A particularly important system in which Eph ephrin signalin
A particularly important system in which Eph-ephrin signaling has been widely studied is neural development, in which downstream signal transduction controls neural stem cell (NSC) proliferation, migration, and survival both during early development and in adulthood (19, 20, 21). Furthermore, Eph-ephrin signaling was recently demonstrated to regulate hippocampal neurogenesis in the adult brain, where ephrin-B2 expressed by astrocytes in the NSC sars-cov induces the neuronal differentiation of NSCs via EphB4 signaling (22, 23). The process of NSC-mediated adult neurogenesis plays important roles in learning, memory, and neurological disease (24, 25, 26, 27, 28). Structural experiments have confirmed that ephrin-B2 binds EphB4 receptors to form heterodimers (29), and a recent study has shown increased downstream activity in NSCs with increasingly multivalent ephrin-B2 ligands (23). Thus, biophysical mechanisms of receptor activation likely play a vital role in this therapeutically relevant system. Motivated by the work on the spatiomechanical sensitivity of the EphA2 receptor and the regulatory role of the EphB4 receptor in NSC signaling, we hypothesized that NSC differentiation may be sensitive to spatial manipulation of EphB4-ephrin-B2 physical organization and clustering. Previous studies have induced EphB4 signaling by artificially preclustering ephrin-B2 in solution to generate multimeric receptor-ligand complexes (22, 23). However, these methods do not recapitulate the physical interactions between membrane-bound receptors and ligands. We therefore turned to supported lipid bilayers (SLBs), a system well suited for studying cell-cell contact-dependent signaling (15, 17, 30, 31, 32, 33). Here, we develop a hybrid system to reconstitute the juxtacrine signaling geometry between NSCs and astrocytes by depositing EphB4 receptor-expressing NSCs onto SLBs displaying laterally mobile, monomeric ephrin-B2 ligands. This system provides a physiologically and spatially relevant microenvironment for studying EphB4-ephrin-B2 signaling. It also allows us to precisely control not only the chemical composition of the ligands and membranes but also the physical geometry of receptor-ligand complexes using the technique of spatial mutation (34). Spatial mutation involves the physical control of the spatial patterning of proteins on a lipid bilayer achieved by nanofabricating metal structures on the underlying glass substrates (35, 36). The resulting features guide the movement of supported membrane molecules as well as any engaged cognate receptors on the live cell, thereby controlling the cluster size and number of receptor-ligand complexes that can form (32, 37, 38). Any cellular microenvironmental perturbation, including the spatial mutation, that alters the movement of cell-surface molecules intrinsically imposes mechanical forces onto the cell. Cellular responses to such perturbations are thus spatiomechanically regulated. This, however, does not imply that the receptor system involved directly senses mechanical force. Using this reconstituted juxtacrine signaling platform, we observe EphB4-ephrin-B2 co-clustering and demonstrate membrane-bound monomeric ephrin-B2 activation of EphB4 signaling and downstream neuronal differentiation. Furthermore, by employing spatial mutation, we discover that EphB4 signaling and NSC differentiation are spatially and potentially mechanically modulated. Restricting the motion of supported membrane ephrin-B2 within arrays of small (∼1 μm) grid-patterned corrals was sufficient to abrogate its effects on NSC differentiation, even though similar levels of ephrin-B2 were available to the cell. This result is similar to the spatiomechanical regulation observed in the EphA2-ephrin-A1 system (17, 18), suggesting that such effects may be general to other Eph-ephrin interactions within the family. This work suggests that physical aspects of the NSC niche may impact differentiation and further indicates that this may be significant in the context of regenerative medicine.