Tetanus toxin is internalized by a sequential clathrin-dependent mechanism initiated within lipid microdomains and independent of epsin1

TeNT H_C uptake is dynamin-dependent

We next used the b-TeNT H_C to test the requirement for dynamin in this process. Dynamin is a GTPase essential for clathrin- and caveolin-mediated endocytosis, as well as for several other endocytic and vesicle-trafficking events. Incubation of MNs with the cell-permeable peptide P4, which inhibits dynamin function (Marks and McMahon, 1998), but not with the scrambled peptide P4S, significantly reduced uptake of TeNT H_C, whereas its overall binding to the neuronal surface was not affected (Fig. 2 A). These results were confirmed by overexpressing the well-characterized dynamin mutant K44A, which is defective in GTP binding and hydrolysis and restrains invaginated pits from pinching off (Fig. 2 B; Damke et al., 1994). We used microinjection to introduce foreign DNA into MNs because lipid-based transfection reagents abolished axonal transport in MNs. In contrast, microinjection of plasmids driving the overexpression of control proteins had no effect on cell viability and retrograde transport (Deinhardt and Schiavo, 2005). Expression of dynamin2^K44A significantly reduced TeNT H_C endocytosis at the level of both the soma and neurites (Fig. 2 B), without affecting its binding to the MN surface (Fig. 2 C). These results indicate that dynamin is a central player in TeNT H_C internalization and rule out differences in the mechanism of uptake of TeNT H_C between axons and the soma. This is important because, topologically, only the axon is physiologically relevant for TeNT H_C uptake and retrograde transport. A total block of TeNT H_C endocytosis by the expression of dynamin2^K44A was seen in >95% of the cells (Fig. 2 D). However, dextran internalization still took place under these conditions (Fig. 2 B, g) or upon P4 treatment (Fig. 2 A, b), confirming that MNs were viable and still capable of endocytosis via dynamin-independent pathways. We chose dextran as a control marker for internalization because we found that cholera toxin subunit B (CTB), which is another widely used marker for clathrin-independent endocytosis, is internalized in a strictly dynamin-dependent fashion in MNs (unpublished data). This was surprising because CTB has been shown to use a dynamin-independent entry pathway in other cell types, such as HeLa and mouse embryonic fibroblasts (Torgersen et al., 2001; Massol et al., 2004; Kirkham et al., 2005; Glebov et al., 2006).

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Figure 2.

Dynamin is required for TeNT H_C uptake into MNs. (A) MNs were incubated with 50 μM of the cell-permeable peptide P4 (top) or its scrambled analogue P4S (bottom) for 2 h before addition of 20 nM Alexa Fluor 488–TeNT H_C and 0.2 mg/ml tetramethylrhodamine-dextran for additional 45 min. Images show confocal sections through the cell body. (B–D) MNs were microinjected with a plasmid encoding Myc-dynamin2^K44A. (B) After 25 h of expression, cells were incubated with 20 nM b-TeNT H_C and 0.2 mg/ml tetramethylrhodamine-dextran for 30 min at 37°C, treated with MESNA, fixed, stained for internal biotin and with an anti-Myc antibody to detect the tagged dynamin2^K44A, and imaged. (top) A control MN; (bottom) a microinjected cell. (C) Expression of dynamin2^K44A does not block the surface binding of TeNT H_C to MNs. 25 h after microinjection of dynamin2^K44A, MNs were incubated with Alexa Fluor 647–TeNT H_C for 30 min at 37°C, fixed, and stained with an anti-Myc antibody. A confocal section of an expressing cell is shown. (D) Quantitative analysis of the effect of dynamin2^K44A overexpression on TeNT H_C internalization. Noninjected MNs or cells microinjected with a GFP-encoding plasmid were taken as controls. Numbers in parenthesis indicate the number of MNs imaged per condition. DIC, differential interference contrast. Bars: (A) 5 μm; (B and C) 10 μm.

TeNT H_C localizes to clathrin-coated pits

In previous studies, TeNT was found in coated pits, as well as in coated and uncoated vesicles in spinal cord neurons (Parton et al., 1987; Lalli et al., 2003b). Furthermore, TeNT is taken up by a clathrin-independent route in nonneuronal cells (Montesano et al., 1982). Therefore, we decided to investigate whether TeNT internalization is strictly clathrin-mediated in cultured MNs. First, we assessed the colocalization of clathrin and TeNT H_C at the light microscopy level in MNs microinjected with GFP–clathrin light chain (CLC). Upon incubation with TeNT H_C at 37°C, we could see only a partial overlap between GFP-CLC and TeNT H_C, more readily in the cell body than in the axon (Fig. 3, A and B).

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Figure 3.

TeNT H_C enters clathrin-coated structures in MNs. (A and B) MNs were microinjected with a plasmid encoding GFP-CLC. After overnight expression, cells were incubated with 30 nM Alexa Fluor 555–TeNT H_C, washed, and imaged. (A) GFP-CLC and TeNT H_C dual-positive structures are visible in an axon (arrows). (B) Confocal section of the bottom plane of a MN soma. A partial overlap between GFP-CLC–positive and TeNT H_C–positive structures is observed (inset). (C) MNs were incubated with HRP–TeNT H_C for 45 min on ice, and then chased for 45 min at 12 (a and b) or 18°C (c). Cells were fixed and incubated with DAB/H₂O₂ to label HRP–TeNT H_C. The electron-dense DAB reaction product was often associated with coated pits and budding vesicles, which were visible at higher magnification (b' and c'). Bars: (A) 2 μm; (B) 5 μm; (C) 0.2 μm.

To verify this colocalization at a fine structural level, we used TeNT H_C coupled with HRP. Just like b-TeNT H_C, binding of this fusion protein to MNs was inhibited by preincubation with an excess of unlabeled TeNT H_C and, upon internalization, Alexa Fluor 488–TeNT H_C and HRP–TeNT H_C showed extensive colocalization (Fig. S2, A and B, available at http://www.jcb.org/cgi/content/full/jcb.200508170/DC1). The relationship between internalized TeNT H_C and clathrin-coated structures was then investigated at early stages of the internalization process. To this end, we synchronized the uptake of HRP–TeNT H_C by preincubating the MNs at 4°C and, subsequently, warming to 12°C. This low-temperature treatment allows the plasma membrane to invaginate but inhibits vesicle–pinching off (unpublished data). The effectiveness of this protocol was confirmed by incubating MNs with either b-TeNT H_C or HRP–TeNT H_C at 12°C and, subsequently, treating cells with MESNA or performing the DAB reaction in the presence of ascorbic acid, which is a membrane-impermeable inhibitor of the HRP staining (Stoorvogel, 1998). Upon MESNA treatment, no biotin was detectable by immunofluorescence (Fig. S2 C). Similarly, when the DAB reaction was performed in the presence of ascorbic acid on cells incubated at 12°C, we observed immunolabeling of clathrin, but no DAB staining (Fig. S2 D). These findings indicated that the coated pits remained open to the external medium at 12°C and confirmed the suitability of this temperature block for the study of the initial stages of endocytosis.

After 12°C incubations, the electron-dense DAB reaction product generated by HRP–TeNT H_C was readily observed in coated pits on the plasma membrane of soma, dendrites, and axons (Fig. 3 C). The nature of these coated domains was confirmed by immunogold staining with clathrin heavy chain (CHC) antibodies, which labeled pits containing HRP–TeNT H_C (Fig. 4 A). The DAB reaction product found in both shallow pits and in deeper invaginations was closely associated with clathrin lattice components (Fig. 3 C, a–b′). After the 12°C block, we allowed MNs to internalize TeNT H_C at 18°C to monitor its intracellular axonal transport. Fine structural analysis suggests that progression along the endocytic pathway was slowed down at this temperature and lead to an increase of early endosomal carriers. At 18°C, HRP–TeNT H_C was found in coated vesicles (Fig. 3 C, c) and other vesicular and tubular structures within the axon.

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Figure 4.

Colocalization of HRP–TeNT H_C and clathrin at EM level. (A) HRP–TeNT H_C–containing structures are labeled with anti-CHC. Cells were loaded with HRP–TeNT H_C at 12°C for 45 min before HRP cross-linking with DAB/H₂O₂. MNs were permeabilized with digitonin before labeling with an anti-CHC antibody and incubated with a rabbit anti–mouse bridging antibody, and then with 10 nm immunogold. (B) Whole-mount transmission EM showing distribution of clathrin in vesicles loaded with HRP–TeNT H_C. (a–d) MNs were incubated with HRP–TeNT H_C at 12°C for 45 min, cross-linked with DAB/H₂O₂, extracted with Triton X-100, and then treated with an anti-CHC antibody followed by a bridging antibody and 10 nm immunogold. HRP–TeNT H_C is found in CCPs and CCVs in axons (a, b, d) and soma (c). Control without the bridging antibody showed negligible labeling (e, arrows). Boxed areas show enlarged examples of TENT H_C–positive endocytic structures. Bars, 0.2 μm.

Gold immunolabeling of coated pits in axons was not easily discerned in thin EM sections, as permeabilization of these structures and access to the antigen appeared to be impaired because of the highly packed cytoskeleton in these areas. Therefore, we decided to prepare whole-cell mounts by extracting neurons with Triton X-100 before fixation, thereby improving the antigen availability and providing a better overview over the total population of TeNT H_C–positive membranes. To stabilize HRP–TeNT H_C–containing pits and protect them from solubilization, DAB cross-linking was performed before detergent extraction. At 12°C, the vast majority of DAB-positive structures located along the axons (Fig. 4 B, a, b, and d) and on the cell body (Fig. 4 B, c) were clathrin positive.

The ganglioside GD1b does not enter coated pits in complex with TeNT H_C

Polysialogangliosides of the b-series, including GD1b and its analogues GT1b and GQ1b, have previously been described as essential components of the TeNT receptor complex (Kitamura et al., 1999). However, these lipids, like other residents of sphingolipid-rich microdomains, are thought not to enter clathrin-coated pits (CCPs; Nichols, 2003). Therefore, we asked where GD1b localizes on the neuronal surface in relation to TeNT H_C and clathrin-coated invaginations. By light microscopy, we were able to confirm colocalization of TeNT H_C and GD1b on the neuronal surface by using a specific anti- GD1b antibody (MOG-1; Fig. 5 A). Furthermore, preincubation of MNs with MOG-1 inhibited the binding of TeNT H_C in a dose-dependent manner (Fig. S3 A, available at http://www.jcb.org/cgi/content/full/jcb.200508170/DC1), confirming that GD1b is an essential component of the TeNT receptor complex. To obtain a higher resolution view of the association between TeNT H_C and polysialogangliosides, we incubated MNs with HRP–TeNT H_C in the presence of noncompeting concentrations of MOG-1 at 12°C (Fig. S3 B) and analyzed the samples by EM. As previously described for other components of lipid microdomains, we found clusters of gold-labeled GD1b on the cell surface, often in close proximity to the DAB precipitate generated by HRP–TeNT H_C (Fig. 5 B). In addition, the DAB precipitate was frequently associated with coated structures (Fig. 5 B, arrows and arrowheads). However, we were unable to detect GD1b in CCPs containing the DAB cross-linking product. Instead, gold-labeled GD1b was frequently found at the edge of HRP–TeNT H_C–positive pits (Fig. 5 B, a–h). Furthermore, internalized gold particles were very rarely detected upon incubation at 37°C, suggesting that GD1b remains surface-bound (Fig. 5 B, i). Under the same conditions, TeNT H_C was identifiable in many vesicles and tubules, all of which were free of GD1b gold label (Fig. 5 B, i). To confirm that the DAB precipitate did not conceal any gold particles in internalized structures, we incubated MNs with HRP–TeNT H_C together with gold-conjugated TeNT H_C. Under these conditions, gold label was clearly visible in all HRP-positive structures (Fig. S2 E).

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Figure 5.

GD1b and TeNT H_C undergo independent sorting on the plasma membrane. MNs were probed with TeNT H_C and an anti-GD1b antibody and analyzed at light and EM levels. (A) Cells were incubated with the antibody MOG-1 (2 μg/ml) and 20 nM Alexa Fluor 555–TeNT H_C for 30 min on ice, followed by 5 min at 22°C, and then were washed, fixed, and imaged. A good overlap of the two signals could be seen both on neurites (a–d) and soma (e–h). DIC, differential interference contrast. (B) EM analysis. MNs were incubated on ice with 60 nM HRP–TeNT H_C and 10 μg/ml MOG-1, followed by 10 nm immunogold. After washing, samples were shifted to 12°C for 45 min (a–h) or 37°C for 15 min (i). MNs were fixed and incubated with DAB/H₂O₂ to label HRP–TeNT H_C. The anti-GD1b gold-labeled antibody is excluded from endocytosed HRP–TeNT H_C vesicles (asterisks), clathrin lattices (arrows), and invaginated coated pits (arrowheads). Bars, 0.2 μm.

Protein labeling

To couple TeNT H_C to HRP, 10 nmol of cysteine-tagged TeNT H_C were incubated with 5 mM EDTA and 6.5 mg EZ-link–activated maleimide-HRP in PBS overnight at 4°C. The conjugate was purified first on ConA–Sepharose (GE Healthcare) and eluted with 0.25 M α-methylmannoside in 10 mM sodium phosphate, pH 7.2. HRP–TeNT H_C was bound to NiNTA-agarose (QIAGEN) and eluted in 20 mM Hepes-NaOH, pH 7.4, 150 mM NaCl, and 500 mM imidazole. Samples containing HRP–TeNT H_C were pooled and dialyzed against PBS. To double label TeNT H_C with an Alexa Fluor dye and HRP, fluorophore labeling was performed first, according to the manufacturer's instructions, using half of the recommended amount of dye and without the addition of glutathione to stop the reaction. Alexa Fluor–labeled TeNT H_C was dialyzed against PBS to remove the excess dye before HRP-conjugation.

Microinjection and internalization assay

MN cultures were prepared and maintained in culture as previously described (Bohnert and Schiavo, 2005). Cells were injected with 0.05 mg/ml of plasmid between 4 and 7 d in vitro. In cases of microinjection of multiple plasmids (e.g., the pTRE-μ2 T156A plasmid that requires a transactivator ptTA for expression; CLONTECH Laboratories, Inc.), 0.04 mg/ml of each construct were mixed before injection. MNs were incubated with 15–20 nM TeNT H_C and then either biotinylated or Alexa Fluor–labeled for 30 min at 37°C. In selected experiments, 20 μg/ml Alexa Fluor 594–transferrin, 10 ng/ml Alexa Fluor 555–CTB, or 0.2 mg/ml tetramethylrhodamine dextran (mol wt 3,000) were mixed with TeNT H_C before addition to the cells. 60 mM KCl was added to the medium just before addition of the ligands to test the effects of depolarization.

In experiments where MNs were pretreated with P4 or P4-scrambled peptide (Marks and McMahon, 1998), 50 μM of peptide was added to the medium at 37°C for 2 h before incubation with 20 nM Alexa Fluor 488–TeNT H_C and 0.2 mg/ml tetramethylrhodamine dextran.

For MESNA treatment, MNs were cooled on ice and then incubated three times for 15 min with 15 mM of ice-cold MESNA in neurobasal medium (Invitrogen), pH 8.3. Cells were washed three times in neurobasal medium and once in PBS, and then fixed.

To test the effect of SV exo/endocytosis on TeNT H_C uptake, MNs were seeded on 13-mm coverslips. At 6 d in vitro, MNs in two wells were incubated with 15 nM BoNT/A and 2 nM BoNT/D for 22 h at 37°C, while control wells were left untreated. Coverslips from treated and untreated wells were then transferred into a new dish and incubated with 20 nM b-TeNT H_C for a further 30 min at 37°C before treatment with MESNA on ice, fixing, and processing as described in the following paragraph. The remaining cells from each well were washed in PBS, scraped, centrifuged, and then resuspended in SDS sample buffer. Proteins were analyzed by Western blotting using standard procedures. Antibodies were used as follows: anti–VAMP-2 (69.1), 1:500; anti–SNAP-25, 1:1,000; anti-actin (AC-40), 1:1,000; and HRP-conjugated secondary antibodies (GE Healthcare), 1:1,000.

Immunofluorescence and confocal microscopy

Cells were fixed in 4% PFA and 20% sucrose in PBS for 15 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 min, blocked in 2% BSA, 10% normal goat serum, and 0.25% fish skin gelatin in PBS for 30 min, and then incubated with the relevant antibodies (anti–VAMP-2 [69.1], 1:300; anti–SNAP-25, 1:300 [Washbourne et al., 1997]; anti-HA [12CA5], 1:1,000; anti-Myc [9E10], 1:250; secondary antibodies, 1:500; or streptavidin 1:500) for 30 min in blocking solution. Cells were mounted in Mowiol-488 and imaged using a LSM 510 laser scanning confocal microscope equipped with a 63×, 1.4 NA, Plan Apochromat oil-immersion objective (both Carl Zeiss MicroImaging, Inc.). Images were processed using LSM 510 software. Images showing GFP-CLC and TeNT H_C colocalization were taken on living cells at 37°C using a laser scanning confocal microscope (IX70; Olympus) equipped with a 60×, 1.2 W, UPlan Apochromat oil-immersion objective and an environmental chamber. Images were captured using the Ultraview Imaging Suite Version 5.5 software (Perkin Elmer) and processed using AQM Advance 6 Kinetic Acquisition Manager software (Kinetic Imaging).

EM

MNs grown on glass coverslips were incubated with 80 nM HRP–TeNT H_C and/or with 10 μg/ml MOG-1 antibody in serum-free neurobasal medium for 45 min at 4°C. Cells were then washed and chased in prewarmed medium at different temperatures for the indicated time. When appropriate, cells were incubated with a 10-nm gold-conjugated anti–mouse antibody (British Biocell International) on ice for additional 30 min and washed before chase in medium alone. Cells were then fixed with 2% PFA and 1.5% glutaraldehyde in 100 mM sodium cacodylate, pH 7.5, for 15 min and treated with DAB (0.75 mg/ml in 50 mM Tris-HCl, pH 7.4) and 0.02% H₂O₂ to cross-link HRP. Samples were postfixed and embedded in Epon as previously described (Stinchcombe et al., 1995). MNs were then sectioned en face, and 60-nm sections stained with lead citrate were viewed in an electron microscope (CM12; Philips).

For whole mounts, MNs were treated as in the pervious paragraph, but instead of being permeabilized with digitonin, they were extracted with 1% Triton X-100 in PBS containing 1 mM MgCl₂ and 0.1 mM CaCl₂ for 10 min at 5°C. After gold labeling, samples were fixed in 4% glutaraldehyde and 1% osmium, dehydrated, and critical-point dried before being prepared for carbon replicas (Hopkins, 1985).

For EM of microinjected cells, MNs were seeded on CELLocate glass-gridded coverslips (Eppendorf). A plasmid encoding ssHRP-KDEL (Connolly et al., 1994) was used as an injection marker. 26 h later, cells were treated with HRP–TeNT H_C and then with DAB, as described in the previous paragraphs. Alternatively, coverslips were incubated with 20 μg/ml human transferrin-HRP (Hopkins et al., 2000) after a 15-min preincubation at 37°C for in serum-free neurobasal medium. After fixation and embedding in Epon, ultrathin sections were cut from the grid area containing the microinjected cells and imaged by EM.

For immunolabeling, samples were incubated with DAB in 50 mM Tris-HCl, 110 mM NaCl, pH 7.4, or with ascorbic acid buffer (20 mM Hepes-NaOH, 70 mM NaCl, and 50 mM ascorbic acid, pH 7.0) at 5°C for 30 min after treatment with HRP–TeNT H_C and chased in medium. Cells were then permeabilized with 40 ng/ml digitonin in permeabilization buffer (25 mM Hepes-KOH, 38 mM aspartate, 38 mM glutamate, 38 mM gluconate, 2.5 mM MgCl₂, and 2 mM EGTA, pH 7.2), fixed in 2% PFA, quenched with 50 mM glycine, and blocked with 1% BSA before treatment with primary antibody in PBS containing 1% BSA for 60 min at room temperature. To enhance the signal, intermediate species-specific antibodies were used. MNs were washed and incubated with an appropriate 10-nm gold-labeled secondary antibody (British Biocell International) in 2% BSA and 2% FCS in PBS for 45 min at room temperature. After washing, cells were fixed and processed for conventional EM. In double- and triple-label experiments, MNs were incubated with 80 nM HRP–TeNT H_C or with 20 μg/ml transferrin-HRP together with TeNT H_C directly conjugated to 10-nm gold particles (as described by Odorizzi et al., 1996) in serum-free neurobasal medium for 45 min at 4°C. Cells were washed and shifted to 12 or 20°C before incubation with DAB/H₂O₂.

We are thankful to S. Tooze, A. Behrens, and members of the Molecular Neuropathobiology laboratory for critical reading of the manuscript.

This work was supported by Cancer Research UK (K. Deinhardt and G. Schiavo), Wellcome Trust 060349 (H.J. Willison), and the Medical Research Council (O. Berninghausen and C.R. Hopkins).