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• W2989273805 abstract "Structural studies reveal molecular details of netrin-mediated clustering of DCC and uncoordinated 5 (UNC5), suggesting how they switch between growth cone attraction and repulsion in axon guidance.Three separate DCC-binding sites have been identified on netrin, and each site can be targeted by competing receptors or cofactors.Environmental cofactors may affect netrin-mediated signaling and adhesion.Structural studies suggest that the auxiliary guidance cue draxin locally expressed in the spinal cord alters netrin–DCC interactions to promote adhesion and axonal fasciculation.We propose that netrin mediates conditional adhesion between cells decorated with netrin receptors. Conditional adhesion is a reversible and highly localized process that can facilitate fasciculation, haptotaxis, and downstream signaling. Netrin is a prototypical axon guidance cue. Structural studies have revealed how netrin interacts with the deleted in colorectal cancer (DCC) receptor, other receptors, and co-factors for signaling. Recently, genetic studies suggested that netrin is involved in neuronal haptotaxis, which requires a reversible adhesion process. Structural data indicate that netrin can also mediate trans-adhesion between apposing cells decorated with its receptors on the condition that the auxiliary guidance cue draxin is present. Here, we propose that netrin is involved in conditional adhesion, a reversible and localized process that can contribute to cell adhesion and migration. We suggest that netrin-mediated adhesion and signaling are linked, and that local environmental factors in the ventricular zone, the floor plate, or other tissues coordinate its function. Netrin is a prototypical axon guidance cue. Structural studies have revealed how netrin interacts with the deleted in colorectal cancer (DCC) receptor, other receptors, and co-factors for signaling. Recently, genetic studies suggested that netrin is involved in neuronal haptotaxis, which requires a reversible adhesion process. Structural data indicate that netrin can also mediate trans-adhesion between apposing cells decorated with its receptors on the condition that the auxiliary guidance cue draxin is present. Here, we propose that netrin is involved in conditional adhesion, a reversible and localized process that can contribute to cell adhesion and migration. We suggest that netrin-mediated adhesion and signaling are linked, and that local environmental factors in the ventricular zone, the floor plate, or other tissues coordinate its function. The precise wiring of neurons during early development is a central issue in neuroscience. Netrin (see Glossary) is a prototypical guidance cue that is involved in the proper wiring of commissural neurons as their axons migrate dorsoventrally from the roof plate (RP) to the floor plate (FP) of the spinal cord, where netrin is secreted in abundance [1Serafini T. et al.Netrin-1 is required for commissural axon guidance in the developing vertebrate nervous system.Cell. 1996; 87: 1001-1014Abstract Full Text Full Text PDF PubMed Scopus (1062) Google Scholar]. There, axons cross the midline to establish bilaterality (Figure 1A) [2Chedotal A. Roles of axon guidance molecules in neuronal wiring in the developing spinal cord.Nat. Rev. Neurosci. 2019; 20: 380-396Crossref PubMed Scopus (54) Google Scholar]. The primary netrin receptors are deleted in colorectal cancer (DCC) and its homolog neogenin [3Keino-Masu K. et al.Deleted in colorectal cancer (DCC) encodes a netrin receptor.Cell. 1996; 87: 175-185Abstract Full Text Full Text PDF PubMed Scopus (876) Google Scholar]. Commissural axons expressing DCC on their surface are attracted to a netrin source [4de la Torre J.R. et al.Turning of retinal growth cones in a netrin-1 gradient mediated by the netrin receptor DCC.Neuron. 1997; 19: 1211-1224Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar]. However, when the uncoordinated 5 (UNC5) receptor is co-present, axons are repelled [5Hedgecock E.M. et al.The unc-5, unc-6, and unc-40 genes guide circumferential migrations of pioneer axons and mesodermal cells on the epidermis in C. elegans.Neuron. 1990; 4: 61-85Abstract Full Text PDF PubMed Scopus (728) Google Scholar]. Therefore, netrin was assigned the role of a bi-functional long-range guidance cue that can attract or repel axons at a distance, depending on the receptors present on the growth cone, the extension tip of a developing neurite [6Kolodkin A.L. Tessier-Lavigne M. Mechanisms and molecules of neuronal wiring: a primer.Cold Spring Harb. Perspect. Biol. 2011; 3: a001727Crossref PubMed Scopus (436) Google Scholar, 7Lim Y.S. Wadsworth W.G. Identification of domains of netrin UNC-6 that mediate attractive and repulsive guidance and responses from cells and growth cones.J. Neurosci. 2002; 22: 7080-7087Crossref PubMed Google Scholar, 8Finci L.I. et al.The crystal structure of Netrin-1 in complex with DCC reveals the bifunctionality of Netrin-1 as a guidance cue.Neuron. 2014; 83: 839-849Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar]. The role of netrin as a long-range guidance cue was challenged recently. When netrin expression was ablated from the FP in mice (Figure 1A), commissural axons still found their way and crossed the midline at the FP. Netrin was in fact prominently expressed by neural progenitors in the ventricular zone (VZ), and was deposited on the pial margins of the spinal cord. It was suggested that netrin acted through an adhesive process called haptotaxis [9Dominici C. et al.Floor-plate-derived netrin-1 is dispensable for commissural axon guidance.Nature. 2017; 545: 350-354Crossref PubMed Scopus (117) Google Scholar,10Varadarajan S.G. et al.Netrin1 produced by neural progenitors, not floor plate cells, is required for axon guidance in the spinal cord.Neuron. 2017; 94: 790-799Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar]. This mechanosensing model for netrin-mediated growth cone migration is consistent with studies that show that netrin can provide traction for a growth cone to navigate [11Moore S.W. et al.Traction on immobilized netrin-1 is sufficient to reorient axons.Science. 2009; 325: 166Crossref PubMed Scopus (78) Google Scholar, 12Moore S.W. et al.Netrin-1 attracts axons through FAK-dependent mechanotransduction.J. Neurosci. 2012; 32: 11574-11585Crossref PubMed Scopus (68) Google Scholar, 13Brankatschk M. Dickson B.J. Netrins guide Drosophila commissural axons at short range.Nat. Neurosci. 2006; 9: 188-194Crossref PubMed Scopus (115) Google Scholar] and alter its trajectory. When netrin was ablated from the VZ, commissural axons also crossed the midline [9Dominici C. et al.Floor-plate-derived netrin-1 is dispensable for commissural axon guidance.Nature. 2017; 545: 350-354Crossref PubMed Scopus (117) Google Scholar,10Varadarajan S.G. et al.Netrin1 produced by neural progenitors, not floor plate cells, is required for axon guidance in the spinal cord.Neuron. 2017; 94: 790-799Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar,14Moreno-Bravo J.A. et al.Synergistic activity of floor-plate- and ventricular-zone-derived Netrin-1 in spinal cord commissural axon guidance.Neuron. 2019; 101: 625-634Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar]. However, the particular ‘tilt’ in the trajectory of the axons that had previously been observed by Ramon y Cajal was only preserved when both FP and VZ netrin were present [14Moreno-Bravo J.A. et al.Synergistic activity of floor-plate- and ventricular-zone-derived Netrin-1 in spinal cord commissural axon guidance.Neuron. 2019; 101: 625-634Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar,15Wu Z. et al.Long-range guidance of spinal commissural axons by Netrin1 and Sonic Hedgehog from midline floor plate cells.Neuron. 2019; 101: 635-647Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar]. This suggested that netrin acts differently depending on the region where it is produced, and that environmental co-factors may also modulate its action. Here, we examine how netrin function could be moderated to act in such a versatile manner, based on recent biophysical and structural studies, and propose a model of conditional adhesion. Netrin comprises a laminin VI domain, a V domain containing three EGF repeats, and a C-terminal netrin-like domain, termed NTR (Figure 1Bi). The VI and V domains that are responsible for receptor binding are highly conserved in sequence [8Finci L.I. et al.The crystal structure of Netrin-1 in complex with DCC reveals the bifunctionality of Netrin-1 as a guidance cue.Neuron. 2014; 83: 839-849Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar]. In axon guidance in vitro assays that test growth cone attraction and repulsion, neurons respond to a truncated netrin construct (sNetrin) covering these VI and V domains in an identical manner to full-length netrin [16Serafini T. et al.The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6.Cell. 1994; 78: 409-424Abstract Full Text PDF PubMed Scopus (1161) Google Scholar]. Broadly expressed throughout the brain, DCC is a single-pass transmembrane protein that has an extensive extracellular region and a 350-residue cytoplasmic tail (Figure 1Ci). The ectofragment comprises four Ig-like domains at the N terminus that fold into a horseshoe configuration [17Chen Q. et al.N-terminal horseshoe conformation of DCC is functionally required for axon guidance and might be shared by other neural receptors.J. Cell Sci. 2013; 126: 186-195Crossref PubMed Scopus (21) Google Scholar], followed by six fibronectin type III (FN) domains. However, the cytosolic portion that does not fold into a defined structure contains three conserved sequence motifs, P1, P2, and P3, responsible for signaling [18Kolodziej P.A. et al.frazzled encodes a Drosophila member of the DCC immunoglobulin subfamily and is required for CNS and motor axon guidance.Cell. 1996; 87: 197-204Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar]. Genetic and biochemical studies demonstrated that the FN4 and FN5 domains of DCC are involved in netrin-binding [19Geisbrecht B.V. et al.Netrin binds discrete subdomains of DCC and UNC5 and mediates interactions between DCC and heparin.J. Biol. Chem. 2003; 278: 32561-32568Crossref PubMed Scopus (84) Google Scholar,20Kruger R.P. et al.Mapping netrin receptor binding reveals domains of Unc5 regulating its tyrosine phosphorylation.J. Neurosci. 2004; 24: 10826-10834Crossref PubMed Scopus (58) Google Scholar]. The other receptor, UNC5, comprises two Ig-like domains followed by two thrombospondin type 1 (Tsp) domains in the ectofragment and a large cytoplasmic tail (Figure 1Cii) [21Leung-Hagesteijn C. et al.UNC-5, a transmembrane protein with immunoglobulin and thrombospondin type 1 domains, guides cell and pioneer axon migrations in C. elegans.Cell. 1992; 71: 289-299Abstract Full Text PDF PubMed Scopus (344) Google Scholar]. The tail folds into three compact domains [22Wang R. et al.Autoinhibition of UNC5b revealed by the cytoplasmic domain structure of the receptor.Mol. Cell. 2009; 33: 692-703Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar]. The Ig-like domains appear to be engaged in netrin-binding [19Geisbrecht B.V. et al.Netrin binds discrete subdomains of DCC and UNC5 and mediates interactions between DCC and heparin.J. Biol. Chem. 2003; 278: 32561-32568Crossref PubMed Scopus (84) Google Scholar]. Two crystal structures of sNetrin in complex with different fragments of DCC ectodomain revealed three DCC-binding sites on netrin [8Finci L.I. et al.The crystal structure of Netrin-1 in complex with DCC reveals the bifunctionality of Netrin-1 as a guidance cue.Neuron. 2014; 83: 839-849Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar,23Xu K. et al.Neural migration. Structures of netrin-1 bound to two receptors provide insight into its axon guidance mechanism.Science. 2014; 344: 1275-1279Crossref PubMed Scopus (120) Google Scholar]. Figure 2A depicts a composite model that combines the two structures into one figure. The sNetrin molecule has a tadpole-like shape with a large laminin VI domain at the N terminus as the head, followed by three consecutive EGF repeats as a relatively rigid straight tail (Figure 2A, the molecule in cyan). One DCC receptor binds through its FN4 domain to the netrin VI domain (Figure 2A,B, site 1) [23Xu K. et al.Neural migration. Structures of netrin-1 bound to two receptors provide insight into its axon guidance mechanism.Science. 2014; 344: 1275-1279Crossref PubMed Scopus (120) Google Scholar]. The second DCC uses its abutting area of FN5–FN6 domains to bind the boundary between the EGF-1 and EGF-2 repeats of the netrin V domain (site 2) [8Finci L.I. et al.The crystal structure of Netrin-1 in complex with DCC reveals the bifunctionality of Netrin-1 as a guidance cue.Neuron. 2014; 83: 839-849Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar]. The third DCC receptor can bind with its FN5 domain to the EGF-3 repeat of the V domain (site 3) [8Finci L.I. et al.The crystal structure of Netrin-1 in complex with DCC reveals the bifunctionality of Netrin-1 as a guidance cue.Neuron. 2014; 83: 839-849Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar,23Xu K. et al.Neural migration. Structures of netrin-1 bound to two receptors provide insight into its axon guidance mechanism.Science. 2014; 344: 1275-1279Crossref PubMed Scopus (120) Google Scholar]. It has also been observed that, at sites 1 and 3, DCC can be replaced by its homologous receptor neogenin [23Xu K. et al.Neural migration. Structures of netrin-1 bound to two receptors provide insight into its axon guidance mechanism.Science. 2014; 344: 1275-1279Crossref PubMed Scopus (120) Google Scholar]. The composite model of the three DCC receptors bound to sNetrin shows that all three receptors are oriented in a roughly parallel fashion, with their C termini all pointing toward the cell membrane, such that the receptors would emanate from the same cell surface (Figure 2B). Here, we renumber the three sites as they occur from the N to C terminus of netrin. Unlike many other receptor–ligand complexes, here the binding of ligand does not appear to bring the three DCC receptors to touch each other on the netrin surface. Therefore, DCC binding is considered to be modular and each site can act independently. The binding site 2 has a unique feature. At this site, seven negatively charged sulfate ions are observed sandwiched in between the sNetrin–DCC interface in the crystal structure [8Finci L.I. et al.The crystal structure of Netrin-1 in complex with DCC reveals the bifunctionality of Netrin-1 as a guidance cue.Neuron. 2014; 83: 839-849Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar]. At the interface, there is a cluster of five positively charged arginines on the sNetrin side. On the DCC side, the binding region is also full of positively charged residues. We called this binding site a generic receptor-binding site because of its electrostatic nature, which is not rigorously specific [8Finci L.I. et al.The crystal structure of Netrin-1 in complex with DCC reveals the bifunctionality of Netrin-1 as a guidance cue.Neuron. 2014; 83: 839-849Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar]. Functional assays confirmed that generic site 2 can also bind UNC5 in a competitive manner to replace DCC (Figure 2B). When both sites 2 and 3 are bound by DCC, it elicits axon attraction. When DCC remains at site 3 on the EGF-3 repeat and is replaced by UNC5 at site 2, it turns attraction into repulsion in an in vitro axon guidance assay [8Finci L.I. et al.The crystal structure of Netrin-1 in complex with DCC reveals the bifunctionality of Netrin-1 as a guidance cue.Neuron. 2014; 83: 839-849Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar] (Figure 2B). UNC5 uses its Ig domains to bind netrin (Figure 2B) [24Grandin M. et al.Structural decoding of the Netrin-1/UNC5 interaction and its therapeutical implications in cancers.Cancer Cell. 2016; 29: 173-285Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar]. We propose that the sulfate ions from crystallization buffer, which mediate interactions between netrin and DCC in the generic binding site seen in the crystal structure, would be replaced in vivo by sulfated glycosaminoglycans (GAGs), acting as an environmental factor to facilitate netrin receptor selection [25Smock R.G. Meijers R. Roles of glycosaminoglycans as regulators of ligand/receptor complexes.Open Biol. 2018; 8: 180026Crossref PubMed Scopus (21) Google Scholar]. These may occur in soluble form in the extracellular matrix, or may be fixed at the cell surface associated with proteoglycans. In this regard, it is interesting that Lon-2, a glypican homolog, has been associated with netrin–DCC signaling in Caenorhabditis elegans [26Blanchette C.R. et al.Glypican is a modulator of netrin-mediated axon guidance.PLoS Biol. 2015; 13: e1002183Crossref PubMed Scopus (36) Google Scholar]. Another structural study on netrin, complemented by site-directed mutagenesis, peptide inhibition, and domain deletion experiments, confirmed that UNC5 also engages the arginine cluster on the generic receptor-binding site 2 of netrin [24Grandin M. et al.Structural decoding of the Netrin-1/UNC5 interaction and its therapeutical implications in cancers.Cancer Cell. 2016; 29: 173-285Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar]. Therefore, we conclude that there is strong structural evidence that netrin acts as a scaffold to cluster different receptors on the cell surface, depending on the receptors expressed and environmental factors that may alter affinity for certain receptors locally. The primary receptor is DCC, but all these three sites can be replaced by other receptors or co-factors. In particular, the generic receptor-binding site allows exchange of DCC by UNC5 (Figure 2B). Other netrin receptors might also bind at this generic binding site, which is modulated by GAGs for distinct functions. For example, amyloid precursor protein (APP) [27Rama N. et al.Amyloid precursor protein regulates netrin-1-mediated commissural axon outgrowth.J. Biol. Chem. 2012; 287: 30014-30023Crossref PubMed Scopus (47) Google Scholar] and Down syndrome cell adhesion molecule (DSCAM) [28Ly A. et al.DSCAM is a netrin receptor that collaborates with DCC in mediating turning responses to netrin-1.Cell. 2008; 133: 1241-1254Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar] act as co-receptors for DCC in netrin-mediated axon guidance, whereas CD146 promotes angiogenesis through netrin binding [29Tu T. et al.CD146 acts as a novel receptor for netrin-1 in promoting angiogenesis and vascular development.Cell Res. 2015; 25: 275-287Crossref PubMed Scopus (90) Google Scholar]. Although the extracellular parts of DCC molecules on the cell surface do not appear to contact each other, they are nevertheless brought close enough by netrin to facilitate their cytoplasmic P3 motif dimerization with the focal adhesion target (FAT) domain of focal adhesion kinase (FAK) [30Xu S. et al.The binding of DCC-P3 motif and FAK-FAT domain mediates the initial step of netrin-1/DCC signaling for axon attraction.Cell Discov. 2018; 4: 8Crossref PubMed Scopus (8) Google Scholar] (Figure 2A,B). Binding between the DCC-P3 motif and FAK-FAT in concert with phosphatidylinositol 4,5-bisphosphate (PIP2) mediates the initial step of signaling by recruiting and activating FAK at the membrane and subsequently assembling with Src to form a multiprotein complex for cytoskeleton-associated axon attraction [30Xu S. et al.The binding of DCC-P3 motif and FAK-FAT domain mediates the initial step of netrin-1/DCC signaling for axon attraction.Cell Discov. 2018; 4: 8Crossref PubMed Scopus (8) Google Scholar,31Goni G.M. et al.Phosphatidylinositol 4,5-bisphosphate triggers activation of focal adhesion kinase by inducing clustering and conformational changes.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: E3177-E3186Crossref PubMed Scopus (88) Google Scholar]. Similarly, when netrin engages DCC together with UNC5, the intracellular DCC-P1 motif comes into close contact with the cytoplasmic DCC-binding motif-containing domain, UPA (derived from a protein interaction domain common in the UNC5, PIDD and Ankyrin families) of UNC5 (Figure 2B), which converts netrin-induced attraction to repulsion [22Wang R. et al.Autoinhibition of UNC5b revealed by the cytoplasmic domain structure of the receptor.Mol. Cell. 2009; 33: 692-703Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar,32Hong K. et al.A ligand-gated association between cytoplasmic domains of UNC5 and DCC family receptors converts netrin-induced growth cone attraction to repulsion.Cell. 1999; 97: 927-941Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar]. Thus, we have obtained a complete structural picture of netrin-mediated oligomerization of DCC and UNC5 both extra- and intracellularly (Figure 2). We have also provided structural evidence that there could be an outside-in signal transferred through the clustering of DCC–DCC or DCC–UNC5 by netrin. The presence of three DCC-binding sites on netrin is puzzling, since all models so far have assumed that netrin-mediated dimerization of DCC is sufficient for its function to induce a signal across the cell membrane [33Lai Wing Sun K. et al.Netrins: versatile extracellular cues with diverse functions.Development. 2011; 138: 2153-2169Crossref PubMed Scopus (311) Google Scholar, 34Gibert B. Mehlen P. Dependence receptors and cancer: addiction to trophic ligands.Cancer Res. 2015; 75: 5171-5175Crossref PubMed Scopus (36) Google Scholar, 35Mehlen P. et al.Novel roles for Slits and netrins: axon guidance cues as anticancer targets?.Nat. Rev. Cancer. 2011; 11: 188-197Crossref PubMed Scopus (203) Google Scholar]. The often implied analogy that netrin–DCC signaling is similar to receptor tyrosine kinase (RTK) signaling [36Ullrich A. Schlessinger J. Signal transduction by receptors with tyrosine kinase activity.Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4609) Google Scholar] is probably incorrect, since netrin is a more complicated ligand than the growth factors that exclusively engage RTKs. Apparently, for netrin, it takes more than two to tango! One candidate protein that appears to become tangled in the netrin/DCC ‘tango’ is dorsal repulsive axon guidance protein (draxin), which is expressed at the dorsal side of the spinal cord, (Figure 1A) [37Islam S.M. et al.Draxin, a repulsive guidance protein for spinal cord and forebrain commissures.Science. 2009; 323: 388-393Crossref PubMed Scopus (127) Google Scholar], in the region where commissural axons would encounter VZ-derived netrin. Draxin was originally identified as a repulsive cue for commissural neurons, but the main phenotype in draxin-knockout mice is defasciculation [37Islam S.M. et al.Draxin, a repulsive guidance protein for spinal cord and forebrain commissures.Science. 2009; 323: 388-393Crossref PubMed Scopus (127) Google Scholar], indicating that draxin has a role in axonal adhesion. Initially, using cell-based binding assays, it was determined that draxin binds the N-terminal Ig-like domains of DCC [38Ahmed G. et al.Draxin inhibits axonal outgrowth through the netrin receptor DCC.J. Neurosci. 2011; 31: 14018-14023Crossref PubMed Scopus (66) Google Scholar]. Unexpectedly, biophysical studies indicated that draxin also directly binds netrin, with a conserved region covering a linear polypeptide of 22 amino acids [39Gao X. et al.A floor-plate extracellular protein-protein interaction screen identifies draxin as a secreted Netrin-1 antagonist.Cell. Rep. 2015; 12: 694-708Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar] (Figure 1Bii). This revealed an interesting triangular relationship that occurs when all three components are present in the spinal cord. DCC as a receptor is able to bind two guidance cues, with netrin at the membrane-proximal FN domains of the receptor and draxin at its membrane-distal N-terminal Ig-like domains. In addition, the two cues bind each other with high affinity (Figure 1Ci) [39Gao X. et al.A floor-plate extracellular protein-protein interaction screen identifies draxin as a secreted Netrin-1 antagonist.Cell. Rep. 2015; 12: 694-708Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar]. Thus, can we disentangle these interactions to understand the role of draxin in netrin–DCC function? Structural studies have now determined that draxin comprises a large unstructured N-terminal polypeptide of 260 residues that includes the 22-residue conserved hydrophobic netrin-binding sequence and an approximate 90-residue cysteine knot domain at the C terminus that binds to DCC at the Ig4 domain (Figures 1Ci and 3B) [40Liu Y. et al.Structural basis for draxin-modulated axon guidance and fasciculation by Netrin-1 through DCC.Neuron. 2018; 97: 1261-1267Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar]. Draxin appears to scramble the netrin–DCC interactions; it can outcompete DCC binding at the binding site 3 to the EGF-3 repeat of netrin (Kd = 10 nM [39Gao X. et al.A floor-plate extracellular protein-protein interaction screen identifies draxin as a secreted Netrin-1 antagonist.Cell. Rep. 2015; 12: 694-708Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar] versus Kd ∼5 μM for netrin–DCC binding [23Xu K. et al.Neural migration. Structures of netrin-1 bound to two receptors provide insight into its axon guidance mechanism.Science. 2014; 344: 1275-1279Crossref PubMed Scopus (120) Google Scholar]), and the binding of DCC and draxin on netrin largely overlap (Figure 3A) [40Liu Y. et al.Structural basis for draxin-modulated axon guidance and fasciculation by Netrin-1 through DCC.Neuron. 2018; 97: 1261-1267Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar]. While forming strong interactions with the netrin molecule, the soluble draxin molecule is thus able to dislodge DCC at this site 3. Therefore, we now have identified two conditions where netrin–DCC interactions are modulated by external factors: DCC can be outcompeted by UNC5 at the generic receptor binding site 2 on netrin, and by draxin at site 3 on the EGF-3 repeat of netrin. It was previously proposed that draxin is a netrin antagonist [39Gao X. et al.A floor-plate extracellular protein-protein interaction screen identifies draxin as a secreted Netrin-1 antagonist.Cell. Rep. 2015; 12: 694-708Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar], but structural work implies a more complex involvement of draxin in netrin biology. The netrin and DCC-binding sites on draxin are in close proximity, and would favor the formation of a draxin-mediated trans-netrin–DCC dimer over a cis-netrin–DCC dimer. This creates a DCC–draxin–netrin–DCC bridge-like configuration between two axons (Figure 3C) [40Liu Y. et al.Structural basis for draxin-modulated axon guidance and fasciculation by Netrin-1 through DCC.Neuron. 2018; 97: 1261-1267Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar]. This model is appealing because, with netrin, draxin forms a molecular bridge between commissural axons that carry DCC at their surface, thus promoting adhesion and fasciculation. Indeed, absence of draxin was shown to cause defasciculation [37Islam S.M. et al.Draxin, a repulsive guidance protein for spinal cord and forebrain commissures.Science. 2009; 323: 388-393Crossref PubMed Scopus (127) Google Scholar]. Recent analysis of netrin derived from the VZ or FP suggested that netrin interactions are adapted by co-factors to act differentially on commissural axons decorated with DCC [14Moreno-Bravo J.A. et al.Synergistic activity of floor-plate- and ventricular-zone-derived Netrin-1 in spinal cord commissural axon guidance.Neuron. 2019; 101: 625-634Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar,15Wu Z. et al.Long-range guidance of spinal commissural axons by Netrin1 and Sonic Hedgehog from midline floor plate cells.Neuron. 2019; 101: 635-647Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar]. Draxin may have a key modulatory role here, because it only occurs in vertebrates and appears to be an environmental factor that has evolved to have a specific role in axon guidance at the spinal cord [39Gao X. et al.A floor-plate extracellular protein-protein interaction screen identifies draxin as a secreted Netrin-1 antagonist.Cell. Rep. 2015; 12: 694-708Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar]. Further experimental evidence will be required to assess the effects of draxin on netrin-mediated adhesion and signaling. Both structural and genetic data indicate that environmental factors may alter netrin–DCC interactions and adapt them to the functional properties of netrin as it engages DCC from the same cell for signaling, or from apposing cells for adhesion. The apposing cells could be axons to form an axonal bundle for fasciculation, or they could be situated on the pial surface of the spinal cord and mediate haptotaxis of the axonal growth cone. We propose to call this environmental engagement of netrin in cell–cell attachment ‘conditional adhesion’ (Figure 4). Cells decorated with DCC adhere to each other on the condition that netrin is present and has been primed by environmental factors (such as draxin [40Liu Y. et al.Structural basis for draxin-modulated axon guidance and fasciculation by Netrin-1 through DCC.Neuron. 2018; 97: 1261-1267Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar] or GAGs [8Finci L.I. et al.The crystal structure of Netrin-1 in complex with DCC reveals the bifunctionality of Netrin-1 as a guidance cue.Neuron. 2014; 83: 839-849Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar,25Smock R.G. Meijers R. Roles of glycosaminoglycans as regulators of ligand/receptor complexes.Open Biol. 2018; 8: 180026Crossref PubMed Scopus (21) Google Scholar] present in the extracellular matrix). Since conditional adhesion is a reversible and highly localized process, it should serve well in a mechanism for haptotaxis. The toggling between cis and trans DCC interactions will create a local force that propels the growth cone in a certain direction [11Moore S.W. et al.Traction on immobilized netrin-1 is sufficient to reorient axons.Science. 2009; 325: 166Crossref PubMed Scopus (78) Google Scholar,12Moore S.W. et al.Netrin-1 attracts axons through FAK-dependent mechanotransduction.J. Neurosci. 2012; 32: 11574-11585Crossref PubMed Scopus (68) Google Scholar]. Another netrin receptor that has been primarily recognized for an adhesive function is DSCAM [41Schmucker D. Chen B. Dscam and DSCAM: complex genes in simple animals, complex animals yet simple genes.Genes. Dev. 2009; 23: 147-156Crossref PubMed Scopus (132) Google Scholar]. Netrin was reported to interact with the stem of the DSCAM ectodomain, covering Ig7-Ig9 (Figure 1Ciii) [28Ly A. et al.DSCAM is a netrin receptor that collaborates with DCC in mediating turning responses to netrin-1.Cell. 2008; 133: 1241-1254Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar,42Liu G. et al.DSCAM functions as a netrin receptor in commissural axon pathfinding.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 2951-2956Crossref PubMed Scopus (117) Google Scholar], although the functional role of DSCAM in netrin-mediated axon guidance is debated [43Palmesino E. et al.Genetic analysis of DSCAM's role as a Netrin-1 receptor in vertebrates.J. Neurosci. 2012; 32: 411-416Crossref PubMed Scopus (38) Google Scholar]. Contrary to DCC, netrin does not appear to dimerize DSCAM close to the cell membrane to promote signaling, because there are six FN domains and one Ig10 domain between the membrane and the netrin-binding site. Netrin binding overlaps with the region of the ectodomain that is involved in homodimerization [44Sawaya M.R. et al.A double S shape provides the structural basis for the extraordinary binding specificity of Dscam isoforms.Cell. 2008; 134: 1007-1018Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar], which has been shown to be critical for DSCAM function [45Hughes M. et al.Homophilic Dscam interactions control complex dendrite morphogenesis.Neuron. 2007; 54: 417-427Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar]. Interestingly, the guidance cue Slit also binds DSCAM at the tip of the ectodomain covering four N-terminal domains that form the horseshoe (Figure1Ciii) [46Dascenco D. et al.Slit and receptor tyrosine phosphatase 69D confer spatial specificity to axon branching via Dscam1.Cell. 2015; 162: 1140-1154Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar]. There is a striking similarity between the DCC–netrin–draxin and the DSCAM–netrin–Slit interactions, where draxin and Slit are interacting at the horseshoe-shaped tip of the receptors (Figure 1Ci,iii). This provides another example where the guidance cues Slit and netrin may regulate conditional adhesion between axons through the cell surface receptor DSCAM, affecting local processes, such as repulsion, target attachment, and haptotaxis. Netrin has traditionally been studied as a long-range cue in axon guidance to guide growth cones through the clustering of its receptors DCC, UNC5, and DSCAM. This model requires further refinement to include the role of netrin in axonal adhesion and haptotaxis. Structural studies reveal three separate DCC-binding sites on the rigid tadpole-like netrin molecule. Therefore, DCC binding appears to be modular and each site can act independently. Each site can be replaced by other receptors and/or co-factors for a variety of functions, depending on the biological setting. Based on structural considerations, draxin may modulate netrin–DCC interactions to promote axonal adhesion. This model can be further extended, considering the variety of netrin receptors with adhesive (DSCAM), repellent (UNC5), and signaling (APP) capabilities (see Outstanding Questions). It also appears that netrin function can be modulated by environmental factors. A new model that considers the role of environmental factors from the extracellular matrix is proposed that also incorporates the mechanoeffector properties of netrin. We hypothesize that netrin may act as an adhesive glue between cells that are decorated with netrin receptors. We call this conditional adhesion, a dynamic process that allows cells to locally adhere and de-adhere, which, in conjunction with movements in the cytoskeleton, may direct a migrating cell along a netrin gradient.Outstanding QuestionsWith signaling and adhesion processes synergized by netrin, how are commissural axons guided spatiotemporally to the floor plate?How is netrin signaling affected by the cis and/or trans-interactions of its receptors? Are these interactions propagated across the cell membrane as a signal for a cell to mobilize its cytoskeleton?How do receptors other than DCC bind to netrin and can they engage in cis and/or trans-interactions, or do they only disrupt netrin–DCC binding?How do UNC5 and other repellent receptors alter netrin–DCC-mediated adhesion? Is there a direct link between de-adhesion and repulsion?Can the structural model for draxin-mediated fasciculation be verified? Does draxin also have a role in netrin signaling? With signaling and adhesion processes synergized by netrin, how are commissural axons guided spatiotemporally to the floor plate?How is netrin signaling affected by the cis and/or trans-interactions of its receptors? Are these interactions propagated across the cell membrane as a signal for a cell to mobilize its cytoskeleton?How do receptors other than DCC bind to netrin and can they engage in cis and/or trans-interactions, or do they only disrupt netrin–DCC binding?How do UNC5 and other repellent receptors alter netrin–DCC-mediated adhesion? Is there a direct link between de-adhesion and repulsion?Can the structural model for draxin-mediated fasciculation be verified? Does draxin also have a role in netrin signaling? This work was supported by the Ministry of Education of China to J-H.W. and Y.Z., the National Science Foundation of China (NSFC) Major Research Grant (91132718) to Y.Z., an NIH grant (P01 HL103526), funds from the Peking-Tsinghua Center for Life Sciences and Claudia Adams Barr Program for Innovative Cancer Research to J-H. W. R.G.S. was supported by the European Research Council (ERC) under a Horizon 2020 MSCA-IF (702346) grant. a nerve fiber that carries impulses away from the cell body. Most of the time, a neuron has only one axon with a long extension to connect and transmit signals to other neurons or muscle cells. pathfinding of an axon from one neuron to accurately connect to another neuron, a process that is aided by guidance cues during neuronal development. a neuron with its axon crossing the midline of the spinal cord to ensure the bilateral symmetry of the nervous system. guidance cue receptors will contribute to cell–cell adhesion, on the condition that the guidance cue is present to promote adhesion. Other components from the extracellular matrix (ECM), such as laminins and glycosaminoglycans, may regulate the engagement of these guidance cues. Conditional adhesion is a reversible and localized process that can contribute to cell adhesion and migration. originally discovered as a colon tumor suppressor gene, DCC was later identified as the primary receptor for netrin-1. As a single-pass transmembrane protein, it is broadly expressed on axon growth cones throughout brain. in anatomy, moving in a direction from the back or upper (dorsal) to the front or bottom (ventral) parts of the spinal cord. a protein secreted in the dorsal part of the spinal cord. As an auxiliary guidance cue, it modulates netrin action, having an adhesive function. in neuroscience, refers to bundling of many axons or neuronal fibers. an integral structure on the ventral midline of the embryonic neural tube, spanning from the midbrain to the tail region of vertebrates. The floor plate secretes high amounts of netrin during development. directional movement or outgrowth of cells along a surface. In the case of axon outgrowth and guidance, axons move along a gradient of guidance cues that are attached to the surface. a prototypical axon guidance cue. This soluble glycoprotein interacts with its receptors to mediate cell migration through attraction, repulsion, adhesion, and haptotaxis. a thin layer of the embryonic neural tube that connects the lateral plates dorsally. a long, thin, tubular structure comprising nervous tissue, which extends from the brainstem to the lumbar region of the vertebrate column. The brain and spinal cord are the major components of the central nervous system. netrin receptor that causes axonal repulsion. a transient embryonic layer of tissue containing neural stem cells of the central nervous system, in this context lying between the roof and the floor plate of the spinal cord." @default.
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• W2989273805 title "Netrin Synergizes Signaling and Adhesion through DCC" @default.
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