Rhotekin regulates axon regeneration through the talin–Vinculin–Vinexin axis in Caenorhabditis elegans

Axon regeneration requires actomyosin interaction, which generates contractile force and pulls the regenerating axon forward. In Caenorhabditis elegans, TLN-1/talin promotes axon regeneration through multiple down-stream events. One is the activation of the PAT-3/integrin–RHO-1/RhoA GTPase–LET-502/ROCK (Rho-associated coiled-coil kinase)–regulatory non-muscle myosin light-chain (MLC) phosphorylation signaling pathway, which is dependent on the MLC scaffolding protein ALP-1/ALP-Enigma. The other is mediated by the F-actin-binding protein DEB-1/vinculin and is independent of the MLC phosphorylation pathway. In this study, we identified the svh-7/rtkn-1 gene, encoding a homolog of the RhoA-binding protein Rhotekin, as a regulator of axon regeneration in motor neurons. However, we found that RTKN-1 does not function in the RhoA–ROCK–MLC phosphorylation pathway in the regulation of axon regeneration. We show that RTKN-1 interacts with ALP-1 and the vinculin-binding protein SORB-1/vinexin, and that SORB-1 acts with DEB-1 to promote axon regeneration. Thus, RTKN-1 links the DEB-1–SORB-1 complex to ALP-1 and physically connects phosphorylated MLC on ALP-1 to the actin cytoskeleton. These results suggest that TLN-1 signaling pathways coordinate MLC phosphorylation and recruitment of phosphorylated MLC to the actin cytoskeleton during axon regeneration.


Introduction
Axon regeneration after neuronal injury is a biologically conserved process that restores functions to the nervous system.Injured axons form new growth cones that pull and extend the axon along the extracellular matrix (ECM).The driving force behind this axonal growth is a non-muscle myosin-dependent contractile force generated by actomyosin interactions [1].This contractile force is transmitted to the ECM via cell-ECM adhesion sites, allowing axons to grow along the ECM [2].Two molecular events take place during this process.One is the phosphorylation of the non-muscle myosin light-chain (MLC), which activates the myosin ATPase activity required for force generation [3].This phosphorylation is mediated by several kinases, including MLC kinase (MLCK), Rho-associated coiled-coil kinase (ROCK) and citron kinase [4][5][6].Another is the recruitment of myosin to the adhesion-associated actin cytoskeleton to transmit the forces generated to the ECM.It is unclear how these events are coordinated in time and space to achieve effective axon regeneration.
The cytoskeletal protein talin plays a central role in force generation by linking the actin cytoskeleton to the ECM [7].Talin activates integrins and vinculin, proteins essential for cell-ECM adhesion.Integrins are receptors that bind to the ECM and normally exist in an inactive conformation with low binding affinity to the ECM [8][9][10].Upon activation by talin, integrins undergo a conformational change from an inactive to an active state and bind tightly to the ECM, where they transduce intracellular signals via protein kinases and GTPases to regulate myosin activity [11].Vinculin is a multi-domain cytoskeletal adaptor protein that, upon binding to talin, interacts and cross-links with F-actin, which then interacts with the talin-integrin-ECM complex to form an adhesion-bound actin cytoskeleton [12,13].Activated vinculin also recruits other cytoskeletal proteins such as vinexin and paxillin to regulate adhesion strength and dynamics [14][15][16][17].
The nematode Caenorhabditis elegans has recently emerged as an attractive model for studying the mechanisms of axon regeneration in the mature nervous system [18,19].Its amenability to genetic analysis has led to the discovery and elucidation of novel signaling pathways involved in axon regeneration.We have previously shown that the RHO-1/RhoA GTPase-LET-502/ROCK-MLC-4/MLC phosphorylation signaling pathway in C. elegans promotes axon regeneration in a manner dependent on the ALP (α-actinin-associated LIM protein)-Enigma protein ALP-1 (Fig 1A; [20]).Genetic analysis revealed that ALP-1 serves as a scaffold for activation of MLC phosphorylation via the RHO-1-LET-502 pathway.In addition, axon injury induces inside-out activation of integrins via the cAMP-EPAC-1/Epac (Exchange protein directly activated by cAMP)-RAP-2/Rap GTPase-TLN-1/talin cascade, leading to activation of the RHO-1-LET-502 pathway (Fig 1A; [21]).Thus, TLN-1 ultimately induces MLC-4 phosphorylation through integrin activation.In addition to its role as an integrin activator, TLN-1 also activates DEB-1/vinculin, an essential linker protein between the actin cytoskeleton and ECM-bound integrins [22,23].Indeed, DEB-1 is required for axon regeneration in C. elegans [21].However, expression of MLC-4(DD), which mimics phosphorylation, does not suppress the deb-1 defect [21], suggesting that the TLN-1-DEB-1 signaling pathway regulates axon regeneration independently of the MLC-4 phosphorylation pathway (Fig 1A ).

SVH-7/RTKN-1 is homologous with mammalian Rhotekin
We have previously isolated a number of candidate genes involved in axon regeneration and named them the svh genes [24].Among these, the svh-7 gene encodes a protein homologous to human Rhotekin (Fig 1B).The svh-7 gene was found to be identical to the rtkn-1 gene [25] and so svh-7 was renamed rtkn-1.Rhotekin was originally identified as a putative target of RhoA [26].Rhotekin contains an N-terminal Rho-binding domain (RBD), a central pleckstrin homology (PH) domain and a C-terminal Proline-rich motif (PxxP) (Fig 1B; [27]).The PH domain has an affinity for phosphoinositide and helps to localize proteins to specific membrane structures [28,29].
To determine whether RTKN-1 functions in axon regeneration, we examined the regeneration of laser-severed axons in γ-aminobutyric acid (GABA)-releasing D-type motor neurons (Fig 1C).In young adult wild-type animals, approximately 70% of axons initiated regeneration within 24 h of axon injury (Fig 1C and 1D, and S1 Table ), whereas the frequency of axon regeneration was reduced in rtkn-1(ok1404) mutant animals (Fig 1C and 1D, and S1 Table ).To confirm that the axon regeneration defect was due to the rtkn-1 mutation, an approximately 14 kb DNA fragment containing the rtkn-1 gene was introduced into animals carrying the rtkn-1(ok1404) mutation.We observed that the rtkn-1 transgene, as an extrachromosomal array, significantly rescued the defect associated with this mutation (Fig 1D and ).These results suggest that RTKN-1 is involved in axon regeneration after laser axotomy.We next investigated whether RTKN-1 acts in a cell-autonomous manner.The rtkn-1 defect was rescued when rtkn-1 was expressed from the unc-25 promoter in D motor neurons, but not when it was expressed from the mec-7 promoter in sensory neurons (Fig 1D and S1 Table).Thus, RTKN-1 functions in a cell-autonomous manner in D-type motor neurons.
Since mammalian Rhotekin is involved in RhoA signaling [26,27,30], we examined whether RTKN-1 functions in the RhoA-ROCK-MLC phosphorylation pathway that regulates axon regeneration.We found that the expression of an active GTP-bound RHO-1, RHO-1(G14V), from the unc-25 promoter in D-type neurons did not suppress the rtkn-1 axon regeneration defect (Fig 1D and S1 Table).Furthermore, expression of MLC-4(DD), which mimics phosphorylation, from the unc-25 promoter was unable to rescue the rtkn-1 defect (Fig 1D and S1 Table ).These results suggest that RTKN-1 does not function in the RHO-1-MLC-4 phosphorylation signaling pathway to regulate axon regeneration.

RTKN-1 interacts with SORB-1/vinexin
Rhotekin has recently been shown to interact with vinexin [27].Mammals have two major vinexin isoforms, vinexin α and vinexin β.Vinexin α is a full-length isoform with one N-terminal sorbin homology (SoHo) domain and three C-terminal Src homology 3 (SH3) domains, whereas vinexin β lacks the SoHo domain (Fig 2A; [31]).Since both isoforms interact with Rhotekin [32], the SH3 domain is responsible for the interaction with Rhotekin.The PxxP motif is an essential module mediating the interaction with the SH3 domain [33].Consistent with this, the SH3 domain of vinexin associates with the PxxP motif and recruits Rhotekin to the midbody during cell division [32,34].C. elegans SORB-1 is most homologous to vinexin (Fig 2A; [35]) and RTKN-1 has a PxxP motif in its C-terminal region (Fig 1B).We observed that SORB-1C, which contains the SH3 domain, was able to associate with RTKN-1 (Fig 2B).These results suggest that the SH3 domain of SORB-1 associates with the PxxP motif of RTKN-1, but this possibility is not yet conclusive.

RTKN-1 interacts with ALP-1
What is the role of RTKN-1 in binding to SORB-1?SORB-1 is highly localized to integrin adhesion complexes and is recruited to the actin-binding, membrane-distal regions of dense bodies in an ALP-1-dependent manner via the C-terminal SH3 domains [35].However, SORB-1 does not directly interact with ALP-1.The SH3 domain of SORB-1 mediates the interaction with RTKN-1 (Fig 2B ), raising the possibility that RTKN-1 binding to ALP-1 may link SORB-1 to ALP-1.We therefore investigated this possibility using yeast two-hybrid analysis.We found that RTKN-1 binds weakly to ALP-1 (Fig 3A).RTKN-1 adopts an autoinhibitory conformation due to intramolecular interactions between the N-terminal RBD region and the C-terminal PH domain [25].This autoinhibitory conformation prevents RTKN-1 from interacting with its appropriate partner.Consistent with this, an RTKN-1 fragment (RTKN-1N) with an N-terminal region (amino acids 1-172) bound more strongly to ALP-1 than fulllength RTKN-1 (Figs 3A and 4D).It is possible that the interaction of the C-terminal PxxP motif of RTKN-1 with the SH3 domain of SORB-1 disrupts the intramolecular self-binding of RTKN-1, resulting in the interaction of RTKN-1 with ALP-1.Taken together, these results suggest that RTKN-1 links SORB-1 and ALP-1.
As the tln-1(e259) allele has a mutation in the C-terminal dimerization domain (Fig 5A ), it is possible that this mutation affects dimer formation.In fact, a recent study reported that the L2509P mutation (Fig 5A ), located within the dimerization domain of human talin-1, disrupts dimer formation and significantly reduces adhesion maturation by integrin activation, but has little effect on its interaction with vinculin [39].These results are consistent with the possibility that the tln-1(e259) mutation is only defective in the PAT-3 pathway and does not affect the DEB-1 pathway.

Discussion
We have previously shown that TLN-1/talin promotes axon regeneration by activating the PAT-3/integrin-RHO-1/RhoA-LET-502/ROCK-MLC-4/MLC phosphorylation pathway in an ALP-1/ALP-Enigma-dependent manner [20,21].The promotion of axon regeneration by TLN-1 also requires the DEB-1/vinculin activation pathway, which is independent of the MLC-4 phosphorylation pathway.Thus, TLN-1 successfully induces axon regeneration by simultaneously activating two different signaling pathways (Fig 6).Mammalian talin has also been shown to be involved in promoting axon growth and regeneration.Overexpression of full-length talin-1 in cultured neurons activates integrins and stimulates axon regrowth [40].Talin consists of two domains, an N-terminal head domain that binds integrins and a C-terminal rod domain that binds vinculin [41][42][43].Interestingly, the N-terminal head domain of talin-1 alone is sufficient to activate integrins but not to stimulate axon regrowth, suggesting that the rod domain of talin-1 is also required for axon regeneration [40].This result is consistent with our model that activation of both integrins and vinculin is required to promote axon regeneration.However, the function of the DEB-1/vinculin signaling pathway in regulating axon regeneration was unclear.In this study, we first attempted to identify the components that function in the TLN-1-mediated signaling pathways required for axon regeneration.As we had previously genetically identified svh genes that regulate axon regeneration [24], we investigated the role of the svh-7/rtkn-1 gene, which encodes a mammalian homolog of Rhotekin [25], in regulating axon regeneration.Mammalian Rhotekin was originally identified as a putative target of RhoA [26], but we found that RTKN-1 does not function in the RhoA-ROCK-MLC phosphorylation pathway that regulates axon regeneration.Thus, RTKN-1 acts independently of RHO-1 in axon regeneration.Mammalian Rhotekin has also been identified as a binding partner for vinexin [32], but its physiological function in this interaction remains unclear.Vinexin has been identified as a vinculin-binding protein and is required to induce a conformational change of vinculin to a form with higher affinity for actin [31,44].The SH3 domain of vinexin binds to Rhotekin [31,32].Similarly, RTKN-1 binds to the SH3 domain of SORB-1.RTKN-1 also binds to ALP-1, which serves as a platform for phosphorylation of MLC-4 by LET-502/ROCK [20].Previously, we found that the alp-1(ok820) mutation reduced axon regeneration and that the phenotype was suppressed by phosphomimetic MLC-4(DD).These results suggest that the region deleted by the alp-1(ok820) mutation (57 amino acids before the LIM1 domain) is important for MLC-4 phosphorylation [20].In contrast, the axon regeneration defect in alp-1(km95) null mutants is not rescued by MLC-4(DD).Thus, phosphorylated MLC-4 still requires ALP-1 to induce axon regeneration.Axon regeneration requires a myosin-dependent contractile force that is transmitted to the ECM via the cell-ECM adhesion site.This requires active myosins to interact with the adhesion-associated actin cytoskeleton, which forms the vinculin-talinintegrin-ECM complex [2].Because RTKN-1 can bind to both SORB-1 and ALP-1, RTKN-1 can act as a bridging molecule between DEB-1-SORB-1 and ALP-1-phosphorylated MLC-4 (Fig 6).This could be a mechanism to localize phosphorylated MLC-4 to the DEB-1-associated actin cytoskeleton to induce axon regeneration.In addition, the DEB-1-SORB-1 signaling pathway and the integrin-MLC phosphorylation pathway are activated by TLN-1.Activation of these two signaling pathways by TLN-1 is therefore a possible mechanism to regulate myosin phosphorylation and localization, thereby promoting axon regeneration.

C. elegans strains
The C. elegans strains used in this study are listed in S2 Table .All strains were maintained on nematode growth medium plates and fed with bacteria of the OP50 strain according to a previously described standard method [38].

Microscopy
Fluorescence images of transgenic animals were observed under a 100x objective lens of a Nikon ECLIPSE E800 fluorescence microscope and photographed with a Zyla CCD camera.Confocal fluorescence images were captured using a Zeiss LSM-800 confocal laser-scanning microscope with a 63x objective.

Axotomy
Axotomies were performed as previously described [24].Animals were axotomized at the young adult stage.Commissures with growth cones or small branches present on the proximal fragment were counted as "regenerated".Proximal fragments showing no change after 24 h were counted as "no regeneration".A minimum of 20 individuals with 1-3 axotomized commissures were observed for most experiments.

Statistical analysis
Statistical analysis was performed as previously described [24].Briefly, confidence intervals (95%) were calculated using the modified Wald method and two-tailed P-values were calculated using Fisher's exact test (https://www.graphpad.com/quickcalcs/contingency1/)and adjusted by Bonferroni correction for multiple tests.

Fig 1 .
Fig 1. SVH-7/RTKN-1 is required for axon regeneration.(A) Regulation of axon regeneration by the TLN-1/talin signaling network.In response to axon injury, cAMP levels are elevated, resulting in the activation of EPAC-1, which then activates RAP-2.GTP-bound RAP-2 interacts with and activates TLN-1, which then activates PAT-3 and DEB-1.Activation of PAT-3 leads to phosphorylation of MLC-4 via the RHO-1-LET-502 pathway.ALP-1 acts as a platform for LET-502-mediated phosphorylation of MLC-4.DEB-1 acts independently of the MLC-4 phosphorylation pathway.(B) Domain structure of RTKN-1 and human Rhotekin.The Rho binding domain (RBD) and pleckstrin homology (PH) domains are shown in red and yellow, respectively.The Pro-rich motif (PxxP) is also shown.Identical and similar residues are highlighted in red and yellow shading, respectively.The bold lines below the RTKN-1 diagram indicate the extent of the deleted regions in the ok1404 and km94 mutants.(C) Representative D-type motor neurons in wild-type and rtkn-1(ok1404) mutant animals 24 h after laser surgery.Yellow and white arrowheads indicate regenerating and non-regenerating growth cones, respectively.Scale bar, 10 μm.(D) Percentage of axons that initiated regeneration 24 h after laser surgery at the young adult stage.The number of axons examined is shown.The error bar represents the 95% confidence interval (CI).*P < 0.05, **P < 0.01, according to Fisher's exact test.NS, not significant.https://doi.org/10.1371/journal.pgen.1011089.g001

Fig 2 .
Fig 2. SORB-1/Vinexin is required for axon regeneration.(A) Domain structure of SORB-1 and human vinexin.The sorbin homology (SoHo) and Src homology 3 (SH3) domains are shown in blue and black, respectively.The bold line below the SORB-1 diagram indicates the extent of the deleted region in the gk304 mutant.(B) Yeast two-hybrid assays for RTKN-1 interaction with SORB-1 and SORB-1C.The reporter strain PJ69-4A was co-transformed with the expression vectors encoding GAL4 DBD-RTKN-1, GAL4 AD-SORB-1 and GAL4 AD-SORB-1C as indicated.Yeasts carrying the indicated plasmids were grown on selective plates lacking histidine and containing 10 mM 5-aminotriazole for 4 days.(C and D) Percentage of axons that initiated regeneration 24 h after laser surgery at the young adult stage.The number of axons examined is shown.The error bar represents the 95% confidence interval (CI).*P < 0.05, **P < 0.01, ***P < 0.001, according to Fisher's exact test.NS, not significant.https://doi.org/10.1371/journal.pgen.1011089.g002