br Materials and Methods br Results
Materials and Methods
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. Ephs and ephrins are known to exhibit a high level of cross talk among family members, so ephrin-B2 on SLBs may interact with other Eph types on NSCs. Indeed, antibody blocking experiments suggested that both EphB4 and EphB2 were responsible for NSC binding, but as concurrent blocking did not completely ablate adhesion, other Ephs were likely interacting as well. In addition to EphB4 and EphB2, ephrin-B2 has been shown to bind EphB1 (52), EphB3 (53), EphB6 (54), and EphA4 (55). However, EphB4 was confirmed to be largely responsible for transducing the biological activity of ephrin-B2 signaling, as blocking EphB4 abrogated neuronal differentiation (22). Using this system, we characterized the spatiomechanical sensitivity of EphB4-ephrin-B2 signaling on induced NSC neurogenesis. On 3-μm grid-patterned substrates but not 5 μm grids or nongrid control patterns, neurogenesis was significantly reduced. Importantly, the patterned substrates all presented roughly the same density of ephrin-B2, and Cr grids served only as LY2940680 barriers to restrict the movement of lipid molecules and ephrin-B2 ligands (Fig. S4). The change in differentiation, therefore, was not due to the quantity of ephrin-B2 available. Additionally, the length scale associated with the spatial mutations was on the order of microns, which is far larger than the nanoscale dimensions of molecular interactions. Hence, molecular-scale clustering of ligand-receptor complexes were likely not disrupted even in the smallest grids. In all corrals, visible microclusters formed, and proximal signaling data revealed that known EphB4-ephrin-B2 induced-phosphorylation cascades were unaffected (Fig. 4 E). In particular, we examined pan-phosphorylated-tyrosine and known EphB4-ephrin-B2 signaling targets, including phosphorylated-ERK and active β-catenin. Furthermore, as is evident from Fig. 4 D, the cell footprints are of similar size across variable grid sizes. Because the grids are substantially smaller in scale than the cell, ligands will not gather from outside the cell footprint. Thus, we conclude that the total amount of ligand exposed to the cells remained unchanged by varying grid size. In summary, we observe a clear effect from physically restricting the movement and assembly of EphB4-ephrin-B2 signaling clusters on NSC differentiation. Although the observed EphB4-ephrin-B2 clusters on all patterned substrates were apparently sufficient to induce downstream activation of several targets (Fig. 4 E), it is possible that restricting cluster size and microscale spatial organization on the cell membrane impacts downstream signaling. We have shown that increased oligomerization on the nanoscale induces higher levels of neurogenesis (23), but the role of microscale clustering remains undetermined. Alternatively, we have shown that the mechanical properties of the cellular microenvironment regulate NSC differentiation (56), and mechanical forces could conceivably play a role in the observed behavior. That is, the spatial mutation method utilized here intrinsically imposes mechanical forces on the receptor-ligand complexes, and a number of studies have demonstrated mechanical regulation of transmembrane receptors due to physical properties of ligand presentation, such as lateral mobility (57, 58) and tugging forces at cell-cell junctions (59). Future work may explore the relative roles in biochemical and/or biomechanical signaling in mediating the effects of spatiomechanical perturbations on downstream NSC behaviors.