Date of Completion

4-27-2018

Embargo Period

4-26-2018

Keywords

Dendritic Filopodia, Dendritic Spines, EphB2, optoEphB2, Eph Receptors

Major Advisor

Ji Yu

Co-Major Advisor

Yi I. Wu

Associate Advisor

Ann E. Cowan

Associate Advisor

Bruce J. Mayer

Associate Advisor

Betty A. Eipper

Field of Study

Biomedical Science

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

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.

Additional Files
Movie 1_1 Cry2 CIBN.avi (978 kB)
Movie 1.1
Movie 1_2 Cry2 Clustering.avi (4050 kB)
Movie 1.2
Movie 2_1 MEF Morphology pJY547 pCL49.avi (1442 kB)
Movie 2.1
Movie 2_2 MEF Local.avi (366 kB)
Movie 2.2
Movie 2_3 MEF Rev Rep.avi (1855 kB)
Movie 2.3
Movie 2_4 Growth Cones.avi (229 kB)
Movie 2.4
Movie 3_1 Single Filopodium Lifeact pQY174_pCL55.avi (278 kB)
Movie 3.1
Movie 3_2 Filopodium Membrane Expansion.avi (228 kB)
Movie 3.2
Movie 3_3 pQY174 pCL55 Protrusions.avi (170 kB)
Movie 3.3
Movie 3_4 LY294002.avi (223 kB)
Movie 3.4
Movie 3_5 LY294002 DMSO Control.avi (154 kB)
Movie 3.5
Movie 3_6 Cytochalasin D.avi (330 kB)
Movie 3.6
Download
COinS