Toxicon 82 (2014) 76–82

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Intracellular trafficking of Clostridium botulinum C2 toxin Masahiro Nagahama a, *, Chihiro Takahashi a, Kouhei Aoyanagi a, Ryo Tashiro a, Keiko Kobayashi a, Yoshihiko Sakaguchi b, Kazumi Ishidoh c, Jun Sakurai a a

Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan b Interdisciplinary Research Organization, Miyazaki University, Miyazaki 889-2192, Japan c Division of Molecular Biology, Institute for Health Sciences, Tokushima Bunri University, Yamashiro, Tokushima 770-8514, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 October 2013 Received in revised form 23 January 2014 Accepted 11 February 2014 Available online 26 February 2014

Clostridium botulinum C2 toxin is a binary toxin composed of an enzymatic component (C2I) and binding component (C2II). The activated binding component (C2IIa) forms heptamers and the oligomer with C2I is taken up by receptor-mediated endocytosis. We investigated the intracellular trafficking of C2 toxin. When MDCK cells were incubated with C2I and C2IIa at 37  C, C2I colocalized with C2IIa in cytoplasmic vesicles at 5 min, and C2I then disappeared (15 min incubation and later), and C2IIa was observed in the vesicles. Internalized C2I and C2IIa were transported to early endosomes. Some of both components were returned to the plasma membrane through recycling endosomes, whereas the rest of C2IIa was transported to late endosomes and lysosomes for degradation. Bafilomycin A1, an endosomal acidification inhibitor, caused the accumulation of C2IIa in endosomes, and both nocodazole and colchicine, microtubule-disrupting agents, restricted C2IIa’s movement in the cytosol. These results indicated that an internalized C2I and C2IIa complex was delivered to early endosomes, and that subsequent delivery of C2I to the cytoplasm occurred in early endosomes. C2IIa was either sent back to the plasma membranes through recycling endosomes or transported to late endosomes and lysosomes for degradation. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Clostridium botulinum C2 toxin Endocytosis Early endosomes Late endosomes Recycling endosomes

1. Introduction Clostridium botulinum produces botulinum C2 toxin, which recruits a binding component (C2II) to deliver the enzymatic component (C2I) to the interior of eukaryotic cells (Barth et al., 2004; Aktories and Barbieri, 2005; Aktories et al., 2012). Each protein has been reported to lack toxic activity when injected alone (Barth et al., 2004). Abbreviations: Bafilomycin A1, BFA; DAPI, 40 ,6-diamidino-2phenylindole; DEME, Dulbecco’s modified Eagle medium; EEA1, early endosome antigen 1; ER, endoplasmic reticulum; GFP, green fluorescent protein; Lamp2, lysosomal-associated membrane protein 2; MDCK cell, Madin–Darby canine kidney cell. * Corresponding author. Tel.: þ81 (0)88 602 8483; fax: þ81 (0)88 655 3051. E-mail address: [email protected] (M. Nagahama). http://dx.doi.org/10.1016/j.toxicon.2014.02.009 0041-0101/Ó 2014 Elsevier Ltd. All rights reserved.

These proteins act in binary combinations to produce toxic, cytotoxic, and lethal effects, and influence vascular permeability (Barth et al., 2004). C2I ADP-ribosylates monomeric actin at arginine-177 in the cytosol (Aktories et al., 1986; Wiegers et al., 1991). This ADP-ribosylation causes the breakdown of F-actin, leading to cell rounding and death. C2II cleavage by trypsin was shown to remove the N-terminal 20 kDa fragment, leading to the activation of C2II (C2IIa) (Barth et al., 2004). C2 toxin belongs to a family of binary actin-ADP-ribosylating toxins that includes Clostridium perfringens iota-toxin (Ia, an enzymatic component and Ib, the binding component), Clostridium spiroforme iota-like toxin, Clostridium difficile ADPribosyltransferase, and vegetative insecticidal protein from Bacillus cereus (Barth et al., 2004; Aktories and Barbieri, 2005; Aktories et al., 2012).

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C2IIa recognizes asparagine-linked carbohydrates on the surface of target cells and forms heptamers that bind C2I (Eckhardt et al., 2000). The toxin-receptor complex was shown to be internalized by receptor-mediated endocytosis and translocated to early endosomes (Barth et al., 2000; Pust et al., 2010). At the acidic pH of the endosomal compartment with vesicular Hþ-ATPase, the C2IIa oligomer was reported to be inserted into the endosomal membrane and formed pores, through which bound C2I was then translocated into the cytosol (Barth et al., 2000). C2IIa pores are essential for the translocation of C2I across endosomal membranes (Blöcker et al., 2003a, 2003b) and C2I translocates in an unfolded conformation through these pores into the cytosol (Haug et al., 2003). Membrane translocation of C2I was shown to be facilitated by the chaperone heat shock protein (Hsp) 90 (Haug et al., 2003) and folding helper enzyme cyclophilin A (CyPA) (Kaiser et al., 2009). CyPA interacts with C2I in intact cells (Kaiser et al., 2009) and Hsp90 directly binds to C2I (Kaiser et al., 2011). Kaiser et al. (2012) also demonstrated that the FK506-binding protein plays a role during membrane translocation of C2I. After translocation to the cytosol, C2I ADP-ribosylates G-actin in the cytosol. This event subsequently causes the depolymerization of actin filaments, breakdown of the actin cytoskeleton, and rounding of the cells (Barth et al., 2004; Aktories and Barbieri, 2005; Aktories et al., 2012). C2IIa and iota-toxin b were shown to induce endocytosis of their receptors via a lipid raftmediated process (Nagahama et al., 2004, 2009, 2012). However, the endosomal trafficking pathway of internalized C2 toxin is unknown. MDCK cells provide a good model system to study the binding and internalization of C2 toxin (Nagahama et al., 2009, 2012). In this study, we examined the intracellular trafficking of C2 toxin using MDCK cells. 2. Materials and methods 2.1. Materials Recombinant C2I and C2II were expressed, fused with glutathione S-transferase (GST) in Escherichia coli BL21, as described previously (Nagahama et al., 2009). To obtain C2IIa, C2II was activated by incubation with trypsin, as described previously (Nagahama et al., 2009). Rabbit antiC2I and anti-C2II antibodies were prepared as described previously (Nagahama et al., 2009). Bafilomycin A1 (BAF), nocodazole, colchicine, mouse anti-Golgi 58K antibody, and anti-mouse IgG–fluorescein isothiocyanate (FITC) were obtained from Sigma (St. Louis, MO). Mouse anti-early endosome antigen 1a (anti-EEA1a) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and the mouse antilysosome-associated membrane protein 2 (anti-Lamp2) antibody was obtained from AbD Serotec (Oxford, United Kingdom). Dulbecco’s modified Eagle’s medium (DEME) and Hanks’ balanced salt solution (HBSS) were obtained from Gibco BRL (New York, NY). Alexa Fluor 568conjugated goat anti-rabbit IgG, Alexa Fluor 488conjugated goat anti-mouse IgG, CellLights lysosomegreen fluorescent protein (GFP), CellLights endoplasmic reticulum (ER)-GFP, and 40 ,60 -diamino-2-phenylindole

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(DAPI) were obtained from Molecular Probes (Eugene, OR). The expression vector for GFP-Rab11 was prepared as described previously (Nagahama et al., 2012). 2.2. Cell culture and assay of cytotoxicity Madin–Darby canine kidney (MDCK) cells were obtained from the Riken Cell Bank (Tsukuba, Japan). Cells were cultured in DEME supplemented with 10% fetal calf serum (FCS), 100 units/ml of penicillin, 100 mg/ml of streptomycin, and 2 mM glutamine (FCS-DEME). All incubation steps were carried out at 37  C in a 5% CO2 atmosphere. Cells for cytotoxicity assays were inoculated in FCSDEME on 48-well tissue culture plates (Falcon, Oxnard, CA). Various concentrations of C2I and C2IIa were mixed in FCSDEME and inoculated onto cell monolayers. Cells were observed for morphological alterations 4 h after inoculation, as described previously (Nagahama et al., 2002). To measure the effects of BAF, nocodazole, and colchicine on the cytotoxicity of C2 toxin, MDCK cells were preincubated with these agents at 37  C for 1 h and then incubated with C2I and C2IIa at 37  C for 4 h. 2.3. Fluorescence labeling of C2I and C2IIa and confocal fluorescence microscopy Fluorescent C2I and C2IIa were labeled with Cy3 (GE Healthcare) and Alexa488 (Invitrogen) according to the manufacturer’s recommendations. No significant loss of biological activity was observed with fluorescent C2I and C2IIa as discerned by the cytotoxicity assay. MDCK cells were plated on a polylysine-coated glass-bottomed dish (Matsunami, Osaka, Japan) and incubated at 37  C in a 5% CO2 incubator overnight in FCS-DEME. Cells were washed twice with PBS and then stained with Hoechst 33258 solution (5 mg/ml) at 37  C for 10 min. After cells were washed again twice with PBS, they were incubated with Cy3-C2I and Alexa488-C2IIa for the indicated time periods at 37  C. These cells subsequently were fixed with 4% paraformaldehyde at room temperature for 10 min, and free radicals were quenched by incubation with 50 mM NH4Cl in PBS. After washing in PBS, cells were observed using a Nikon A1 laser scanning confocal microscope (Tokyo, Japan). 2.4. Immunofluorescence analysis Cells were plated on a polylysine-coated glass-bottomed dish as described above. To study the internalization of C2IIa, C2IIa (1 mg/ml) was incubated with cells at 4  C for 1 h in FCS-DEME. After three washes in cold FCS-DEME, cells were transferred to FCS-DEME prewarmed to 37  C and incubated at the same temperature for various periods. They were washed four times with cold phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde at room temperature. Dishes were incubated at room temperature for 15 min in 50 mM NH4Cl in PBS and in PBS containing 0.1% Triton X-100 at room temperature for 20 min for antibody labeling. After being washed with PBS containing 0.02% Triton X-100, the dishes were incubated at room temperature for 1 h with PBS containing 4% BSA, followed by the

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primary antibody (rabbit anti-C2II antibody or anti-C2I antibody, mouse anti-EEA1 antibody, mouse anti-Lamp2 antibody, or mouse anti-Golgi58K antibody) in PBS containing 4% BSA at room temperature for 1 h. They were then washed with PBS containing 0.02% Triton X-100, incubated with the secondary antibody (Alexa Fluor 568-conjugated anti-rabbit IgG or anti-mouse IgG-FITC) in PBS containing 4% BSA at room temperature for 1 h, washed extensively with PBS containing 0.02% Triton X-100, and analyzed with confocal fluorescence microscopy (Nagahama et al., 2011a, 2012). Nuclei were stained with DAPI. To study the localization of Lysosome-GFP and ER-GFP, MDCK cells were seeded and grown at 37  C for 24 h on polylysine-coated glass-bottomed dishes before transfection with CellLightsÔ Lysosome-GFP or CellLightsÔ ER-GFP according to the manufacturer’s instructions. They were then treated with C2I plus C2IIa, fixed, permeabilized, and blocked as described above. Cells were transfected with Nucleofector (Amaxa, Koln, Germany) for experiments with GFP-Rab11 according to the following program: MDCK cells, Kit L, program A24. Briefly, 1  106 MDCK cells were pelleted and resuspended in 0.1 ml of solution L, and electroporated with 2 mg of the pGFP-Rab11 plasmid. Electroporated cells were resuspended in 0.35 ml of complete medium. Of this solution, 0.1 ml was seeded on polylysine-coated glassbottomed dishes and incubated at 37  C. After 24 h, they were treated with C2I plus C2IIa, fixed, permeabilized and blocked as described above. All images represent a single section through the focal plane. Images shown in the figures are representative of at least three independent experiments and were produced with Adobe Photoshop (Nagahama et al., 2011a, 2012). Quantitative analysis of colocalization was performed using imageJ software. 3. Results 3.1. Internalization of C2I and C2IIa into MDCK cells To investigate the internalization of C2 toxin into MDCK cells, Cy3-labeled C2I and Alexa488-labeled C2IIa were incubated with MDCK cells in DEME containing 10% fetal bovine serum at 37  C for various times (Fig. 1A and B). C2IIa colocalized with C2I in cytoplasmic vesicles after 5 min. C2I disappeared after 15 min and later, and C2IIa was then localized in the cytoplasmic vesicles. These results indicate that the C2I/C2IIa complex is internalized via endocytosis and C2I rapidly translocates into the cytosol. 3.2. Endocytic trafficking of C2IIa To examine the endocytic trafficking of C2IIa, we investigated whether endocytic markers were associated with intracellular compartments containing C2IIa (Fig. 2A and B). Incubation of MDCK cells with C2IIa at 4  C resulted in plasma membrane staining consistent with the binding of toxin components to a cell-surface receptor (Fig. 2A). C2IIa was internalized upon the transfer of cells from 4  C to 37  C. C2IIa colocalized with early endosome antigen 1 (EEA1), a marker of early endosomes, after 5 min, but not after 15 min. C2IIa partially colocalized with GFP-Rab11 for 5 min, but not 15 min in cells that had been transfected

Fig. 1. Intracellular trafficking of fluorescent C2I and C2IIa in MDCK cells. (A) MDCK cells prestained with Hoechst 33528 were incubated with Cy3labeled C2I (1 mg/ml) and Alexa488-labeled C2IIa (1 mg/ml) in DEME containing 10% fetal calf serum at 37  C for the period indicated. Cells were fixed. C2I (red), C2IIa (green), and the nucleus (blue) were viewed with a confocal microscope. The experiments were repeated three times, and a representative result is shown. Bar, 7.5 mm. (B) Quantification of colocalizations. The percent of C2I/C2IIa colocalization represents the ratio of the number of endosomal structures stained for C2I and C2IIa to the total number of endosomal structures stained for C2IIa. The percent of colocalization was determined for each cell and the results represent the average  SEM of several cells (n > 10) obtained from at least three independent experiments.

with a plasmid encoding GFP-Rab11, a marker of recycling endosomes. We then examined the trafficking of C2IIa to late endosomes and lysosomes. C2IIa did not colocalize with lysosomal-associated membrane protein 2 (Lamp2), a marker of late endosomes and lysosomes, after 15 min, but did so after 30 min. C2IIa also colocalized with GFPlysosomes, a lysosome marker, after 30 min, but did not with Golgi58K, a Golgi marker, or ER-GFP, an endoplasmic reticulum marker. In contrast, negative control preparations with the addition of Alexa568 conjugated-anti rabbit IgG, FITC conjugated-anti mouse IgG, and DAPI failed to detect fluorescence signals except for DAPI (data not shown). These results indicate that internalized C2IIa is delivered to early endosomes, in which sorting occurs and that some C2IIa is transported to the plasma membrane in Rab11-positive endosomes. Next, we examined the recycling of C2IIa. Cells were incubated with C2IIa for 60 min at 4  C to enable binding of C2IIa. After washing, these cells were incubated at 37  C for various time periods to trigger internalization and recycling of C2IIa. When C2I is added at different time points to these cells, the uptake of C2I by recycled C2IIa were monitored by the C2I-induced cell-rounding (Fig. 2C). 100% of cells were round after 4 h when C2I was added immediately after washing (0 min). Most cells were round after 4 h when C2I was applied after 15 min of incubation. On the other hand, the addition of C2I after 30 min decreased the cell rounding activity, and the addition of C2I after 60 min did not cause cell rounding. As shown in Fig. 2A, most C2IIa internalized and dose not remain on cell surface after 5 min incubation. From these results, the uptake of C2I by recycled C2IIa occurred within 30 min.

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Fig. 2. Colocalization of C2IIa and endosome markers in MDCK cells. (A) MDCK cells were incubated with C2IIa (1 mg/ml) at 4  C for 1 h, washed, and incubated at 37  C for the period indicated. Cells were fixed, permeabilized, stained with DAPI and antibodies to EEA1, Lamp2, Golgi58K, and C2II. MDCK cells were transiently transfected with pGFP-Rab11, pGFP-lysosome, or pER-GFP. After 24 h of transfection, the transfected cells were incubated with C2IIa as described above. Cells were fixed, permeabilized, and stained with the anti-C2II antibody and DAPI. C2IIa (red), endosome markers (green), and the nucleus (blue) were viewed with a confocal microscope. The experiments were repeated three times, and a representative result is shown. Bar, 5 mm. (B) Quantification of colocalizations. The percent of C2IIa/endocytic marker colocalization represents the ratio of the number of endosomal structures stained for C2IIa and for endocytic markers to the total number of endosomal structures stained for endocytic markers. The percent of colocalization was determined for each cell and the results represent the average  SEM of several cells (n > 10) obtained from at least three independent experiments.

3.3. Endocytic trafficking of C2I To investigate the endocytic trafficking of C2I, MDCK cells were preincubated with C2IIa at 4  C for 60 min, washed, and incubated with C2I at 37  C for various periods

(Fig. 3A and B). C2I colocalized with EEA1 and GFP-Rab11 after 5 min, but did not with Lamp2, GFP-Lysosome, Golgi58K, or ER-GFP. These results indicate that C2I rapidly translocates across endosomal membranes into the cytosol after endocytosis.

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Fig. 3. Colocalization of C2I and endosome markers in MDCK cells. (A) MDCK cells were incubated with C2IIa (1 mg/ml) at 4  C for 1 h, washed, and incubated with C2I (1 mg/ml) at 37  C for the period indicated. Cells were fixed and permeabilized and stained with DAPI and antibodies to EEA1, Lamp2, Golgi58K, and C2I. MDCK cells were transiently transfected with pGFP-Rab11, pGFP-lysosome, or pER-GFP. After 24 h of transfection, the transfected cells were incubated with C2IIa and C2I as described above. Cells were fixed, permeabilized, and stained with the anti-C2I antibody and DAPI. C2I (red), endosome markers (green), and the nucleus (blue) were viewed with a confocal microscope. The experiments were repeated three times, and a representative result is shown. Bar, 5 mm. (B) Quantification of colocalizations. The percent of C2I/endocytic marker colocalization represents the ratio of the number of endosomal structures stained for C2I and for endocytic markers to the total number of endosomal structures stained for endocytic markers. The percent of colocalization was determined for each cell and the results represent the average  SEM of several cells (n > 10) obtained from at least three independent experiments.

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3.4. Effect of endocytosis inhibitors on the intracellular trafficking of C2IIa

endosomes to the cytosol and that C2 toxin is trafficked by microtubule-based transport.

Inhibitors that interfere with known intracellular trafficking pathways were utilized to investigate the route of C2IIa. Gibert et al. (2007) reported that the treatment of Vero cells with C2 toxin in the presence of bafilomycin A1 (BAF), which inhibits vacuolar-type Hþ-ATPase and prevents the acidification of endosomes, resulted in the decreased translocation of C2 toxin from endosomes into the cytosol. Treating of MDCK cells with C2IIa in the presence of DMSO vehicle had no effect on the internalization of C2IIa at 37  C (Fig. 4A). The accumulation of C2IIa in the cytoplasmic vesicles was markedly higher with BAF than in the absence of an inhibitor. Disruption of the microtubules with nocodazole and colchicine inhibited transport from early to late endosomes (Nagahama et al., 2011a, 2011b). As shown in Fig. 4A, nocodazole and colchicine disrupted the perinuclear localization of C2IIa, and C2IIa-containing vesicles were scattered in the cytosol. In contrast, BAF inhibited the rounding of cells induced by C2I plus C2IIa (Fig. 4B). However, nocodazole and colchicine did not inhibit C2 toxin-induced cell rounding. These inhibitors themselves did not exhibit any morphological effects under our experimental conditions (data not shown). These results indicate that C2I is translocated from acidic

4. Discussion

Fig. 4. Effect of inhibitors on the intracellular trafficking of C2IIa. (A) MDCK cells were incubated with DMSO, bafilomycin A1 (50 nM), nocodazole (20 mg/ml), or colchicine (50 mM) at 37  C for 1 h and were then rinsed. C2IIa (1 mg/ml) was added and incubated at 37  C for 30 min. Cells were fixed, permeabilized, and stained with the anti-C2IIa antibody and DAPI. C2IIa (red) and the nucleus (blue) were viewed with a confocal microscope. The experiments were repeated three times, and a representative result is shown. Bar, 7.5 mm. (B) MDCK cells were pretreated with DMSO, bafilomycin A1 (50 nM), nocodazole (20 mg/ml), or colchicine (50 mM) at 37  C for 1 h. The cells were incubated with C2I (100 ng/ml) and C2IIa (100 ng/ml) at 37  C for 4 h. Pictures were taken. The total number of cells and number of round cells were counted from the pictures and the percentage of round cells was calculated. Values are given as the mean  S.D. (n ¼ 3).

In the present study, we demonstrated the dynamics of the intracellular trafficking of C2I and C2IIa. After internalization of the C2I/C2IIa complex into cells, C2I was released from acidic early endosomes to the cytosol, and C2IIa was trafficked to lysosomes through an endocytic pathway. C. botulinum C2 toxin entered host cells and induced toxicity by exploiting endocytic trafficking (Barth et al., 2004; Aktories et al., 2012). C2IIa recognized asparaginelinked carbohydrates on the surface of target cells and formed heptamers on the lipid rafts of plasma membranes (Eckhardt et al., 2000). The binding of C2I to the C2IIa oligomer on lipid rafts triggered activation of the PI3K-Akt signaling pathway, and, in turn, the initiation of clathrindependent receptor-mediated endocytosis (Nagahama et al., 2009). C2I and C2IIa were transported to early endosomes, in which acidification promoted the cytosolic entry of C2I. Translocation of C2I was facilitated by the activities of the host cell chaperone Hsp90 and the peptidyl-prolyl cis/trans isomerase activities of cyclophilin A (Haug et al., 2003; Kaiser et al., 2011) and FK506-binding protein 51 (Kaiser et al., 2012). In the cytosol, C2I ADPribosylated G-actin, resulting in actin depolymerization and cell rounding. However, the intracellular route of C2I and C2IIa has not been described in detail. In the present study, we investigated the internalization of C2I and C2IIa by endocytosis. When MDCK cells were incubated with fluorescent C2I and C2IIa at 37  C, a rapid internalization of the two components was observed in the intracellular vesicles. The maximum level of colocalization between the two components was observed after 5 min of cells incubating at 37  C. C2I rapidly disappeared (15 min incubation and later) and C2IIa was evident in cytoplasmic vesicles in the cytosol, which showed that C2I was translocated to the cytosol. When C2IIa was incubated with MDCK cells at 37  C, it colocalized with EEA1 after 5 min, which indicated that it reached the early endosomes. C2IIa no longer localized with EEA1 at 15 min. After 5 min, it colocalized with Rab11 (Gruenberg, 2001), which showed that some C2IIa was delivered to recycling endosomes. The addition of C2I to cells preincubated with C2IIa at 37  C for 15–30 min caused cell rounding. From this result, C2IIa recycled back to the plasma membrane, where C2IIa was capable of rebinding to C2I. Recycling may be important for C2IIa to extend the entry of C2I. After 30 min, C2IIa colocalized with Lamp-2 and GFP-lysosomes, indicating that C2IIa moved to late endosomes and lysosomes. These results demonstrate that C2IIa is endocytosed and sorted from early endosomes to recycling endosomes or late endosomes and lysosomes. We investigated the internalization of C2I by endocytosis. When C2I was incubated with MDCK cells in the presence of C2IIa at 37  C, it colocalized with EEA1 after 5 min, which suggested that it reached early endosomes with C2IIa. C2I no longer localized with EEA1 at 15 min. C2I faded in the cytosol after 15 min or more. These results

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demonstrate that C2I is endocytosed and released from early endosomes to the cytosol. Bafilomycin A1, an inhibitor of vacuolar-type ATPases, inhibited cell rounding in response to C2 toxin and caused the accumulation of C2IIa in endosomes. This result was attributed to its inhibition of intracellular acidification. After the acidification of early endosomes, C2IIa was shown to form channels that mediate the passage of C2I into the cytosol as previously reported (Schmid et al., 1994; Barth et al., 2000). Bafilomycin A1 was also previously shown to block endocytic transport (Huotari and Helenius, 2011). Therefore, since the inhibition of endosome acidification with bafilomycin A1 does not perturb endocytic trafficking, endosomal acidification is required for the translocation of C2I and endocytic transport of C2IIa. In this experiment, cell rounding by C2 toxin was not inhibited by nocodazole or colchicine. As the disruption of microtubules by nocodazole and colchicine does not inhibit the initial acidification of endosomes (Huotari and Helenius, 2011), C2I is translocated from endosomes to the cytosol. However, treating the cells with nocodazole and colchicine restricted the transport of C2IIa towards the perinuclear region, in which the majority of lysosomes are localized, and caused the dispersal of C2IIa-containing vesicles, which indicated that the intracellular movement of C2IIa was impaired. These results confirm that C2I is translocated from acidic endosomes to the cytosol after endocytosis and that C2IIa is trafficked from early endosomes to late endosomes and lysosomes via microtubule-based transport. In conclusion, we demonstrated the intracellular routes of C2I and C2IIa. The C2I/C2IIa complex is internalized, transported to early endosomes. C2I is translocated from early endosomes to cytosols, while C2IIa is sorted into recycling endosomes and late endosomes. Ethical statement We certify that human subjects were not used in this work.

Acknowledgments We thank M. Higuchi and K. Kashiwazaki for their technical assistance. This work was supported by a Grantin-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, MEXT.SENRYAKU, 2012. Conflict of interest The authors declare that there are no conflicts of interest.

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Intracellular trafficking of Clostridium botulinum C2 toxin.

Clostridium botulinum C2 toxin is a binary toxin composed of an enzymatic component (C2I) and binding component (C2II). The activated binding componen...
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