- and ring-shaped structures. This assembly is time-dependent and arcs, slits and rings are observed to reassemble dynamically into larger rings. Our experiments also show that the lipid composition of the membrane affects the assembly of the LLO oligomers into larger structures. We further observe that each of the oligomeric LLO structures can form transmembrane pores of very different size, which explains the step-wise conductivity increase observed in electrophysiological measurements. Whether or not these structures are able to form pores depends on the cholesterol and the phospholipid composition of the membrane. We show that pore formation by LLO is not accompanied by a vertical collapse of the prepore, such as previously reported for another CDC the perfringolysin O (PFO) from Clostridium perfringens.36


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RESULTS and DISCUSSION Tracking the oligomeric states of LLO. To understand the LLO mechanism, it is essential to characterize the individual steps of pore formation. These steps are (1) membrane binding, (2) oligomerization, (3) ring formation, and (4) membrane insertion. To investigate these individual steps in a native lipid environment, we incubated hemoglobin-free erythrocyte ghosts with LLO and visualized the resulting structures by negative stain EM.

Upon binding to the erythrocyte ghost membranes, LLO oligomerized and assembled into arc-, slit- or ring-shaped structures of variable diameter (Figure 1A-C). The presence of fragmented arcs, slits, and rings next to the erythrocyte ghosts indicate that LLO oligomers can detach from the ghosts. Possibly detachment occurred before LLO oligomers were able to insert into the membranes and form complete rings. The region inside the rings and slits was darker, indicating a stain-filled deformation or hole of the membrane, but the region inside the arcs did not differ from the surrounding membrane. This observation suggests that the slits and rings form transmembrane pores whereas the arcs do not (Figure 1A-C). This observation is consistent with our previous model35 by which only closed LLO rings can form transmembrane pores, in accordance with the growing-pore mechanism.29

Biological membranes, such as the erythrocyte ghosts used in our studies,

can

oligomerization

vary and

in

lipid

pore

composition, formation.

We

which

affects

therefore

LLO

binding,

investigated

the

oligomerization of LLO on artificial planar lipid monolayers composed of dioleoyl phosphatidylcholine (DOPC) and cholesterol at a molar ratio of 1:1 (Figure 1DF). We observed considerably more ring- and arc-shaped structures compared to erythrocyte ghosts. The ring diameter (54 ± 3 nm, average ± SD; n = 43) showed less variation compared to rings on erythrocyte ghosts (51 ± 8 nm, n = 37). In addition to complete rings, we also observed incomplete rings and arcs of various curvature radii and dimensions. The stain density did not convincingly indicate the presence or absence lipids within the rings or arcs and therefore did not show reliably whether transmembrane pores were formed. ACS Paragon Plus Environment

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Furthermore, although the arcs often assembled into clusters, they rarely formed slits as observed on erythrocyte ghost membranes. The oligomeric state of LLO varies with time. Our observations so far show that LLO oligomers can form arcs, slits, and rings on erythrocyte ghosts and planar lipid layers. The same structures were found in membrane-free regions on the EM grids. We therefore asked whether their formation is a dynamic process or whether LLO oligomers preassemble and insert into membranes where they remain static. To answer this question, we investigated the timedependent membrane binding and oligomerization of LLO on planar lipid monolayers (Figure 2). After only five minutes at 37°C, we observed the formation of LLO arcs and rings on the lipid monolayers. Arcs often formed chains. The stain density around these chains or inside the rings did not differ from the surrounding lipid areas, suggesting that these oligomers did not form transmembrane pores. After 15 minutes the number of rings increased but most of them were fragmented or deformed. Following 30 minutes of incubation, the lipid layers were densely covered with arcs and rings, which were frequently fragmented. Some of the rings, including a number that were incomplete or deformed, were darker on the inside. However, because of possible staining artifacts the EM images do not show conclusively whether or not there were lipid layers within these rings.

Cholesterol- and lipid-dependent assembly of LLO oligomers and transmembrane pore formation. To characterize the assembly of the transmembrane pores convincingly we applied atomic force microscopy (AFM).33,37 We incubated unresealed hemoglobin-free erythrocyte ghosts with LLO at 37°C and visualized the resulting structures at 37°C by AFM (Supporting Information, Figure S1). The initial evaluation of the AFM topographs showed arc-, slit- and ring-shaped LLO structures, which confirmed the EM observations. However, these experiments do not show how LLO assembles into oligomers and forms transmembrane pores. To investigate this process systematically we imaged LLO assembly and pore formation on supported lipid membranes (SLMs). In an initial control experiment we adsorbed LLO to freshly cleaved mica, which we used as sample support in our AFM studies


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(Supporting Information, Figure S2). LLO readily adsorbed to the negatively charged hydrophilic mica surface as monomers or small oligomers but did not assemble into arcs or rings. After this, we adsorbed vesicles composed of palmitoyl-oleoyl phosphatidylcholine (POPC) or E. coli polar lipids onto freshly cleaved mica, where they fused into homogeneous SLMs.38,39 In this and in the following SLM preparations we took particular care that the lipid membranes showed no apparent defects such as holes, cracks or exposed rims, which might cause artificial protein insertion or assembly. LLO binding was not observed in the AFM topographs of these cholesterol-free SLMs, even after prolonged incubation with LLO for 30 minutes at 37°C (Supporting Information, Figure S2).

Next, we assembled SLMs from porcine brain total lipid extract and incubated the SLMs with LLO at 37°C for 30 minutes. LLO bound to these SLMs and formed densely packed patches of arc- and slit-shaped oligomers (Figure 3A,B). Occasionally, arcs and slits combined into ring-shaped structures, which, however, were not perfectly circular. Height analysis indicates that these structures protrude 7.7 ± 0.8 nm (average ± SD; n = 264) from the SLM surface and that they occasionally form transmembrane pores (Figure 3C, example 1). The brain total lipid extract contains phospholipids and cholesterol. However, ≈ 60% (wt) of its lipid content is unknown. To examine the role of cholesterol in LLO membrane binding, oligomerization, and transmembrane pore formation, we thus supplemented to the brain total lipid extract with 30 wt% cholesterol, prepared SLMs, and incubated them with LLO as described above. The AFM topographs show that increasing the percentage of cholesterol results in more arc- and slit-shaped LLO oligomers, whereas ring-like oligomers are rarely observed (Figure 3D,E). The height of 7.7 ± 0.4 nm (n = 199) of the LLO oligomers is similar to that found on brain total lipid extract SLMs without supplementing cholesterol. The height profile also shows a perturbed membrane around these oligomers, but no indications that they form transmembrane pores (Figure 3F).

In comparison to brain total lipid extract, synthetic lipids offer the advantage of preparing lipid mixtures having defined compositions. Therefore,


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to better address the role of lipids in LLO binding, oligomer and pore formation, we prepared SLMs of POPC and of cholesterol at a molar ratio of 7:3. These SLMs were then incubated with LLO at 37°C. AFM topographs show that LLO assembles into densely packed arc-, slit- and occasionally ring-shaped oligomers on these SLMs (Figure 3G,H). Ring-shaped structures appeared in shape comparable to those observed on SLMs of brain total lipid extract (Figure 3A-E). The structures protruded 8.4 ± 0.8 nm (n = 186) from the POPC SLM, similar to those observed before. In contrast to the SLMs prepared with brain total lipid extract (Figure 3C,F), the height profiles of these arc- and slitshaped structures show more frequently the formation of transmembrane pores in the POPC SLMs (Figure 3I). An increase in cholesterol concentration from 30 to 50 mol% of the POPC SLMs resulted in a five-fold increase in the number of ring-shaped structures (Figure 3J,K), which protruded 8.0 ± 0.9 nm (n = 57) from the SLM surface (Figure 3L), comparable to those observed on SLMs with different lipid and cholesterol compositions (Figure 3C,F,I). The height analysis of these arc-, slit- and ring-shaped structures further showed that the majority of them form pores through the SLMs (Figure 3L).

We further investigated the interaction of LLO with SLMs composed of different lipid mixtures, namely with SLMs composed of POPC, palmitoyl-oleoyl phosphatidylserine (POPS) and cholesterol at a molar ratio of 1:1:2 and with SLMs composed of POPS and cholesterol at a molar ratio of 1:1. As described in the previous experiments the POPS containing SLMs were incubated with LLO at 37°C. Compared to the experiments made on SLMs before (Figure 3AL), in SLMs composed of POPC, POPS and cholesterol the average number of arcs, slits and rings decreased considerably (Figure 3M,N). Nevertheless, irrespective of their low occurrence, these oligomeric structures could form transmembrane pores (Figure 3O). In the absence of POPC, on SLMs prepared only with POPS and cholesterol (Figure 3P,Q), we observed even fewer LLO oligomers. We could not observe these oligomeric structures having formed transmembrane pores in the SLMs (Figure 3R).

Our AFM experiments show that binding and assembly of LLO oligomers depends not only on cholesterol, but also on other lipids. It has been reported


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that cholesterol is crucial for the binding of CDCs to the lipid membrane.20,22 Indeed, we here observe that LLO does not bind cholesterol-free SLMs like those composed of E. coli polar lipids or of pure POPC, and requires cholesterol to bind to POPC. Furthermore, supplementing brain total lipid extract with cholesterol increases LLO binding. Once bound to the lipid membrane LLO readily assembles into arc-, slit- and ring-shaped oligomers. Each of these LLO oligomers can form transmembrane pores. Apart from this cholesterol dependence, we observe that the phospholipid POPC promotes the formation of transmembrane pores. It is worth to note that although on brain lipid extract the total number of LLO oligomers is much higher than on SLMs made from POPC and cholesterol, they form fewer transmembrane pores. On the other hand we observed that even in the presence of cholesterol the phospholipid POPS reduces the membrane binding and oligomerization of LLO, as well as the subsequent pore formation in the membranes. Time-lapse imaging of LLO assembly and pore formation. To follow the time course of LLO oligomerization and transmembrane pore formation, we imaged SLMs composed of POPC and cholesterol (1:1 molar ratio) by timelapse AFM while incubating the SLMs with LLO at 37°C. To ensure complete and defect-free SLMs we imaged the SLMs before adding LLO. Within 30 minutes of incubation LLO formed arc-, slit-, and ring-shaped structures on the SLM (Figure 4). Over time these arcs, slits and rings fused to form larger ringshaped structures. Such time-lapse AFM experiments show the self-assembly of LLO into ring-shaped structures (Supporting Information, Figures S3 and S4). After sufficiently long time intervals the number of LLO arcs decreased and that of ring-shaped structures increased. In rare cases we observed the fragmentation and disassembly of ring-shaped structures into arcs and slits. More frequently it was observed that ring-shaped structures fused with arcs, slits or other ring-shaped structures to form larger rings. Such fusion processes were largely independent of the fact whether the arc- or ring-shaped structures had already formed transmembrane pores. However, with increasing time not only the transition of arc- and slit-shaped structures into rings increased but also the percentage to which these structures formed transmembrane pores.


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The time-lapse topographs of LLO oligomers also revealed that arcshaped (7.8 ± 0.8 nm, n = 139), slit-shaped (7.7 ± 0.8 nm, n = 72) and ringshaped (7.9 ± 0.8 nm, n = 42) structures protruded by similar heights from the membrane surface. During the entire course of the time-lapse AFM experiments, the majority of the LLO structures did not change height within the accuracy of the measurements. This suggests that the fusion process of arcs and slits into rings and of rings into larger rings as well as the formation of transmembrane pores is not associated with a vertical height change in the LLO monomers. LLO oligomers form pores in the absence of a vertical collapse. Transmembrane pore formation in the membrane of target cells or phagosomes and their subsequent lysis is the main function of LLO and other CDCs from pathogenic bacteria. In addition to their role in forming large membrane pores, the CDCs are involved in various other processes during host cell infection. Among many other functions (reviewed in ref.40) LLO induces influx of extracellular calcium31 and phagosome maturation by pH uncoupling.35,41 These processes would require pores or channels much smaller than the large pores required for lysis. Recently, we showed by EM that LLO forms arc- and slitshaped structures on erythrocyte membranes.42 These structures might form smaller pores or channels. Similarly, the CDCs suilysin, pneumolysin and streptolysin also form arc-shaped structures of different size, as shown by AFM and EM.26,28 Our time-lapse high-resolution AFM topographs show that not only the ring-shaped structures, but also the arc- and slit-shaped structures, which LLO forms on the membranes, can be considered as prepores that are able to form transmembrane pores having very different sizes (Figures 3 and 4). The LLO prepores on the membrane surface appear to be less mobile than recently characterized suilysin prepores.28 One reason for this difference may be that in these previous AFM-based studies suilysin has been locked by introducing a disulfide bridge in domain 3.28 Apparently, suilysin with a locked domain 3 diffuses fast and appears as streaks in AFM topographs until the disulfide bridge is reduced. On the other hand this result may indicate that a conformational change of the suilysin domain 3 – or more precisely the flexibility of the membrane-inserting helices in domain 3 – is required to form a proper


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prepore. Thus, the cysteine-locked mutant of suilysin might represent a membrane-attached conformation, which upon reduction of the cysteines converts to the prepore conformation (Figure 3F in ref.28) and subsequently to the transmembrane pore. Consequently, if we consider the "prepore" state of PFOY181C as a membrane-attached state,36 we would interpret the reported vertical collapse of PFOS190C/G57C,36 as prepore formation rather than pore formation. Indeed, the absence of a vertical collapse would be in good agreement with our observation that the height of unmodified, wild-type LLO protruding from the membrane is the same for transmembrane pores and prepores. It might also indicate that the cysteine-mutations may prevent proper formation of the prepores and lock PFO in a conformation that might be artificial and does not occur in the pore-forming process of the native toxins. However, we cannot exclude that different members of the CDC family insert into membranes in different ways, one of which may include a vertical height collapse whereas the other does not. High-resolution AFM imaging of LLO oligomers. To further investigate the LLO self-assembly into oligomers we recorded high-resolution AFM topographs of membrane-bound LLO oligomers. To reduce lateral mobility during AFM imaging, we wanted to densely pack the LLO oligomers. We thus prepared SLMs from brain total lipid extract supplemented with 30 wt% cholesterol. The SLMs were incubated for 30 minutes with LLO at 37°C before the temperature was reduced to room temperature during data collection. This reduced the mobility of the LLO oligomers and improved the quality of the AFM topographs, which nicely show substructures of the arc- and slit-shaped LLO oligomers. In average these substructures showed a lateral distance of 2.6 ± 0.3 nm (n = 59), which matches the distance of LLO monomers in the linear rows found in 3D crystals.35 Based on structural insights from AFM and EM we created a model of LLO oligomerization and pore formation on target membranes (Figure 5). According to this model, LLO binds to the membranes and forms arc- slit-, and ring-shaped oligomers, which are able to insert into the membrane and form transmembrane pores. These LLO oligomers can fuse with each other into larger rings.


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CONCLUSION Our time-lapse AFM images show that arc- and slit-shaped LLO oligomers can fuse into larger rings. It was also observed that rings fuse with other arc-, slitand ring-shaped structures to grow and thereby to increase the size of the prepore or transmembrane pore. This fusion of arc-, slit- and ring-shaped prepores and pores was proposed as a combined model43 and represents a hybrid model of the two alternative mechanisms of “growing pore” and “preporeto-pore” transition.29 It may be necessary to extend these models, as our data show that the arc- and slit-shaped LLO oligomers can also form transmembrane pores. Thus, the prepore-to-pore transition is not restricted to complete ringshaped prepores but appears general to all arc-, slit- and ring-like oligomers formed by LLO. Whether or not LLO oligomers can form transmembrane pores clearly depends on the lipid composition of the membrane. We show the probability of observing transmembrane pores is highest in membranes having high cholesterol and POPC content (Figure 3). Furthermore, the high-resolution time-lapse AFM images suggest that the transition of arc-, slit- and ring-shaped prepores or pores to form larger rings is a kinetic process. The ratio of arcs, slits and rings changes with time in favor of the ring-shaped structures. The lipid composition of the membrane appears to shift this equilibrium, which suggests a thermodynamic component. We observed a maximum of ring-shaped structures in erythrocyte membranes and in POPC membranes supplemented with 50 mol% cholesterol.

The presence of small pores that might be formed by arcs, slits and rings could explain some observations, such as the formation of transient LLO pores of different size31 that permit extracellular Ca2+ influx into the cell. At sublytic concentration, smaller arc-, slit- or ring-shaped pores may not find each other easily to form the larger ring-shaped pores necessary for cell lysis. Our findings are in good agreement with electrophysiological properties of the CDCs in artificial planar lipid membranes44 and in membranes of nucleated cells45 where the CDCs form pores of different conductivities.


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A profound knowledge about the mechanisms by which bacteria utilize CDCs in virulence is indispensable for the development of drugs and vaccines to combat these pathogens. Here we extend the proposed mechanisms for the pore formation by CDC into a combination of the “growing-pore” and the “prepore-to-pore” mechanism. Future experiments will show whether all CDC share a general mechanism, whether some members prefer one or the other mechanism, or whether the preferred mechanism for the pore formation is determined by environmental factors.


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MATERIALS and METHODS

Protein Expression and Purification. LLO was heterologously expressed and purified as described.35 The LLO gene containing an N-terminal His6-tag as well as a thrombin cleavage site but lacking the secretion signal sequence (1–24) was cloned into the pET15b vector and expressed in E. coli BL21 (DE3) cells, which were grown in TB medium. After cell disruption with a microfluidizer (M110L, Microfluidics Corp, Westwood, MA, USA) and a centrifugation step at 12.000 rpm for 1 h, the supernatant containing the His6-tagged LLO was loaded onto a Ni2+-NTA affinity column. The protein was eluted through the addition of thrombin over night at 4°C. LLO was further purified by size exclusion chromatography using a Superdex 200 column and 150 mM NaCl, 50 mM TrisHCl, pH 7.0, as a running buffer. The LLO purity was controlled by SDS-PAGE and the pure protein was concentrated to 5 mg ml–1. Specimen Preparation, Electron Microscopy and Image Processing. Hemoglobin-free resealed ghosts were prepared as described before.46 Briefly, the ghosts were diluted (1:20) in resealing buffer (130 mM KCl, 10 mM NaCl, 3 mM MgCl2, 11 mM Tris-HCl, pH 7.6) and incubated for 30 minutes at 37°C. Afterwards, the ghosts were centrifuged for 20 minutes at 12.000g and the pellet was resuspended in reaction buffer (150 mM NaCl, 50 mM Na3PO4, 5 mM DTT, pH 6.6). The ghosts were then incubated with LLO (0.5 µg ml–1) for 30 minutes at 37°C. For negative staining the ghosts were pipetted onto a carboncoated EM grid, washed twice with water and incubated with 1% uranyl acetate.

Planar lipid layers and LLO were incubated in Teflon well plates. Therefore 6 µg ml–1 LLO in reaction buffer (150 mM NaCl, 50 mM Na3PO4, 5 mM DTT, pH 6.6) was transferred into one of the wells and a droplet of a DOPC and cholesterol (1:1 molar ratio, 0.5 mg ml–1) solution in chloroform was put on top of it. The lipid layer was formed through chloroform evaporation and covers the droplet at the liquid air interface. After the specified incubation time at 37°C in a humid environment a carbon-coated 400 mesh copper EM grid was used to lift the lipid layer from the droplet surface.


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After washing twice with water, the membranes were stained with 1% uranyl acetate. The grids with the ghosts and membranes were imaged with a FEI Tecnai Spirit (120 kV) transmission electron microscope equipped with a Gatan 2K x 2K CCD camera at a magnification between 17.000–45.000x and 0.8–1.0 µm defocus. Atomic Force Microscopy (AFM). Force-distance curve-based AFM (FDbased AFM)47 was performed using a Nanoscope Multimode 8 (Bruker, Santa Barbara, USA) operated in the PeakForce Tapping mode. The AFM was equipped with a 120 µm piezoelectric scanner and fluid cell. AFM cantilevers used (BioLever mini BL-AC40, Olympus Corporation, Tokyo, Japan) had a nominal spring constant of 0.1 N m–1, a resonance frequency of ≈110 kHz in liquid and sharpened silicon tip with a nominal radius of 8−10 nm. If not stated otherwise the FD-based AFM topographs were recorded in buffer solution (150 mM NaCl, 11 mM citric acid, 19 mM H2NaPO4, 3 mM TCEP, pH 5.5) as described.48 The maximum force applied to image the samples was 70 pN, the oscillation frequency and oscillation amplitude of the cantilever was set to 2 kHz and 40 nm, respectively. The AFM was placed inside a home built acoustic isolation box having an integrated temperature control. Liposome Preparation for AFM. Unilamellar liposomes were prepared at room temperature (≈23°C) by hydration of lipid films and extrusion through polycarbonate filters with 0.1 µm pore diameter (Nucleopore®Polycarbonate, Whatman) according to the method described by Avanti Polar Lipids (www.avantilipids.com). All lipids and the extruding equipment used for liposome preparation were purchased form Avanti Polar Lipids, Alabaster, AL. The composition of the liposomes was: brain total lipid extract, brain total lipid extract with 30 wt% cholesterol, POPC and cholesterol with molar ratios of 1:1 and 7:3, POPS, POPC and cholesterol with a molar ratio of 1:1:2, and POPS and cholesterol with a molar ratio of 1:1. Liposomes (10 mg ml–1) were stored at –80°C in buffer solution (150 mM NaCl, 20 mM Hepes, pH 7.25). Preparation of Supported Lipid Membranes (SLMs) for AFM. Supported lipid membranes (SLM) were prepared by fusion of unilamellar liposomes on


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mica. The liposome solution was sonicated (25 kHz, 500 W, Transsonic TI-H-5, ELMA) at room temperature in 150 mM NaCl, 40 mM CaCl2, 20 mM Hepes, pH 7.25 for 5 minutes and immediately adsorbed onto freshly cleaved mica.49 POPC containing liposomes were prepared using the same conditions but without CaCl2. After ≈60 minutes of adsorption, the SLM were gently rinsed with the imaging buffer (150 mM NaCl, 11 mM citric acid, 19 mM H2NaPO4, 3 mM TCEP, pH 5.5). The SLM was imaged by FD-based AFM to check whether the lipid bilayers completely covered the mica and showed no defects. Only if the SLM covering the mica surface showed no defects, LLO was applied. Buffer solutions were freshly made using nano-pure water (18.2 MOhm cm–1) and pro analysis (>98.5%) purity grade reagents from Sigma-Aldrich and Merck. Each experimental condition characterized by AFM was reproduced at least six times. Time-Lapse AFM of LLO Binding, Assembly and Pore Formation. SLMs were incubated with LLO at final concentration of 14 mg ml–1 in imaging buffer at 37°C for 30 min. In all experiments the FD-based AFM imaging force was limited to ≈70 pN. For time-lapse experiments, the SLMs were prepared and the FD-based AFM was thermally equilibrated at 37°C for ≈60 min. To ensure that the mica is defect-free and completely covered by lipid bilayer, the SLMs were imaged by AFM before they were incubated with LLO in the AFM fluid cell. FDbased AFM images were acquired every 10–30 minutes for 6 h over a selected area. During time-lapse AFM, imaging buffer and LLO was refreshed every ≈100 minutes using the fluid exchange channels of the AFM fluid cell. AFM images were analyzed using the AFM (Bruker) software.


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ASSOCIATED CONTENT Supporting Information AFM topographs showing LLO oligomers bound to erythrocyte ghosts (Figure S1). Control experiments showing the unspecific adsorption of LLO to mica and that LLO does not bind to supported lipid membranes (SLMs) in the absence of cholesterol (Figure S2). Time-lapse AFM experiments showing the time course of LLO oligomerization and transmembrane pore formation (Figures S3 and S4).

AUTHOR INFORMATION Corresponding Authors *Email: (D.J.M) [email protected] and (Ö.Y.) [email protected]

Author Contributions All authors designed the experiments; E.M. and S.A.M. conducted the AFM experiments; K.v.P expressed and purified the protein, conducted the EM experiments; all authors analyzed the data and wrote the paper. D.J.M and Ö.Y. coordinated the project. #

E.M. and K.v.P. contributed equally to this work.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS We thank Werner Kühlbrandt for continuous support and discussion of our paper, Sabine Häder and Heidi Betz for technical assistance, Deryck Mills for EM support. This work was funded by the Max Planck Society, the Frankfurt International Max Planck Research School (IMPReS), the Swiss National Science Foundation (Grant 200021_134521/1) and the European Union Marie Curie Actions program through the ACRITAS Initial Training Network (FP7PEOPLE-2012-ITN, Project 317348). ACS Paragon Plus Environment

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(46) Kant, J. A.; Steck, T. L. Specificity in the association of glyceraldehyde 3phosphate dehydrogenase with isolated human erythrocyte membranes. J. Biol. Chem. 1973, 248, 8457-8464. (47) Dufrene, Y. F.; Martinez-Martin, D.; Medalsy, I.; Alsteens, D.; Muller, D. J. Multiparametric imaging of biological systems by force-distance curve-based AFM. Nat. Methods 2013, 10, 847-854. (48) Pfreundschuh, M.; Martinez-Martin, D.; Mulvihill, E.; Wegmann, S.; Muller, D. J. Multiparametric high-resolution imaging of native proteins by forcedistance curve-based AFM. Nat. Protoc. 2014, 9, 1113-1130. (49) Müller, D. J.; Amrein, M.; Engel, A. Adsorption of biological molecules to a solid support for scanning probe microscopy. J. Struct. Biol. 1997, 119, 172188.


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FIGURES and LEGENDS

Figure 1. Negative stain EM of LLO on erythrocyte ghosts and artificial lipid membranes. (A-C) Unresealed erythrocyte ghosts incubated with LLO. LLO forms mainly ring-like structures (red arrows) or slits (green arrows) while arcs (green circles) and incomplete rings (red circles) were found near the ghosts. (D-F) Planar lipid layers incubated with LLO, negatively stained with uranyl acetate and imaged by EM. The lipid layer composed of DOPC and cholesterol (molar ratio 1:1) is completely covered with LLO arcs (green circles) and rings (red arrows). At higher magnification most of the rings appear fragmented. Arcs often form clusters (blue circles). Scale bar corresponds to 50 nm.


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Figure 2. Time-dependent oligomerization of LLO. Lipid layers composed of DOPC and cholesterol (molar ratio 1:1) were incubated for 5, 15, and 30 minutes with LLO. Two images are shown for each time point. After 5 minutes mostly arcs, which cluster and form chain-like structures (red arrows) and occasionally rings are observed. After 15 minutes the number of rings increases and the arcs are less clustered. Some of the rings show a higher internal density (blue arrows). After 30 minutes arcs and rings completely cover the lipid layers. Scale bar corresponds to 50 nm.


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Figure 3. AFM topographs of LLO-bound to lipid bilayers. LLO bound to supported lipid membranes (SLMs) of brain total lipid extract (A-C), brain total lipid extract supplemented with 30 wt% cholesterol (D-F), POPC and cholesterol at molar ratio of 7:3 (G-I), POPC and cholesterol at molar ratio of 1:1 (J-L), POPS, POPC and cholesterol at molar ratio of 1:1:2 (M-O), and POPS and cholesterol at molar ratio of 1:1 (P-R). The high-resolution AFM topographs (B, E, H, K, N and Q) are taken from different areas than those shown in the overview topographs (A, D, G, J, M and P). (C, F, I, L, O and R) Height profiles of LLO oligomers (red curves) were measured along the red lines in the topographs. Zero lines (0 nm) indicate the SLM surface, black lines (≈–5 nm) indicate the surface of the mica support. The full-range color scales of the AFM topographs correspond to a height of 20 nm. Each experiment was reproduced at least 3 times using independent SLM and LLO preparations. Scale bars of topographs correspond to 50 nm and of height profiles to 10 nm.


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Figure 4. Time-lapse AFM of LLO assembly and pore formation. Two examples of time-lapse topographs showing LLO self-assembly on SLMs of POPC and cholesterol (molar ratio 1:1). Topographs were taken 30, 150, and 330 minutes after adding LLO to the buffer solution. Green and red height profiles taken along the green and red lines show that the arc- and ring-shaped pores and prepores fuse into larger ring-like pores (5, 6, 11, and 12). Brown lines of the height profiles indicate the SLM surface, black lines (≈–5 nm) indicate the surface of the mica support. The full-range color scales of the topographs correspond to a height of 22 nm. Scale bars of topographs correspond to 50 nm and of height profiles to 20 nm.


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Figure 5. High-resolution AFM topograph of self-assembled LLO and model of LLO self-assembly and pore formation. (A) High-resolution AFM topograph of LLO oligomers assembled on SLMs of brain total lipid extract supplemented with 30 wt% cholesterol. LLO was incubated for 30 minutes at 37°C and the topograph was recorded at room temperature. The full-range color scale of the topograph corresponds to a height of 22 nm. Scale bar corresponds to 50 nm. (B) Model of LLO oligomerization and transmembrane pore formation. LLO monomers bind to cholesterol containing membranes and oligomerize to arc-, slit- and ring-shaped structures. LLO oligomers, which represent LLO prepores (red), either grow by fusion to form larger prepores or change

their

conformation

to

form

transmembrane

pores

(blue).

Transmembrane pores fuse with other pores and grow larger. With time the oligomers form larger assemblies, indicating that the self-assembly process is kinetically driven. With increasing cholesterol and POPC content more pores are formed. LLO prepores and transmembrane pores do not change significantly the height by which they protrude above the lipid membrane.


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Graphic for Table of Contents 34x13mm (300 x 300 DPI)


Directly Observing the Lipid-Dependent Self-Assembly and Pore

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