Báo cáo khoa học: Passage through the Golgi is necessary for Shiga toxin B subunit to reach the endoplasmic reticulum

Passage through the Golgi is necessary for Shiga toxin B subunit to reach the endoplasmic reticulum Jenna McKenzie1, Ludger Johannes2,3, Tomohiko Taguchi4 and David Sheff1

1 Department of Pharmacology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA 2 Institut Curie, Centre de Recherche, Laboratoire Traﬁc, Signalisation et Ciblage Intracellulaires, Paris, France 3 CNRS UMR144, Paris, France 4 Department of Biochemistry, Osaka University Graduate School of Medicine, Japan

Keywords endosomes; Golgi; membrane trafﬁc; retrograde trafﬁc; Shiga toxin

Correspondence D. Sheff, Department of Pharmacology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242-2600, USA Fax: +1 319 335 8930 Tel: +1 319 335 7705 E-mail: david-sheff@uiowa.edu

) treatment,

(Received 11 November 2008, revised 4 January 2009, accepted 7 January 2009)

doi:10.1111/j.1742-4658.2009.06890.x

Both Shiga holotoxin and the isolated B subunit, navigate a retrograde pathway from the plasma membrane to the endoplasmic reticulum (ER) of mammalian cells to deliver catalytic A subunits into the cytosol. This route passes through early ⁄ recycling endosomes and then through the Golgi. Although passage through the endosomes takes only 30 min, passage through the Golgi is much slower, taking hours. This suggests that Golgi passage is a key step in retrograde trafﬁc. However, there is no empirical data demonstrating that Golgi passage is required for the toxins to enter the ER. In fact, an alternate pathway bypassing the Golgi is utilized by SV40 virus. Here we ﬁnd that blocking Shiga toxin B access to the entire temperature block or subcellular surgery Golgi with AlF4 prevented Shiga toxin B from reaching the ER. This suggests that there is no direct endosome to ER route available for retrograde trafﬁc. Curiously, when Shiga toxin B was trapped in endosomes, it entered the cytosol directly from the endosomal compartment. Our results suggest that trafﬁck- ing through the Golgi apparatus is required for Shiga toxin B to reach the ER and that diversion into the Golgi may prevent toxin escape from endo- somes into the cytosol.

Shiga toxin (Stx) is a bacterial exotoxin responsible for an estimated 165 million annual cases of severe dysen- tery worldwide [1]. The toxin attacks cytosolic targets in mammalian cells. To reach these targets, the toxin navigates a retrograde pathway that passes sequentially through the plasma membrane, endosomes, Golgi and endoplasmic reticulum (ER) [2–5]. Passage through the Golgi appears to be rate limiting on this pathway, resulting in prominent labeling of this organelle. How- ever, such prominent labeling may be misleading. Recycled transferrin was assumed to pass sequentially through the early endosomes (EEs) and recycling endo- somes (REs) based on prominent labeling of the RE at

later time points [5,6]. It later became evident that the majority of transferrin actually bypasses the RE. The same may be true for Golgi passage of Stx. Empirical data supporting a requirement for passage through the Golgi is lacking. Indeed, treatment with brefeldin A provides protection against the holotoxin, suggesting involvement of the Golgi. However, that protection is incomplete, suggesting that Golgi passage may be favored but not required [7,8]. Furthermore, other toxins, such as diphtheria toxin, bypass the Golgi and ER by escaping the endosomal compartment directly into the cytosol [9]. SV40 virus is internalized into a spe- cialized compartment which can communicate directly

Abbreviations BFA, brefeldin A; EE, early endosome; ER, endoplasmic reticulum; MEM, minimal Eagle’s medium; PDI, protein disulﬁde isomerase; RE, recycling endosome; Stx, Shiga toxin; StxB, Shiga toxin B; Tfn, transferrin; TfnR, transferrin receptor; TGN, trans-Golgi network; WGA, wheatgerm agglutinin.

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[10]. with the ER, bypassing endosomes and Golgi There may even be alternative retrograde pathways between the endosomes and ER that either include or bypass the Golgi, where the majority of trafﬁc nor- mally passes through the Golgi. To investigate these possibilities, we examined the fate of Stx where access to the Golgi was blocked.

regulated vesicular trafﬁcking process [25,26]. In addi- tion, components of the retromer complex, speciﬁcally sorting nexins 1 and 2 and Vps26 are required for traf- ﬁc of Stx through the endosomes, but it is still unclear if this mediates an intra-endosomal step or if they are required for delivery to the TGN [27–29]. Delivery to the TGN does appear to involve the GARP complex, ﬁrst identiﬁed in yeast as mediator of retrograde trafﬁc into the Golgi [30]. It is clear that Stx does not pass through the late endosomes [24]. Instead, direct trans- port to the TGN is mediated by syntaxin 5, syntaxin 6, and syntaxin 16, a pathway that is shared by the endogenous protein TGN38, which cycles between Golgi and plasma membrane via REs [31,32]. Unlike TGN38, trafﬁc of StxB from endosomes to Golgi is dependent upon the Golgin, GCC185 [31,33–35].

Here we examine the trafﬁc of StxB which follows the same route as the holotoxin through the retrograde trafﬁcking pathway [15,36]. We perturbed access to the ) treatment, temperature block and Golgi by an AlF4 subcellular there exit surgery to examine whether routes for StxB to bypass the Golgi while trafﬁcking from endosomes to ER. Using these systems, we deter- mined that Golgi transit is required for trafﬁcking to the ER.

Results

StxB co-localizes with transferrin-positive endosomes

is

co-localization studies,

Stx is secreted by Shigella dysenteriae. It is highly homologous to the Shiga-like toxins (also termed vero- toxins) secreted by enterohemorrhagic strains of Esc- herichia coli. Stx is a member of the A-B5 family of toxins, which are composed of one enzymatic A sub- unit, noncovalently bound to a B subunit composed of a homopentamer of B fragments [11]. The Stx A sub- unit is an rRNA N-glycosidase, which stops protein synthesis and causes cell death [12]. The A subunit must be delivered to the host-cell cytosol to encounter its ribosomal substrate. To reach this destination, it is carried by a homopentameric B subunit (StxB) along a retrograde pathway from the plasma membrane through the EE ⁄ RE to the Golgi and the ER. Stx takes advantage of trafﬁcking through the Golgi to the catalytic facilitate cleavage and activation of A subunit by trans-Golgi network (TGN) resident furin protease [13]. The catalytic domain remains attached to the anchor domain by a disulﬁde bridge that is cleaved when the complex enters the cytosol. Entry of the catalytic A subunit into the cytosol is via retrotranslocation [14–16]. The B subunit initially gains entry to cells by binding the neutral glycosphingolipid, globotriaosyl ceramide (Gb3 or CD77) at the cell surface [17]. Bound toxin is endocytosed via both clathrin-dependent and -independent mechanisms and is delivered to EEs [18–20]. There is no known protein receptor for Stx B subunit (StxB), and the mechanism by which it recruited into clathrin-coated pits remains unknown. StxB binding to Gb3 at the cell sur- face induces changes in plasma membrane topology resulting in the formation of tubular invaginations that facilitate internalization [21]. It remains to be deter- mined whether this toxin-induced pathway or clathrin- mediated endocytosis is predominant in normal cells. In both cases, newly internalized StxB appears to be delivered to EEs. StxB binding is not a passive process. Binding and endocytosis of the toxin is accompanied by activation of Syk kinase and activation of microtu- bule networks, which facilitate transport into the cell [22,23].

We ﬁrst sought to establish a time-line for retrograde trafﬁc of StxB in green monkey kidney BSC-1 cells. These cells were selected due to their distinct endo- somal and Golgi morphologies that allow ready visual identiﬁcation. Like HeLa cells, different strains of BSC-1 cells show different afﬁnities for StxB. Our lab- oratory strain (a gift from I. Mellman) binds StxB readily. Another strain reported by Spooner et al. [37] does not. For cells were infected with adenovirus containing human transferrin receptor (TfnR), a well-studied marker of the endo- cytic recycling pathway [5,38]. This infection did not alter the morphology of internalized StxB observed in uninfected cells (not shown). Cells were labeled on ice with both Cy3–StxB and Alexa 488–transferrin (Tfn) for 30 min. Internalization of both labels was per- formed at 37 (cid:2)C in label-free medium for the indicated times (Fig. 1A). After 5 min, Tfn was in peripheral puncta representing EEs (Fig. 1A; 5 min) [5]. StxB the EEs. This co-localized with Tfn throughout suggested that although internalization of StxB may be through clathrin-dependent or -independent mechanisms,

suggesting that

to the Golgi,

this

Passage of Stx through the EEs ⁄ REs is well docu- mented and involves many proteins that are now being identiﬁed [3,9,24]. Two Rab GTPases, Rab11a and Rab6A¢, regulate retrograde trafﬁc of Stx from the is a EEs ⁄ REs

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A

B

C

Fig. 1. Trafﬁcking of StxB in BSC-1 cells. Cy-3 StxB was bound to BSC-1 cells on ice and internalized at 37 (cid:2)C for the times shown. (A) StxB passes through Tfn-positive endosomes. Alexa 488 Tfn and StxB bound to BSC-1 cells expressing human Tfn receptor on ice and then warmed for times shown. Both co-localized up to 20 min. By 45 min Tfn (green) and StxB (red) had separated. Arrow indicates perinuclear endosome. StxB remained in a Golgi-like ribbon for the remainder of the Tfn ⁄ StxB time-course 60–180 min. (B) Internalized StxB (red) with cells ﬁxed and immunolabeled for Golgi marker GM130 (green). Note co-localization (yellow). (C) Internalized StxB (red) with cells ﬁxed and immunolabeled for ER marker PDI (green). Note co-localization at 240 min. Inset is indicated area magniﬁed. Bars = 10 lM.

they converge on the EEs [39,40]. After 10 min, StxB and Tfn co-localized in both the peripheral EEs and a perinuclear organelle, identiﬁed by Tfn pulses as the RE (Fig. 1A; 10 min) [41]. After 30 min, Tfn primarily labeled the endosomes, although the signal was weaker due to recycling of Tfn into the media, whereas StxB had entered a separate perinuclear structure (Fig. 1A; 30 min). This structure had the appearance of a Golgi ribbon in these cells. The difference in localization was more obvious after 45 min (Fig. 1A; 45 min arrow the indicates

transferrin-containing endosomes). At

later times, Tfn had recycled out of the endosomes and was no longer clearly visible although StxB remained in Golgi morphology (Fig. 1A; 60, 120 min) [38,42]. At 180 min, the internalized StxB took on a lacy the ER (Fig. 1A), suggesting appearance typical of that a substantial amount of the toxin had been deliv- ered to the ER [43]. Thus, endocytosed StxB was deliv- ered into the endocytic recycling pathway within endosomes transferred to perinuclear 5 min, was within 10–20 min, and then was delivered to the Golgi within 30–45 min of internalization.

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StxB is delayed in the Golgi before entering the ER

at

longer

StxB internalization

after

pathway.

Surprisingly,

structures

in the cytosol. The GDI

We next characterized the passage of StxB through the Golgi of BSC-1 cells under normal cell culture condi- tions (Fig. 1B,C). The distribution of StxB at various time points was compared with that of the cis ⁄ medial Golgi marker GM130, or the ER marker protein disul- ﬁde isomerase (PDI) [44,45]. Cells were labeled with StxB as before and ﬁxed for immunoﬂuorescence. StxB initially partly co-localized with the cis ⁄ medial Golgi marker GM130 after 20 min (Fig. 1B), and co-localiza- tion increased up to 120 min (Fig. 1B). This conﬁrmed that StxB passes from the transferrin-positive endo- somes to the Golgi rather than to another compart- ment such as late endosomes [24]. Passage through the Golgi was slow, as observed elsewhere [2]. To deter- mine how long it took for StxB to enter the ER, we internalized StxB for up to 4 h and labeled the cells for the ER marker, PDI (Fig. 1C). StxB remained in a perinuclear ribbon (Golgi, as shown by co-localization in Fig 1B) up to 120 min. StxB began to co-localize with PDI at 150 min (not shown) and 180 min (not shown). By 240 min (Fig 1C), StxB was localized to the ER as shown by co-localization with the ER resi- dent, PDI. These data support the observation that passage through the Golgi is the slowest step in the retrograde pathway, requiring up to 120 min [46]. Taken together, Fig. 1A–C established a normal time- course of StxB trafﬁc in BSC-1 cells. We used this time-course as a basis for our further experiments.

as

Passage through the Golgi is required for StxB to reach the ER in cytoplasts

lacking a Golgi apparatus

ized with Tfn in endosomal structures (Fig. 2A; 10 min yellow arrow). After 30 min, Tfn and StxB continued to co-localize with Tfn in endosomes (compare Fig. 2A; 30 min to Fig. 1A). Because Tfn recycles out of times cytoplasts (120 min), it was necessary to add Tfn to the media for 5 min and chase in unlabeled media for the ﬁnal 25 min of the assay before ﬁxation to illuminate the 120 min, endocytic although the majority StxB (red arrows) remained inside the cytoplasts, it did not co-localize with endo- somal (labeled with Tfn, green arrow). Rather, it appeared in a diffuse cytosolic-like pattern (red arrows, Fig. 2A; 120 min). To ensure that Golgi was not inadvertently included in the cytoplasts, we immunolabeled cytoplasts for GM130 and found it to be absent from the cytoplast, but readily visible in the karyoplast (Fig. 2B). To identify which compartment the StxB had entered, we chased StxB into cytoplasts for 120 min and labeled the plasma membrane (wheat- germ agglutinin, WGA; Fig. 2C), ER (PDI; Fig. 2D), and cytosol (Rho GDI; Fig. 2E). StxB (red arrows) did not co-localize with WGA (green arrows; Fig. 2C) and thus had not recycled to the plasma membrane. Nor did it co-localize with the ER marker, even when allowing 240 min for co-localization with PDI (green arrows; Fig. 2D). However, StxB did co-localize with cytosolic GDI (yellow arrow; Fig. 2E), suggesting that StxB was immunolabel required methanol ﬁxation, which causes a grainy cast to cytosolic proteins. Cytosolic depletion using SLO or saponin proved unfeasible treated cytoplasts detached from the coverslip. Together, these results suggest that when the Golgi was absent, StxB did not enter the ER. Furthermore, under these conditions the toxin was able to escape the endosomes directly into the cytosol. At no time was an ER morphology or co-localization with PDI of StxB observed. While it is possible that some remnant Golgi, below the threshold of visualization, was present in the cytoplast, it was clearly insufﬁcient to mediate StxB trafﬁc to the ER. This phenomenon may occur to some extent during normal transit of the endosomes, although the amount of toxin available for escape may be minimal as the toxin passes rapidly through the endosomes to the Golgi. It may, however, correspond to the brefel- din A-resistant toxicity reported elsewhere [8].

Aluminum ﬂuoride traps StxB and Tfn in perinuclear endosomes

We wished to test directly if passage through the Gol- gi ⁄ TGN was required for StxB entry into the ER. To accomplish this, we made use of subcellular surgery to [47]. create cytoplasts Peripheral extensions of adherent BSC-1 cells were cleaved using a glass micro-pipette to create cytoplasts (peripheral areas lacking a nucleus) and karyoplasts, containing the nucleus, the Golgi apparatus and the REs [48]. Cytoplasts generated in this manner lack a Golgi apparatus, and importantly, cannot regenerate one [47]. By contrast, cytoplasts can regenerate func- tional REs from peripheral EEs, as we have previously demonstrated. Recycling of Tfn in cytoplasts is com- plete and follows the same kinetics in cytoplasts as in whole cells [6]. Cytoplasts and karyoplasts were labeled with StxB and Tfn for 5 min at 37 (cid:2)C (rather than on ice to avoid releasing the cytoplast from the coverslip) and both ligands were chased into the cytoplasts for various times (Fig. 2A). After 10 min, StxB co-local-

We wished to conﬁrm the requirement for Golgi pas- sage and to quantify escape of StxB from endosomes

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A

B

E

C

D

Fig. 2. StxB cannot access the ER in BSC-1 cytoplasts. BSC-1 cells were manually cut with a glass needle to create karyoplasts (k) contain- ing both the nucleus and Golgi and cytoplasts (c). All cytoplasts and karyoplasts were labeled with Cy-3 StxB (red) that was internalized for times shown. (A) Shiga and Tfn (green) internalized together for 10 min then chased for 10 or 30 min. For 120 min, Tfn was internalized for the ﬁnal 25 min. (B) Cytoplast with StxB (red) immunolabeled for Golgi marker GM130 (green). (C) Cytoplast stained for plasma membrane with wheat germ agglutinin (green). Note that the cytoplast has moved next to the karyoplast but the two remain separate. (D) Cytoplast labeled for ER marker PDI (green), at various times of StxB (red) internalization. Note exclusion of StxB from ER. (E) Cytoplast labeled for cytosolic marker GDI (green) note co-localization (yellow) with StxB (red). Insets are cytoplasts presented in single channels with larger inset showing a magniﬁed view of the combined channels. Red arrows indicate StxB, green arrows indicate other compartment markers as indicated. Bars = 10 lM.

))

normally have recycled out of the cell, and StxB would normally have moved to the Golgi. Both remained in the endosomes of treated cells after 60 and even 120 min (Fig. 3; 60 min, 120 min, yellow arrows). Although this result is based on the fortunate effects ) treatment on these two pathways, it does not of AlF4 necessarily imply that the same drug target is involved in both retrograde and recycling pathways. It does, however, present a unique opportunity. As in the cytoplast, StxB is prevented from reaching the Golgi, and it is trapped inside of the endosomes. This allowed us to quantify the consequences of trapping StxB in endosomes.

StxB leaks into the cytosol when trapped at endosomes

into the cytosol. However, cytoplasts are extremely small, and must be made individually, making frac- tionation impossible. Therefore, we used a pharmaco- is an logical approach. Aluminum ﬂuoride (AlF4 activator of small GTPases and is well-documented to block recycling of Tfn from the RE [5,49]. Although ) at the RE is not known, the precise target of AlF4 the effect of this drug on Tfn recycling is immediate and remarkably speciﬁc to recycling out of the RE in nonpolar cells and to basolateral recycling from the RE in polarized cells [5]. Treatment for > 1 h results in dispersal of the Golgi although both the TGN and the ER remain functional [50,51]. Because StxB co-localized extensively with Tfn in perinuclear REs, ) might also block retrograde we suspected that AlF4 StxB from the endosomes to the TGN just as it did for recycling trafﬁc to the plasma membrane. Fortu- itously, both StxB and Tfn were trapped together in ) treatment (Fig. 3). This was the REs following AlF4 especially apparent after 60 min, when Tfn would

)-treated cells StxB trapped in the endosomes of AlF4 took on a diffuse cytosolic appearance at later time points following internalization (Fig. 3; 120 min, red

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Fig. 3. Aluminum ﬂuoride traps Stx in endosomes. Both StxB (red) and Tfn (green) were bound to BSC-1 cells on ice. Both were internalized ). Yellow arrows indicate where both Tfn and StxB have been trapped in a perinuclear at 37 (cid:2)C for times shown in the presence of AlF4 endosome. Red arrows indicate StxB in diffuse distribution. Inset is magniﬁcation of indicated area. Bar = 10 lM.

the cytoplasts. This suggested that StxB alone could escape endosomes if it was not sequestered into the Golgi. It was not completely clear if the extra-endoso- mal StxB was in a membrane or cytosolic fraction. It was also unclear if endosomal escape was speciﬁc ) treatment altering the for StxB or resulted from AlF4 endosomal membranes to allow escape of all cargo.

arrows). RE-associated versus peripheral ﬂuorescence was measured using NIH image in 20 cells; 20 ± 7% was found in the periphery. The diffuse material did ) is not have an ER morphology, however, AlF4 known to disperse the medial Golgi in some cells after extended treatment (> 120 min) [50]. To determine which cellular compartment StxB had entered, we per- formed a series of co-localizations (Fig. 4). The distri- bution of StxB was compared to the cis ⁄ medial Golgi marker GM130 in BSC-1 cells in the presence of ). Although dispersed, the Golgi remained clearly AlF4 visible as structures surrounding StxB-labeled endo- somes at times up to 240 min (Fig. 4A). GM130 did not co-localize with the StxB in punctate (endosomal) structures nor did it co-localize with the diffuse StxB ) treatment (Fig. 4A). This conﬁrmed both that AlF4 prevented access to the Golgi and that StxB was not in the fragmented Golgi.

It was possible that AlF4

To differentiate between these possibilities, we used a cell fractionation approach to separate cytosol from membrane-bound organelles. Iodinated StxB was bound to BSC-1 cells on ice, washed, then warmed in ) to initiate internaliza- the presence or absence of AlF4 tion. Cells were harvested after 120 min of chase, then homogenized in a ball-bearing cell homogenizer so as to recover intact organelles. Membrane and cytosolic fractions were separated via Opti-prep step-gradients. In these experiments, (Fig. 5A) cytosol was collected from the top of the gradient, and all membranes were collected from an Optiprep cushion at the bottom of the gradient. As a control for rupture of endosomes, 125I-labeled Tfn was bound to the cell surface and then chased into the endosomal compartments of control cells. Because Tfn normally recycles rapidly out of the cell, it was chased for 20 min in control cells or for )-treated cells. This ensured that Tfn 120 min in AlF4 would be in the REs [5,6]. Because Tfn is released

) treatment may have allowed StxB to bypass the Golgi and enter the ER. However, despite changes in ER morphology (Fig. 4B), StxB did not co-localize with the ER marker PDI, even at 240 min (Fig. 4B). As with the cytoplasts, StxB ) did not prevented from reaching the Golgi by AlF4 access the ER. A fraction of the internalized StxB appeared to escape the endosomes as had occurred in

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A

B

Fig. 4. Aluminum ﬂuoride traps StxB in endosomes. (A) StxB (red) bound to BSC-1 cells and internalized for times shown in the presence of ). Cells were stained for Golgi marker GM130 (green). Note diffuse StxB. Red arrows indicate StxB in endosomal structure, Green AlF4 arrows indicate the Golgi. (B) Cells labeled as in A but stained for ER marker PDI (green). Green arrows indicate ER structures. ER morphol- ogy is altered (compare with Fig. 1). Bar = 10 lM.

A

B

Fig. 5. StxB trapped in the endosomes leaks into the cytosol. (A) Quantiﬁcation of StxB in cytosol. 125I-labeled StxB or 125I-labeled Tfn inter- nalized into BSC-1 cells expressing transferrin receptor for 120 min. Cells were harvested and homogenized. Total cytosol was separated from total membranes using an Optiprep 0 ⁄ 8 ⁄ 25% step gradient. Average values for the percent of each ligand in the cytosol with and with- ) are shown. Error bars are SD. *Signiﬁcant change. n = 15 for StxB conditions, n = 9 for Tfn conditions. (B) Cell fractionation of out AlF4 BSC-1 organelles to identify those containing internalized ligands on preformed linear 8–25% Optiprep gradients. Top of gradient is to the ) for 1 h. Bar 1 indi- left. (I) 125I-labeled StxB internalized in the absence (dark red, closed circles) or presence (orange, open circles) of AlF4 cates cytosolic fractions. Bars 2 and 3 indicate endosome or Golgi associated peaks in treated cells. Bar 4 indicates a peak of membrane bound StxB found only in treated cells. (II) Positions in the gradient of cytosol (red), plasma membrane (orange), lysosomes (yellow), REs (green), Golgi (light blue), ER (dark blue) and cell debris (purple). Supporting data are given in Fig. S1. (III) 125I-labeled Tfn internalized for ) treated cells (treat- 25 min in control cells to locate RE (at 1 h it is recycled out of the cell), Bar 6 (dark blue closed squares) and 1 h in AlF4 ment prevents recycling) (light blue, open squares). Note that some Tfn remains at the plasma membrane Bar 5 (n = 1, results typical).

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with the cytoplast results, this suggests that when StxB is trapped within the endosomes, it can ‘escape’ into the cytosol, as previously suggested for dendritic cells and macrophages [52,53]. A similar escape has also been observed for the Stx A subunit [8].

A temperature block separates StxB and Tfn

) (Fig. 5A; StxB vs. StxB + AlF4

from its receptor at the neutral pH of the gradient, it acts as a sensitive indicator of endosomal rupture dur- ing handling. It also serves as a speciﬁc indicator of ) treatment. changes in endosomal fragility due to AlF4 Figure 5A shows that there was no difference in cyto- )-treated solic transferrin between control and AlF4 )). Thus, endo- cells (Fig. 5A; Tfn and Tfn + AlF4 somes were not made more fragile by the drug treat- ment. By contrast, the amount of StxB found in the cytosol was signiﬁcantly larger in the presence of )). The differ- AlF4 ence was statistically signiﬁcant with P < 0.0001 (Stu- dent’s t-test). This cytosolic escape was not observed when StxB was internalized for only 20 min (data not shown), a time at which StxB remained in endosomes and was not visualized in the cytosol in intact cells.

lengths of

discriminated

can

It remained possible that StxB was equally capable of entering the cytosol from any organelle along the ret- rograde pathway. We tested this possibility by using a temperature block to trap StxB within the TGN for a time. In HeLa cells, it has been reported that maintain- ing the cells at 20 (cid:2)C traps StxB along with Tfn in the endosomes [24]. We too observed this effect in HeLa cells (Fig. S2). However, reducing the temperature to 20 (cid:2)C in BSC-1 cells had a surprising and useful effect. Both Tfn and StxB were bound to BSC-1 cells and internalized at 20 (cid:2)C for various time (Fig. 6A). As expected, Tfn did not recycle out of the endosomes, but remained in the perinuclear region. After 30 min, StxB appeared to co-localize with Tfn in the majority of cells. However, after 60 min, StxB sep- arated into another perinuclear structure that did not co-localize with Tfn. This distribution was maintained up to 180 min. We suspected that this other structure might be part of the Golgi due to the ribbon-like appearance. Fortunately, in BSC-1 cells, the TGN and cis ⁄ medial Golgi visually be (although there is slight overlap) using the TGN marker TGN46 and the cis ⁄ medial marker GM130 (Fig. 6B). We therefore compared the localization of StxB with that of TGN46 and GM130 after 120 and 180 min of internalization at 20 (cid:2)C. There was striking co-localization of StxB with the TGN marker at both time points (Fig. 6C) suggesting that at 20 (cid:2)C in BSC-1 cells, StxB was trapped in the TGN. This was very dif- ferent from the situation at 37 (cid:2)C (Fig. 6D) where StxB co-localized with both TGN and cis ⁄ medial Golgi in as little as 90 min.

We wished to conﬁrm that we were seeing co-locali- zation in the TGN and not at ER exit sites. StxB inter- nalized at 20 (cid:2)C co-localized with TGN46 but clearly did not co-localize with the ER exit site marker Sec31, again suggesting that StxB was trapped speciﬁcally at the TGN at this temperature (Fig. 6E).

Cytosolic StxB accounted for 12% of the radiola- beled StxB, but analysis of cell ﬂuorescence had found that 25% of internalized StxB was not in the endo- somes. To resolve this difference, we utilized a differ- ent density gradient protocol to fractionate organelles within the cell. 125I-labeled StxB cell homogenates )-treated cells were applied to from control and AlF4 preformed linear 8–25% Optiprep gradients. We have previously described the use of these gradients for the fractionation of cellular organelles [5]. Gradients were characterized by locating fractions containing alkaline phosphodiesterase activity (plasma membrane), B-hex- osaminidase activity (lysosomes) radiolabeled Tfn internalized for 25 min (REs), added phenol red (cyto- sol), GM130 (Golgi) and PDI (ER). The position of the peak activity for each organelle is shown in the color-coded bar in Fig. 5B (II), and in Fig. S1. StxB internalized for 60 min in control cells co-localized with Golgi and REs (which could not be readily distin- ) treatment guished in this gradient). However, AlF4 shifted the distribution of both StxB and Tfn into a doublet of peaks at, and just below, the density of REs (Fig. 5B). In this representative experiment, 9% of StxB was observed in the cytosol in AlF-treated cells compared with 3% in control cells. Also, 24% of StxB was observed in a dense fraction (compared with 8% in control cells) that did not co-segregate with any of the characterized organelles. Although this could not be identiﬁed, we speculate that it may represent transport vesicles derived from the endosomes, unable to reach the Golgi. This fraction would account for the difference between non-endosomal StxB observed microscopically and that observed in the cytosolic frac- tion of step gradients above.

These results suggest that AlF4

) can block retro- grade trafﬁc at the endosomes and that StxB is able to escape the endosomes to the cytosol. Taken together

These results suggested that BSC-1 cells, unlike HeLa cells, hold StxB in the TGN at 20 (cid:2)C. This difference between HeLa and BSC-1 cells provided a natural experiment in BSC-1 cells to test if StxB could escape into the cytosol when it was held in the TGN instead of the endosomes. Notably, no escape of StxB into the cytosol was seen in the cells held at 20 (cid:2)C

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B

D

E

Fig. 6. A 20 (cid:2)C temperature block traps Stx in the TGN in BSC-1 cells. (A) StxB (red) and Tfn (green) were internalized for times shown at 20 (cid:2)C. Yellow arrows indicate co-localization at earlier times, red arrows indicate StxB differently distributed than Tfn. (B) anti-GM130 for cis ⁄ medial Golgi (green) and anti-TGN46 for TGN (red) are visually resolved, two BSC-1 cells are shown. (C) StxB (red) co-localizes with TGN46 (blue) but not with GM130 (green) when internalized at 20 (cid:2)C. Purple arrows indicate co-localization of StxB and TGN46. Red bor- dered inset shows magniﬁcation of cytosol in a cell adjacent to the labeled Golgi. Note lack of cytosolic StxB. (D) Same as (C) but internal- ized at 37 (cid:2)C. Yellow arrow indicates partial co-localization of GM130 and StxB. (E) StxB (red) co-localizes with TGN46 (blue) but not with ER exit point marker Sec31 (green) when internalized at 20 (cid:2)C. Upper insets are enlarged versions of regions indicated. Lower insets are Sec31 (green), StxB (red) and a merge of only Sec31 and StxB. Purple arrows indicate co-localization of StxB and TGN46. Green and red arrows indicate locations of ER exit sites and StxB respectively. Bar = 10 lM.

even after 180 min (Fig. 6C; red box and E). Taken together these results suggest that StxB cannot escape the TGN into the cytosol, and that escape may be

dependent upon some property of the endosomes such as low pH found in EEs or membrane composition [54,55].

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Discussion

Stx is an A–B5 toxin that binds to the plasma mem- brane lipid Gb3. In this respect it is like cholera toxin and SV40 virus, both of which bind to the ganglioside GM1 [56,57]. Both toxins, and the virus are trans- ported to the ER after internalization [58]. However, whereas cholera toxin appears to pass through endo- somes and the Golgi, SV40 bypasses both the normal endocytic organelles and the Golgi, trafﬁcking directly from a specialized endocytic population to the ER [10]. These examples demonstrate that binding to a glycolipid receptor, and even trafﬁcking to the ER do not guarantee passage through the Golgi. Further- more, even if passage through the Golgi is a normal component of the retrograde pathway, it does not automatically follow that this pathway is exclusive of other routes.

We sought to determine whether retrograde trafﬁc of StxB required passage through the Golgi to reach the ER. A morphological examination of retrograde trafﬁc, as performed here, suggests that StxB appears to progress sequentially from the plasma membrane to the endosomes to the Golgi and then to the ER. How- ever, just as the majority of Tfn actually bypasses the RE, a fraction of StxB may actually bypass the Golgi [5]. Alternatively, the Golgi transit route could be pre- ferred and mask a lower ﬂux endosome to ER route.

EE compartments. The most obvious would be to recycle out of the endosomes and return to the plasma membrane along with Tfn. However, StxB did not reappear at the plasma membrane in any visible amount. A second possibility would be for StxB to be shunted into late endosomes (also found in cytoplasts) [6]. However, we did not see co-localization with lyso- somal proteins (J. McKenzie & D. Sheff, unpublished observations). To our surprise, StxB appeared to enter the cytosol directly from the endosomes in cytoplasts. Because cytoplasts are extremely small and must be formed manually, we were unable to perform biochemi- cal analysis or cell fractionation on these preparations. ) treat- However, we were fortunate in ﬁnding that AlF4 ment blocked exit of StxB from the endosomes. Despite being a generalized inhibitor of GTPase function in ) is speciﬁc for the endocytic recycling pathway, AlF4 ), we were able to con- egress from REs [5]. Using AlF4 ﬁrm that 12% of internalized StxB accessed the cytosol directly from the endosomes. This result is inconsistent with prior ﬁndings that a small percentage of Stx A subunit (5%) can effectively reach its target even when trafﬁc along the postendosomal retrograde path- way is impaired by disruption of the Golgi with brefel- din A [8]. It is also consistent with the prior ﬁnding that 10% of cell-associated StxB reaches the cytosol in human monocyte derived macrophages [53]. This is particularly relevant because monocytes-derived macro- phages internalize StxB, but do not support retrograde trafﬁc of StxB from endosomes to the Golgi [52]. Fur- thermore, our ﬁnding that StxB trapped in the TGN could not enter the cytosol, suggests that endocytic membranes are particularly susceptible to penetration by StxB. Thus toxicity in the monocyte-derived macro- phages may paradoxically result from the inability of the cell to transport toxin out of the endosomes and into the Golgi (provided that the toxin would then not be able to exit the Golgi).

We used cytoplasts to physically separate endosomes and ER from the Golgi in an isolated piece of living cells. We had previously determined that these cytop- lasts are able to regenerate a fully functional endocytic system with both EEs and REs [6]. They also contain ER, as demonstrated here. However, they are unable to regenerate a Golgi, which allowed us to test directly whether Golgi transit was required for StxB to pro- gress from endosomes to the ER [47]. In the absence of a Golgi, StxB was unable to access the ER in cytop- lasts. Had a slower or lower ﬂux pathway connected the endosomes directly to the ER, we would have seen StxB in the ER of the cytoplasts. Although it remains possible that such a pathway exists for some endo- genous proteins, it is clearly not accessed by StxB.

Penetration of the endosomal membrane by StxB was surprising in light of the normal retrograde path- way taken by StxB. However, endosomal escape is known not only for bacterial toxins such as diphtheria toxin, but also for ﬁbroblast growth factor following endocytosis [54, 60–62]. Such translocation may repre- sent another physiological pathway that is subverted by StxB when the Golgi pathway is unavailable.

Our ﬁndings raise the question of why StxB should take such a circuitous path from plasma membrane to cytosol. Other toxins such as diphtheria toxin and anthrax toxin take advantage of low endosomal pH to penetrate the endosomal membrane and enter the cyto- sol directly [59]. Our results may shed some light on the host–pathogen interaction that has developed around the retrograde trafﬁc of StxB. In cytoplasts, StxB was trapped at the endosomes. There are several possible alternative routes that would be available out of the

Clearly StxB cannot be present in the cytosol while membrane bound. This suggested that the toxin was able to dissociate from Gb3 while inside the endosome. Such dissociation is unlikely to be a result of low pH because acid wash of bound StxB does not remove it from the plasma membrane. However, StxB can bind up to 15 Gb3 molecules, mediated through three

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grade transport out of the Golgi into the ER. While this process is also slow, it is quantitative, and results in delivery of the bulk of the toxin to the ER where the A subunit can retrotranslocate into the cytosol by exploiting the translocon [16,66]. In this case, the cur- rent retrograde transport of StxB and of holotoxin may have developed as a series of host–pathogen inter- actions and adaptive responses. Although our data cannot be conclusive in this respect it is at the least supportive of the possibility.

Materials and methods

Cells and reagents

this effect

different sites on each B subunit of the pentamer [63]. The overall Kd of the StxB ⁄ Gb3 complex is 10)9 m. This results from the combined associations of all sites with Gb3 molecules in the membrane. However, binding to site II formed by the cleft between monomers is much weaker that that of site I [63]. Thus if Gb3 is present at lower concentrations in the endosomes than at the plasma membrane, one would expect dissociation of the cleft-bound Gb3 from the complex, and dissociation of the entire molecule when Gb3 concentrations become sufﬁciently low. Recently, it has been demonstrated that decreasing the concentration of Gb3 available at the plasma membrane in Vero cells results in a precipitous decline in StxB binding [64]. We would suggest that the concentration of Gb3 may be signiﬁcantly lower within endosomes than at the plasma membrane, providing the possibility for some StxB to no longer be bound to the receptor [53]. Further examination of is beyond the scope of this study.

The ability of StxB to penetrate the endosomal membrane raised the important question of whether the endosomal membrane was uniquely permeable to StxB. If StxB can cross any membrane, then it would be expected that it could directly penetrate the plasma membrane, and this is not the case, as demonstrated lines lacking Gb3 which are resistant to the by cell toxin [65]. Our use of BSC-1 cells allowed use to take advantage of a unique feature of the 20 (cid:2)C tempera- ture block in these cells. Rather that trapping StxB at the endosomes as in HeLa cells, StxB was able to traf- ﬁc to the TGN in these cells. Although all trafﬁcking is slowed (or stopped in some cases) at this tempera- ture, this would still allow for escape of the StxB into long incubation times. We did not the cytosol at observe StxB entering the cytosol under these condi- tions, suggesting that the TGN membrane is less permeable to StxB than that of the endosomes.

BSC-1 cells clone CCL-26 from ATCC (Manassas, VA, USA) were cultured in minimal Eagle’s medium (MEM) sup- plemented with 10% fetal bovine serum, 1% nonessential amino acids, 1% sodium pyruvate, 100 unitsÆmL)1 penicillin and 100 lgÆmL)1 streptomycin in 5% CO2, 95% air. NaF and AlCl3 were obtained from Fisher (Fair Lawn, NJ, USA). Recombinant wild-type StxB derived from S. dysenteriae was produced from clone in pSU108. The protein was induced and puriﬁed as previously described [24,67]. Puriﬁed StxB was labeled using Alexa-dye protein labeling kits from Invitrogen (Carlsbad, CA, USA) according to manufacturers directions. Polyclonal rabbit anti-Sec31 serum (kind gift from the Warren Lab, Max F. Perutz Laboratories, Vienna, Austria); mouse anti-GM130 mAb from BD Biosciences (San Jose, CA, USA); mouse anti-PDI mAb from Nventa (San Diego, CA, USA); polyclonal sheep anti-TGN46 from Serotec (Raleigh, NC, USA); polyclonal rabbit anti-(Rho GDI clone K-21) from Santa Cruz (Santa Cruz, CA, USA); Alexa 488-labeled WGA, Alexa488 human holotransferrin, Alexa 488 goat anti-mouse IgG, Alexa 488 goat anti-rabbit IgG and Alexa 633 donkey anti-sheep IgG were obtained from Invitrogen (Carlsbad, CA, USA). 125I-conjugated Tfn and StxB were made using Iodo-Gen (Pierce ⁄ Thermo Scien- tiﬁc, Rockford, IL, USA) as previously described for Tfn [5].

StxB and Tfn labeling

BSC-1 cells were grown on glass coverslips. For cytoplasts, square gridded coverslips (Belco) were used and cells for live images were grown on glass-bottom 35 mm dishes. For Tfn uptake experiments, cells were preincubated in serum- free media for 30 min at 37 (cid:2)C to clear Tfn from the cell. Cells were chilled on ice and labeled with a 1 : 200 dilution of 0.22 mgÆmL)1 Cy3 StxB and a 1 : 100 dilution of 5 mgÆmL)1 Alexa 488–Tfn in NaCl ⁄ Pi. Internalization was performed by placing the cells in 37 (cid:2)C MEM for indicated times. Tfn was omitted as indicated. When noted, 50 lm aluminum chloride ⁄ 30 mm sodium ﬂuoride was included during internalization. For 20 (cid:2)C block studies, cells were

One tempting possibility is that the difference in membrane permeability has driven host–pathogen interactions over the course of evolution. Some toxins, such as anthrax toxin have evolved mechanisms to avoid recycling out of the endosomes by rapidly enter- ing the cytosol. Our data suggest that while StxB can accomplish this penetration, the process is slow and inefﬁcient. Host cells may have acquired the ability to sequester the toxin in the Golgi, thereby preserving the cell so that over time the toxin may be degraded. Such a possibility is supported by the rapid and near quanti- tative diversion of StxB from the endocytic pathway into the Golgi. The slow passage of the toxin through the Golgi also argues in favor of this possibility. The response of the pathogen to this sequestration appears to have been to develop a mechanism to exploit retro-

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described previously [5]. ER was detected by western blot for PDI. Endosomes were identiﬁed by the presence of 125I-labeled Tfn internalized for 25 min.

labeled in MEM (without fetal bovine serum) at 20 (cid:2)C instead of on ice, for 5 min, media was then replaced with MEM. Cytoplasts are sensitive to cold and so were labeled for 5 min at 37 (cid:2)C in MEM followed by unlabeled MEM at 37 (cid:2)C.

Immunoﬂuorescence

Cytoplast creation

BSC-1 cells were grown on gridded coverslips for 2 days to allow cells to spread out. Glass needles were prepared using 1.0 mm o.d., 0.50 mm i.d., 10 cm length capillaries from Sutter Instruments (Novato, CA, USA) on a Sutter Instru- ments P-97 micropipette puller. For microsurgery, cells were transferred to bicarbonate-free MEM buffered with 10 mm Hepes pH 7.4. Cellular microsurgery was performed using the glass needle mounted in a Sutter Instruments MP-285 micromanipulator with a joystick controller under manual control. Cuts took an average of 5 min. Cytoplasts were allowed to recover for at least 1 h after microsurgery prior to labeling. Cytoplasts were ﬁxed as for whole cells. Cells were washed with NaCl ⁄ Pi, ﬁxed in 3% PFA at room temperature for 15 min. Cells were permeabilized with 0.05% (w ⁄ v) saponin in NaCl ⁄ Pi with 3% BSA for 1 h then washed three times in blocking buffer (0.05% saponin, 3% BSA, NaCl ⁄ Pi) for 5 min. For PDI labeling, permeabi- lization was performed by 0.04% Triton X-100 in NaCl ⁄ Pi for 4 min then washed three times in blocking buffer (3% BSA, NaCl ⁄ Pi) for 5min. For GDI labeling, cells were rinsed with cold NaCl ⁄ Pi then ﬁxed for 5 min in 100% methanol at )20 (cid:2)C. For immunolabeling, cells were incu- bated with a 1 : 200 dilution of antibodies (except PDI, 1 : 500) at room temperature for 45 min. Cells were visual- ized with appropriate Alexa-conjugated secondary anti- bodies at 1 : 200 dilution for 30 min at room temperature, washed and mounted.

Membrane and cytosol fractionation

Image analysis

All images were acquired on a Zeiss 200M inverted micro- scope equipped with a Hamamatsu ER camera operated by Openlab from Improvision (Coventry, UK) on a Macintosh G4 computer from Apple (Cupertino, CA, USA). Low exposure and high exposure images of all cells were obtained. For cytoplasts, low exposures are shown for kar- yoplast and cytoplast, and the inset is of the higher expo- sure due to the low levels of signal in cytoplasts. Contrast in images was optimized using photoshop from Adobe (San Jose, CA, USA) on an Apple G5 computer.

Acknowledgements

This work supported by a grant to JM from the American Heart Association #081365G as well as to DS from American Heart Association #035078N. Support for TT was provided by 21st Century Center of Excellence Program from the Ministry of Educa- tion, Culture, Sports, Science and Technology of Japan, The Core Research for Evolutional Science and Technology (CREST), JSPS (Japan Society for the Promotion of Science) Core-to-Core Program Grants-in-aid for Scientiﬁc Research (18050019), and Senri Life Science Foundation Grants. Many thanks to Graham Warren for discussions about the Golgi and to Lawrence Pelletier for discussions about cytoplasts and Stx. We would also like to thank Mark Stamnes, Heidi Hehnly, Naava Naslavsky and Steve Caplan for their thoughtful discussions and suggestions.

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BSC-1 cells were grown to near conﬂuency in 35 mm dishes. For Tfn labeling, BSC-1 cells were infected with human Tfn receptor expressing adenovirus at an MOI of > 50, 24 h prior to use. Cells were serum starved for 30 min then labeled on ice with 125I-labeled Tfn or StxB for 30 min. Cells were then rinsed with chilled NaCl ⁄ Pi and ). warmed in MEM to 37 (cid:2)C in presence or absence of AlF4 For control cells (Tfn, no AlF), the radiolabeled Tfn was taken up warm for 5 min then chased for 20 min, because it would otherwise recycle back out to the cell surface. Cells were then harvested and homogenized via a ball-bearing cell homogenizer. Post nuclear supernatants were then gen- erated by centrifugation at 1000 g for 5 min. Two different fractionations were performed. To separate cytosol from membranes, the supernatant was overlaid onto a layer of 8% Optiprep which was in turn overlaid onto a layer of 25% OPTI-Prep (diluted with ICT buffer: 78 mm KCl, 4 mm MgCl2, 8.37 mm CaCl2 10 mm EGTA, 50 mm Hepes ⁄ KOH pH 7.0) [6,68]. Cytosol remained in the top fraction, while membranes and organelles were recovered from the 8 ⁄ 25% interface after centrifugation at 100 000 g for 120 min at 4 (cid:2)C. CPM in each fraction was counted in a gamma counter for 5 min and the data compiled. Data were normalized to the total counts in each tube. Each con- dition was repeated at least four times. For density gradient organelle fractionation, the supernatant was overlaid onto an 8–25% preformed linear gradient of Optiprep created using a Gradient master from Biocomp (Toronto, Canada) and centrifuged at 100 000 g for 20 h to allow density equilibration of organelles in the gradient. Cytosol was identiﬁed visually using phenol red. Plasma membrane, lysosomes and Golgi were identiﬁed by enzyme assay as

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Supporting information

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The following supplementary material is available: Fig. S1. Fractionation of BSC-1 organelles on an 8–25% Optiprep gradient. Fig. S2. A 20 (cid:2)C temperature block in HeLa cells.

This supplementary material can be found in the

online version of this article.

61 Sorensen V, Wiedlocha A, Haugsten EM, Khnykin D, Wesche J & Olsnes S (2006) Different abilities of the four FGFRs to mediate FGF-1 translocation are linked to differences in the receptor C-terminal tail. J Cell Sci 119, 4332–4341. 62 Haugsten EM, Sorensen V, Brech A, Olsnes S &

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