1

TETANUS TOXIN IS TRANSPORTED IN A NOVEL NEURONAL

COMPARTMENT CHARACTERISED BY A SPECIALISED PH REGULATION Stephanie Bohnert and Giampietro Schiavo

Molecular NeuroPathobiology Laboratory, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln’s Inn Fields, London, WC2A

3PX, UK Running title: pH regulation of the axonal retrograde pathway

Address correspondence to: Giampietro Schiavo, Molecular NeuroPathobiology Laboratory, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln’s Inn Fields, London, WC2A 3PX, UK, Tel. +44-20-7269-3300; Fax +44-20-7269-3417; E-Mail: Giampietro.Schiavo@cancer.org.uk Tetanus toxin binds specifically to motor neurons at the neuromuscular junction. Here, it is internalised into vesicular carriers undergoing fast retrograde transport to the spinal cord. Despite the importance of this axonal transport pathway in health and disease, its molecular and biophysical characterisation is presently lacking. We sought to fill this gap by determining the pH regulation of this compartment in living motor neurons using a chimera of the tetanus toxin binding fragment (TeNT HC) and a pH-sensitive variant of the green fluorescent protein (ratiometric pHluorin). We have demonstrated that moving retrograde carriers display a narrow range of neutral pHs, which is kept constant during transport. Stationary TeNT HC-positive organelles instead exhibit a wide spectrum of pHs, ranging from acidic to neutral. This distinct pH regulation is due to a differential targeting of the vacuolar (H+) ATPase, which is not present on moving TeNT HC compartments. Accordingly, inhibition of the vacuolar (H+) ATPase under conditions that completely abolish the intracellular accumulation of acidotrophic dyes, does not affect axonal retrograde transport of TeNT HC. However, a functional vacuolar (H+) ATPase is required for early steps of TeNT HC trafficking following endocytosis, and it is localised to axonal vesicles containing TeNT HC. Altogether, these findings indicate that the vacuolar (H+) ATPase plays a specific role in early sorting events directing TeNT HC to axonal carriers,

but not in their subsequent progression along the retrograde transport route, which escapes acidification and targeting to degradative organelles.

INTRODUCTION The clostridial neurotoxin family is formed by tetanus (TeNT1) and seven serotypes of botulinum neurotoxins (BoNTs, named A-G). They all share an identical structural organisation, comprising of a 100 kDa heavy (H) chain and a 50 kDa light (L) chain, linked via a disulfide bond and other non covalent interactions (1,2) (Fig. 1a). The carboxy-terminal part of the heavy chain (HC, 50 kDa) is responsible for membrane binding and internalisation, whereas the amino-terminal domain (HN, 50 kDa) mediates membrane translocation of the L chain into the cytosol (1,3). The L chain is a highly specific zinc-endoprotease, which is responsible for the cleavage of synaptic SNARE proteins necessary for neurotransmitter release (2). TeNT binds to motor neurons (MNs) at the neuromuscular junction. Upon internalisation, TeNT is retrogradely transported towards the MN cell body, where it is released into the extracellular medium and enters adjacent inhibitory interneurons (4). In these cells, TeNT blocks the release of inhibitory neurotransmitters by cleaving VAMP/synaptobrevin, a member of the SNARE superfamily (1,3). We have recently developed an assay to follow the retrograde transport of TeNT in MNs using fluorescently-tagged versions of the non-toxic TeNT HC binding

http://www.jbc.org/cgi/doi/10.1074/jbc.M506750200The latest version is at JBC Papers in Press. Published on October 18, 2005 as Manuscript M506750200

Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

2

fragment (5). In these cells, TeNT and TeNT HC are internalised and transported in morphologically identical organelles with overlapping speed distributions (6). These carriers are powered for their retrograde movement by distinct molecular motors, including cytoplasmic dynein and myosin Va (6-8). TeNT HC shares this compartment with the nerve growth factor and the low affinity neurotrophin receptor p75NTR (5). These findings validate TeNT HC as an ideal tool for dissecting the molecular machinery controlling axonal retrograde transport and the trafficking of neurotrophin receptors and their ligands in living MNs. To date, the precise characterisation of the intraluminal pH of this axonal retrograde pathway and its relevance in the regulation of transport in MNs remain unclear. These features are particularly important since acidic pH triggers a conformational change in TeNT and other CNTs, allowing the active subunit to translocate through the endosomal membrane into the cytosol (9,10). Moreover, acidic pH is known to drive the dissociations of ligands, including growth factors, from their receptors and influence their signalling and intracellular targeting (11,12). We sought to address these important questions by preparing chimeras of TeNT HC and ratiometric pHluorin, a pH-sensitive green fluorescent protein (GFP) mutant (13). Several pH-sensitive variants of GFP have been developed by independent mutagenesis approaches and used as pH reporters to follow vesicular transport dynamics (14,15). Whereas the wild-type GFP has a largely unaltered spectrum between pH 5.5 and 10, the excitation spectrum of the ratiometric pHluorin shows two main peaks at 395 nm and at 475 nm, the intensity of which reciprocally varies as a function of pH due to the protonation of its chromophore (13). In this study, we demonstrate that moving axonal retrograde TeNT HC carriers display a neutral pH, which is kept constant during transport. In contrast, stationary TeNT HC organelles located in axons and in somas exhibit a wide range of pHs. The distinct pH regulation of these compartments is due to a differential

targeting of the vacuolar (H+) ATPase (vATPase), whose activity is required for early events in the formation and/or sorting of axonal TeNT HC vesicles, but not for their following axonal transport.

MATERIALS AND METHODS Chemicals and fluorescent probes - Chemicals were purchased from Sigma and BDH unless stated otherwise. Restriction endonucleases and DNA-modifying enzymes were from New England Biolabs. pGEX-4T-VSV-G-Kin-TeNT HC encoding the residues 855-1,314 of TeNT fused at the N-terminus with a protein kinase A phosphorylation site, the vesicular stomatitis virus protein G (VSV-G) epitope and glutathione-S-transferase (GST) (3), was linearised with NdeI. This vector and the insert encoding ratiometric pHluorin, obtained by treating the pGEX-2T-AF058694 construct (13) with AgeI, were blunt-ended using (3’→5’ exo-) Klenow fragment of DNA polymerase I, purified and then ligated. The resulting pGEX-4T-3-VSV-G-Kin-pHluorin-TeNT HC construct was shown to correspond to the published TeNT HC and pHluorin sequences, except for the absence of the mutation L220F in the latter (13). After transformation into FB810 competent bacteria, GST-VSV-G-Kin-pHluorin-TeNT HC and GST-pHluorin were expressed and purified as previously described (16). Cysteine-tagged TeNT HC was expressed using pGEX-4T3-Cys-TeNT HC and labelled with AlexaFluor488-maleimide (Molecular Probes) as previously described (5). Fluorescent probes contained on average 1.8 moles of dye per mole of TeNT HC. Immunofluorescence and Western Blot analysis - Primary MNs were isolated from Sprague-Dawley E14 rat embryos and cultured following described protocols (17). Embryo-tested bovine serum albumin was dialysed at 4˚C against phosphate-buffered saline (PBS) for 24 h and then for further 48 h against Leibovitz L-15 medium (GIBCO), pH 7.3, using Spectra/Por membranes with a 25 kDa cut-off (Pierce). All experiments were

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

3

performed using MNs differentiated for 5-8 days in vitro (DIV 5-8). For immunofluorescence experiments (IF), MNs were incubated with either 15 µM [3-(2,4-dinitroanilino)-3’-amino-N-methyldipro-pylamine] (DAMP) (Oxford Biomedical Research; stock solution 3mM) and/or 15 nM TeNT HC-Alexa488 for 2 or 45 min at 37°C. Cells were then fixed in 4% paraformaldehyde, 20% sucrose in PBS, permeabilised with 0.1% Triton X-100 and blocked with 2% embryo-tested bovine serum albumin, 0.25% porcine skin gelatine, 0.2% glycine, 15% foetal calf serum in PBS. Primary antibodies were diluted in blocking solution and incubated with the fixed cells for 45 min at room temperature. DAMP was detected using monoclonal α-DNP for 45 min at room temperature (Oxford Biomedical Research). After rinsing, AlexaFluor goat α-mouse, α-rabbit or α-chicken IgG (Molecular Probes) were diluted 1:300 in blocking solution and incubated for 30 min. After extensive washing, the coverslips were mounted with Mowiol 4-88 (Harco). Fluorescent images were acquired using a Zeiss LSM 510 confocal microscope equipped with a Zeiss x63, 1.40 NA DIC Plan-Apochromat oil-immersion objective. A region of interest was chosen and simultaneously excited using the 488 nm and 543 nm lines of a krypton-argon and a helium-neon laser, respectively. Images were collected by averaging eight times at a single focal plane. The polyclonal rabbit α-31kDa vATPase (a kind gift from S. Breton, Massachusetts General Hospital, MA) was used 1:300 for immunofluorescence (IF) and 1:4,000 for Western Blot (WB). The monoclonal α-60kDa vATPase antibody (2E7; a kind gift from H. Sze, University of Maryland, MD) (18) was diluted 1:10 for IF and 1:100 for WB. The chicken α-75kDa vATPase (ATP6E; GenWay) was used 1:100 for IF and 1:1000 for WB. Polyclonal rabbit α-116kDa vATPase (SYSY) was used 1:300 for IF and 1:3000 for WB. Rat brain extracts (100 µg/lane) were incubated for 10 min at room temperature

with loading buffer, separated on a 12.5% SDS gel and transferred onto nitrocellulose (Schleicher & Schuell). After blocking for 1 h in PBS containing 5% skimmed milk, membranes were incubated with primary antibody for 1 h in the same buffer, washed with PBS and incubated with HRP-conjugated secondary antibody. Immunoreactivity was detected using enhanced chemiluminescence (ECL, Amersham Biosciences). Endocytosis assays - MNs were treated with 0.5 nM BafA1 for 15 min at 37°C and then incubated for 30 min with VSV-G-Kin-TeNT HC (20 nM) or TeNT HC labelled with disulfide-linked biotin (30 nM). After VSV-G-Kin-TeNT HC incubation, cells were fixed in 4% paraformaldehyde in PBS and blocked with 2% embryo-tested bovine serum albumin, 0.25% porcine skin gelatine, 0.2% glycine, 15% foetal calf serum in PBS. Rabbit α-VSV-G tag antibody (Biogenes) diluted 1:80 in blocking solution was incubated with the fixed cells for 30 min at room temperature. Cells were washed twice with PBS and then permeabilised with 0.1% Triton X-100 in blocking solution and blocked for further 30 min. MNs were incubated with a primary mouse α-VSV-G (Cancer Research UK) for 30 min, washed twice in PBS and incubated for 30 min with AlexaFluor goat α-mouse and α-rabbit IgG (Molecular Probes), diluted 1:300 in blocking solution. After extensive washing with PBS, coverslips were mounted with Mowiol 4-88. Alternatively, MNs incubated with TeNT HC-biotin (Deinhardt K. and Schiavo G., unpublished results) were cooled on ice and then treated three times with ice-cold 20 mM MESNA in neurobasal medium (Gibco), pH 8.3 for 15 min to cleave the biotin moiety from surface-bound TeNT HC. Cells were washed three times in neurobasal medium pH 8.3, once in PBS, fixed in 4% paraformaldehyde in PBS, permeabilised with 0.1% Triton X-100 and finally blocked in blocking solution. TeNT HC-biotin was revealed using streptavidin-AlexaFluor488 1:500 in blocking solution.

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

4

Cells were extensively washed in PBS and mounted in Mowiol 4-88. Fluorescence excitation spectra of pHluorin-TeNT HC in vitro and in vivo - Samples containing 0.5 µM pHluorin-TeNT HC or GST-pHluorin in 50 mM sodium cacodylate, 100 mM NaCl, 1 mM CaCl2, 1 mM MgCl2 were adjusted to the indicated pHs with NaOH or HCl. Excitation spectrums (350 to 500 nm) were acquired using a 710 Photomultiplier Detection System (Photon Technology International) at a fixed emission of 508 nm. As expected, the fluorescence excitation spectrum of GST-pHluorin was identical to that of pHluorin-TeNT HC. MNs were incubated with 40 nM pHluorin-TeNT HC in neurobasal medium for 30 min at 37°C and 7.5% CO2. The medium was then replaced with a buffer containing 120 mM KCl, 20 mM NaCl, 0.5 mM CaCl2, 0.5 mM MgSO4, 20 mM HEPES, adjusted to a pH between 5.0 and 8.0 with NaOH or HCl. 20 min before imaging, the ionophores monensin and nigericin (both 10 µg/ml; Calbiochem), were added to equilibrate the intracellular pH to that of the external buffer (19). The emission intensities of pHluorin-TeNT HC compartments in axons and soma were measured for each pH at 508 nm upon excitation at 405 and 488 nm. The background was then subtracted and the corrected emission values for the 405 nm excitation were divided by the corresponding emission intensities obtained by exciting at 488 nm (R405/488). Microscopy and data quantification - To monitor fluid phase uptake, MNs were incubated with 6 µM GST-pHluorin in neurobasal medium (GIBCO) for 1 h at 37°C and 7.5% CO2, washed with non-fluorescent Dulbecco’s minimum essential medium without phenol red, riboflavin, folic acid, penicillin/streptomycin and supplemented with 30 mM HEPES-NaOH, pH 7.3 (DMEM–), and immediately imaged by low-light time-lapse microscopy. Alternatively, MNs were transferred to neurobasal medium, incubated for further 30 min, washed and then imaged.

MNs were incubated with 40 nM pHluorin-TeNT HC or 40 nM TeNT HC-Alexa488 in complete medium for 30 min at 37°C, followed by three washes with DMEM–. Cells were placed in a humidified chamber maintained at 37°C, and after 20 min imaged every 5 s. In selected samples where axonal transport of TeNT HC-Alexa488 was observed, concanamycin A (ConA) (Alexis; stock solution 10 µm) or bafilomycin A1 (BafA1) (Alexis; stock solution 100 nM) were added at a final concentration of 10 and 0.5 nM, respectively, and the same cell was imaged after 5, 15 and 30 min incubation. The ConA analysis shown in Fig. 4 is representative of 5 independent experiments, using MNs from two different preparations, whereas the BafA1 effects have been evaluated in three independent experiments using MNs isolated from the same preparation. The functionality of the proton-pump inhibitors was tested by applying 50 nM Lysotracker Red DND-99 (Molecular Probes) for 15 min on untreated MNs, or MNs pre-incubated with either 10 nM ConA for 15 min or 0.5 nM BafA1 for 10 min and then imaged by confocal microscopy. For quantification purposes, the colocalisation macro of the LSM510 software (Zeiss) has been used following manufacturer’s instructions. For pHluorin-TeNT HC, images were taken every 5 s with a Nikon Diaphot 300 inverted microscope equipped with a Nikon x100, 1.3 NA Plan Fluor oil-immersion objective. Specimens were sequentially excited at 488 nm and then at 405 nm for 333 ms using the excitation filters 485DF15, 4000DF10, the dicroic filter 490-575DBDR, and the emission filter 528-633DBDR (Omega Optical). Images were acquired with a Hamamatsu C4742-95 Orcal cooled charge-coupled device camera (Hamamatsu Photonic Systems) controlled by the Kinetic Acquisition Manager 2000 software (Kinetic Imaging). Two channel series were processed with Lucida 4.0 (Kinetic Imaging). For time-lapse low-light microscopy of TeNT HC-Alexa488-treated cells, images were acquired every 5 s with a Nikon Diaphot 200 inverted microscope

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

5

equipped with Nikon x100, 1.25 NA Plan DIC oil-immersion objective, using a standard Nikon FITC B-2A filter and exposure times between 111 and 222 ms. The Motion Analysis software (Kinetic Imaging) was used for carrier tracking. Only moving carriers that could be tracked for at least four time points were analysed. Single movements of TeNT HC carriers, which are described by their progress between two consecutive frames, have been used to determine the speed of the carriers. Statistical analysis and curve fitting were performed using Microsoft Excel®. Single frame-images were processed for presentation in Adobe® Photoshop® 5.5. Kymographs were generated using MetaMorph (Universal Imaging Corporation) after rotation of the image stack to align the neuronal process vertically. Vertical single line-scans through the centre of each process were plotted sequentially for every frame in the time series.

RESULTS Axonal TeNT HC carriers have recently been shown to avoid accumulating the acidotropic probe Lysotracker Red DND-99 in their lumen, suggesting that these organelles escape acidification (5). However, no conclusions could be drawn from previous work about the pH dynamics of the retrograde transport compartment or its regulation in the axon and soma. To address this issue, we chose a strategy based on bacterially-expressed ratiometric pHluorin fused with TeNT HC. First of all, we tested if pHluorin still retains its described optical properties (13) once tagged to TeNT HC by diluting it with buffers ranging from pH 5.0 to 8.0 and monitoring its excitation spectrum at an emission wavelength of 508 nm (Fig. 1b). As expected, the fluorescence spectrum of pHluorin-TeNT HC shows two peaks at 395 and 475 nm. The 395 nm peak was more intense at a neutral pH and decreased at acidic pHs, whereas the 475 nm peak showed an opposite behaviour. To establish a pH-calibration curve in this system, MNs were pre-treated with 40 nM pHluorin-TeNT HC at 37°C for 30 min,

washed and then incubated with buffers at different pHs containing monensin and nigericin. Following treatment with these ionophores, all cellular compartments have been observed to re-equilibrate to the pH of the external medium (19). Ratiometric images were taken and the 405/488 ratios for each pH, averaged from randomly-chosen pHluorin-TeNT HC positive organelles in axons and somas (n=24 to 52), were used to build a cellular calibration curve for pHluorin-TeNT HC (Fig. 1c). To test the ability of pHluorin to monitor pH in living cells, we incubated MNs with an excess of GST-pHluorin (6 µM), under conditions allowing the labelling of all endocytic compartments by fluid phase uptake. Following incubation for 1 h at 37˚C, MNs were washed and imaged immediately, or after a 30 min chase. In absence of chase, the labelled organelles (n=119) show a broad pH spectrum, ranging from acidic to neutral (Fig. 1d), which is in agreement with the pH heterogeneity observed experimentally in the endosomal pathway (20). This distribution drastically changed after a 30 min chase, when mainly acidic structures were observed (n=96). About 70 % of the organelles had a luminal pH below 5.5 (Fig. 1d), which reflects the accumulation of GST-pHluorin in late endosomes and lysosomes (20). These combined results indicate that pHluorin can be used as a functional pH reporter to assess the pH of axonal retrograde carriers in our cellular system. To this end, we incubated rat MNs with 40 nM pHluorin-TeNT HC at 37°C for 30 min. Binding experiments confirmed that pHluorin-TeNT HC binds specifically to the MN surface (data not shown), as reported for untagged TeNT HC (21). The cells were then washed and after 20 min, low-light time-lapse imaging started in the 488 nm channel, followed by the 405 nm channel. Ratiometric images (R405/488) were obtained from these two time-series (Fig. 2a; see also videos 1a-c). The movement of a retrograde pHluorin-TeNT HC carrier is indicated by arrowheads, whereas a stationary compartment is labelled with asterisks (Fig. 2a). TeNT and TeNT HC have been shown to undergo

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

6

axonal retrograde transport with a similar speed range both in vivo (0.8-3.6 µm/s) (4), and in vitro (0.2-3.6 µm/s) (5,6). To investigate whether pHluorin-TeNT HC carriers are transported at the same rate as the TeNT compartments, we determined their speed distribution profile. Both the average speed and the overall curve shape observed for pHluorin-TeNT HC carriers fit well with the previously reported data (5,6). In particular, the average speed observed for round vesicles (n=33, 402 movements) was 0.82 ± 0.25 µm/s, whereas tubules (n=34, 334 movements) had an average speed of 1.18 ± 0.29 µm/s (Fig. S1). The pH dynamics of axonal retrograde carriers were then followed by monitoring the 405/488 emission ratio of TeNT HC compartments during transport. As shown in Fig. 2b, the pH of both types of carriers remained relatively constant during transport. Based on this finding, we then measured the pH of these organelles by ratiometric imaging only in the first frame of the videos, thus avoiding possible artefacts due to phototoxicity and photobleaching. To unravel a possible correlation between intraluminal pH and the average speed of axonal pHluorin-TeNT HC compartments, the velocity of the single movements (n=735; 67 carriers) was plotted against their corresponding pH (Fig. 2c). The large majority of moving organelles were neutral, their pH falling in the 7.0-7.5 (42% of the total) or 7.5-8.0 range (26% of the total). This neutral population of axonal carriers included both round vesicles and tubules and presented a speed distribution profile overlapping with that observed previously (compare Fig. 2c with Fig. S1) (5,6). Only a few pHluorin-TeNT HC-positive compartments displayed a pH below 7.0 (14% of the total), whereas, no moving carriers having an acidic pH (≤ 6.0) were detected. In sharp contrast, stationary pHluorin-TeNT HC positive organelles in somas and axons displayed a wide range of pHs, from acidic to neutral (Fig. 2d). This result was confirmed by the partial co-localisation of these stationary compartments in the soma with Lysotracker Red DND-99 using time-lapse

confocal microscopy (Fig. S2). Organelle acidification in the cell body has also been observed in real time, although with extreme low frequency (data not shown). What is the cause of the different pH distribution in moving and stationary pHluorin-TeNT HC positive organelles? At least three formal explanations might account for this phenomenon: i. the acidification machinery is excluded from or not acquired by transported organelles; or ii. its activity is specifically inhibited; or iii. the axonal retrograde carriers containing TeNT HC rapidly dissipate the pH gradient due to a high permeability to protons. A major cellular complex involved in organelle acidification is the vATPase. vATPases are found on a variety of intracellular compartments, including clathrin-coated vesicles (CCV), synaptic vesicles (SV) and secretory granules (22), and are responsible for the acidification of degradative organelles, such as lysosomes and phagosomes (22,23). We tested the presence of the vATPase on axonal and somatic organelles containing TeNT HC using antibodies against the subunits B and E in the V1 domain of the vATPase and the a subunit in the V0 domain (Fig. 3a). All antibodies specifically recognised their corresponding subunits in rat brain extracts (Fig. 3b). Immunofluorescence experiments performed by fixing MNs upon incubation for 45 min with TeNT HC-Alexa488 revealed that vATPase and TeNT HC-positive transport compartments were morphologically very different both in axons (Fig. 3c) and soma (Fig. 3d). A very limited overlapping of the two staining pattern was seen (≤ 6% at late internalisation points; see Fig. 5e), suggesting that the vATPase is not present on TeNT HC carriers. The frequency of the structures in which vATPase colocalised with TeNT HC (Fig. 3) resembled those observed for the stationary TeNT HC organelles, which can be acidified (Fig. 2d). We were also able to partially localise the vATPase with DAMP, an acidic granule marker (Fig. S3a and b), and with the SV protein VAMP1 (Fig. S3c), thus confirming that these antibodies are able

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

7

to detect functional vATPase on acidic organelles in neurons (22). A major caveat of an experimental approach based on immunolocalisation is the limit imposed by the sensitivity of the staining antibodies. To exclude a role for the vATPase activity in axonal TeNT transport, we specifically blocked its catalytic function using ConA and BafA1. Both drugs bind with high affinity to the subunit c of the vATPase V0 domain (IC50=10 nM for ConA and 0.5 nM for BafA1) (24). At these concentrations, ConA and BafA1 are effective in blocking Lysotracker Red DND-99 accumulation in acidic organelles in MNs (Fig. 4a), demonstrating that the activity of the vATPase was inhibited under these experimental conditions. However, direct comparison of the speed distribution profile of TeNT HC carriers in untreated MNs (Fig. 4; solid line) with those of treated MNs at different time points (Fig. 4; dotted lines) indicate that neither of the drugs alter axonal retrograde transport (see also Videos 2a-e and Fig. S4a). TeNT HC transport was also unaltered by long-term incubation with ConA (≥90 min). In treated MNs, we observed a slight reduction in speed values and a decrease in carrier frequency at later time points (Fig. 4; empty circles). We attribute these minor variations to the repetitive, long term imaging of the same axon, since untreated MNs showed the same overall alterations of AlexaFluor488-TeNT HC transport (Fig. S4b). To test whether the vATPase is involved in an early step of TeNT HC trafficking, we modified our experimental protocol by pre-treating MNs for 15 min with BafA1 and ConA before adding AlexaFluor488-TeNT HC (Fig. 5a). Imaging started 20 min after removal of the fluorescent TeNT HC (t=0 min). Only a faint, rather homogeneous, membrane staining of MN axons was observed at t=30 min (Fig. 5b). As shown by the kymographs in Fig. 5b, no moving TeNT HC carriers or stationary TeNT HC-positive compartments were detected, strongly indicating that this probe was not sorted to the retrograde transport pathway. In contrast, kymographs of MNs treated with vATPase

inhibitors after endocytosis and sorting of TeNT HC following the protocol described in Fig. 4b (t=30 min) displayed several progressing carriers (Fig. 5c; arrowheads) and stationary compartments (Fig. 5c; asterisks). Interestingly, we were able to observe a significant overlap between AlexaFluor488-TeNT HC and vATPase at early time points of internalisation (t=2 min; Fig. 5d and e), in sharp contrast to that observed at late time points (t=45 min; Fig. 5e). Is TeNT HC internalisation impaired by treatment with vATPase inhibitors? Two independent lines of evidence shown in Fig. 6 argue against this possibility. Treatment with 0.5 nM BafA1 prior to incubation with VSV-G tagged TeNT HC, followed by sequential staining in the absence and the presence of permeabilisation (Fig. 6a), revealed that a pool of TeNT HC is internalised under these conditions. Furthermore, BafA1 application did not impair the entry of biotinylated-TeNT HC into MNs (Fig. 6b). These findings indicate that a functional vATPase is not required for TeNT HC endocytosis, but plays a specific role in an early sorting step(s) targeting TeNT HC to axonal transport carriers.

DISCUSSION In this study, we monitored the pH dynamics of TeNT HC positive compartments in real time using a pHluorin-TeNT HC fusion protein. Chimeras of pHluorin have been used as pH probes in a variety of biological systems. In neurons, pHluorin-based sensors have been exploited to follow the presynaptic activity within neuronal networks (14), to report synaptic vesicle fusion (25) and to assess the molecular identity of different synaptic compartments during SV recycling (26). On this basis, we chose a novel fusion protein between ratiometric pHluorin and TeNT HC as a non-invasive method to investigate the pH of the fast axonal retrograde compartment in MNs. Here, we showed that these endocytic carriers have a neutral pH, which is kept constant during axonal movement. This finding is in

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

8

contrast with the pH dynamics described for the classical endosomal pathway, which undergoes a rapid acidification upon internalisation (20). The entry of TeNT in a neutral endocytic compartment has important mechanistic effects on the pathogenesis of tetanus. In fact, acidic pH triggers a conformational change of TeNT, which allows its membrane insertion and the translocation of the L chain into the cytosol, where it cleaves VAMP (2). This phenomenon occurs in hippocampal neurons in vitro (27), and in spinal cord inhibitory interneurons in vivo (1,28). In artificial liposomes, the pH initiating the membrane insertion of TeNT is about 5.0 (29), which is within the pH range of the endosomal pathway. By demonstrating that the fast axonal retrograde compartment in MNs displays a neutral pH, we provide an explanation on the entrapment of TeNT in the lumen of the retrograde carrier during axonal transport to the soma, a journey shared with neurotrophins and their receptors (5). The lack of acidification of the fast retrograde transport carriers might contribute to the low degradative capability of this compartment, which ensures the integrity of its endogenous and exogenous cargoes. This is in agreement with the long half-life of internalised TeNT in vivo (≥ 5 d in mouse sciatic nerve) (28). Several pathogens and virulence factors have been shown to either transit or accumulate in non-acidic cellular compartments. This is the case for Shiga-like (SLT-1B) and Cholera toxin (CT-B) B subunits, which have been extensively used as probes to uncover Golgi-dependent and independent pathways from the plasma membrane to the ER (30,31). C. pneumoniae, an intracellular parasite, inhabits a non-acidic vacuole, which is distinct from late endosomes and lysosomes (32). Furthermore, Echovirus 1 and simian virus 40 (SV40) enter a non-acidic compartment after leaving the plasma membrane in caveolin-enriched structures, termed caveosomes, which lack markers for endosomes, lysosomes, ER or Golgi (33-35). In contrast with the retrograde transport carriers, stationary TeNT HC-positive structures, which are

distributed in soma and neurites, have a much broader pH range spanning from pH 5.0 to pH 7.5. This observation is in agreement with the reported trimodal frequency distribution of the endocytic organelle pH in axon shafts of sympathetic neurons (36). Furthermore, it suggests that the machinery determining the intralumenal pH of these axonal compartments is strictly regulated and coordinated to the activity of the motor complexes responsible for their retrograde movement (37). What is the mechanistic basis of this differential pH regulation? A likely possibility is that this is due to the sorting of the vATPase away from this compartment. Acidification is essential for diverse cellular processes, such as protein targeting along the secretory pathway, recycling of receptors to the plasma membrane, and protein degradation in the endomembrane lumen (22,38). Inhibition of the vATPase activity perturbs some, if not all these functions (39,40). Consequently, we tested for the presence of the vATPase by using several antibodies against different subunits of its V1 and V0 domains. The experiments revealed very little colocalisation between TeNT HC and vATPase-positive organelles, suggesting that the lack of acidification is due to the absence of the vATPase from the retrograde carriers and not to an altered proton permeability of their membrane bilayer (41). In contrast, the vATPase is likely to be present on stationary organelles, which can be acidified. An inducible, organelle-specific vATPase sorting has been previously demonstrated for phagosomes containing M. avium, which fail to acidify due to the exclusion of vATPase from their delimiting membranes (42). The notion that vATPase activity is dispensable for fast retrograde transport in MNs has been confirmed by the lack of effect on this process of vATPase inhibitors used at concentrations abolishing the accumulation of acidotrophic dyes. In contrast, pre-treatment of MNs with vATPase inhibitors blocked TeNT accumulation in endosomal compartments and its sorting to the retrograde transport

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

9

route. Accordingly, we found that TeNT organelles may contain vATPase at very early time points after their endocytosis. This is in agreement with previous findings where the vATPase has been detected in CCV (22). Interestingly, TeNT has been found in CCV in spinal cord neurons (43) and it is internalised via a clathrin-dependent pathway in isolated MNs (Deinhardt K., unpublished results). CCV and other early endocytic organelles, which may coincide with the stationary TeNT HC structures, might act as initial sorting stations where axonal carriers are formed and their content sorted for transport. Mild acidic pHs might play a role in early steps of the endocytic pathway of TeNT by inducing a partial conformational change, which, although not sufficient to trigger the insertion of the molecule in the inner core of the lipid bilayer, might promote an enhanced interaction with the membrane surface and facilitate the initial sorting of TeNT HC. In conclusion, we demonstrated that a functional vATPase is transiently required for the initial sorting of TeNT HC to its retrograde transport route and it is then sorted away from the axonal carriers at later stages. As a consequence, these

retrograde organelles display a neutral luminal pH, which might protect the native conformation of acid-labile cargoes and stabilise receptor-ligand interactions. In particular, these conditions might allow the sustained interaction of some neurotrophins such as nerve growth factor, which have been found to enter TeNT HC carriers (5), and its receptors, providing additional basis for the long-range axonal neurotrophin signalling (44). Future experiments will directly address the molecular composition of these retrograde carriers and the activation status of neurotrophin-receptor cargo complexes during transport.

ACKNOWLEDGEMENTS We would like to thank A. de Bettignies for the pGEX-4T-3-VSV-G-Kin-pHluorin-HC cloning, H. Sze for the α-60kDa 2E7 antibody, S. Breton for the α-31kDa vATPase antibody, K. Deinhardt for the biotinylated TeNT HC, G. Hammond for scientific discussions, A. Brady for assistance in data analysis, F. Batista and S. Tooze for critical reading of the manuscript and for helpful comments.

REFERENCES

1. Schiavo, G., Matteoli, M., and Montecucco, C. (2000) Physiol. Rev. 80, 717-766 2. Humeau, Y., Doussau, F., Grant, N. J., and Poulain, B. (2000) Biochimie 82, 427-446 3. Herreros, J., Lalli, G., Montecucco, C., and Schiavo, G. (2000) J. Neurochem. 74,

1941-1950 4. Stöckel, K., Schwab, M., and Thoenen, H. (1975) Brain Res. 99, 1-16 5. Lalli, G., and Schiavo, G. (2002) J. Cell Biol. 156, 233-240 6. Lalli, G., Gschmeissner, S., and Schiavo, G. (2003) J. Cell Sci. 116, 4639-4650 7. Hafezparast, M., Klocke, R., Ruhrberg, C., Marquardt, A., Ahmad-Annuar, A.,

Bowen, S., Lalli, G., Witherden, A. S., Hummerich, H., Nicholson, S. et al. (2003) Science 300, 808-812

8. Deinhardt, K., and Schiavo, G. (2004) Biochem. Soc. Symp., in press 9. Montecucco, C., Papini, E., and Schiavo, G. (1994) FEBS Lett. 346, 92-98 10. Williamson, L. C., and Neale, E. A. (1994) J. Neurochem. 63, 2342-2345 11. Tycko, B., Keith, C. H., and Maxfield, F. R. (1983) J. Cell Biol. 97, 1762-1776 12. Balbis, A., Baquiran, G., Dumas, V., and Posner, B. I. (2004) J. Biol. Chem. 279,

12777-12785 13. Miesenbock, G., De Angelis, D. A., and Rothman, J. E. (1998) Nature 394, 192-195 14. Yuste, R., Miller, R. B., Holthoff, K., Zhang, S., and Miesenbock, G. (2000) Methods

Enzymol. 327, 522-546 15. Ashby, M. C., Ibaraki, K., and Henley, J. M. (2004) Trends Neurosci. 27, 257-261

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

10

16. Lalli, G., Herreros, J., Osborne, S. L., Montecucco, C., Rossetto, R., and Schiavo, G.

(1999) J. Cell Sci. 112, 2715-2724 17. Arce, V., Garces, A., de Bovis, B., Filippi, P., Henderson, C. E., Pettmann, B., and

deLapeyriere, O. (1999) J. Neurosci. Res. 55, 119-126 18. Ward, J. M., Reinders, A., Hsu, H., and Sze, H. (1992) Plant Physiol. 99, 161-169 19. Awaji, T., Hirasawa, A., Shirakawa, H., Tsujimoto, G., and Miyazaki, S. (2001)

Biochem. Biophys. Res. Commun. 289, 457-462 20. Weisz, O. A. (2003) Int. Rev. Cytol. 226, 259-319 21. Herreros, J., Lalli, G., and Schiavo, G. (2000) Biochem. J. 347, 199-204 22. Stevens, T. H., and Forgac, M. (1997) Annu Rev Cell Dev Biol 13, 779-808 23. Nelson, N. (2003) J. Bioenerg. Biomembr. 35, 281-289 24. Mattsson, J. P., and Keeling, D. J. (1996) Biochim. Biophys. Acta 1280, 98-106 25. Bozza, T., McGann, J. P., Mombaerts, P., and Wachowiak, M. (2004) Neuron 42, 9-

21 26. Mitchell, S. J., and Ryan, T. A. (2004) J. Neurosci. 24, 4884-4888 27. Matteoli, M., Verderio, C., Rossetto, O., Iezzi, N., Coco, S., Schiavo, G., and

Montecucco, C. (1996) Proc. Natl. Acad. Sci. U S A 93, 13310-13315 28. Habermann, E., and Dreyer, F. (1986) Curr. Top. Microbiol. Immunol. 129, 93-179 29. Schiavo, G., Boquet, P., Dasgupta, B. R., and Montecucco, C. (1990) J Physiol

(Paris) 84, 180-187 30. Lencer, W. I., Strohmeier, G., Moe, S., Carlson, S. L., Constable, C. T., and Madara,

J. L. (1995) Am. J. Physiol. 269, G548-557 31. Schapiro, F. B., Lingwood, C., Furuya, W., and Grinstein, S. (1998) Am. J. Physiol.

274, C319-332 32. Al-Younes, H. M., Rudel, T., and Meyer, T. F. (1999) Cell Microbiol 1, 237-247 33. Pelkmans, L., Kartenbeck, J., and Helenius, A. (2001) Nat. Cell Biol. 3, 473-483 34. Pelkmans, L., Burli, T., Zerial, M., and Helenius, A. (2004) Cell 118, 767-780 35. Pietiainen, V., Marjomaki, V., Upla, P., Pelkmans, L., Helenius, A., and Hyypia, T.

(2004) Mol. Biol. Cell 15, 4911-4925 36. Overly, C. C., and Hollenbeck, P. J. (1996) J. Neurosci. 16, 6056-6064 37. Gunawardena, S., and Goldstein, L. S. (2004) J. Neurobiol. 58, 258-271 38. Nelson, N., and Harvey, W. R. (1999) Physiol. Rev. 79, 361-385 39. van Weert, A. W., Dunn, K. W., Gueze, H. J., Maxfield, F. R., and Stoorvogel, W.

(1995) J. Cell Biol. 130, 821-834 40. Schoonderwoert, V. T., Holthuis, J. C., Tanaka, S., Tooze, S. A., and Martens, G. J.

(2000) Eur. J. Biochem. 267, 5646-5654 41. Wu, M. M., Grabe, M., Adams, S., Tsien, R. Y., Moore, H. P., and Machen, T. E.

(2001) J. Biol. Chem. 276, 33027-33035 42. Sturgill-Koszycki, S., Schlesinger, P. H., Chakraborty, P., Haddix, P. L., Collins, H.

L., Fok, A. K., Allen, R. D., Gluck, S. L., Heuser, J., and Russell, D. G. (1994) Science 263, 678-681

43. Parton, R. G., Ockleford, C. D., and Critchley, D. R. (1987) J. Neurochem. 49, 1057-1068

44. Howe, C. L., and Mobley, W. C. (2004) J. Neurobiol. 58, 207-216 45. Inoue, T., and Forgac, M. (2005) J. Biol. Chem. 280, 27896-27903

FOOTNOTES 1The abbreviations used are: TeNT, tetanus toxin; BafA1, bafilomycin A1, BoNT, botulinum neurotoxins; CCV, clathrin-coated vesicles; ConA, concanamycin A; DAMP, [3-(2,4-dinitroanilino)-3’-amino-N-methyldipropylamine]; DIC, difference interference contrast; DIV, days in vitro; GFP, green fluorescent protein; GST, glutathione S-transferase; IF, immunofluorescence; MESNA, sodium 2-mercapto-ethanesulfonate; MNs, motor neurons; p75NTR, neurotrophin receptor p75; PBS, phosphate-buffered saline; SV, synaptic vesicles;

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

11

TeNT HC, carboxy-terminal 50 kDa domain of tetanus toxin; vATPase, vacuolar (H+) ATPase; VSV-G, vesicular stomatitis virus G protein; WB, Western Blot. Laboratory work by the authors is supported by Cancer Research UK.

FIGURE LEGENDS

Fig1. Spectral and biophysical characterisation of pHluorin-TeNT HC. a) Scheme of clostridial neurotoxins and the ratiometric pHluorin-TeNT HC fusion protein. This chimera is expressed as recombinant GST fusion protein containing a thrombin cleavage (arrow). b) pH-dependence of the fluorescence excitation spectra of pHluorin-TeNT HC in vitro. Samples containing 0.5 µM pHluorin-TeNT HC at the indicated pHs were excited with wavelengths ranging from 350 nm to 500 nm, and emission was monitored at 508 nm. c) Calibration curve of pHluorin-TeNT HC in MNs. MNs were incubated with 40 nM pHluorin-TeNT HC in neurobasal medium for 30 min at 37°C. The medium was then exchanged with buffers at the indicated pHs, prior to addition of monensin and nigericin. Ratiometric images were taken for every pH and processed as described in Experimental Procedures. 405/488 ratios (R405/488; ± SEM) of pHluorin-TeNT HC compartments (n=24-52) were averaged for each pH. The resulting calibration curve is best described by the trend line y=-0.0006x3+0.0088x2-0.0159x+0.1163 (R2=0.999). d) GST-pHluorin is a functional probe to monitor the pH of endosomal compartments in MNs. MNs were incubated with 6 µM GST-pHluorin in neurobasal medium for 1 h at 37°C. Cells were washed and immediately imaged as described in Experimental Procedures (filled bars). Alternatively, the incubation medium was replaced by neurobasal medium and incubated for other 30 min (empty bars) prior to imaging. The endocytic compartments detected by fluid phase uptake of GST-pHluorin displayed a broad range of pHs, whereas upon 30 min chase, the probe is shifted to acidic compartments. Fig. 2. pHluorin-TeNT HC is retrogradely transported along axons in MNs. a) MNs were incubated with 40 nM pHluorin-TeNT HC for 30 min at 37°C, washed and imaged by low-light microscopy. Two representative 488 and 405 nm time series of a MN axon and its ratio (R405/488) are shown. 405 nm frames have been inverted to improve their contrast. The cell body is located out of view at the bottom of the image. Intervals between single frames are 5 s. A retrograde carrier (arrowheads) and a stationary compartment (asterisks) are indicated. The single frame image size is 2.6 µm x 14.6 µm. See also Videos 1a-c. Bar, 2 µm. b) The pH of pHluorin-TeNT HC carriers remains constant during transport. The 405/488 ratios of a representative pHluorin-TeNT HC-positive round vesicle and tubule were measured every 5 s, converted to pH (right axis) and plotted as a function of time. c) Moving TeNT HC carriers display an overall neutral pH. Shown is the speed and frequency of single movements (n=736) of pHluorin-TeNT HC carriers and their corresponding pHs. Neutral carriers (pH≥7.0) account for the large majority of the total. d) Comparison between the pH distribution of stationary organelles containing pHluorin-TeNT HC and axonal retrograde compartments. The stationary organelles (white bars) include compartments in both axons and soma (n=171), whereas the carriers (black bars) include axonal round vesicles and tubules (n=67). Fig. 3. The vATPase is excluded from axonal retrograde carriers containing TeNT HC-Alexa488. a) Schematic structure of the vATPase (45). The antibodies used for the WB analysis and in IF experiments were raised against the subunits B and E of the V1 domain and the a subunit of the V0 domain. b) The antibodies recognised single bands (α-31kDa, α-60kDa and α-116kDa) and a doublet (α-75kDa) at their expected molecular weights in unboiled rat brain extracts. IF showed very limited co-localisation between vATPase (in red) and TeNT HC-positive organelles (in green) in MN axons c) and soma d). Note the different morphology of vATPase and TeNT HC compartments. Bar, 5 µm.

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

12

Fig. 4. ConA and BafA1 inhibit acidification and do not alter axonal TeNT HC transport. a) DIC (left) and Lysotracker Red DND-99 staining (right) are shown for control, ConA-treated and BafA1-treated MNs. MNs were pre-incubated with vehicle (DMSO) or 10 nM ConA (15 min) or 0.5 nM BafA1 (10 min) prior to addition of 50 nM Lysotracker Red DND-99. After 15 min incubation, cells were washed and imaged by confocal microscopy without fixation. Bar, 20 µm. MNs were incubated with 40 nM TeNT HC-Alexa488, washed and imaged by low-light time-lapse microscopy (filled circles). Retrograde transport in the same axon was measured at 5 min (empty triangles), 15 min (empty squares) and 30 min (empty circles) after application of 10 nM ConA (b) or 0.5 nM BafA1 (c). A scheme of the experiment is shown on top of panel b. The number of single movements monitored for each condition is indicated in the right hand tables. Fig. 5. A functional vATPase is required for TeNT HC sorting to the retrograde transport route. a) Schematic time-scale of the experiment. MNs were treated with 10 nM ConA or 0.5 nM BafA1 and then incubated with 40 nM TeNT HC-Alexa488, washed and imaged by low-light time-lapse microscopy. b) Epifluorescence of MN axons at t=30 min of imaging. The corresponding kymographs are shown below. c) Kymographs of MN axons treated as described in Fig. 4c (t=30 min). Arrowheads indicate retrograde TeNT HC-Alexa488 carriers, whereas asterisks mark stationary organelles. In b) and c) the MN soma is located out of view on the right. d) At early time points of TeNT HC-Alexa488 internalisation (t=2 min), a partial overlap between TeNT HC-Alexa488 (in green) and vATPase (116 kDa subunit; in red) can be detected. e) Relative colocalisation between the vATPase, stained with an antibody against the 116 kDa subunit, and TeNT HC at early (t=2 min) and late (t=45 min) internalisation points in axons and soma (n=6 to 8). Vertical bar, 40 s; horizontal bar, 5 µm. Fig. 6. BafA1 does not inhibit TeNT HC internalisation. a) MNs were treated with 0.5 nM BafA1 for 15 min at 37°C, followed by 30 min incubation with VSV-G-Kin-TeNT HC. Cells were fixed and surface-bound TeNT HC was detected using an α-rabbit VSV-G antibody (in green). MNs were then permeabilised and internalised TeNT HC was detected using an α-mouse VSV-G antibody (in red). See Material and Methods. A scheme of the experiment is shown on top. Bar, 5µm. b) After BafA1 treatment (0.5 nM), cells were incubated with TeNT HC labelled with disulfide-linked biotin at 37°C for 30 min, washed and incubated with a membrane impermeable reducing agent (MESNA) on ice. Internalised TeNT HC was detected using Streptavidin-Alexa488. For the control, MNs were incubated with TeNT HC-biotin on ice for 20 min prior to MESNA treatment. Time-scale of the experiment is on top. Bar, 10 µm.

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

db

-COOH

ratiometricpHluorin

GST HC

cleavage

S-S-COOHH2N-

L HHCHN

intracellularactivity translocation binding

a c

no chase30 min chase

0

0.2

0.6

0.4

pH

Figure 1 Bohnert and Schiavo

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

0-0.4 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2

7

6

5

4

3

2

1

0

6.0-6.56.5-7.07.0-7.57.5-8.0>8.0

pH

Speed (µm/s)

movingstationary0.4

0.3

0.2

0.1

05.0 5.5 6.0 6.5 7.0 7.5 8.0

pH

488 nm 405 nm Ratio 405/488

vesicletubule0.24

0.22

0.20

7.5

0 5 10 15 20 25 30 35time (s)

pH

8.0

a

Figure 2 Bohnert and Schiavo

b d

c retrogradeantero-grade

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

membrane

lumen

cytoplasm

V0

V1

AB

A

BA

B

G2

d

DF

c’cc

c”c

2H+

ATP

ADP+Pi

α−60kDa

α−31kDa

α−75kDa

α−116kDa

vacuolar (H+) ATPasea

75

50

25

kDaα−60kDa

100120

160

908070

kDaα−116kDa

25

375075

kDaα−75kDa

302015

4060kDa

α−31kDa

b

TeNT HC-Alexa488

vATPase

merge

α−60kDaα−31kDa α−75kDa α−116kDac

α−60kDaα−31kDa α−75kDa α−116kDad

TeNT HC-Alexa488

vATPase

merge

Figure 3 Bohnert & Schiavo

E

a

H C

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

-0.8 -0.4 0 0.4 0.8 1.2 1.6 2.0 2.4 2.80

0.05

0.10

0.15

0.20

0.25

Speed (µm/s)

262229303220

no drug5 min15 min30 min

singlemovementsBafA1

c

487280372263

no drug5 min15 min30 min

singlemovements

-0.8 -0.4 0 0.4 0.8 1.2 1.6 2.0 2.4 2.80

0.05

0.10

0.15

0.20

0.25

Speed (µm/s)

ConAb

retrogradeantero-grade

TeNT HC-Alexa488 wash drug

5 15 30 t (min)nodrug

0-60 -30DIC LysoTracker

ConA

control

BafA1

a

Figure 4 Bohnert and Schiavo

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

ConABafA1

TeNT HC-Alexa488 wash

30 t (min)0-60 -30

drug

-75

imaging

a

b

c

dvATPase mergeTeNT Hc

Figure 5 Bohnert and Schiavo

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

axon soma axon soma

vATPase over total TeNT HcTeNT Hc over total vATPase

e

late (45 min) early (2 min)

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

TeNT Hc-VSVG wash/fix/block

12515 45

BafA1

0

α-rabbitVSVG

155

perme-abilise/block

195

α-mouseVSVG

240

2ndanti-body

225 270 t (min)

α-rabbit VSVG α-mouse VSVG merge

0.5 nM BafA1 pretreatment4°C control

b

a

TeNT Hc-biotin onice

225 t (min)11015 45

drug

0 65 135

perme-abilise/block

195

Strept-avidin

115

3 xMESNA fix

Figure 6 Bohnert and Schiavo

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

13

SUPPLEMENTAL MATERIAL

Fig. S1. Quantitative kinetic analysis of pHluorin-TeNT HC carriers. The distribution of single speed values observed for vesicles (n=33, empty bars) and tubules (n=34, filled bars) from six independent experiments is shown. Retrograde movement is conventionally shown as positive. Similar numbers of single movements for the two carriers (vesicles, 402; tubules, 333) have been analysed. Fig. S2. TeNT HC enters acidic compartments in a MN soma. TeNT HC-Alexa488 and Lysotracker Red DND-99 display a partial co-localisation in stationary organelles in a MN soma. MNs were incubated with 40 nM TeNT HC-Alexa488 and 100 nM Lysotracker Red DND-99 for 30 min at 37°C, washed and imaged without fixation by confocal microscopy. Panels show single frames (TeNT HC in green, Lysotracker Red DND-99 in red) and the merge image from a dual colour confocal movie. The inset displays a high magnification of the indicated area. Bar, 10 µm. Fig. S3. vATPase partially localises with acidic granules and synaptic vesicle markers. a) DAMP (in green) shows a partial overlap with vATPase-positive compartments (in red) in a MN soma. b) Relative colocalisation between the vATPase (stained with an antibody against the 116 kDa subunit) and DAMP at early stages of internalisation (t=2min). c) The SV protein VAMP1 (in green) partially localised with the vATPase (in red) at synapse-like structures in MN axons. Bar, 5 µm. Fig. S4. Long term imaging affects axonal retrograde transport in control and ConA-treated MNs. a) Cultured MNs were incubated with 40 nM TeNT HC-Alexa488 for 30 min at 37°C, washed and then imaged by low-light time-lapse microscopy as detailed in Experimental Procedures. ConA (10 nM) was added to the sample only after axonal transport was detected. Representative time series of moving TeNT HC-Alexa488 carriers (arrowheads) and a stationary organelle (asterisk), before and 5, 15 and 30 min after ConA treatment are shown in a MN axon. The MN soma is located at the bottom. Frame size is 3.12 µm x 16.9 µm and intervals between single frames are 5 s. Bar, 2 µm. b) Retrograde TeNT HC-Alexa488 transport was measured in the same axon 5 min (full circles), 15 min (empty squares) and 30 min (empty circles) after exposure to 488 nm light.

VIDEOS Video 1a-c. pH analysis of axonal retrograde transport carriers. MNs were incubated with pHluorin-TeNT HC for 30 min at 37°C, washed and imaged as detailed in Experimental Procedures. Samples were consecutively excited at 488 nm (Video 1a) and 405 nm (Video 1b). Video 1b has been inverted to improve the contrast. The ratiometric movies R405/488 (Video 1c) were assembled using Lucida 4.0. The MN cell body is located at the bottom. Frames were taken every 5 s with an exposure time of 333 ms and correspond to a field of 2.6 µm x 14.6 µm. Videos consist of 5 frames played at 10 frames/s. Video 2a-e. Axonal retrograde transport is unaffected by vATPase inhibitors. Video 2a shows a still phase image of a MN axon incubated with TeNT HC-Alexa488 for 30 min at 37°C. The same field has been imaged by low-light time-lapse microscopy to monitor retrograde axonal transport of TeNT HC at t=0 (Video 2b, untreated sample) and at 5 min (Video 2c), 15 min (Video 2d) and 30 min (Video 2e) after treatment with 10 nM ConA. The MN soma is located at the bottom. Frames were taken every 5 s with an exposure time of 111 ms and correspond to a field of 5.98 µm x 64.2 µm. The videos consist of 40 frames played at 10 frames/s.

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

vesiclestubules

0

0.4

0.8

0.12

0.16

-0.4 0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2

Speed (µm/s)

Figure S1 Bohnert and Schiavo

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

Figure S2 Bohnert and Schiavo

LysotrackerTeNT Hc merge

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

DAMP mergeα-116kDavATPase

a

Figure S3 Bohnert and Schiavo

VAMP1 α-75kDa vATPase mergec

0.1

0.2

0.3

0.4

0.5

0.6

0vATPaseover totalDAMP

DAMPover totalvATPase

b

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from

15 min ConA 30 min ConA

5 min ConAcontrol (no ConA)

-0.4 0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.20

0.05

0.10

0.15

0.20

Speed (µm/s)

5 min15 min30 min

singlemovements

198318271

no drug

b

a

Figure S4 Bohnert and Schiavo

retrogradeantero-grade by guest on A

pril 9, 2020http://w

ww

.jbc.org/D

ownloaded from

Stephanie Bohnert and Giampietro Schiavospecialised pH regulation

Tetanus toxin is transported in a novel neuronal compartment characterised by a

published online October 18, 2005J. Biol. Chem. 

  10.1074/jbc.M506750200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

Supplemental material:

  http://www.jbc.org/content/suppl/2005/10/24/M506750200.DC1

by guest on April 9, 2020

http://ww

w.jbc.org/

Dow

nloaded from