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2414 Research Article O clearance through sequestration into blebs that bud passively from the plasma membrane

Peter A. Keyel1,*, Lyussiena Loultcheva2, Robyn Roth2, Russell D. Salter3, Simon C. Watkins4, Wayne M. Yokoyama1 and John E. Heuser2,5,‡ 1Howard Hughes Medical Institute, St Louis, MO 63110, USA 2Department of Cell Biology and Physiology, Washington University in Saint Louis, St Louis, MO 63110, USA 3Department of Immunology, University of Pittsburgh, Pittsburgh, PA 15260, USA 4Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, PA 15260, USA 5Department of Cell Biology and Physiology, Washington University in Saint Louis, Box 8228, 660 S Euclid Avenue, St Louis, MO 63110, USA *Present address: Department of Immunology, University of Pittsburgh, Pittsburgh, PA 15260, USA ‡Author for correspondence ([email protected]) Accepted 14 March 2011 Journal of Cell Science 124, 2414-2423 © 2011. Published by The Company of Biologists Ltd doi:10.1242/jcs.076182

Summary Cells survive exposure to bacterial pore-forming , such as streptolysin O (SLO), through mechanisms that remain unclear. Previous studies have suggested that these toxins are cleared by . However, the experiments reported here failed to reveal any evidence for endocytosis of SLO, nor did they reveal any signs of damage to endosomal membranes predicted from such endocytosis. Instead, we illustrate that SLO induces a characteristic form of plasma membrane blebbing that allows cells to shed SLO by the process known as ectocytosis. Specifically, ‘deep-etch’ electron microscopy of cells exposed to SLO illustrates that the is rapidly sequestered into domains in the plasmalemma greatly enriched in SLO pores, and these domains bleb outwards and bud from the cell surface into the medium. Such ectocytosis is even observed in cells that have been chemically fixed before exposure to SLO, suggesting that it is caused by a direct physical action of the toxin on the , rather than by an active cellular reaction. We conclude, therefore, that ectocytosis is an important means for SLO clearance and hypothesize that this is a primary method by which cells defend themselves generally against pore-forming toxins.

Key words: Blebbing, Ectocytosis, Pore-forming toxin, Cytolysin

Introduction because its activity is pH dependent (Geoffroy et al., 1987).

Journal of Cell Science Many strains of secrete pore-forming toxins (PFTs) that Likewise, other bacteria have evolved complex strategies to ensure are important for (Awad et al., 1995; Cossart et al., 1989; endocytosis of their pathogenic products, such as that of the AB Hirst et al., 2004; Limbago et al., 2000; Portnoy et al., 1988). family of toxins, where one subunit triggers uptake and delivery of These PFTs generally bind to in target-cell membranes a second, toxic subunit. For example, the AB-toxin of Bacillus and oligomerize into complexes of 35–50 subunits that form ~30- anthracis possesses an ‘A’ subunit (the protective ) that nm pores in their membranes (Rossjohn et al., 2007; Tweten, promotes tyrosine phosphorylation of its own receptor in order to 2005). By doing so, they can severely permeabilize cells and cause provoke uptake of the ‘B’ subunit (the lethal factor) (Abrami et al., lysis and death. However, cells can survive exposure to low sublytic 2010). However, B. anthracis also expresses the PFT anthrolysin doses of bacterial PFTs, although the mechanism(s) for how they O (Shannon et al., 2003), leaving it unclear why an AB mechanism do so remain largely unknown; all that is known for certain is that would be necessary if anthrolysin O itself were capable of triggering cell survival depends upon a Ca2+-dependent mechanism (Walev endocytosis. et al., 2001). Proposals for what this mechanism might be (reviewed These varieties of bacterial attack, in which endocytosis of the by Aroian and van der Goot, 2007) have included: active pore- toxin is designed by nature to promote cellular damage, illustrate disassembly, pore-blockade through the formation of some type of why there might be a serious problem with cells using endocytosis cytoplasmic ‘clot’, and endocytosis of the pores. The current model as a mechanism of toxin clearance. In an effort to reconcile these for the inactivation of the archetypal bacterial PFT streptolysin O potential problems, some observers have proposed that internalized (SLO) is that endocytosis serves as the primary mechanism (Idone toxins are promptly destroyed by autophagy (Gutierrez et al., 2007) et al., 2008; Thiery et al., 2010). or rapidly sorted away from lysosomes, perhaps by entering Endocytosis has also been implicated in eliminating other PFTs multivesicular bodies (Husmann et al., 2009). However, no from the plasmalemma, including aureus -toxin experimental evidence to support either of these alternatives has and Vibrio cholera cytolysin (Gutierrez et al., 2007; Husmann et been uncovered to date. al., 2009). However, if endocytosis were the primary survival An alternative possibility is that bacterial toxins are shed directly mechanism that cells used to clear PFTs, cells would probably from the plasma membrane, a process termed ectocytosis (Eken et have to deal with the problem of potentially permeabilizing their al., 2008; Gasser and Schifferli, 2005; Stein and Luzio, 1989). endosomal membranes, which could expose them to toxic Indeed, there is already evidence that SLO-treated cells do shed hydrolases from their lysosomal systems. Indeed, the PFT SLO along with some cellular (Babiychuk et al., 2009; does not act until after it is endocytosed, simply Walev et al., 1995; Walev et al., 2000; Walev et al., 1996; Xie and SLO induces membrane shedding 2415

Low, 1995). However, to date it has been thought that cells shed only monomeric SLO (Walker et al., 1995), and most attention has focused on the cellular proteins that are shed, including glycosylphosphatidylinositol (GPI)-anchored proteins, L-selectin, CD14 and interleukin (IL)-6 receptor (Babiychuk et al., 2009; Walev et al., 2000; Walev et al., 1996; Xie and Low, 1995). In fact, the conclusion that SLO was shed in monomeric form came exclusively from sucrose-gradient sedimentation studies, but no ultrastructural studies were performed to confirm this conclusion. Here, we tested the hypothesis that SLO pores are eliminated from the cell through ectocytosis, rather than being internalized by endocytosis. To do this, we re-examined the mechanism of membrane repair in cells treated with SLO by ‘deep-etch’ electron microscopy (EM) and we designed experiments to test whether blocking endocytosis rendered cells more vulnerable to SLO. We show that cells exposed to SLO sequester the toxin into distinct blebs that bulge from the plasma membrane and are promptly shed from cells, thereby satisfying the definition of ectocytosis. Additionally, and surprisingly, such ectocytosis occurs readily in chemically fixed cells, suggesting that it is a physical process, probably involving physicochemical changes in the plasma membrane. When this process occurs in living cells, it mediates the shedding of SLO from the cell surface, most probably promoting cell survival and serving to prevent the entry of pathogenic molecules into the cell.

Results Microscopic titration of SLO action We first confirmed, as previously reported (Walev et al., 2001), that the absence of external Ca2+ heightens cell permeability and mortality following SLO treatment (supplementary material Fig. Fig. 1. Endocytosis is not required for plasma membrane repair. S1A). We used this Ca2+ dependence to experimentally uncouple (A–H) CHO cells in Ca2+-free Ringer’s solution in the presence of FM1-43 SLO-induced membrane permeabilization from Ca2+-induced (green, marks transiently permeabilized cells) were given a bolus of SLO to resealing, as previously described (Babiychuk et al., 2009; Walev the center of the dish. SLO was allowed to diffuse across the dish for 5 minutes at 37°C. Cells were then given Ca2+-containing full medium (A–D) or et al., 2001). To create a gradient of cell damage, we first cultured full medium plus 0.45 M sucrose (E–H) for 10 minutes at 37°C, followed by Journal of Cell Science cells on glass-bottomed dishes, washed them and removed all PI to mark cells that failed to reseal. The panels show various regions of the 2+ medium except for a thin film of Ca -free Ringer’s solution. We dish, ranging from the edge, where no permeabilization occurred (A,E), then gently applied a small bolus of SLO to the center of the dish, through to regions where cells were permeabilized but resealed (B,C,F,G) and which created a concentration gradient of SLO across the dish to the center of the dish, where cells were permanently permeabilized by SLO through diffusion (Fig. 1A–H). We documented the (D,H). (I–P) HeLa cells were pretreated with 2 mg/ml HRP for 4 minutes at permeabilization by including the membrane-impermeant dye FM1- 37°C, permeabilized for 3 minutes at 37°C with a bolus of SLO to the center 43, which remains in the plasmalemmae of undamaged cells but of the coverslip in Ca2+-free Ringer’s solution with HRP, incubated in full enters and stains the organelles of SLO-permeabilized cells. After medium with HRP for 11 minutes at 37°C and either fixed immediately (I–L) 5 minutes of this exposure to SLO, we washed the dishes with or incubated for 1 hour in full medium without HRP at 37°C and fixed (M–P). At 1 hour, HRP internalization can be seen as punctate structures (arrowheads) complete medium containing normal levels of Ca2+ and incubated in unpermeabilized (M) and transiently permeabilized cells (N,O), whereas them for 10 minutes to allow the cell membranes to reseal, if they cells that failed to reseal show residual HRP staining of the nuclear envelope could. This also washed the FM1-43 out of the membranes of non- and endoplasmic reticulum (L,P). Nuclear HRP is indicative of transient permeabilized cells, leaving this dye only in cells that had been permeabilization. (Q–T) CHO cells were washed and challenged with SLO at permeabilized during the 5-minute exposure to SLO. Finally, we 0 units per ml (Q), 31.25 units per ml (R), 62.5 units per ml (S) or 125 units added propidium iodide (PI) to mark cells that failed to reseal. per ml (T) in the presence of 2.5 mg/ml dx647 (red) for 4 minutes at 37°C. Typically, we observed that the cells at the edge of these dishes Then the cells were washed in PBS, fixed and stained with DAPI (blue). had not permeabilized at all (as indicated by their total lack of FM Micrographs show representative fields of cells from one of three independent 1-43 or PI staining; Fig. 1A), whereas more proximally along the experiments. Scale bars: 10m. SLO gradient, some cells had permeabilized but resealed successfully (as shown by their retention of FM1-43 but exclusion of PI; Fig. 1B). By contrast, at the center of the dish, where SLO Endocytosis does not protect cells from SLO attack concentration was maximal, all cells had permeabilized and very Next, we used the above protocol to test whether endocytosis was few had been able to recover successfully (as shown by their necessary for membrane repair during or after SLO exposure. We minimal FM1-43 staining and positive PI staining; Fig. 1C,D). reasoned that, if endocytosis was necessary for plasma membrane Thus, this method did indeed permit us to observe the full spectrum repair, then blocking this process during and after SLO exposure of SLO effects in each culture, even although SLO activity can should prevent or retard the resealing and recovery of SLO- vary uncontrollably between experiments (a well-known problem). permeabilized cells. To block endocytosis abruptly, we placed cells 2416 Journal of Cell Science 124 (14)

in a hypertonic sucrose medium (Heuser and Anderson, 1989; In contrast to the above results, the phenotype of cells at 1 hour Keyel et al., 2004). In Chinese-hamster ovary (CHO) cells, such after recovery from SLO in the presence of HRP was quite different. hypertonic sucrose treatment reduced internalization of dextran Unperturbed cells at the periphery of each dish looked the same as and/or BSA to 30% of normal levels (supplementary material Fig. at 10 minutes and contained the expected number of HRP- S2A–E). When we treated CHO cells with SLO and allowed them containing given their earlier 10-minute exposure to to recover in the presence of 0.45 M sucrose, we observed the HRP (Fig. 1M, arrowheads). However, cells in the regions of the same range of cell phenotypes as those observed in the absence of dishes that had shown uptake and retention of HRP into their sucrose (Fig. 1E–H). Importantly, the same proportion of cells, and at 10 minutes now showed clear and strong DAB in the same distribution, were FM1-43-positive but PI-negative, staining of their nuclei, indicating that HRP was transferred from indicating that they had been transiently permeabilized by the SLO the cytoplasm to the nucleus (Fig. 1N,O). but had managed to reseal by the time PI was added. These data This sequestration of HRP into the nucleus has never been show that membrane repair can occur normally under conditions reported before and deserves further analysis for its own sake. In that impair endocytosis and thus cast doubt on the notion that addition, it served two purposes here. First, it demonstrated that endocytosis is important for toxin clearance. the transiently permeabilized cells remained viable for the full hour following exposure. Permanently permeabilized cells exposed Viability of SLO-treated cells to HRP for prolonged periods never showed such nuclear retention; Given the above evidence that CHO cells can recover from SLO- all they showed was a faint positive staining of their residual mediated permeabilization within 10 minutes, both with or without membranes (Fig. 1P). Second, the nuclear uptake allowed us to sucrose, it next became important to determine whether they assess the level of endocytosis in these cells because it cleared the remained viable at later time points. We tested this by performing cytoplasm of HRP. We could therefore count the number of HRP- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide positive endosomes in cells that were transiently permeabilized by (MTT) cell viability assays after SLO treatment (supplementary SLO and compared with this number in control cells at the edges material Fig. S1B). Even at SLO exposures of up to ~65 units per of the dishes (Fig. 1M–P, arrowheads). We scored over 120 cells ml, >90% of our CHO cells remained viable. Moreover, we in each condition, counting all the HRP-positive vesicles observed determined by fluorescence-activated cell sorting (FACS) that, at within each cell, and found 9±4 endosomes in non-permeabilized such doses of SLO, many of the cells that remained viable had cells near the edges of the dishes compared with 7±2 endosomes been permeabilized. We observed a substantial population of cells in transiently permeabilized cells more centrally. Thus, we found that were weakly PI-positive when PI was added before, but not a small but significant decrease in endocytosis in transiently after, SLO exposure (supplementary material Fig. S1C–F). This permeabilized cells compared with that in control cells (P0.057, was in addition to the population of strongly PI-positive cells that as determined by a Student’s t-test). We observed no sign of any always appeared after SLO treatment, regardless of when PI was increase in endocytosis of HRP, not even in cells that were viable added – indicating the population of cells permanently damaged enough after SLO-exposure to move HRP from their cytoplasm to by the SLO – and which correlated with the percentage of dead their nuclei. These results do not fit well with the notion that cells determined by the MTT assay. Taken together, these data endocytosis is important for recovery from, or for resistance to,

Journal of Cell Science indicate that the majority of cells that recover from a transient SLO (Idone et al., 2008). permeabilization by SLO remain viable at later time-points thereafter. To examine further the possibility that SLO exposure stimulates some sort of compensatory endocytosis, we repeated the protocols Levels of endocytosis in SLO-treated cells originally used to develop the idea (Idone et al., 2008). We If endocytosis were triggered in transiently permeabilized cells incubated CHO cells simultaneously with a lysine-fixable 10-kDa following SLO challenge, we would have expected to observe an dextran conjugated to Alexa-Fluor-647 and various concentrations increase in their uptake of extracellular tracers, such as horseradish of SLO for 4 minutes at 37°C and then examined these cells by peroxidase (HRP) or dextran, if we added such tracers along with fluorescence microscopy. We found that untreated control cells the SLO. To examine this possibility, we incubated CHO cells with showed an abundance of fluorescent dextran-containing punctae, HRP during the initial SLO permeabilization in Ca2+-free Ringer’s presumably representing endosomes (Fig. 1Q). By contrast, CHO solution, continued their exposure to HRP during their first 10 cells treated with sublytic amounts of SLO exhibited a variable minutes of recovery in complete medium and then either fixed the degree of permeabilization (as seen by variable retention of dextran cells then or let them recover for a full hour before fixation (Fig. within their cytoplasm; Fig. 1R,S), but displayed 50% fewer 1I–P). fluorescent dextran-containing punctae than did control cells At 10 minutes after SLO exposure in the presence of HRP, cells (supplementary material Fig. S2F). Interestingly, at SLO doses that on the edge of the dishes were again unperturbed, and again served killed 40% of the cells (supplementary material Fig. S1F), cells as controls for monitoring the background level of HRP generally appeared to be flooded with cytoplasmic dextran, but endocytosis. By contrast, cells more central on the dishes were still displayed curious dextran-positive structures that did not look transiently permeabilized, given that they showed uptake of HRP like bona fide endosomes because they were generally much larger into their rather than into their endosomes (this is like the and more tightly grouped about the nucleus than normal endosomes uptake and retention of FM1-43 described above; Fig. 1I–K). (Fig. 1T). These structures could have been interpreted as being Finally, cells in the very center of the dishes, exposed to the endosomes in earlier studies. However, EM studies of cells highest doses of SLO, were again irreparably lysed, as shown by damaged this severely by SLO in the presence of HRP suggested the faint diaminobenzidine (DAB) staining of their nuclear that these structures are inverted fragments of plasmalemma envelopes but their inability to retain observable amounts of HRP that passively trap extracellular materials as they form (data not in their cytoplasm (Fig. 1L). shown). SLO induces membrane shedding 2417

Fig. 2. SLO remains on the cell surface. CHO cells were treated with 0 units per ml (A), 62.5 units per ml (B), 125 units per ml (C) or 250 units per ml (D) SLO and fixed. Cells were stained without chemical permeabilization with antibodies against SLO (green), giantin (red) and DAPI (blue). Giantin marks the Golgi and only stains those cells that lost plasma membrane integrity, indicated by asterisks, whereas arrowheads denote SLO punctae on non- permeabilized cells. (E)Mean (±s.e.m.) intensity of anti-SLO antibody staining on the cell surface of the non-permeabilized cells from the experiments described in A–D, normalized to the maximum intensity. Over 75 cells per treatment in three independent experiments were analyzed. Intensity changes were significant (*P0.083; **P0.029) between treatments. Micrographs show representative fields of cells from one of three independent

Journal of Cell Science experiments. Scale bar: 10m.

Lysosomal exocytosis is not provoked by SLO exposure Fig. 3. SLO provokes blebbing from living cells. (A)CHO cells were treated for 30 minutes with 500 units per ml SLO at 37°C, fixed and prepared for Our counts of HRP-positive endosomes in cells exposed to SLO DEEM. Black arrowheads mark the ring-shaped SLO pores present on blebs. could have been grossly misleading if exocytosis of endosomes White arrowheads denote coated pits. (B–E) HeLa cells were treated with and/or lysosomes occurred in recovering cells at the very same 125,000 units per ml SLO for 1 minute, fixed and either prepared for DEEM time as endocytosis, which is known to occur in some forms of (B,BЈ) or freeze-fracture EM (C–E). Blebs (C,D) or plasma membrane (E) are membrane damage (Reddy et al., 2001). Conceivably, such shown. The fracture region is depicted in gray for clarity. Arrowheads mark the exocytosis could have perfectly counterbalanced whatever increase assembled ring-shaped SLO pores. Micrographs are representative of two in HRP endocytosis occurred, and left us with the mistaken independent experiments. Scale bars: 100 nm (A,D,E); 50 nm (B,BЈ,C). impression that no change in numbers had occurred. To rule out this possibility, we first preloaded the endosomal-lysosomal system of cells with HRP, by giving them one full hour of exposure to exogenous HRP, and then applied SLO. Importantly, this did not Antibodies against the Golgi giantin (also known as golgin lead to any sign of decline or diminution in the number of HRP- subfamily B member 1) were included to delineate those cells positive endosomes in any of our cells, in any region of the dishes permanently permeabilized by SLO (Fig. 2, asterisks). This showed – damaged or undamaged (data not shown). Thus, we conclude that, at all doses, a substantial fraction of cells were SLO-positive that lysosomal exocytosis was not masking any increase in HRP but giantin-negative, indicating that these cells remained alive but endocytosis. retained SLO on their surface (Fig. 2B–D). Curiously, this labeling of SLO of giantin-negative cells was often punctate, as observed Localization of SLO to the surface of cells by light microscopy (Fig. 2, arrowheads), and staining intensity If endocytosis was not actively involved in SLO clearance, then increased in a dose-dependent manner (Fig. 2E). Such punctae SLO would be expected to remain on the surface of cells. To test might have been taken to be endosomes in earlier studies (Idone et for this, we fixed SLO-treated CHO cells and immunostained them al., 2008); however, we would not expect endosomes to stain with for SLO without detergent permeabilization (Fig. 2A–D). antibodies against SLO in cells where the Golgi complex did not 2418 Journal of Cell Science 124 (14)

stain with antibodies against giantin, indicating that they had not colleagues (Idone et al., 2008), probably represents SLO-induced been permeabilized. Rather, the observed SLO punctae are likely blebbing rather than SLO endocytosis. to be surface structures – possibly clumps of SLO pores on the cell surface. Shedding of SLO-enriched vesicles from treated cells The above EM results are also entirely consistent with earlier EM localization of SLO on the surface of cells biochemical evidence that SLO induces the shedding of small To gain further insight into this punctate distribution of SLO on the membrane vesicles from the plasma membrane (Xie and Low, cell surface, we turned to deep-etch EM (DEEM), which resolves 1995). Xie and Low concentrated on the release of GPI-linked individual SLO pores, in order to determine their distribution on proteins in SLO-treated cells, but they did not consider the the cell surface. This required titrating the dose of SLO to a level possibility that SLO itself was released along with these other where cells were not grossly permeabilized, which we determined proteins (Xie and Low, 1995). Other studies have examined the by phase-contrast light microscopy before EM. direct shedding of material from the plasma membrane and named Cells treated with sublytic doses of SLO displayed by DEEM the process ectocytosis (Eken et al., 2008; Gasser and Schifferli, plasma membranes enriched in pores; but these pores were not 2005; Stein and Luzio, 1989). The present finding of SLO on distributed uniformly or randomly on the cell surface. Rather, they characteristic membrane blebs thus raised the possibility that were clustered into discrete highly enriched domains; in fact they SLO pores are shed through ectocytosis. To test for this directly, were so enriched that they typically appeared to exclude most we collected the supernatants from cells exposed to sublytic other membrane proteins (Fig. 3A, black arrowheads). doses of SLO and analyzed these supernatants, both biochemically Quantification of the number of pores revealed an average of 24 and ultrastructurally. To ensure that we did not collect just cell pores per m2 on blebs, whereas pores were undetectable on the debris from ruptured cells, we kept the dose of SLO sublytic, and plasma membrane (P0.004, as determined by a Student’s t-test). we spun the supernatant at 1200 g for 5 minutes to sediment any Importantly, we never observed these clusters of SLO pores on any large cell fragments or any whole cells that might have been indentations of the plasma membrane that might have been incipient released into the supernatant. The supernatant remaining after endosomes, despite the fact that the openings of -coated this low-speed spin was then concentrated by ultracentrifugation pits and caveolae were always readily apparent in our deep-etch at 100,000 g for 40 minutes, resuspended in a small volume of replicas, even when they were very shallowly indented (e.g. when buffer, adsorbed to glass coverslips and prepared for DEEM. We they are presumably at a very early stage of formation; Fig. 3A, observed closed membranous vesicles that were often almost white arrowheads). Rather, all the clusters of SLO we observed completely saturated with SLO pores (Fig. 4A). As a control in occurred on eversions of the plasma membrane, on structures that these experiments, we treated our cleared cell supernatants with could only be called ‘blebs’. Triton X-100 before high-speed centrifugation. This greatly This is not to say, however, that such eversions or blebs looked reduced the amount of SLO in the pellet and left nothing at all to like the standard reactive blebs that cells form when they are see by DEEM, confirming that the shed structures were indeed irritated or injured (Charras et al., 2008; Charras et al., 2006; membranous in nature. Charras et al., 2005; Mitchison et al., 2008; Straight et al., 2003). Parallel biochemical analyses of these putative ectosomes

Journal of Cell Science They did not. The latter sorts of blebs are larger, more plump and confirmed that they were indeed greatly enriched in SLO (Fig. their membranes bear a perfectly normal complement of proteins, 4B,C). Quantification of these immunoblots indicated that ~60% as judged by the appearance of the small ‘bumps’ seen in replicas of the SLO was shed in the first 5 minutes. Along with SLO, of the outside surface of cells (supplementary material Fig. S3A,B). ectosomes from RMAS cells stably transduced with the GPI-linked By contrast, the SLO-enriched blebs were smaller, flatter, more viral protein m157 (Tripathy et al., 2006) contained a rich pleomorphic and tended to exclude the normal plasma membrane complement of m157. This is consistent with the important previous ‘bumps’ and ‘blobs’ that are generally considered by freeze-etch study demonstrating SLO-induced release of GPI-anchored proteins practitioners to be the exofacial portions of intramembrane proteins. (Xie and Low, 1995). By contrast, we did not detect the lysosomal protein Lamp1 in our ectosome fraction, suggesting that SLO- ‘Quick freezing’ of living cells reveals SLO pores in blebs treated cells do not shed any of their lysosomal contents into the To confirm that SLO also concentrates into blebs in living cells, medium (Fig. 4C). Our ectosomes were heterogeneous in size, we next treated HeLa cells with SLO and immediately quick-froze ranging from ~200 nm up to ~2 m, which was similar in size and them without any chemical fixation. In order to view the surfaces diversity to the blebs we observed protruding from the of these cells by EM, they had to be freeze-fractured and then plasmalemma of whole cells (Fig. 3). This is considerably larger deep-etched, rather than simply freeze-dried as in the experiments than the usual size of exosomes, the other major type of vesicle above (this is because the medium on top of living cells obscures released from cells by exocytosis of multivesicular bodies (Raposo them if they are directly freeze-dried). We could readily observe, et al., 1996; Stoorvogel et al., 2002; Thery et al., 1999). on these freeze-fractured cells, similar-looking SLO structures to those described above, which were again concentrated into SLO localizes to blebs on chemically fixed cells membrane blebs and eversions (Fig. 3B–D, arrowheads). By Having found that the SLO-enriched structures on the surfaces of contrast, the number of SLO rings in the rest of the membrane of cells (and the structures shed from SLO-treated cells) were these cells appeared to be exceedingly low (Fig. 3E). Additionally, relatively depleted of other membranous proteins, we next needed the freeze-fractured cells illustrated that many of the SLO rings to consider the possibility that SLO caused some sort of phase- formed large pores straight through the plasma membrane (Fig. separation in the plasma membrane and that this was somehow 3C,D, gray indicates the fracture faces and arrowheads indicate coupled to the membrane eversion and blebbing observed. To test pores). Taken together, these results suggest that the punctate for this, we took advantage of the observation that SLO can bind distribution of SLO observed here, and previously by Idone and to, and polymerize into rings, on aldehyde-fixed cells (Duncan and SLO induces membrane shedding 2419

Fig. 5. SLO localizes to and induces blebs in fixed cells. HeLa cells were fixed, then treated with either 12,500 units per ml (A) or 125,000 units per ml Fig. 4. Cells shed SLO-rich microvesicles. (A)Platinum replicas of pellets SLO (B–D), fixed and prepared for DEEM. (A)Pore segregation into blebs is formed after high-speed ultracentrifugation (high-speed pellet) from RMAS- observed at low magnification (insets show higher magnification images m157 cells treated with 250 units per ml SLO were visualized by DEEM. contrasting the plasma membrane and bleb). Arrowheads indicate SLO pores. (B,C)CHO (B) or RMAS-m157 (C) cells were incubated with the indicated (B,C)A high-magnification three-dimensional anaglyph view of plasma concentration of SLO (in units per ml) for 5 minutes at 37°C and the cells membrane (B) or a two-dimensional view of bleb membrane (C) reveals an sedimented. The pellets formed after low-speed cell centrifugation (cell overabundance of pores on the bleb membrane. (D)SLO pores are observed in Journal of Cell Science pellets) were washed and solubilized in SDS sample buffer, whereas the smooth phase-separated regions of the plasma membrane (colored gray for supernatants were ultracentrifuged at 107,000 g for 40 minutes at 4°C to pellet contrast). Micrographs are representative of four independent experiments. any shed membranes. Triton X-100 was added to the supernatant marked Tx Scale bars: 500 nm (A); 100 nm (B–D). just before the high-speed spin. Cell pellets (C), high-speed supernatants (S) and high-speed pellets (P) were resolved by SDS-PAGE and probed with the HAB-003 anti-SLO monoclonal antibody, the 6H121 anti-m157 monoclonal antibody or the anti-Lamp1 rat antibody. SLO was detectable in the high-speed When we increased SLO concentrations far beyond lytic levels, we pellet along with the GPI-linked m157. Micrographs show representative cells continued to observe preferential segregation of SLO into or structures from one of three independent experiments, whereas blots are plasmalemmal blebs, with up to 320 pores per m2 on some blebs, representative of ten experiments. Scale bar: 100 nm. although the general plasma membrane also began to display an average of 2 pores per m2 (Fig. 5A–C). We could also observe a phase separation in small portions of the plasma membrane. These Schlegel, 1975). Indeed, we found a great abundance of SLO- portions of the plasma membrane were characterized by smooth enriched blebs on cells that had been prefixed either in membrane, again lacking observable intramembrane particles, and formaldehyde, glutaraldehyde, or both (Fig. 5). Importantly, we these regions supported SLO polymerization (pore formation) (Fig. never observed any such abundance of blebs in control cells that 5D). were fixed comparably but not treated with SLO (supplementary material Fig. S3C,D). These experiments also helped us distinguish Ca2+ is required for the shedding, but not the formation, of between ectosomes and exosomes; the latter could not possibly be SLO-enriched blebs released from fixed cells because their discharge requires processing Given the importance of Ca2+ for both membrane repair and the in multivesicular bodies before their exocytic release from within formation of large blebs (Babiychuk et al., 2009; Charras et al., these bodies, both of which are living processes that do not continue 2008; Walev et al., 2001), we next sought to determine whether after chemical fixation (Raposo et al., 1996; Stoorvogel et al., Ca2+ plays any role in the generation of the blebs observed here or 2002; Thery et al., 1999). the localization of SLO to these blebs using the prefixed cell Prefixation of cells before exposure to SLO also permitted the system. To do this, we prefixed cell cultures in the presence of use of a higher concentration of SLO than was possible for living Mg2+ and 3 mM EGTA (in place of the usual 2 mM Ca2+ in our cells; thus, we could study pore formation itself in greater detail. fixatives), and then exposed the cells to SLO without changing 2420 Journal of Cell Science 124 (14)

their cation environment. SLO pores and SLO-enriched blebs were Interestingly, we also observed blebs that did not release from just as abundant on cells fixed and treated in Mg2+ and EGTA as cells, yet these blebs accumulated Sytox Green (Fig. 6E; in those fixed in the presence of Ca2+, with a global average of 7.4 supplementary material Movie 3), suggesting that blebs can lose pores per m2 in the presence of Ca2+ and 6.7 pores per m2 in communication with the cytoplasm even before they are shed. This Mg2+ and EGTA (Fig. 6A,B). This demonstrated that Ca2+ is not has been previously described for the PFT perforin (Keefe et al., necessary for the insertion of SLO into membranes, polymerization 2005), suggesting that this is a common mechanism among PFTs. into pores in the membrane or for the segregation of SLO into Under Ca2+-free conditions, however, we did not observe any plasmalemmal blebs. Thus, SLO-induced bleb formation is ectocytosis (Fig. 6F; supplementary material Movie 4). Instead, we independent of Ca2+. observed the formation, and wiggling about, of long filopodial We next performed three-dimensional confocal imaging on 3T3 structures (Fig. 6F, arrowhead). This suggests that Ca2+ is required cell cultures labeled with the membrane dye Cellmask Orange to for the final scission event. determine whether the shedding of SLO-enriched blebs in living To determine whether Ca2+ plays a role in the final step of cells was Ca2+ dependent. Control cells labeled with Cellmask ectocytosis, we collected the 107,000 g pellet from clarified Orange excluded Sytox Green, showed normal cellular activity supernatants of cells that had been prefixed and then treated with and did not undergo blebbing, indicating that the dye was nontoxic SLO in the presence of either Ca2+ or Mg2+ and EGTA and analyzed (Fig. 6C; supplementary material Movie 1). Next, 3T3 cultures them for SLO content. We found a dramatic increase in the amount were treated with moderate doses of SLO in the presence of a of SLO recovered from cells that had been treated in the presence normal concentration of Ca2+, and blebbing and release of vesicles of Ca2+ (supplementary material Fig. S4). Because SLO partitions was observed (Fig. 6D; supplementary material Movie 2). into blebs independently of Ca2+ (Fig. 6), the results provide a Journal of Cell Science

Fig. 6. Ca2+-dependent SLO shedding. (A,B)Three- dimensional DEEM anaglyphs illustrate CHO cells that were prefixed, treated with 4,000 units per ml SLO for 4 minutes at 37°C, and fixed, all in the absence of Ca2+. Black arrowheads denote SLO rings present in blebs. White arrowheads mark clathrin-coated pits. SLO pores are pseudo-colored gray in the higher magnification view of the boxed area (shown in B) for clarity. (C–F) 3T3 cells labeled with Cellmask Orange (red), in the presence of Sytox Green (green), were untreated (C) or treated with 250 units per ml SLO in the presence (D,E) or absence (F) of Ca2+. The lower panels show a higher magnification view of the boxed area in the upper panels. Arrowheads in C–D denote blebs separating from the plasma membrane, whereas arrowheads in F denote filopodia. The time following SLO addition is displayed in minutes:seconds. Micrographs are representative of three independent experiments. Scale bars: 100 nm (A); 200 nm (B); 10m (C–F); 5m (C–F, lower panels). SLO induces membrane shedding 2421

measure of the amount of vesicles recovered. Taken together, we glomerulonephritis (Cunningham, 2000). Ectocytosis might also conclude that Ca2+ is needed for the scission event of ectocytosis. be the mechanism through which cellular proteins, such as alkaline phosphatase, the IL-6 receptor and CD14 (Walev et al., 1996; Xie Discussion and Low, 1995), are shed in response to SLO. Here, we provide evidence that a prototypical PFT, streptolysin O, The evidence we provide here that chemically fixed cells also provokes a cell-survival mechanism that involves shedding of the undergo ectocytosis when exposed to SLO argues that the toxin, not internalization and degradation. Specifically, we show mechanism could be a physicochemical response of the that sublytic SLO doses, which create pores in the plasma plasmalemma to perturbations of its lipids, which are not fixed by membrane, provoke a cellular reaction in which the pores rapidly aldehydes. Because the lipid composition of cells can be modified become segregated into plasmalemmal blebs, and the blebs become both genetically and post-translationally (Bishop and Bell, 1988; rapidly shed into the surrounding medium, in the process currently Schujman and de Mendoza, 2005), it is highly probable that cells termed ectocytosis. will vary in their ability to respond to toxins in this manner. Contrary to earlier reports, we find no evidence for sequestration Various cell types do show differential sensitivity to PFTs (Keefe of SLO pores into membrane invaginations or into incipient et al., 2005), which could be the result of different propensities to endosomes, and we observe no increase in endocytosis in SLO- engage in ectocytosis. Additionally, cell survival after exposure to treated cells (Idone et al., 2008; Thiery et al., 2010). Furthermore, the PFT aerolysin involves the caspase-1-dependent upregulation we find that blocking endocytosis does not compromise the ability of sterol regulatory protein and other lipid-modifying of cells to survive SLO. From these results, we conclude that (Gurcel et al., 2006). Thus, ectocytosis might be an important endocytosis is not likely to be the primary mechanism of SLO mechanism for cell survival. clearance, but that ectocytosis probably is. Ectocytosis could also protect cells from the deleterious effects While this manuscript was under review, Babiychuk et al. 2+ of internalized bacterial PFTs. Bacterial toxins that reach the described a Ca -dependent cell blebbing that is important for secretory granules of T cells kill the cells through permeabilization membrane repair, although they did not link this blebbing to of secretory granules containing granzymes (Carrero et al., 2008). sequestration and removal of SLO pores (Babiychuk et al., 2010). 2+ Even toxins that act at the surface, such as anthrolysin O, can be However, the active blebbing that occurs in a Ca -dependent active at acidic pH (Wei et al., 2005), underscoring the importance fashion is the abrupt protrusion of big (micron-sized) cytoplasmic for cell survival of preventing these toxins from having access to protrusions depleted of cortical cytoskeleton and filled with readily the endosomal-lysosomal system. Thus, we describe a repair mobile cytoplasmic components (most notably ribosomes) (Zeligs response that is beneficial to the cell because it permits cell survival, and Wollman, 1977). This blebbing also occurs during mitosis eliminates the bacterial toxin and prevents the toxin from further (zeiotic blebbing), and upon exposure to oxidizing and reducing manipulating the cell or ‘sneaking into’ the endosomal-lysosomal agents, as well as occuring generally when the cortical cytoskeleton system of the cell. of the cell is constitutively weakened, such as in filamin-deficient melanoma cells (Charras et al., 2008; Charras et al., 2006; Charras Materials and Methods et al., 2005; Cunningham et al., 1992; Mitchison et al., 2008; Plasmids and antibodies

Journal of Cell Science Straight et al., 2003). Such ‘active’ blebs display a characteristic The pBAD-gIII plasmid encoding His-tagged SLO was a gift from Michael Caparon behavior; they not only protrude extremely abruptly, like herniations (Washington University in St Louis, St Louis, MO) (Magassa et al., 2010). The pmx- of the plasmalemma generated by internal turgor pressure (which m157 plasmid, encoding m157, was as previously described (Smith et al., 2002). The anti-giantin polyclonal antibody was from Covance, CD19 conjugated to they probably are), but also retract actively after a minute or so, allophycocyanin (APC), CD86-APC, the anti-Lamp1 rat antibody specific to murine coincident with (and consequent to) the reconstruction of an Lamp1 and the anti-Lamp1 monoclonal antibody H4A3 specific for human Lamp1 actomyosin-rich cortex immediately under their membranes were from BD Biosciences, the anti-SLO monoclonal antibody HAB-003 was from (Mitchison et al., 2008). Abcam (Cambridge, MA) and anti-m157 monoclonal antibody 6H121 was as previously described (Tripathy et al., 2006). Fluorescently labeled secondary The blebs we describe here are different from these ‘reactive antibodies were from Invitrogen and HRP-conjugated antibodies were from Jackson blebs’ in several ways: they are generally considerably smaller; ImmunoResearch. they do not contain normal cytoplasmic components but appear to Cell culture be isolated from the cytoplasm almost from the moment of their HeLa cells were cultured in DMEM supplemented with 10% fetal calf serum (FCS) formation; they do not bear the normal complement of membrane and 23 L-glutamine, CHO cells were cultured in similarly supplemented Ham’s F12 proteins that occur elsewhere in the plasmalemma (‘reactive blebs’ medium and YAC-1 cells in similarly supplemented RPMI. 3T3 cells were cultured do contain these proteins) but appear to be considerably depleted in HeLa medium supplemented with sodium pyruvate and non-essential amino acids. RMAS-m157 cells were generated by retroviral transduction of pmx-m157, of cytoplasmic proteins and often lack them entirely (membrane as previously described (Smith et al., 2002), and cultured in RPMI with 10% FCS, proteins being largely replaced here with SLO pores); and bleb 23 L-glutamine and 4 g/ml puromycin. formation can occur independently of Ca2+, although shedding is 2+ Reagents Ca dependent. How the formation and scission of nascent All reagents were from Sigma unless otherwise specified. SLO was either purified ectosomes coordinates with internal membrane fusion events from pyogenes (Sigma) or induced in pBAD-gIII SLO-transformed (McNeil and Kirchhausen, 2005; McNeil et al., 2000) remains to Escherichia coli strain BL21 (Agilent Technologies) and purified on nickel-NTA be determined. agarose beads (Qiagen). SLO from Sigma was reconstituted in water and 10 mM DTT and snap-frozen on dry ice in aliquots. We observed a ~50% reduction in SLO These ectosomes might induce host inflammatory effects, activity following each round of freeze-thaw, and rapid inactivation at 37°C, and although any immunomodulatory functions remain to be hence reported activity, rather than protein concentration, for SLO. Both proteins determined. It is possible that ectosomes might aid in the generation were used at a similar hemolytic activity, and this hemolytic activity corresponded of anti-toxin antibodies. Anti-SLO antibodies are known to form to the same cytolytic activity in both sources of SLO. Any endotoxin present in the toxin preparations did not affect cell killing (data not shown). The SLO sensitivity during group-A streptococcal infections and are important in the of MyD88–/– cells was comparable to that of wild-type cells (data not shown), ruling diagnosis of scarlet fever, rheumatic fever and post-infectious out any role of SLO binding to toll-like receptor 4 (Park et al., 2004) in our system. 2422 Journal of Cell Science 124 (14)

Purification of SLO out SLO and adding full medium, with 2 mg/ml HRP, for a further 11 minutes at SLO was induced in exponentially growing cells with 0.2% arabinose for 3 hours at 37°C. Cells were washed and either fixed in 2% glutaraldehyde or incubated for 1 25°C. Cells were collected, centrifuged, frozen at –80°C and purified as described hour in full medium at 37°C before fixation. HRP was visualized using standard previously (Mishra et al., 2005). Purity was assessed by SDS-PAGE, protein techniques (Graham and Karnovsky, 1966). Cells were imaged and prepared for concentration by BCA assay (Thermofisher Scientific) and hemolytic activity by thin-section EM as previously described (Heuser, 2005). For HRP preloading erythrocyte lysis. One unit was the amount of SLO causing 50% lysis of a 2% experiments, cells were incubated in full medium supplemented with 5 mg/ml HRP suspension of human erythrocytes in 10 mM HEPES pH 7.4, 2 mM CaCl2, 0.3% for 2 hours before the SLO challenge. BSA in PBS after 30 minutes at 37°C, as determined by the A405 of the supernatant, and matched the hemolytic activity reported by Sigma. Following one freeze-thaw, Ultracentrifugation commercially available SLO typically had an activity >15,700 units per ml (517,200 Cells were harvested, spun, washed in RPMI with 2 mM CaCl2 and resuspended in units per mg of protein), whereas His-tag-purified SLO had an activity of 650,000 RPMI with 2 mM CaCl2 with SLO for 5 minutes at 37°C and centrifuged at 1000 g units per ml (260,000 units per mg of protein). for 5 minutes. Cell pellets were washed in PBS and solubilized at 95°C in SDS- sample buffer; supernatants were spun at 107,000 g (30,000 r.p.m., Sw50.1 Ti) for FACS assay 40 minutes at 4°C to pellet all membranes. The supernatant was saved, whereas the Cells were harvested, centrifuged and resuspended in R/B medium (RPMI, 2 mM pellet was washed, spun at 107,000 g for 40 minutes at 4°C and resuspended in ϫ 6 CaCl2 and 0.5% BSA), with 20 g/ml PI, at 2 10 cells per ml. Cells were incubated either Ringer’s solution, for EM, or in 95°C SDS-sample buffer, for SDS-PAGE. For with SLO for 5 minutes at 37°C. PI incorporation was immediately assessed by prefixed cells, cells were grown to confluency in 10-cm dishes, washed in PBS, FACS using a FACScan (BD Biosciences). For some experiments, the incubation fixed for 8 minutes in 2% formaldehyde with 0.01% glutaraldehyde in Ringer’s was extended to 30 minutes, PI was not added until after incubation with SLO, solution, washed in PBS, and incubated with 1000 units per ml SLO in Ringer’s immediately before FACS, or cells were challenged with SLO in the presence of 2 solution for 4 minutes at 37°C. Supernatants, together with one wash were collected, mM EGTA. Cell viability was assessed using the Vybrant MTT cell proliferation clarified by centrifugation at 300 g for 5 minutes at 4°C and ultracentrifuged at assay (Invitrogen). 2+ 103,000 g for 40 minutes at 4°C. Ca -free preparations used MgCl2 instead of Electron microscopy CaCl2 and included 3 mM EGTA. The pellet was resuspended and either dissolved CHO or HeLa cells were grown on coverslips, treated with SLO at 37°C in Ringer’s in SDS-sample buffer or spinoculated onto glass coverslips for DEEM. solution (155 mM NaCl, 3 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 3 mM NaH2PO4, 5 mM HEPES pH 7.2, and 10 mM glucose) for 4 or 30 minutes, washed and fixed We thank members of the Yokoyama laboratory for technical in 2% glutaraldehyde in 100 mM NaCl, 30 mM HEPES pH 7.2, 2 mM CaCl2 and assistance and helpful discussion and the Heuser laboratory for 150 mM sucrose, (NaHCa-Suc) for 1 hour. To measure SLO binding in fixed cells, transcribing dictations. This work was supported by the Howard CHO or HeLa cells grown on coverslips were fixed in 2% glutaraldehyde in NaHCa- Suc for 8 minutes at room temperature, washed in Ringer’s solution, incubated in Hughes Medical Institute and grants from the National Institutes of the indicated concentration of SLO at 37°C in Ringer’s solution for 1 or 4 minutes Health. The authors declare that they have no competing financial and fixed again in 2% glutaraldehyde in NaHCa-Suc. To eliminate Ca2+, Mg2+ interest. P.K., W.Y. and J.H. devised the research approach. P.K., L.L., replaced the Ca2+ present in all the buffers used and buffers were supplemented with R.R., R.D.S., S.W. and J.H. performed experiments. R.R. and J.H. EGTA. Platinum replicas were prepared as described previously (Heuser, 2000), performed electron microscopy. P.K., R.R., J.H. and W.Y. analyzed examined in a 100CX microscope (JEOL, Peabody, MA) and imaged with an AMT digital camera (Danvers, MA). data. P.K., W.Y. and J.H. wrote the manuscript. Deposited in PMC for release after 6 months. Fluorescence microscopy Supplementary material available online at CHO cells were treated with SLO at the indicated concentrations for 5 minutes at 37°C, fixed in 2% paraformaldehyde, probed with anti-SLO and anti-giantin http://jcs.biologists.org/cgi/content/full/124/14/2414/DC1 antibodies followed by goat Alexa-Fluor-488-conjugated anti-mouse-IgG and goat Alexa-Fluor-568-conjugated anti-rabbit-IgG, DAPI stained, mounted in gelvatol and References imaged using a 1.40 NA, 633 Plan-Apochromat objective on an Eclipse 80i Abrami, L., Kunz, B. and van der Goot, F. G. (2010). toxin triggers the

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