Optogenetic Control of EphB Signaling in Dendrites

Abstract

EphrinB receptors (EphBs) are receptor tyrosine kinases that promote dendritic spine morphogenesis by altering local actin polymerization at axo‐dendritic contacts. Soluble ligands have been used with neuronal cultures to activate EphBs, though local stimulation is more desirable to model local contact and study downstream signaling dynamics. Since EphB signaling requires receptor clustering, we designed an optogenetic tool, named optoEphB, based on an optimized mutant of the plant protein cryptochrome 2 (Cry2olig, residues 1‐498 with E490G mutation), which shows blue light‐induced clustering. OptoEphB consists of the EphB2 cytoplasmic domain fused to Cry2olig and mCherry at the C‐terminus with myristoylation at the N‐terminus. Global and focal blue light illumination of HEK293 cells expressing optoEphB showed reversible optoEphB clustering and retraction of membrane protrusions, indicative of EphB2 signaling. In hippocampal neurons, focal illumination of dendrites produced transient, dynamic protrusions that resembled dendritic filopodia. We observed recruitment of the pleckstrin homology domain of Akt in illuminated regions, suggesting phosphoinositide 3‐kinase (PI3K) activation in this process. Our results demonstrate that optoEphB locally activates EphB signaling and thus represents a novel optogenetic method of Eph receptor activation. Further experiments will determine PI3K‐dependence of filopodial protrusions and use biosensors to measure the spatiotemporal dynamics of downstream Rho GTPase activity, implicated in EphB‐driven spine morphogenesis. These results will bring new insights into current models of dendritic spine development.