Fig 1: In vivo inhibitory interactions between RGMA and NET1(A–D) In vivo inhibitory effects of RGMA and NET1 on embryonic mouse cortical neuron migration are silenced in the presence of both cues.(A) Graphical overview of the in utero electroporation (IUE) experiment. Embryos were electroporated at E14 with a GFP construct in addition to (combinations of) different expression vectors (RGMA, NET1, or shRNA). At E15, pregnant mothers were injected with EdU to label the population of cortical neurons born at E15. At E17, migration of Edu+ neurons was quantified in the cortical plate (CP) in 4 different bins (1–4).(B) Immunohistochemistry showing NET1 expression in the deep part of the E16 cortex following co-electroporation of GFP and NET1-mCherry.(C) EdU staining on E17 coronal sections of the mouse cortex to visualize migrating neurons born at E15, one day after IUE of the VZ at E14. Scale bar, 100 μm.(D) Quantification of Edu+ neuron migration using the bins shown in (C). Upper graph, IUE of RGMA and NET1 constructs reduced migration of EdU+ neurons, an effect silenced when RGMA and NET1 are co-electroporated. Lower graph, reduced migration of neurons following NET1 electroporation is partly rescued by knockdown of NEO1 (shNEO1). One-way ANOVA followed by Sidak’s multiple comparisons: RGMA vs. GFP bin 4 p = 0.0094, NET1 vs. GFP bin 4 p < 0.0001, NET1 vs. RGMA+NET1 bin 1 p = 0.0231, NET1 vs. RGMA+NET1 bin 4 p < 0.0001, NET1+shSCR vs. GFP bin 2 = 0.0366, NET1-shSCR vs. GFP bin 4 p < 0.0001, NET1+shNEO1 vs. GFP bin 4 p = 0.0108. GFP, RGMA, and NET1+ RGMA: n = 6 embryos, NET1 and NET1+shSCR: n = 4 embryos, NET1+shNEO1: n = 7 embryos. i.p., intraperitoneally; E, embryonic day; VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; MZ, marginal zone.(E and F) Model for NEO1 signaling via the NET1 and RGM guidance molecules in trans.(E) NET1-induced clustering of NEO1 at the cell surface via Interface-1 and -2 interactions can lead to NEO1 intracellular interactions, inducing e.g. attractive guidance and outgrowth (left panel). In contrast, RGM binding to potentially pre-clustered NEO1 results in NEO1 dimerization in a signaling compatible conformation (Bell et al., 2013) (right panel). This architecture leads to activation of downstream signaling resulting in repulsive guidance (e.g., growth cone collapse), a process that can be potentiated by BMP morphogens (Healey et al., 2015).(F) Combined binding of RGM and NET1 to NEO1 results in “trimer-of-trimers” super-complexes, preventing cell surface clustering, thereby inhibiting both RGM-mediated repulsive but also NET1-mediated attractive signaling. See also Figure S5, Figure S7, Figure S8.
Fig 2: Structural and functional analysis of the ternary NEO1-NET1-RGM complex, related to Figures 2, 3, and 4(A, B) Surface representations of NET1-NEO1 interactions The NEO1-NET1 Interface-1, formed by the NEO1 FN4-NET1 LN interaction is shown in A. Interface residues are mapped onto solvent accessible surfaces displayed in open-book view (blue, left panel in A). Residue conservation calculated with ConSurf server (https://consurf.tau.ac.il/) is mapped onto the protein surfaces according to a white-to-black gradient (right panel in A). Surfaces are highlighted with a line. The NEO1-NET1 Interface-2, formed by the NEO1 FN5-NET1 LE3 interaction is shown in B. Presentation is as in A. (C-G) Sugar sites identified on the ternary NEO1-NET1-RGMB crystal structure. (C) Ribbon presentation of the NEO1-NET1-RGMB protomer with the 4 N-linked N-acetylglucosamine (NAG; yellow) and 4 sucrose-octasulfate (SOS; light blue) molecules depicted as sticks. (D-G), Close-up views of the 4 SOS-binding sites with residue side chains within hydrogen-bonding distance shown in stick representation and labelled. Potential hydrogen bonds are displayed as dashed black lines. (H) NET1-RGM interaction analysis in the ternary trimer-of-trimers complex determined by X-ray crystallography. Overall 1:1:1 trimer architecture is displayed on the left. The close-up shows the interface between NET1 and RGMB. The sigmaA-weighted 2Fo-Fc map of the final refinement in AUTOBUSTER is displayed and contoured at 1σ. RGMB is ordered to residue D323 and a dashed line denotes disordered residues linking to a putative helical stretch of Ala residues, which were built into this density as the sequence could not be unambiguously assigned. (I) Non-reducing SDS-PAGE of purified RGMAECD and RGMBECD used as analytes for SPR injections. (J) Schematics of the experimental SPR set up. NET1ΔNTR (ligand) was attached to a streptavidin-coupled sensor chip via a biotinylated C-terminal Avi-tag. RGMECD and NEO1FN456 (analytes) were injected to probe interactions. (K, L), SPR equilibrium binding curves for NET1ΔNTR binding experiments with NEO1FN456 (K and L; same measurement for comparison), RGMBECD (K) and RGMAECD (L). (M, N) SPR equilibrium binding curves for the NEO1-NET1 interaction. A schematic of the experiment (NEO1: red, NET1: blue) and the calculated Kd values are shown. The maximal response for the wild type NEO1FN456:NET1ΔNTR interaction represents 100% binding. Sensorgrams for NEO1:NET1ΔNTR interactions, corresponding to Figure 3B and Figure S6J are shown in (B).
Fig 3: Interface analysis of the ternary NEO1-NET1-RGMB super-complex(A) Close-up views of the observed NET1-NEO1 interfaces (right: interface 1, left: interface 2). Residues are displayed in stick representation and labelled according to domain color-coding. A Ca2+ ion bound to NET1 LN (grey sphere) and hydrogen bonds (dashed black lines) are displayed. Mutated residues are in bold and underlined.(B) SPR equilibrium binding curves for the NET1-NEO1 interaction. A schematic of the experiment and the calculated Kd values are shown.(C) AUC analysis of the NEO1FN456-NET1ΔNTR-RGMBECD complex, using NET1ΔNTR WT and mutants. Both NET1 interface-1 and -2 mutants abolish the 3:3:3 stoichiometry of the NEO1-NET1-RGMB super-complex.(D) Overlapping expression of NET1 RNA (in situ hybridization), and NEO1 and RGMB protein (immunohistochemistry) in consecutive coronal sections of E16 mouse striatum. Boxed area is shown at higher magnification for NEO1 and RGMB. Scale bar, 100 μm.(E) RGMB immunoprecipitation (IP) from adult mouse cortex was followed by immunoblotting. Input samples (lane 1), IP using control non-specific IgGs (cntrl) (lane 2), and anti-RGMB IP (lane 3). NEO1 and NET1 co-IP with RGMB from adult mouse brain lysates.(F and G) Functional analysis of the effect of NET1 on RGMA-mediated growth cone collapse.(F) Representative examples of growth cones from mouse P0 cortical neurons. Neurons were stained with the microtubule marker Tuj1 (green) and F-actin marker phalloidin (red). Scale bar, 10 μm.(G) Quantification of growth cone collapse. Growth cones were treated with control or RGMA alone and in combination with different NET1 variants. Proportions of collapsed growth cones relative to control are displayed. n = 3 experiments, one-way ANOVA followed by Tukey’s multiple comparison test. ∗p < 0.05. Data are shown as means ± SEM.(H–J) Comparison of binary NEO1-RGM (PDB ID 4BQ6 [Bell et al., 2013]) and the ternary NEO1-NET1-RGMB complexes shown as ribbons. The ternary NEO1-NET1-RGMB protomer complex architecture (I) clashes with the NEO1-RGM dimer-of-dimers signaling conformation (H) when superimposed on NEO1 (marked with an asterisk) (J). See also Figure S3, Figure S4, Figure S5.
Fig 4: SEC, MALS and SDS-PAGE analysis of the ternary NEO1-NET1-RGM complexes, related to Figure 2(A) SEC of the ternary NEO1FN456-NET1ΔNTR-RGMBECD complex. The SEC fraction (elution volume ~9.8-10.1 ml) indicated with a red line was analyzed using MALS (panel B) and cryo-EM. SEC fractions indicated with a blue line (elution volume ~8-12 ml) were analyzed on SDS PAGE (panels C and D). (B) SEC-MALS analysis of the NEO1FN456-NET1ΔNTR-RGMBECD complex. Calculated MW of 1:1:1 mol:mol:mol complex is 144.4 kDa (129.35 kDa of protein plus 15.06 kDa of seven Asn-linked Man9GIcNAc2 glycans). Calculated MW of 3:3:3 complex is 433.24 kDa. The NEO1-NET1-RGMB complex eluted as two peaks with corresponding MW of 422.7 kDa and 117.9 kDa (indicated with red lines). (C, D) SDS PAGE analysis of SEC fractions. Fractions were heated (100 °C, 10 minutes) in the presence or absence of 2-mercaptoethanol (panels C and D, respectively). (E) NEO1FN456 co-elutes with extracellular domain of RGMA (RGMAECD) on SEC, suggesting that NEO1 and RGMA form a binary complex. SEC fractions were analyzed using SDS-PAGE under non-reducing and reducing conditions. Under reducing conditions, the RGMAECD dissociates into two fragments (labelled N-term. and C-term.) due to an autocatalytic cleavage mechanism. SEC fractions containing the binary NEO1-RGMA complex used to form the ternary NEO1-NET1-RGMA complex are indicated. SEC running buffer: 150 mM NaCl, 10 mM HEPES pH 7.5, 2 mM CaCl2, 0.02% NaN3 (flow rate 0.3 ml/min; Superose 6 Increase 10/300 GL column; 21 °C). (F) SDS-PAGE analysis (non-reducing and reducing conditions) of NET1 and NEO1-RGMA used to assemble the ternary NEO1-NET1-RGMA complex for SEC-MALS analysis. Traces corresponding to absorbance at 280 nm, light scattering and molecular masses derived from SEC-MALS are shown in black, blue and red, respectively. Calculated molecular masses based on protein amino acid sequences: NET1ΔNTR, 49.2 kDa plus 3 Asn-linked glycans, 5.6 kDa; FN domains 4–6 of NEO1, 39.2 kDa plus 2 Asn-linked glycans, 3.8 kDa; RGMA, 42.2 kDa plus 3 Asn-linked glycans, 5.6 kDa. Thus, calculated mass of the glycosylated NEO1-NET1-RGMA ternary 3:3:3 complex is 437.0 kDa. The ternary complex dissociated on SEC-MALS as suggested by a major peak corresponding to 79.97 kDa. However, an additional peak corresponding to 444.4 kDa, which is consistent with the NET1:NEO1:RGMA 3:3:3 mol:mol:mol complex, was also observed. (G) FN domains 4–6 of NEO1 co-elute with the full-length extracellular domain of RGMC (RGMCECD) on SEC, suggesting that NEO1 and RGMC form a binary complex. SEC fractions were analyzed using SDS-PAGE under non-reducing and reducing conditions. Under reducing conditions, a fraction of RGMCECD dissociates into two fragments (labelled N-term. and C-term.) as observed for RGMAECD (E). (H) SEC and SDS-PAGE analysis of the ternary NET1–NEO1–RGMC complex. The ternary NEO1-NET1-RGMC complex elutes as two major peaks (12.5 and 13.9 ml peaks) at lower elution volume compared to the binary NEO1-RGMC complex (16.3 ml, G) or NET1 in isolation, suggesting that the NEO1-NET1-RGMC ternary complex forms in solution. SEC running buffer: 150 mM NaCl, 10 mM HEPES pH 7.5, 2 mM CaCl2, 1 mM sucrose octasulfate, 0.02% NaN3 (flow rate 0.3 ml/min; Superose 6 Increase 10/300 GL column; 21 °C). SEC input was 0.6 ml of the ternary complex at 2.6 mg/ml.
Fig 5: Structural analysis of RGM interactors and consequences for the ternary NEO1-NET1-RGM complex, related to Figures 3 and 7.(A-C) Model for BMP2-dependent clustering of the ternary 3:3:3 NEO1-NET1-RGM complex. (A) Ribbon presentation of the ternary NEO1-NET1-RGM complex, with modelled RGMB N-terminal domain based on the full-length RGMB structure (PDB ID. 4UI2). One of the three RGMB N-terminal domains essential for BMP binding is marked with a dotted circle. (B) The ternary complex containing full-length RGMB harbors three distinct binding sites for the disulfide linked BMP dimer (green) (here shown for BMP2). (C) Further addition of the ternary NEO1-NET1-RGM complex and the dimeric BMP2 morphogen can lead to clustering and a continuous arrangement in with RGMB bridges the dimer of BMP2 and the ternary complex. Asterisks mark the “free” RGMB-binding sites on BMP2. (D, E) The RGMB VLK phosphorylation site mapped onto the ternary NEO1-NET1-RGM complex structure. (D) Ribbon representation of the NEO1-NET1-RGMB protomer structure (color coded as in Figure 1). RGMB tyrosine 268 (Y268) that was previously shown to be phosphorylated by VLK is colored in purple and highlighted. (E) Ribbon representation of the NEO1-NET1-RGMB trimer-of-trimers complex. RGMB-Y268 is facing the inside of the ternary complex, and is likely shielded for VLK access.
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