The work described by Dong Groves and co workers tested
The work described by Dong, Groves, and co-workers (11) tested the hypothesis that neural stem cell development is sensitive to the clustering and mechanical state of the EphB4:ephrin-B2 receptor-ligand complex. They show that the quality of ephrin-B2-ligand signaling depends on its biophysical state, which controls the fate of neural stem cells. This type of regulation transcends the more conventional dogma of RTK signaling, in which ligand binding alone regulates signaling. Rather, this work suggests that physical processes need to be considered because of their distinct roles in regulating the signaling outputs of this class of receptors. It was previously shown that to trigger EphB4 activation, the clustered soluble ectodomain of ephrin-B2 was required (9, 12). However, this type of ligand departed from the native ligand, which is presented on the plasma membrane of an apposed cell. To better capture this geometry, Groves and co-workers employed supported lipid bilayers (SLBs) encoded with ligands as a “surrogate” astrocyte for neuronal stem cell binding (3, 5, 6, 7, 8, 13) (Fig. 1). This artificial system mimics the signaling geometry between neural stem ecopipam kinase and astrocytes because the receptor-ligand complexes formed here are laterally mobile and thus allow for long-range rearrangements. To prevent rapid dissociation of the ligand from the SLB, the authors developed a versatile chemical conjugation strategy, DNA-SNAP-tag coupling, to tether the ephrin-B2 ligand to the synthetic SLB for long-term ligand display with significantly improved stability. In this approach, ephrin-B2 is genetically encoded with the SNAP-tag enzyme, which covalently links with benzylguanine-conjugated oligonucleotides at room temperature and with high yield. The DNA oligonucleotide can anchor the ephrin-B2 to the SLB, provided a complementary oligonucleotide is present on the SLB. For reference, the most common strategy to tether proteins onto phospholipid membranes is through nickel-NTA-polyhistidine chelation, which is simple to perform but suffers from limited stability in typical cell-culture media (13). Therefore, the DNA-SNAP-tag offers a highly efficient strategy to anchor proteins to phospholipid membranes for 12–24 h under cell-culture conditions. Next, the authors decided to investigate the role of receptor clustering in its signaling functions. In a key experiment, the authors used “spatially mutated” SLBs (3). This is an interesting strategy in which the SLB is assembled onto a glass slide that is nanofabricated with a grid of 100-nm-wide metal lines (1). The metal grid is flush with the surrounding SLB but functions as a barrier to the diffusion of the phospholipids, and it therefore confines the ephrin-B2 ligand into specific boxes. Lateral clustering of the EphB4:ephrin-B2 complex is, in turn, limited because of the geometry of the metal grid. Using these spatially mutated EphB4 complexes, the authors found that the activation of the receptor and its downstream signaling targets were not impacted by the diffusion barriers that impaired clustering. Specifically, Western blotting showed that phosphor-extracellular-signal-regulated-kinase and active β-catenin levels were not changed on nanopatterned substrates that limit lateral clustering of the EphB4 receptor. However, hindering receptor clustering with smaller grid spacing showed a significant reduction in neural stem cell differentiation 5 days post seeding. This result suggests that although immediate downstream signaling (phosphorylation of targets) was not directly altered with receptor clustering, the “quality” of the ephrin-B2 signal was modulated by its spatial organization and potentially by the magnitude of the mechanical resistance imposed on the ligand-receptor complex. The importance of the work is twofold. First, the work points to the potential general principles of how biophysical processes are intimately linked with the signaling functions for juxtacrine receptor-ligand interactions. Thus, this particular ligand-receptor pair may represent the tip of the iceberg, and many more and different receptor-ligand pairs that partake in juxtacrine signaling may be regulated by physical mechanisms of spatial organization, confinement, clustering, and mechanical tension. Second, the article provides a set of tools that can be broadly adopted by the community to investigate these biophysical mechanisms of signal regulation. The integration of chemical biology approaches for linking proteins to supported lipid membranes along with the nanolithography and microscopy offer an important toolset to elucidate the role of physical perturbations in Eph signaling, in particular, and more broadly defined juxtacrine signaling. The integration of newly developed probes to quantify molecular forces (14, 15) onto the EphB4 complex will ultimately reveal the contribution of mechanics and clustering to this type of signal regulation.