Previous studies have induced EphB

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 AP-III-a4 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.
Materials and Methods
Results
Discussion The spatial properties of receptor-ligand interactions can influence receptor activation and signal propagation, but studying this phenomenon requires the development of systems capable of recapitulating complex biophysical traits. In this study, we simulated the juxtacrine geometry of Eph-ephrin signaling transduced by ephrin-B2-presenting astrocytes in contact with EphB4-expressing NSCs (22). By displaying laterally mobile monomeric ephrin-B2 on SLBs, we mimicked the membrane presentation of ephrin-B2. Furthermore, we were able to probe the role of membrane receptor spatial organization in NSC signaling and differentiation using the technique of spatial mutation. The key technical advance enabling these days-long studies was the development of a DNA-SNAP-tag conjugation method providing stable ligand presentation for the duration of bilayer stability. In our hands, bilayers remained intact for 12–24 h.