Title: FAK–MAPK-dependent adhesion disassembly downstream of L1 contributes to semaphorin3A-induced collapse
Abstract: Article8 May 2008free access FAK–MAPK-dependent adhesion disassembly downstream of L1 contributes to semaphorin3A-induced collapse Ahmad Bechara Ahmad Bechara Université de Lyon, Centre de Génétique Moléculaire et Cellulaire (CGMC) UMR CNRS 5534, Villeurbanne, France Search for more papers by this author Homaira Nawabi Homaira Nawabi Université de Lyon, Centre de Génétique Moléculaire et Cellulaire (CGMC) UMR CNRS 5534, Villeurbanne, France Search for more papers by this author Frédéric Moret Frédéric Moret Université de Lyon, Centre de Génétique Moléculaire et Cellulaire (CGMC) UMR CNRS 5534, Villeurbanne, France Search for more papers by this author Avraham Yaron Avraham Yaron University of San Francisco, Genentech Inc., San Francisco, CA, USA Search for more papers by this author Eli Weaver Eli Weaver The Miami Project to Cure Paralysis, University of Miami, Miami, FL, USA Search for more papers by this author Muriel Bozon Muriel Bozon Université de Lyon, Centre de Génétique Moléculaire et Cellulaire (CGMC) UMR CNRS 5534, Villeurbanne, France Search for more papers by this author Karima Abouzid Karima Abouzid Université de Lyon, Centre de Génétique Moléculaire et Cellulaire (CGMC) UMR CNRS 5534, Villeurbanne, France Search for more papers by this author Jun-Lin Guan Jun-Lin Guan Cornell University, Ithaca, NY, USA Search for more papers by this author Marc Tessier-Lavigne Marc Tessier-Lavigne University of San Francisco, Genentech Inc., San Francisco, CA, USA Search for more papers by this author Vance Lemmon Vance Lemmon The Miami Project to Cure Paralysis, University of Miami, Miami, FL, USA Search for more papers by this author Valérie Castellani Corresponding Author Valérie Castellani Université de Lyon, Centre de Génétique Moléculaire et Cellulaire (CGMC) UMR CNRS 5534, Villeurbanne, France Search for more papers by this author Ahmad Bechara Ahmad Bechara Université de Lyon, Centre de Génétique Moléculaire et Cellulaire (CGMC) UMR CNRS 5534, Villeurbanne, France Search for more papers by this author Homaira Nawabi Homaira Nawabi Université de Lyon, Centre de Génétique Moléculaire et Cellulaire (CGMC) UMR CNRS 5534, Villeurbanne, France Search for more papers by this author Frédéric Moret Frédéric Moret Université de Lyon, Centre de Génétique Moléculaire et Cellulaire (CGMC) UMR CNRS 5534, Villeurbanne, France Search for more papers by this author Avraham Yaron Avraham Yaron University of San Francisco, Genentech Inc., San Francisco, CA, USA Search for more papers by this author Eli Weaver Eli Weaver The Miami Project to Cure Paralysis, University of Miami, Miami, FL, USA Search for more papers by this author Muriel Bozon Muriel Bozon Université de Lyon, Centre de Génétique Moléculaire et Cellulaire (CGMC) UMR CNRS 5534, Villeurbanne, France Search for more papers by this author Karima Abouzid Karima Abouzid Université de Lyon, Centre de Génétique Moléculaire et Cellulaire (CGMC) UMR CNRS 5534, Villeurbanne, France Search for more papers by this author Jun-Lin Guan Jun-Lin Guan Cornell University, Ithaca, NY, USA Search for more papers by this author Marc Tessier-Lavigne Marc Tessier-Lavigne University of San Francisco, Genentech Inc., San Francisco, CA, USA Search for more papers by this author Vance Lemmon Vance Lemmon The Miami Project to Cure Paralysis, University of Miami, Miami, FL, USA Search for more papers by this author Valérie Castellani Corresponding Author Valérie Castellani Université de Lyon, Centre de Génétique Moléculaire et Cellulaire (CGMC) UMR CNRS 5534, Villeurbanne, France Search for more papers by this author Author Information Ahmad Bechara1, Homaira Nawabi1, Frédéric Moret1, Avraham Yaron2, Eli Weaver3, Muriel Bozon1, Karima Abouzid1, Jun-Lin Guan4, Marc Tessier-Lavigne2, Vance Lemmon3 and Valérie Castellani 1 1Université de Lyon, Centre de Génétique Moléculaire et Cellulaire (CGMC) UMR CNRS 5534, Villeurbanne, France 2University of San Francisco, Genentech Inc., San Francisco, CA, USA 3The Miami Project to Cure Paralysis, University of Miami, Miami, FL, USA 4Cornell University, Ithaca, NY, USA *Corresponding author. University of Lyon, CGMC-UMR-CNRS 5534, Universite Claude Bernard Lyon1, 43 bd 11 Novembre, Villeurbanne 69622, France. Tel.: +33 0472 43 26 91; Fax: +33 0472 43 26 85; E-mail: [email protected] The EMBO Journal (2008)27:1549-1562https://doi.org/10.1038/emboj.2008.86 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Axonal receptors for class 3 semaphorins (Sema3s) are heterocomplexes of neuropilins (Nrps) and Plexin-As signalling coreceptors. In the developing cerebral cortex, the Ig superfamily cell adhesion molecule L1 associates with Nrp1. Intriguingly, the genetic removal of L1 blocks axon responses of cortical neurons to Sema3A in vitro despite the expression of Plexin-As in the cortex, suggesting either that L1 substitutes for Plexin-As or that L1 and Plexin-A are both required and mediate distinct roles. We report that association of Nrp1 with L1 but not Plexin-As mediates the recruitment and activation of a Sema3A-induced focal adhesion kinase–mitogen-activated protein kinase cascade. This signalling downstream of L1 is needed for the disassembly of adherent points formed in growth cones and subsequently their collapse response to Sema3A. Plexin-As and L1 are coexpressed and present in common complexes in cortical neurons and both dominant-negative forms of Plexin-A and L1 impair their response to Sema3A. Consistently, Nrp1-expressing cortical projections are defective in mice lacking Plexin-A3, Plexin-A4 or L1. This reveals that specific signalling activities downstream of L1 and Plexin-As cooperate for mediating the axon guidance effects of Sema3A. Introduction Class 3 semaphorins (Sema3s) are secreted guidance cues whose repulsive activity restricts the trajectory of a great number of axon subsets to prevent their aberrant growth during the development of neuronal connectivity (He et al, 2002). Axonal receptors for Sema3s comprise binding subunits, the Nrps, complexed with signalling co-receptors, the Plexin-As, which mediate the guidance activity of Sema3s by activating various intracellular pathways, including RhoGTPases, CRMPs, mitogen-activated protein kinase (MAPK), PI3K and Src kinase families (Kruger et al, 2005). Ig superfamily cell adhesion molecules (IgSFCAMs) have also been found to contribute to Sema3 responses, but their function has remained elusive so far (Castellani and Rougon, 2002; Falk et al, 2005). The IgSFCAM L1 associates with neuropilin (Nrp)1 and was proposed to be a component of the Sema3A receptor (Castellani et al, 2000; Castellani, 2002; Itoh et al, 2004). Similarly, another member of the L1-CAM subgroup, NrCAM, was found to associate with Nrp2 and to be required for growth cone responses to Sema3B and Sema3F in vitro (Falk et al, 2005). In both cases, the interaction is mediated by the ectodomain of IgSFCAMs and Nrp proteins. During the development of cortical connectivity, Sema3A is thought to guide axons at several levels, from their initial growth out of the cortical plate of the cerebral cortex, through the corpus callosum and during their navigation to subcortical targets in the internal capsule and at the entry of the spinal cord (Bagnard et al, 1998; Polleux et al, 1998; Castellani et al, 2000; Gu et al, 2003). Interestingly, the genetic removal of L1 also disturbs these cortical pathways, causing midline crossing defects and size reduction of the corpus callosum (Dahme et al, 2007; Cohen et al, 1998; Demyanenko et al, 1999; Castellani et al, 2000). Disorganization of fibres in the internal capsule and defective pyramidal decussation have also been reported in L1−/y mice (Dahme et al, 2007; Ohyama et al, 2004). The contribution of Plexin-As to the guidance effects of Sema3A on cortical axons has not yet been investigated, but all plexin-A members are detected at the mRNA level in the developing cerebral cortex over the period of cortical neuron migration and axon extension (Murakami et al, 2001). Intriguingly, experiments conducted with explants from mice lacking L1 showed that cortical axons are unresponsive to Sema3A, indicating that Plexin-As cannot compensate for the lack of L1 in vitro (Castellani et al, 2000). These data lead to the question of the precise role of L1 in the Sema3A receptor and why Plexin-As cannot compensate their loss. Four Plexin-A members have been identified and most of them are able to confer a morphological retraction to COS cells exposed to Sema3A when coexpressed with Nrp1, although the ligand binding affinity differs between the complexes (Togashi et al, 2006). Co-culture assays with DRG explants from mice lacking Plexin-A3 and Plexin-A4 have shown that Sema3A-responsive axons primarily utilize Plexin-A4 for their repulsive behaviour to Sema3A (Suto et al, 2005; Yaron et al, 2005). However, cooperative effects exist between different Plexin-As as some DRG axons from Plexin-A4−/− mice only exhibited reduced sensitivity to Sema3A but were fully desensitized by additional genetic removal of Plexin-A3 (Yaron et al, 2005). In the context of the cerebral cortex, L1 and Plexin-A subunits could both be required in a common receptor to perform distinct and/or redundant roles. Alternatively, L1 could mediate Sema3A signalling and thus substitute to Plexin-As in these neurons. Finally, L1 and Plexin-As could also segregate in subpopulations of cortical axons to form distinct receptor complexes. The goal of this study was to address these possibilities to gain insights into the role of L1 in the response of cortical neurons to Sema3A. We report that interaction of L1 but not Plexin-As with Nrp1 specifically mediates the activation of a focal adhesion kinase (FAK)–mitogen-activated protein kinase (MAPK) signalling pathway controlling a crucial process of the repulsive behaviour, the disassembly of adhesion points of the growth cones. We provide evidence from biochemical experiments, culture assays and analysis of mutant mice lacking Plexin-A3 that Plexin-A4 and L1 are coexpressed and initiate distinct signalling cascades that cooperate for mediating the growth cone responses to Sema3A. These data assign a specific function to L1 in Sema3A signalling during axon guidance and elucidate the lack of functional compensation for L1 by Plexin-A proteins in L1-deficient mice. Results FAK is required for the growth cone collapse response to Sema3A The first issue was to determine whether L1 shows signalling activity in the Sema3A receptor. The FAK acts downstream of integrin activation by extracellular matrix components and has a major role in the dynamics of focal adhesions (Mitra et al, 2005). Interestingly, FAK also participates in the signalling of guidance cues including Sema3B (Miao et al, 2000; Carter et al, 2002; Li et al, 2004; Liu et al, 2004; Ren et al, 2004; Falk et al, 2005) although its recruitment to Plexins has not been reported (Barberis et al, 2004). We investigated whether FAK participates in Sema3A signalling in cortical neurons and if so if it could be recruited by L1. Immunocytochemistry performed on E15 embryonic brain sections showed that L1, Nrp1 and FAK are expressed by post-mitotic neurons in the cortical plate of the developing cerebral cortex, consistent with some previous observations (Contestabile et al, 2003), and distributed over the cell body layer and the intermediate zone containing cortical efferents (Figure 1A). FAK and pFAK could also be detected in cultured cortical neurons (Figure 1A and data not shown). To address whether FAK mediates Sema3A signaling, different FAK variants were electroporated into cortical neurons. These included variants with a kinase-dead domain (FAKΔkin), a mutation on the Y397, which is the autophosphorylation site of FAK (FAKY397), deletion of the FERM domain rendering FAK constitutively active (FAKΔFERM; Cohen and Guan, 2005; Mitra et al, 2005) or wild-type FAK (FAKWT) as control. Collapse assays were performed on electroporated cortical neurons (Figure 1B, a total of 80 growth cones assessed per condition from 12 neonatal brains). Overexpression of FAKWT did not interfere with the collapse response observed for wild-type neurons. FAKΔFERM significantly increased the basal level of collapse observed under control conditions. Nevertheless, Sema3A application further increased the level of collapsed growth cones (Figure 1B). In contrast, FAKΔkin and FAKY397 forms had no detectable effects under control treatment but totally abrogated the collapse normally exerted by Sema3A (Figure 1B). Thus, FAK is required for Sema3A to collapse neuronal growth cones. Figure 1.FAK contributes to Sema3A-induced growth cone collapse. (A) FAK, L1 and Nrp1 are expressed by neurons residing in the cortical plate and detected in the intermediate zone where their efferents navigate to exit the cortex. Scale bar: 100 μm. Axonal localization of pFAK, L1 and Nrp1 in cultured cortical neurons is shown. Scale bar: 50 μm. (B) Microphotographs illustrating neurons labelled with antibodies to HA-tagged overexpressed FAK and magnification of the growth cone morphology under control and Sema3A-treated conditions. Overexpression of FAKY397 and FAKkinΔ but not FAKWT and FAKΔFERM prevents the growth cone collapse. Western blot shows the expression of FAK proteins from the plasmids used in electroporation. The histogram shows the percentage of growth cone collapse under control and Sema3A-treated conditions showing that the response is abolished by FAKY397 and FAKkinΔ. (C) Western blot showing FAK precipitation with Nrp1 and L1 from the purified membrane fraction of neonatal mouse cortex. Sema3A stimulation of fresh cortical tissue induces Y397 FAK phosphorylation. lpIP: lysate post immunoprecipitation; NS, nonsignificant; *P<0.001, χ2 test. Download figure Download PowerPoint FAK recruitment to the Sema3A receptor is mediated by L1/Nrp1 complex formation and is ligand-induced Next, we investigated the mechanisms underlying the contribution of FAK to Sema3A effects. First, to assess whether FAK is recruited to the Sema3A receptor, neonatal cortical tissue was collected and then processed for membrane purification and precipitation experiments with antibodies to Nrp1. Western blot analysis showed that FAK co-precipitates with Nrp1 and L1 from this tissue (Figure 1C). To examine FAK activation, FAK was precipitated from control and Sema3A-stimulated fresh cortical tissue. Western blot analysis showed that Sema3A induced Y397 phosphorylation of FAK (Figure 1C). Second, to search for the partners of the Sema3A receptor that mediate the recruitment of FAK, 293 cells were transfected to express various combinations of receptor subunits (Figure 2A and D). The signalling properties of the L1/Nrp1 complex were compared with that of the prototypal Sema3A receptor Plexin-A1/Nrp1 (Toyofuku et al, 2005; Togashi et al, 2006). Interestingly, FAK co-precipitated with L1 from L1/Nrp1-expressing cells but not with Plexin-A1 from Nrp1/Plexin-A1-expressing cells. Likewise, when coexpressed with Nrp1, L1/FAK association could be observed by precipitation with antibodies to FAK and L1, whereas antibody to neither FAK nor to the VSV tag of Plexin-A1 could allow the detection of FAK/Plexin-A1 interaction (Figure 2A and B). This was not due to deficiency of Plexin-A1 construct, as expression of this construct with Nrp1 in COS7 cells conferred upon them a collapse response to Sema3A (data not shown). Figure 2.L1/Nrp1 mediates Sema3A-induced recruitment and activation of FAK. (A, B) FAK precipitates with L1 from L1/Nrp1-expressing 293 cells but not with Plexin-A1VSV (plexVSV) from Plexin-A1/Nrp1-expressing 293 cells. Sema3A treatment (3A) stimulates L1/FAK co-precipitation. The L1/FAK complex could be detected in the control condition by precipitation with anti-L1 but not anti-FAK antibodies. (C, D) FAK does not precipitate with Nrp1, plexVSV or L1 when expressed alone whereas it precipitates with L1 from cells expressing L1 complexed with Nrp1 truncated from its cytoplasmic domain (Nrp1Δcyt). (E) Sema3A induces FAK autophosphorylation detected with anti-py397 in cells expressing L1/Nrp1 but not Nrp1/PlexVSV or PlexVSV. None of the Plexin-A2-A3-A4/Nrp1 complexes mediate FAK phosphorylation upon Sema3A stimulation. (F) Sema3A stimulates FAK/Src association in L1/Nrp1-expressing cells. (G) Precipitation of L1 in Sema3A-stimulated HEK cells expressing L1, Nrp1 and FAK forms for detection of FAK. FAKΔkin, FAKY397, FAKWT but not FAKΔFERM co-precipitated with L1. The photographs show bands at 125 kDa for FAKΔkin, FAKY397 and FAKWT and at 70 kDa for FAKΔFERM. (H) Precipitation of FAK in Sema3A-stimulated HEK cells expressing various truncated L1 forms lacking one or several functional domains of L1 and Nrp1 for the detection of L1. L11176, L11180, L1ΔRSLE but not L11146 co-precipitated with FAK. ERM: binding site to ERM proteins; AP-A: binding site for AP-2/chlatrin-dependent endocytosis; ANK: binding site to ankyrin. Download figure Download PowerPoint These data suggest that FAK associates with L1. An alternative is that FAK is recruited by Nrp1, but only in an L1/Nrp1 complex and not when Nrp1 is complexed with Plexin-A1. This latter possibility was invalidated by the finding that an Nrp1/FAK interaction could not be detected in cells expressing only Nrp1 or Plexin-A1 (Figure 2C). Furthermore, co-precipitation of L1 with FAK from cells expressing a complex of L1 and a cytoplasmic deleted form of Nrp1 could be detected, which demonstrates that L1 is the subunit that triggers the recruitment of FAK (Figure 2C). Notably, we also observed that FAK/L1 association was almost undetectable in cells expressing L1 alone, indicating that the recruitment of FAK is conditional, based on the formation of the L1/Nrp1 complex (Figure 2D). Moreover, Sema3A treatment increased the recruitment of FAK to the L1/Nrp1 complex, as shown by precipitation with antibodies to L1 and FAK (Figure 2A and B). L1/Nrp1 complex formation triggers Sema3A-induced FAK activation Although the recruitment of FAK to the Sema3A receptor was found to depend on L1 but not Plexin-A1, its activation could require Plexin-A in the complex or result from signalling events downstream of Plexin-A. The phosphorylation of FAK was assessed with an antibody that selectively recognizes the pY397. Phosphorylation at Y397 could be detected in Sema3A-treated L1/Nrp1-expressing cells but not in cells expressing Plexin-A1 alone or complexed with Nrp1 (Figure 2E). The recruitment of FAK upon L1/Nrp1 complex formation was not sufficient to induce strong activation, as pY397 FAK was only weakly detected in the untreated condition. As described above, cortical neurons express several Plexin-As other than Plexin-A1 that could share with L1 the capacity of recruiting and activating FAK. We thus examined this possibility by immunoprecipitation of FAK from Sema3A-treated-transfected HEK cells and found that none of the Nrp1/Plex-A2, Nrp1-Plex-A3 and Nrp1/Plex-A4 complexes mediated FAK recruitment and activation (Figure 2E and Supplementary Figure 1). Thus, the L1/Nrp1 but not the Plexin-As/Nrp1 complex could mediate FAK phosphorylation. FAKY397 autophosphorylation creates a binding site for src (Mitra et al, 2005). Consistently, we found in L1/Nrp1-expressing cells that Sema3A stimulates FAK/src co-precipitation (Figure 2F). To delineate the domains of FAK and L1 required for their association, HEK cells expressing L1/Nrp1 with FAKΔkin, FAKΔFERM, FAKY397 or FAKWT were stimulated with Sema3A and processed for precipitation with antibodies to L1 (Figure 2G). Western blot analysis showed that the removal of FAK kinase domain and the Y397 mutation did not affect FAK recruitment to L1, whereas deletion of the FERM domain totally abolished it (Figure 2G). Next, we tested L1 constructs having various cytoplasmic truncations removing key functional domains: L1-1146 truncated after the fourth cytoplasmic residue, L1-1176 lacking the binding domain for the cytoskeletal adaptor ankyrin and the AP-2/chlatryn-dependent internalization motif but still containing the binding site for ezrin–radixin–moeisin (ERM) proteins, L1-1180 lacking only the Ank domain and L1-RSLE lacking only the RSLE motif (Cheng et al, 2005). FAK was precipitated from Sema3A-stimulated L1/Nrp1-expressing cells for western blot detection of L1. We found that L1-1180 but not L1-1146 could recruit FAK. FAK/L1 co-precipitation was also detected with L1-1176 and L1-RSLE. Thus, FAK recruitment to L1 requires a sequence comprising 1146–1176 amino acids, including the ERM binding domain but excluding the RSLE and ankyrin binding domains (Figure 2H). Sema3A-induced FAK-dependent erk1/2 phosphorylation downstream of L1/Nrp1 complex FAK has complex roles during the turnover of adherent contacts. Recent work established that FAK initiates a signalling cascade leading to activation of the MAPK pathway, which controls the disassembly of adherent contacts (Webb et al, 2004). Interestingly, MAPK activation downstream of L1 also mediates some of the major functions of L1 in cell adhesion and axon growth (Kamiguchi and Lemmon, 2000). Moreover, MAPK activation is already linked to Sema3A-induced growth cone collapse, although the nature of the process affected by this signalling remains unclear (Campbell and Holt, 2003). As expected, we could detect erk1/2 phosphorylation in neonatal cortical tissues stimulated with Sema3A (Figure 3A). We thus examined whether Sema3A could induce an MAPK signalling in L1/Nrp1-expressing cells. Stimulated cells were processed for immunoprecipitation and western blot analysis. Sema3A could trigger erk phosphorylation in cells expressing L1/Nrp1 but not Nrp1 alone (Figure 3A). To determine whether FAK signalling is required for MAPK activation, cells were first transfected with L1, Nrp1 and either egfp or dominant-negative FAKΔkin. We found that FAKΔkin but not egfp coexpression abolished Sema3A-induced erk1/2 phosphorylation (Figure 3B). Second, cells were transfected with Nrp1 and L1−1146 mutant lacking FAK recruitment and in this condition Sema3A-induced erk phosphorylation was also abrogated (Figure 3C). Next, we examined whether constitutively active FAK although uncoupled to L1 is sufficient for inducing erk phosphorylation. Cells were transfected with L1, Nrp1 and either FAKWT or FAKΔFERM. Western blot analysis showed that erk phosphorylation was still dependent on stimulation by Sema3A (Figure 3D). This ligand-dependent erk phosphorylation was not due to the recruitment of endogenous FAK to L1, as FAKΔFERM overexpression resulted in loss of association of L1 with both FAKΔFERM and endogenous FAK (Figures 2F and 3E). Thus, Sema3A-induced FAK recruitment and activation by L1 can be overcome by uncoupled active FAK, which is nevertheless insufficient for erk phosphorylation. Additional L1-dependent signalling induced by Sema3A might also occur to trigger erk phosphorylation. Consistent with this hypothesis, Sema3A-induced erk phosphorylation was lost in cells expressing Nrp1, FAKΔFERM and the L1−1146 mutant lacking almost all the cytoplasmic domain (Figure 3F). Figure 3.L1/Nrp1 mediates Sema3A-induced erk phosphorylation. (A) Stimulation by Sema3A of fresh cortical tissue induces erk1/2 phosphorylation, which is reproduced in L1/Nrp1- but not Nrp1-expressing HEK cells. (B) Introduction of dominant-negative FAKΔkin abrogates Sema3A-induced erk1/2 phosphorylation in L1/Nrp1-expressing cells. (C) Sema3A-induced erk phosphorylation is lost in cells expressing Nrp1 and L1−1146 mutant lacking FAK recruitment. (D) Sema3A still triggers erk phosphorylation in cells overexpressing constitutively active FAK (FAKΔFERM) even though this construct blocks the recruitment of endogenous FAK. (E) The blots show bands at 125 kDa (endogenous FAK, FAKWT, FAKΔkin) and at 70 kDa (FAKΔFERM). (F) Sema3A triggers erk phosphorylation in cells expressing L1WT/FAKWT and L1WT/FAKΔFERM but not L1−1146/FAKΔFERM. (G) Introduction of dominant-negative Plex-GPI does not abrogate Sema3A-induced erk1/2 phosphorylation in L1/Nrp1-expressing cells. Download figure Download PowerPoint Finally, we examined whether the cascade downstream of L1 requires Plexin-As signalling because HEK cells express endogenous Plexin-As. However, erk1/2 phosphorylation was still detected in HEK cells transfected with L1/Nrp1 and dominant-negative truncated form of Plexin-A1 in which the cytoplasmic domain has been removed and replaced by a GPI anchor (Plex-A1GPI; Toyofuku et al, 2004; Figure 3G). L1-mediated FAK and MAPK activation is required for Sema3A-induced growth cone collapse To address whether L1-mediated MAPK pathway is required for cortical responses to Sema3A, we disturbed this activation. Two L1 serine residues, mutated in the L1-related human neurodevelopmental disease, have been found to alter the ability of L1 to trigger erk1/2 phosphorylation (Needham et al, 2001). The pathological substitution of the serines S1194 and S1224 by leucine prevented neither L1 cell surface expression nor interaction with Nrp1 (Supplementary Figure 1D). When coexpressed with Nrp1 in HEK cells, these mutated L1 constructs still mediated Sema3A-induced autophosphorylation of FAK. In contrast, erk1/2 phosphorylation was totally lost (Figure 4A). L1WT, L1−1146, L1S1194L and L1S1224L IRES EGFP vectors were overexpressed in cortical neurons in collapse assays. Overexpression of L1−1146 but not L1WT totally blocked Sema3A-induced growth cone collapse (Figure 4B, a total of 320 cones from 12 neonatal brains). Similarly, the L1 serine mutations partially (for L1S1224L) and totally (for L1S1194L) abrogated Sema3A-induced growth cone collapse (Figure 4B, a total of 383 cones from 12 neonatal brains). To confirm that L1 signalling is required for the guidance of cortical axons, we took advantage of the previously developed slice overlay assay, assessing axon trajectory of dissociated cortical neurons grown on top of cortical slices. In normal conditions, about 65% of the axons are directed towards the white matter by Sema3A repulsive activity emanating from the pial side (Polleux et al, 1998). Using this assay, we found that overexpression of FAKΔkin and L1S1194L profoundly disorganized axon trajectory, as only 21 and 27% of axons were properly guided towards the white matter side, respectively (Supplementary Figure 2). Figure 4.L1 mutants interfere with Sema3A-induced erk phosphorylation, release of Paxillin/L1 interaction and collapse response. (A) The L1 serine mutations 1194 and 1224 abolished the Sema3A-induced erk1/2 phosphorylation in L1/Nrp1-expressing cells. Y397 FAK phosphorylation is still detected in cells expressing complexes of Nrp1 and L1S1194L and L1S1224L. (B) Collapse assays with cortical neurons overexpressing the L1 variants L1S1224L, L1S1194L and L1−1146. Microphotographs illustrate the loss of collapse response due to these overexpressions. Histograms depict the percentage of growth cone collapse showing defective responsiveness of neurons expressing L1S1224L, L1S1194L, L1−1146 but not L1WT. *P<0.001 with χ2 test. Scale bar: 25 μm. (C) Co-precipitation of L1 with Paxillin but not talin and vinculin, indicating that L1 complexes in the cerebral cortex contain paxillin. lpIP: lysate post immunoprecipitation. Paxillin/L1 co-precipitation is decreased by Sema3A. (D) Sema3A induces Paxillin release from L1 in L1/Nrp1-expressing cells. (E) L1/Nrp1 mediates Sema3A-induced paxillin phosphorylation at Y31. (F) Sema3A-induced release of Paxillin/L1 interaction is not prevented by the application of the MEK1 inhibitor U0126. Download figure Download PowerPoint L1-mediated FAK–MAPK signalling controls the disassembly of paxillin+ adhesion points induced by Sema3A The nature of the process regulated by this L1-dependent FAK–MAPK activation found required for Sema3A-induced growth cone collapse remained undetermined. The FAK–MAPK cascade was shown to control focal adhesion disassembly in migrating cells (Webb et al, 2004). Moreover, other recent work described that Sema3A at a non-collapsing dose destabilizes adherent points of the growth cones, visualized by the focal adhesion marker Paxillin (Woo and Gomez, 2006). Paxillin is an adaptor protein recruited to dynamic focal contacts and has a crucial role in adhesion turnover. Interestingly for the present context, Paxillin forms a scaffold for activation of erk at focal contacts (Ishibe et al, 2003). It constitutively binds MEK. On phosphorylation by FAK in response to extracellular stimuli, Paxillin recruits inactive erk and active Raf, resulting in local erk activation. This prompted us to investigate first the links between L1 and Paxillin and second the possibility that L1-dependent FAK–MAPK activation mediates Sema3A-induced adhesion disassembly. First, fresh cortical tissues were stimulated with control and Sema3A treatment and L1 was immunoprecipitated for western blot analysis of Paxillin. Strong interaction of L1 with Paxillin but not other proteins of focal adhesions such as talin and vinculin was found in control condition, indicating that L1 might be associated with focal adhesion complexes (Figure 4C). Notably, this Paxillin-L1 association was strongly decreased by Sema3A (Figure 4C). Likewise, quantification of the bands showed that the ratio between precipitated L1 and Paxillin was reduced by 51 and 71% for each Paxillin band in the Sema3A condition compared to the control condition. Second, this Sema3A-induced regulation of Paxillin/L1 interaction could be fully recapitulat