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Endocytic trafficking of membrane proteins is essential for neuronal structure and function. We show that Transient Axonal Glycoprotein1 (TAG1 or CNTN2), a contactin-related adhesion molecule, plays a central role in the differential trafficking of components of the semaphorin3A receptor complex into distinct endosomal compartments in murine spinal sensory neuron growth cones. The semaphorin3A receptor is composed of Neuropilin1 (NRP1), PlexinA4 and L1, with NRP1 being the ligand-binding component. TAG1 interacts with NRP1 causing a change in its association with L1 in the Sema3A response, such that L1 is lost from the complex following Sema3A binding. Initially, however, L1 and NRP1 endocytose together and only become separated intracellularly, NRP1 becoming associated with endosomes enriched in lipid rafts and co-localising with TAG1 and PlexinA4. When TAG1 is missing, NRP1 and L1 fail to separate and NRP1 does not become raft-associated; co-localisation with PlexinA4 is reduced and Plexin signalling is not initiated. These observations identify a novel role for TAG1 in modulating the intracellular sorting of signalling receptor complexes.
Trafficking of membrane proteins is critically important to neuronal development and function (e.g. Kawauchi 2010; Sheen et al 2004; Lowe, 2005; Lee and Gao 2008; Collinet 2010). Endocytosis clears receptors from the cell surface and the endocytic route followed by receptors, affects receptor signalling, turnover and recycling (Di Guglielmo et al., 2003; Sorkin and Von Zastrow, 2009), thus enabling essential processes such as desensitization and adaptation.
Much is known of the intracellular components that exist to control trafficking pathways (Conner and Schmid, 2003), but how membrane receptor composition affects pathway selection is less well-studied. The transmembrane protein Neuropilin1 (NRP1) is the ligand-binding component of receptor complexes for distinct families of extracellular ligands, class 3 semaphorins (Sema3A-F) and vascular endothelial growth factors (VEGF-A-E) (Pellet-Many et al., 2008; Tran et al., 2007). In endothelial cells, NRP1 signalling stimulated by Sema3s is distinct from that stimulated by VEGFs (Pellet-Many et al., 2008) due to the association of NRP1 with different co-receptor partners, Plexins and VEGF receptors respectively (Pellet-Many et al 2008). These associations lead to internalisation of NRP1 via different endocytic pathways (Salikhova et al., 2008).
However, Sema3A can evoke distinct responses from different classes of cortical neurons, even when they express the same receptor complex components: NRP1, PlexinA4 and L1 (Carcea et al., 2010; Castellani et al., 2000, 2002). This appears to be because responsive neurons internalise Sema3A via a lipid raft-mediated, rather than clathrin-mediated endocytic pathway (Carcea et al., 2010). However, since both L1 and PlexinAs have been shown to promote clathrin-mediated NRP1 endocytosis (Castellani et al., 2004), the mechanism controlling this differential trafficking remains unclear.
Embryonic spinal sensory afferents also show differential responsiveness to Sema3A (Messersmith et al., 1995; Puschel et al., 1996; Fu et al., 2000; Pond et al., 2002), which correlates with expression of the GPI-linked, L1-related molecule, TAG1, loss of which results in premature entry of presumptive nociceptive fibres into the dorsal horn and loss of their response to Sema3A in vitro (Law et al., 2008). Sensory growth cones lacking TAG1 fail to clear NRP1 and L1 from their surfaces after Sema3A treatment, suggesting that TAG1 may be required for endocytosis of the Sema3A receptor complex (Law et al., 2008).
Here we show that TAG1 interacts directly with NRP1 and can be found complexed with L1 and NRP1 at the cell surface. We demonstrate that TAG1 is required for several events that occur after Sema3A treatment, including increased association of NRP1 with PlexinA4 and stimulation of clathrin-mediated endocytosis of L1. However, we find that although L1 and NRP1 are endocytosed together after Sema3A treatment, L1 dissociates from the receptor complex and is trafficked in a pathway distinct from NRP1, which instead co-localises with TAG1 and PlexinA4. Consistently, this separation does not occur when TAG1 is missing and phosphorylation of CRMP2, an early indication of Sema3A signalling (Uchida et al., 2005) does not occur. Thus, TAG1 has a critical role in trafficking NRP1 and L1 into distinct endocytic pathways, which appears necessary for Sema3A signal generation.
Maintenance of TAG1 null mutant mice on C57Bl6 background was as reported (Poliak et al., 2003). Animals were maintained with appropriate UK Home Office and Local Ethical Committee approval.
Antibodies used were as reported (Law et al., 2008) with the addition of anti-TAG1 polyclonal (I. Horresh/E. Peles); L1-ICD and 74-5H7 mAb (V. Lemmon); pCRMP (Y. Goshima); PlexinA4 (H. Fujisawa) ; L1 Fab fragment was prepared from L1-324 by Bioserv UK Ltd.
DNA expression constructs as reported (Law et al., 2008) with the addition of full-length FLAG-tagged neuropilin 1 (NRP1; A. Kolodkin) (Kolodkin et al., 1997). Sema3A protein was produced by transfection of Sema3A in COS7 cells as described in Law et al., 2008. Alexa Fluor-555 CTxB was purchased from Molecular Probes.
Dissected dorsal root ganglia (DRG) from embryonic day 13.5 embryos of either sex were cultured on PDL-coated coverslips in Opti-MEM supplemented with 50ng/ml nerve growth factor (NGF) for 14-18h and immunodetection carried out as described (Law et al., 2008).
TritonX-100 extraction of soluble membranes was adapted from Guirland et al., 2004 and protocols used to fix cytoskeletal proteins (Letourneau, 1983). DRG explants treated with mock or Sema3A conditioned-medium were quickly chilled and extracted in ice cold microtubule stabilising buffer (2mM MgCl2, 10mM EGTA, 60mM Pipes, pH 7.0) containing 0.1% TritonX-100 for 1 min followed by fixation with 4% paraformaldehyde (PFA) on ice and then room temperature (RT) for 15 min each, then immunostained as above. The non-specific total protein dye 5-(4,6-dichlorotriazin-2yl) aminofluorescein (DTAF, Sigma) was applied (0.0002% w/v) for 2 hours to detect the total protein content and outline of growth cones.
DRG neurons treated with mock or Sema3A-conditioned medium for 5 minutes were rinsed in ice cold L15 medium followed by 1mg/ml cholera toxin B in L-15 medium for one hour at 12°C (Guirland et al., 2004). Cells were then rinsed 2x in PBS and fixed with 4% paraformaldehyde for 1 hour at RT to fix the lipid rafts (Mayor et al., 1994), then immunostained as above.
The protocol for ligand endocytosis was adapted from Salikhova et al., 2008. DRG neurons in culture were cooled on ice, the medium removed and Alexa Fluor 555-cholera toxin-B (1mg/ml) or L1 Fab (25mg/ml) added in fresh Opti-MEM for 20 min on ice. Cells were then rinsed in Opti-MEM and returned to warm culture medium, together with mock or Sema3A-conditioned medium, and incubated at 37°C for various intervals to allow endocytosis of cholera toxin-B or L1 Fab. Then cells were placed on ice to stop endocytosis and ice cold glycine stripping buffer (100 mM glycine, 100 mM NaCl, pH 2.5) added for 2 min to remove all surface-bound ligands. Then cells were washed 2x in ice cold PBS, fixed with 4% PFA on ice and then at RT for 15 min each, then immunostained as above.
Images were acquired on an Olympus BX61 microscope with GRID confocal using Volocity software version 5.5 (Improvision) or Zeiss AxioImagerZ1 with apotome. 100X UPlan Apo oil objective with a numerical aperture (NA) of 1.4 was used and images were acquired as Z-stacks with 0.2mm spacing. This gives a voxel size of 60 nm in the X- and Y-axes, and 200nm in the Z-axis. 20 images were acquired for each experimental treatment/time-point. Within an experiment, the exposure times for each fluorescence channel were kept constant order to enable the comparison of fluorescence intensities across the various conditions.
All post-acquisition analysis of images was done using Volocity software (Perkin-Elmer). The region of growth cone used for analysis was the distal 30μm of axon. For determination of total protein levels, the total fluorescence intensity in the region of interest (ROI) was measured and background subtracted. The average of ‘total’ fluorescence intensity for each treatment/time-point (+ s.e.m) was used for analysis. For co-localisation analyses, z-stacks were deconvolved using iterative restoration (confidence limit 98%, maximum of 25 iterations). Thresholds were set for each channel to 20-30% of the maximum intensity and input in the co-localisation function to determine the co-localisation coefficient for each channel. The mean co-localisation coefficient (+ s.e.m) for each treatment/time-point was used for comparison.
Compiled data sets from at least two experiments were analysed for statistical significance in Prism software (Graphpad) using two-tailed unpaired t-test with Welch’s correction for unequal variances (2 groups of data) or one-way ANOVA with Bonferroni’s multiple comparison test (more than 2 groups of data). Confidence interval used was set to 95%, with a p-value less than 0.05 taken as statistically significant. The p-values obtained were summarised using asterisks as * : P<0.05, ** : P<0.01, *** P<0.001.
The endocytosis of NRP1 from the cell surface following Sema3A binding has been shown to require the presence of L1, at least in cos7 cells (Castellani et al 2004). However, although L1 is known to be endocytosed in growth cones during L1-stimulated axon growth (Kamiguchi et al., 1998; Kamiguchi et al., 2000), and in cos7 cells after Sema3A treatment (Castellani et al 2004), formal proof that L1 endocytosis is stimulated in growth cones after Sema3A treatment is lacking. To follow L1 endocytosis directly, Fab fragments of anti-L1 monoclonal antibody (mAb 324) were applied in cold medium to live wild type growth cones for 10 mins on ice. The cold medium was then replaced with fresh medium at 37°C, with or without Sema3A but containing no anti-L1 Fab, for different periods before fixation and visualisation of internalised Ab (Kamiguchi et al., 1998). In control conditions, the fluorescence intensity due to internalised L1 Fab remained constant over 20 mins (Figure 1A), consistent with previous studies showing that L1 is constitutively endocytosed and recycled during axon growth (Kamiguchi et al., 2000). However, the intensity of this fluorescence increased significantly 10 mins after Sema3A exposure, reflecting a large increase in the number of Fab-containing internalised particles. By 20 mins the internalised particles become concentrated, usually into a single large aggregate (Figure 1A, arrowhead). At the same time the overall fluorescence appeared to have fallen, suggesting that L1 may be recycled or degraded, though it is possible that saturation of the fluorescence signal caused us to underestimate the amount of L1 present in the aggregate. Such aggregation of internalised Fab was never seen in untreated growth cones.
In L1-stimulated axon growth, L1 is endocytosed via an AP-2, clathrin and dynamin-dependent pathway (Kamiguchi et al., 98; Kamiguchi and Yoshihara 2001). Consistent with the idea that Sema3A-induced L1 endocytosis uses a similar pathway, we found that the co-localisation of L1 with clathrin heavy chain increases 5 mins after Sema3A treatment (Figure 1B). The clathrin adaptor, AP-2 binds to L1 via the YRSL motif present in the cytoplasmic tail of L1, which is required for L1 endocytosis, but this binding is inhibited by phosphorylation of the leading tyrosine (Y1176; Schaefer et al., 2002). Phosphorylation of Y1176 also blocks binding of the mAb 74-5H7 (Schaefer et al., 2002). 74-5H7 immunoreactivity is found associated with L1 in endocytic vesicles and can be used to detect L1 that is available for AP-2-dependent endocytosis (Schaefer et al., 2002; Kamiguchi et al., 98). We therefore used 74-5H7 to determine whether the enhanced L1 endocytosis seen after Sema3A treatment is likely to be AP-2 dependent. Consistent with this idea, we found that the ratio of dephosphorylated (74-5H7 immunoreactive) to total L1 (mAb 324 immunoreactive) increased almost two-fold after Sema3A treatment, compared to mock-treated growth cones, peaking at 10 min but still remaining significantly higher at 20 min (Figure 1C). However, since mAb 324 recognises an extracellular epitope (Itoh et al., 2004), it is also possible that the 324/74-5H7 ratio rises because the extracellular domain is cleaved (e.g. Mechtersheimer et al., 2001). That this is not the case is indicated by the fact that the ratio between 324 fluorescence and that from an antibody against the intracellular domain of L1 (L1-ICD; Nakamura et al., 2010), did not change after Sema3A treatment (not shown).
Together these data confirm that Sema3A induces a net increase in L1 endocytosis in growth cones that peaks around 10 mins after treatment, most likely in an AP-2/clathrin-dependent manner. Moreover, the aggregation of the internalised particles 20 mins after Sema3A treatment, but not in control growth cones, suggests that Sema3A signalling may change the endocytic pathway followed by L1.
Although the data above and previous studies (Castellani et al., 2004) suggest that NRP1, through being complexed with L1, should undergo clathrin-mediated endocytosis (CME), disruption of cholesterol-rich membranes (‘lipid rafts’) in neurons (Guirland et al., 2004; Carcea et al., 2010), leukemic T cells (Moretti et al., 2008) and endothelial cells (Salikhova et al., 2008) inhibits NRP1-mediated signalling and, in a subset of cortical neurons, Sema3A internalisation (Carcea et al., 2010). Indeed, growth cone responses to a range of guidance cues, both attractive and repellant, have been shown to be dependent upon the integrity of lipid rafts, and receptors for the relevant cues, including NRP1, are known to translocate to cholesterol-enriched membrane fractions during the response (Guirland et al., 2004; Marquardt et al 2005). Together these results have been interpreted to indicate that the Sema3A receptor complex undergoes lipid raft-mediated endocytosis (RME) in neurons that collapse in response to Sema3A (Carcea et al., 2010). However, these studies largely depended on pharmacological endocytic inhibitors, the specificity of which is controversial (Ivanov, 2008), and focussed on the fate of internalised NRP1 at late (e.g. 30 min) time points, or on the co-patching of NRP1 with lipid rafts on the cell surface (Guirland et al., 2004), and did not directly address the association of NRP1 with endocytosed L1.
Since, NRP1 has been observed to disappear from the surface of Xenopus retinal growth cones within 5 min of Sema3A treatment (Piper et a., 2005), we determined whether this is also true of mouse DRG growth cones. Levels of surface NRP1 dropped to less than half that of control growth cones within 5 mins of treatment and remained at low levels until at least 10 mins after (Figure 2A), most likely remaining low until at least 45 mins after treatment (Fournier et al., 2000; Law et al., 2008). Thus, like L1, NRP1 also rapidly disappears from the growth cone surface after Sema3A treatment.
To test directly whether NRP1 endocytosis with L1 increases after Sema3A treatment, we followed the co-localisation of NRP1 with internalised L1 using anti-L1 Fabs as above. Five minutes after Sema3A addition, more than 30% of internalised Fab could be found co-localised with NRP1, a two-fold increase over control (Figure 2B). Strikingly, however, by 10 mins this co-localisation reduced to control levels and by 20 mins less than 15% of internalised Fab could be found associated with NRP1, despite the fact that overall levels of internalised anti-L1 Fab rise over the same period (Figure 1A). Because these experiments follow the fate of a single pulse of internalised anti-L1 Fab, this suggests that NRP1 initially internalises with L1, but that at later stages the molecules become separated inside the cell.
The observation that NRP1 initially endocytoses with L1 and that Sema3A-induced L1 endocytosis appears to be clathrin-dependent was unexpected, given that disruption of lipid rafts blocks responses to Sema3A (Guirland et al., 2004; Carcea et al., 2010). One possibility is that, despite the increased co-localisation of L1 with clathrin and the dephosphorylation of the AP-2 binding site, in this instance NRP1 associated with L1 undergoes RME, as has been suggested (Carcea et al., 2010).
We attempted to address the endocytic route taken using classical CME and RME inhibitors (monodansylcadaverine (MDC) and filipin, respectively) but found that the inhibitors alone induced a severe reduction of NRP1 cell surface expression on control growth cones (Figure 3A), suggesting that the drugs affect the steady-state recycling or export of NRP1 to the cell surface and precluding their use in the study of Sema3A-induced endocytosis.
Instead, therefore, we addressed whether NRP1 becomes endocytosed with rafts after Sema3A treatment by labelling cells with un-crosslinked Alexa Fluor 555-tagged cholera toxin B subunit (CTxB), which binds raft-associated GM1 gangliosides (Harder et al., 1998), and followed the association of NRP1 with internalised CTxB in a manner similar to the anti-L1 Fab experiments above. In control growth cones, many CTxB-positive endocytic vesicles were found also to label for NRP1 (Figure 3B), indicating that as much as 25% of total NRP1 protein undergoes RME in control conditions. However, after 5 minutes of Sema3A treatment, the co-localisation between NRP1 and CTxB dropped to ~18%, significantly lower than control growth cones at the same stage (p<0.05, unpaired t-test). Following this initial decrease, co-localisation of CTxB and NRP1 then rose to 31% at 10mins and 34% at 20 mins, whereas control levels remained constant (p<0.05, unpaired t-test). Because this experiment follows the fate of a single pulse of internalised CTxB, this suggests that, in response to Sema3A, NRP1 is not initially internalised with CTxB, but subsequently becomes associated with vesicles containing internalised CTxB, presumably bound to raft-associated GM1 gangliosides. Since the initial decrease in NRP1 co-localisation with endocytic CTxB occurs coincident with the disappearance of NRP1 from the cell surface (Figure 2A) and an increase in overall CTxB uptake (Figure 3C), this strongly suggests that a large proportion of the endocytosis of NRP1 that occurs after Sema3A treatment, does so in membranes that do not bind CTxB.
Taken together, these observations indicate that the association of NRP1 with endocytosed L1 after Sema3A treatment is inversely related to its association with endocytosed CTxB, and are consistent with the idea that NRP1 is initially endocytosed with L1 in non-CTxB binding vesicles, but by 10 mins the two proteins separate and NRP1 is trafficked into intracellular compartments enriched in CTxB binding sites.
Previously, we showed that the disappearance of L1 and NRP1 from the surface of neuronal growth cones after Sema3A exposure is TAG1-dependent. To determine whether loss of TAG1 affects the early or late phase of NRP1 trafficking described above, we repeated the experiments that follow NRP1 co-localisation with internalised anti-L1 Fab, or with internalised CTxB, on growth cones from DRG from TAG1 knockout (TAG1ko) mice. Remarkably, although as in WT there was an initial increase in co-localisation of L1 Fab with NRP1, instead of falling with time, in the absence of TAG1 co-localisation was sustained for at least 20 mins after Sema3A treatment with up to 38% of internalised Fab being co-localised with NRP1 at 10 mins, compared to just 20% in controls (Figure 2C). When instead we followed the co-localisation of endocytic CTxB with NRP1, we found that the increase in the association of NRP1 with internalised CTxB that occurs in WT growth cones after 10 mins (Figure 3B), does not occur in TAG1ko growth cones (Figure 3D). Thus, these data support the idea that TAG1 is required for the separation of NRP1 from L1 and the accompanying translocation of NRP1 to CTxB-binding vesicles, rather than the initial endocytosis of the complex from the cell surface.
The involvement of TAG1 in the translocation of NRP1 to CTxB-binding vesicles led us to consider how TAG1 might affect the Sema3A receptor complex. In principle, TAG1 could exert its influence through its interactions with L1 (Buchstaller et al., 1996; Malhotra et al., 1998; Rader et al., 1996). However, the known association of TAG1 with GM1 gangliosides (Kasahara et al., 2002; Loberto et al., 2003) led us to explore whether TAG1 might play a role in NRP1 translocation through more direct interactions with NRP1.
To understand better the relationship between the three molecules, triple labelling experiments were performed on permeabilised wild type growth cones before and after Sema3A treatment (Figure 4A). On untreated growth cones, TAG1 shows the most restricted localisation and appears largely to be localised to the periphery of the growth cone, most notably on filopodia (Figure 4A, arrows). Quantitation indicates that overall only ~5% of NRP1 and 15% of L1 is associated with TAG1. However, in the peripheral regions where TAG1 is concentrated there is clear overlap with both L1 and NRP1 and ~32% of all TAG1 is L1-associated and 20% NRP1-associated. Ten minutes after Sema3A treatment, however, the association of TAG1 with NRP1 doubles, while that with L1 remains the same. At the same time, mirroring the anti-L1 Fab results above, the colocalisation of L1 and NRP1 has begun to drop and by 20 mins less than 10% of L1 is NRP1-associated. L1 association with TAG1 also falls to almost half untreated levels, while the association of TAG1 with NRP1 has returned to the same as untreated levels. As with the internalised anti-L1 Fab experiments, after Sema3A treatment the bulk of L1 labelling is in a large aggregate; TAG1 and NRP1 labelling does not follow this pattern.
Together, the data above support that L1 becomes separated from NRP1 after Sema3A treatment, apparently entering a distinct intracellular structure. By contrast, the association of NRP1 with TAG1 increases during the course of the Sema3A response. Thus, although previously we had been unable to find evidence for an interaction of NRP1 and TAG1 in trans (Law et al., 2008), this raised the possibility that TAG1 and NRP1 might interact in cis.
To test for interactions between TAG1 and NRP1, we co-expressed full length cDNAs for all three molecules in combinations in cos7 cells (Figure 4B). As expected, immunoprecipitation of NRP1 co-precipitated L1 (Castellani et al., 2000), whether or not TAG1 was present (Figure 4B ‘control’, lanes 7 and 8, bottom panel). Surprisingly, however, NRP1 also co-precipitated TAG1, even when L1 was absent (lanes 6 and 8, middle panel). Thus, in cos7 cells, TAG1 appears to be part of the Sema3A receptor complex and to interact with NRP1 independently of L1.
Our co-localisation studies suggested that the association of NRP1 with L1 might change upon Sema3A binding. To test this, we examined the associations of NRP1, L1 and TAG1 30 mins after Sema3A treatment of transfected cos7 cells; previous studies have shown that these cells respond to Sema3A when transfected with appropriate NRP1 and L1 or PlexinA combinations (Takahashi et al., 1999, Zanata et al., 2002, Castellani et al., 2004). Immunoprecipitation of NRP1 from Sema3A-treated cells co-transfected with NRP1 and L1 (Figure 4B ‘Sema3A’, lane 7, bottom), or NRP1 and TAG1 (lane 6, middle), co-precipitated L1 and TAG1 respectively, similar to untreated cells (Figure 4B ‘control’). However, immunoprecipitation of NRP1 from Sema3A-treated cells transfected with cDNAs for all three proteins (NRP1/L1/TAG1) only co-precipitated TAG1, not L1, which now appeared to be completely absent from the complex (Figure 4B ‘Sema3A’ lane 8, asterisks highlight comparison). Thus, Sema3A binding to NRP1 on cos7 cells results in its dissociation from L1, but only when TAG1 is present. Although earlier time points were not assayed, that NRP1 and L1 are separated 30 mins after Sema3A treatment in cos cells is consistent with the observation that this separation occurs between 5 and 10 mins after treatment in growth cones.
Taken together with the co-localisation data from mutant and wild type growth cones, these data indicate that TAG1 regulates the segregation of the Sema3A receptor components, NRP1 and L1 into different membrane microdomains after Sema3A binding.
Although our observation that NRP1 is trafficked intracellularly into CTxB-binding, presumably raft-enriched membranes is consistent with previous observations that disruption of lipid-rafts blocks Sema3A-induced growth cone collapse (Guirland et al., 2004; Carcea et al., 2010), these latter studies suggested that NRP1 translocated to rafts on the cell surface. The key evidence for this was the increase, after Sema3A treatment, of NRP1 co-localisation to membrane patches induced by the cross-linking of CTxB bound to the surface of live Xenopus retinal growth cones (Guirland et al., 2004). Indeed, using the same technique with mouse DRG growth cones, we also found that the amount of NRP1 fluorescence associated with CTxB-induced patches almost doubled after just 5 mins of Sema3A treatment (Figure 5A), suggesting that as much as 20% of the accessible NRP1 becomes raft-associated after Sema3A treatment. To determine whether L1 also becomes raft-associated in this response, as has been hypothesised (Carcea et al., 2010), we followed L1 localisation in CTxB-induced patches. In contrast to NRP1, the amount of L1 associated with CTxB did not change significantly, remaining similar to control levels even after Sema3A treatment (Figure 5A). To corroborate this result, we used cold detergent extraction to isolate membrane rafts in situ (Guirland et al., 2004). In untreated or mock-treated growth cones, little or no NRP1 remained in growth cones after extraction, whereas substantial amounts became TX100-resistant as early as 5 mins after Sema3A treatment (Figure 5B). Similarly low levels of L1 were found in TX100-extracted control growth cones, but by contrast these did not change after Sema3A treatment (Figure 5B). Thus, whereas a significant amount of NRP1 becomes associated with CTxB-induced membrane patches after Sema3A treatment in spinal sensory neurons, consistent with previous studies, L1 does not, nor does it become enriched in TX100-resistant membranes. This latter result is consistent with L1 being endocytosed via a non-raft associated pathway rather than via RME.
The association of NRP1 with CTxB-induced patches at the cell surface seems at odds with our internalisation data. However, it is important to note that more than 50% of cell surface NRP1 has already internalised (Figure 2A; Piper et al 2005) by the 5 min timepoint at which the CTxB patching experiments were performed, suggesting that the raft-associated NRP1 at the cell surface may represent a separate pool. Moreover, when we assessed whether the co-patching technique exclusively monitors cell surface CTxB, in fact we found that a substantial proportion of the CTxB detected was internalised (not shown). This is perhaps not surprising as the technique involves incubating the living cells at 12°C for 1 hour after the Sema3A treatment to allow CTxB patching to occur (Guirland et al., 2004). This suggests that the pool of cell surface CTxB-associated NRP1 may be even smaller than these experiments suggest. Consistently, as for the association of NRP1 with internalised CTxB, co-patching of NRP1 with CTxB was also TAG1 dependent (Figure 5A), as was the enrichment of NRP1 in TX100-resistant membranes (Figure 5B). Thus, either there are two pools of CTxB-associated NRP1, or these experiments in fact identify internalised NRP1 that has already started to become CTxB associated via the route described above.
The trafficking of NRP1 away from L1 after Sema3A treatment raises the important question of how this is involved in Sema3A signalling. Although NRP1 is considered to be the key ligand-binding component of the Sema3A holoreceptor complex, PlexinA4 is thought to be the main signal-generating component and is essential for Sema3A responses in DRG neurons (Suto et al 2005; Yaron et al., 2005). Critical questions, therefore, are whether PlexinA4 is trafficked similarly to NRP1 or to L1, whether its trafficking is also TAG1 dependent and therefore whether changes in PlexinA4-generated signalling can account for the loss of Sema3A-induced repulsion seen in TAG1ko growth cones (Law et al., 2008). To address these questions, we followed the localisations of NRP1 and PlexinA4 relative to each other, and to other complex components, on growth cones fixed and permeabilised at different time points after Sema3A treatment. As shown in Figure 6A, as might be expected, the amount of NRP1 that is co-localised in growth cones with PlexinA4 in control conditions is relatively high (34% of detectable NRP1), confirming that these molecules form pre-existing complexes (Takahashi et al., 1999). After addition of Sema3A, the association of NRP1 with PlexinA4 increases so that ~60% of total NRP1 is associated with PlexinA4 by 10 mins post-treatment, rising to nearly 70% after 20 mins. This is in distinct contrast to the co-localisation of NRP1 with L1, which falls significantly over the same period, so that by 20 mins post-treatment less than 8% of NRP1 is associated with L1 (Figure 4A). This indicates that PlexinA4 segregates with NRP1 rather than L1 after Sema3A treatment. Consistent with PlexinA4 being trafficked with NRP1, we found that the Sema3A-induced co-localisation of NRP1 with PlexinA4 does not occur in TAG1ko growth cones (Figure 6A) and that in wild type growth cones there is a significant increase in the proportion of TAG1 protein that is co-localised with PlexinA4, as well as with NRP1 (Figure 6B).
Finally, to determine whether TAG1 is required for the initiation of signals by PlexinA4, we monitored the phosphorylation of Collapsin response mediator protein 2 (CRMP2) using an antibody which recognises the phosphorylation of CRMP2 at Ser522, a known readout of Sema3A signalling (Uchida et al., 2005); CRMPs are thought to interact indirectly with the C-terminal region of PlexinAs through direct interactions with MICALs (molecules interacting with CasL) which bind to PlexinAs (Schmidt et al., 2008). Consistent with previous observations (Uchida et al., 2005), in wild type growth cones we saw a significant increase in the absolute levels of phospho-CRMP (pCRMP) staining 10 mins after Sema3A treatment (Figure 6C), coincident with the separation of NRP1 from L1 and its transition into CTxB-enriched intracellular vesicles (Figure 3B). By contrast, this increase does not occur after Sema3A treatment of TAG1ko growth cones (Figure 6C). Together, these data indicate that TAG1-dependent trafficking of NRP1 also affects the trafficking and signalling of PlexinA4, thus accounting for the loss of collapse seen in growth cones lacking TAG1 (Law et al., 2008).
We showed that TAG1 is a critical component of the Sema3A holoreceptor in DRG neurons, required for differential trafficking of receptor components into specific intracellular pathways and essential for signal generation. TAG1 interacts with NRP1 directly and renders the inclusion of L1 in the holoreceptor Sema3A-sensitive: Sema3A binding induces NRP1 and L1 to endocytose together, but intracellularly NRP1 is routed away from L1 into vesicles enriched in CTxB binding sites (Figure 7A) and its association with PlexinA4, the signalling component of the receptor, increases. Concurrently, CRMP2 phosphorylation, an indicator of PlexinA signalling, increases and growth cone collapse is initiated. However, when TAG1 is absent, although NRP1 is still endocytosed with L1, it fails to segregate into CTxB-enriched vesicles appearing instead to be recycled to the cell surface (Figure 7B); increased association with PlexinA4 does not occur, CRMP2 phosphorylation does not increase and collapse is attenuated. These observations indicate that TAG1 plays a critical role in the trafficking of semaphorin receptors into specific endocytic pathways and is required to activate PlexinA signalling. Together with previous observations (Castellani et al., 2004, Law et al., 2008), this suggests that adhesion molecule interactions at the cell surface can modulate responses to diffusible signals by altering the endocytic fate of their receptors.
Endocytosis is a key component of many signalling pathways and the endocytic pathway taken by receptors is recognised to affect signalling outcome (Sorkin and von Zastrow, 2009). How pathway selection is controlled remains poorly characterised, especially in neurons where, given their size and complexity, endocytic sorting is crucially important (Von Bartheld and Altick, 2011). Responses mediated by semaphorin-binding neuropilins are known to vary by cell type (Salikhova et al., 2008; Carcea et al., 2010) and in dendrites versus axons (Polleux et al., 2000; Shelly et al., 2011). Responses vary even among neurons expressing similar receptor components (Carcea et al., 2010), but the molecular basis for this is not clear. A key difference between Sema3A responsive and non-responsive sensory afferents is the expression of the adhesion molecule TAG1 (Law et al., 2008). Here we showed that the GPI-linked protein TAG1 is required to segregate NRP1 from L1 and traffic it to an endosomal subpopulation enriched in lipid rafts, as defined by CTxB binding. GPI-linked proteins are delivered via clathrin-independent endocytosis into GEECs (GPI-enriched endosomal compartments) in a GPI-linkage-dependent manner (Sabheranjak et al., 2002; Mayor and Pagano, 2007). Although the GPI-linked protein Cripto has been shown to localise the precursor of the secreted ligand Nodal to lipid rafts for processing (Blanchet et al., 2008), we believe this is the first demonstration of an endogenous GPI-linked protein driving sorting of a membrane receptor into raft-enriched intracellular membranes. Moreover, in the absence of TAG1-mediated segregation of NRP1, downstream signalling by Plexin (CRMP2 phosphorylation; Goshima et al., 1995) does not occur and collapse is attenuated. Although it has been assumed that the segregation of membrane proteins into lipid rafts occurs at the cell surface (Mayor and Pagano, 2007), our results indicate that this can occur intracellularly, consistent with evidence for the existence of endosomal subpopulations specialised for cell signalling (Zoncu et al., 2009).
These and other results suggest that Sema3A binding generates signals at, at least three different points in the NRP1 trafficking pathway: Sema3A raises intracellular cGMP and Ca2+ levels within 2 mins of treatment, which is required for growth cone repulsion (Togashi et al., 2008). Although Ca2+-induced repulsion requires clathrin-dependent endocytosis, Sema3A-induced Ca2+ fluxes are endocytosis-independent (Tojima et al., 2010), indicating that signalling is initiated at the cell surface (Figure 7A). Endocytosis of NRP1 is dependent on its association with either L1 or PlexinA (Castellani et al., 2004) and L1 is essential for the activation of Focal Adhesion Kinase (FAK), which is required for growth cone collapse and disassembly of focal adhesions (Bechara et al., 2008). FAK is only co-precipitated with L1 when NRP1 is present and this association is enhanced by Sema3A treatment. However, FAK does not associate with PlexinAs and, although PlexinA inhibition prevents collapse in response to Sema3A, focal adhesions still disassemble, indicating PlexinAs and FAK activate different aspects of the response (Bechara et al., 2008; Barberis et al., 2004). The initial increase in L1/NRP1 association followed by separation and subsequent increase in NRP1/PlexinA4 association fits with these observations and suggest a model in which the activation of FAK and Plexin are spatially and temporally separated (Figure 7A). The endocytic route taken by L1 after Sema3A treatment appears qualitatively different from that followed during steady-state recycling: after Sema3A, L1 disappears from the cell surface and appears in large intracellular aggregates not seen in the steady-state.
The increase of NRP1 association with endocytosed L1 within 5 mins of Sema3A treatment is not dependent on TAG1. This appears to conflict with the requirement for TAG1 in the increase in anti-L1 Fab uptake that occurs 10 mins after Sema3A addition. However, it is possible this late increase in L1 endocytosis reflects the general increase in endocytosis that accompanies collapse (Fournier et al., 2000), which only becomes evident around 10 mins. Thus, TAG1 may be required in the initiation of collapse rather than for L1 endocytosis per se. CRMP2, through its ability to bind Numb, is known to regulate L1 endocytosis (Nishimura et al., 2003), raising the possibility that L1 uptake is stimulated by feedback from CRMP2 during Sema3A signalling. Enhanced L1 endocytosis in turn may reflect the general disassembly of focal adhesions that accompanies collapse (Bechara et al., 2008).
The endocytic route that mediates Sema3A-induced responses remains controversial. Although Sema3A signalling can be blocked by agents that disrupt lipid raft integrity (Guirland et al 2004; Salikhova et al., 2008; Carcea et al., 2010), an immediate early endocytic response to Sema3A is blocked by dominant-negative mutant endocytic proteins which disrupt CME (Tojima et al., 2010). Although our results are consistent with NRP1 initially entering the cell via CME rather than RME, we cannot rule out that a non-CME pathway such as macropinocytosis is used (Fournier et al., 2000). Since we show that endocytosed NRP1 dissociates from L1 and becomes associated with CTxB-binding membranes, raft-disrupting reagents may inhibit Sema3A signalling (Guirland et al., 2004; Salikhova et al., 2008; Carcea et al., 2010) because they interfere with this latter intracellular step. Nonetheless, RME-inhibiting reagents apparently block Sema-induced endocytosis of Sema3A or NRP1 in some cortical neurons (Carcea et al., 2010) or endothelial cells (Salikhova et al., 2008), whereas CME inhibitors do not. It is possible that different cell types traffic NRP1 differently. However, raft-disrupting agents may appear to block Sema3A-induced endocytosis because they prevent the intracellular diversion of NRP1 into CTxB-binding vesicles and instead receptors are recycled to the surface, leaving net surface levels unaffected (Figure 7B).
We clearly show that NRP1 traffics into CTxB-binding membranes intracellularly. However, we cannot rule out that NRP1 also becomes associated with rafts at the cell surface. Sema3A treatment of retinal neurons results in rapid NRP1 disappearance from the cell surface, coincident with desensitization, followed by reappearance and resensitization (Piper et al., 2005). This resensitization does not occur if protein synthesis is blocked, even though surface NRP1 levels are restored by recycling. One possibility is that cell surface raft-associated NRP1 represents the pool of recycled, but not reactivated receptor, consistent with our observation that L1 does not become raft-associated after Sema3A treatment. The possibility that intact rafts may be required for NRP1 recycling is consistent with our observation that filipin treatment leads to disappearance of NRP1 from the surface of DRG growth cones in the steady-state (Figure 3A, Figure 7C,D), whereas L1 surface levels are not affected (not shown).
In other cell types, the switch between clathrin-dependent and raft-dependent pathways may nonetheless occur at the cell surface and be regulated differently. For example, in endothelial cells the NRP1 endocytic pathway is apparently determined by the ligand: binding of VEGF leads to CME, whereas binding of Sema3C leads to RME (Salikhova et al., 2008). This may explain why the TAG1 knockout only partially mimics the Sema3A and NRP1 knockout phenotypes (Law et al., 2008); TAG1 may only be required for a subset of Sema responses.
In DRG, however, TAG1 expression clearly correlates with Sema3A responsiveness. Initially, both presumptive proprioceptive and nociceptive axons are repelled by Sema3A (Messersmith et al., 1995; Fu et al., 2000; Pond et al., 2002) and express L1 and TAG1 (Perrin et al., 2001; Law et al., 2008). However, proprioceptive axons lose this sensitivity coincident with TAG1 downregulation and project into the spinal cord (Fu et al., 2000; Pond et al., 2002; Law et al., 2008). TAG1 loss leads to premature nociceptive entry into the dorsal horn (Law et al., 2008), mimicking the effects of loss of NRP1 (Gu et al., 2003). Thus, in DRG neurons TAG1 determines the outcome of Sema3A signalling by altering the intracellular trafficking of the core receptor components. That such a role is assumed by an adhesion molecule suggests this is a mechanism by which growth cones modulate their responses to guidance cues according to cellular context, an idea strongly supported by the observation that cross-linking of the receptor complex with L1 inhibits Sema3A-induced growth cone collapse (Castellani et al 2000; Castellani et al., 2002).
We thank D. Felsenfeld, Y. Goshima, T. Jessell, S. Kenwrick, A. Kolodkin, V. Lemmon, H. Fujisawa, A. Puschel, F. Rathjen, L. Reichardt, M. Tessier-Lavigne and A. Yaron for reagents. This work was supported by a University of Sheffield studentship and a Wellcome Trust VIP Award to P.D. and grants to A.J.W.F from the BBSRC. Thanks to the University of Sheffield Light Microscopy Facility (Wellcome grant GR077544AIA) and to M. Placzek for microscopy facilities.