Fig 2: Stimulation with the ephrinB2 specific receptor EphB4 prevents denervation-induced spine loss and rescues GRIP1 deficiency(A) Timeline of prevention and rescue experiments. EphB4-Fc treatment (B4).(B) Representative pictures of dendritic stretches from dentate granule cells in denervated Ctrl and Grip1 T956 > A OTCs following the rescue paradigm.(C) Quantification of mushroom spine density as ratio to DPL0 (upper graph) corresponding to (B). Significance levels shown for Ctrl B4 ND relative to Ctrl Fc ND (black asterisks), for T956 > A Fc D to T956 > A Fc ND (red asterisks) and for T956 > A B4 D to T956 > A Fc D (green asterisks). Values corresponding to DPL21 are represented in the lower graph; n = 6–13 neurons per condition, 3 experiments.(D) Representative pictures of dendritic stretches from dentate granule cells in denervated Ctrl and Grip1 T956 > A OTCs following the prevention paradigm.(E) Quantification of mushroom spine density as ratio to DPL0 (left graph). Significance levels shown for T956 > A Fc D to T956 > A Fc ND (red asterisks) and for T956 > A B4 D to T956 > A Fc D (green asterisks). Values corresponding to DPL2 are represented in the right graph; n = 4–10 neurons per condition, from 2–3 experiments.(F) Representative reconstructions of mushroom spines from Fc- or EphB4-Fc (B4)-treated ND and D Ctrl or Grip1 T956 > A OTCs at DPL21.(G) Quantification of surface GluA2 represented as percentages of the total GluA2 found in a mushroom spine (n = 5 neurons per condition, 2 experiments).Scale bars: 2 μm (B and D), 1 μm (F). Graphs show means ± SEMs. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Exact p values in Table S1.See also Figure S4.

Fig 3: EphrinB2 mediates GRIP1 function in homeostatic plasticity and promotes surface AMPARs(A) Representative pictures of dendritic stretches from ND and D efnB2 S-9 > A and Ctrl OTCs.(B) Quantification of spine (upper graph) and mushroom spine density (lower graph) as ratios to DPL0 corresponding to (A). Significance levels shown for efnB2 S-9 > A D to Ctrl D; n = 5–7 neurons per condition, 3 experiments).(C–F) Representative reconstructions of mushroom (C), thin (D), and stubby (E) spines, and shafts (F) from OTCs (at 21 days in culture) stimulated with EphB4-Fc (B4) or Fc control.(G) Quantification of surface GluA2 in EphB4-Fc stimulated OTCs, represented as percentages of total GluA2 per compartment, corresponding to (C)–(E); n = 5 neurons per condition, 2 experiments.Scale bars: 2 μm (A), 1 μm (C)–(F). Graphs show means ± SEMs. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Exact p values in Table S1.See also Figure S4.

Fig 4: EphrinB2 interacts with VEGFR2 in hippocampal neurons.(A) Cell lysate from primary wild type hippocampal neurons 14 DIV was used for co-immunoprecipitation. To pulldown ephrinB2, the beads were coupled with EphB4 receptor ectodomain fused to Fc (EphB4-Fc). Fc fragment served as control. Western blots performed using anti-VEGFR2 and anti-ephrinB2 antibodies show interaction of ephrinB2 and VEGFR2. (B) Wild type primary hippocampal neuron cultures were cultured for 14 DIV transfected with EGFP and proximity ligation assay (PLA) was performed using EphB4-Fc and an anti-VEGFR2 specific antibody. Magenta puncta represent amplified PLA signal and reflect the interaction between VEGFR2 and ephrinB2. PLA puncta localize to dendritic spine heads and necks labeled with EGFP (green) (indicated by arrowheads). Scale bar: 10 µm. (C) Labeling of hippocampal neuron cultures at 14 DIV with antibodies against PSD95 (green) revealed localization of the PLA signals representing the VEGFR2-ephrinB2 complex (magenta) to postsynaptic sites (indicated by arrowheads). Scale bar: 10 µm. (D–E) Primary wild type hippocampal neurons at 14 DIV were stimulated with VEGF for 30 min and PLA was performed using the EphB4-Fc and an anti-VEGFR2 specific antibody. VEGFR2-ephrinB2 complex (PLA signal, magenta) localizes to early endosomes labeled using antibodies against the early endosomal compartment protein EEA1 (green) and stimulation with VEGF leads to increased localization of the VEGFR2-ephrinB2 complex to early endosomes. Representative images are shown in (D) and quantification of the percentage of PLA puncta colocalizing with EEA1 labeled endosomes is shown in (E) (n = 3 experiments). Scale bar: 10 µm. Data are represented as mean ± SEM. *p<0.05.

Fig 5: EphrinB2 and EphB4 regulate integrin β1 signaling.(Left) Knockdown of ephrinB2 or EphB4 expression resulted in integrin β1 activation defect. HEK293 cells were transduced by lentivirus containing sh-ephrinB2, sh-EphB4, or sh-scrambled RNA control. Puromycin-resistant cells were assessed for 9EG7 binding by FACS analysis. Results represent the mean ± SEM (n = 3) (**, P < 0.01; one-way ANOVA). (Right) Protein expression of ephrinB2 and EphB4 was verified by immunoblotting of SDS–PAGE-fractionated cell lysates with anti-ephrinB2, anti-EphB4, or anti-α-tubulin.Source data are available for this figure.