Effects of Localized EphB2 Stimulation on Dendritic Filopodia of Hippocampal Neurons

Dendritic filopodia are thin, dynamic neuronal protrusions that make contact with axons. Following contact formation, they are hypothesized to transform into dendritic spines, the mushroom-shaped post-synaptic compartments that are believed to function in learning and memory formation. EphB2 is a receptor tyrosine kinase important for dendritic spine formation and thought to mediate the transition from dendritic filopodia to dendritic spines. The goal of this study is to understand how EphB2 signaling affects the motility and morphology of dendritic filopodia to aid in this transition. An optogenetic tool for EphB2 signaling, optoEphB2, was developed to achieve localized activation of EphB2 signaling at dendritic filopodia. In response to photoactivation, optoEphB2 displayed increased tyrosine phosphorylation, recruited SH2 domains known to bind EphB2, and caused an expected cell collapse phenotype in 3T3 cells and growth cones, thereby validating optoEphB2 function. In rat hippocampal neurons, optoEphB2 induced branch formation in filopodia via the actin nucleators Arp2/3 and N-WASP. Additionally, de novo filopodia formation was observed following optoEphB2 stimulation on dendritic shafts, dependent on Arp2/3, N-WASP, PI3K, and Arg. These downstream signaling mediators are known to independently regulate dendritic spine formation, and Arp2/3 and N-WASP specifically mediate actin branching. Thus, EphB2 signaling may contribute to dendritic spine formation via the formation of new filopodia and may directly stimulate formation of the highly-branched actin network that characterizes the dendritic spine head. Since EphB2 signaling is also important for multiple cell processes, including cell migration and oncogenesis, optoEphB2 will be valuable to study many other biological processes