Date of Completion

1-9-2018

Embargo Period

7-8-2018

Keywords

Plexin-B1, Optogenetics, Contact Inhibition of Locomotion, Osteoblasts, Osteoclasts

Major Advisor

Yi I. Wu

Co-Major Advisor

Carol C. Pilbeam

Associate Advisor

John Carson

Associate Advisor

Leslie Loew

Associate Advisor

Ann Cowan

Associate Advisor

Kimberly Dodge-Kafka

Field of Study

Biomedical Science

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Bone remodeling is critical for maintenance of bone health and requires a balance of bone resorption by osteoclasts followed with formation by osteoblasts. These opposing activities are spatially and temporally segregated and coordinated on the bone surface. Chemoattraction between osteoclasts and osteoblasts has been extensively investigated and has been proposed to direct osteoblasts to the site of bone resorption. However, the mechanism by which osteoclasts and osteoblasts are spatially segregated on the bone surface remains unknown. In this thesis, I demonstrate that osteoclasts induce contact inhibition of locomotion in migrating osteoblasts, which may enable spatial segregation of these cells. I identify Semaphorin 4D, expressed on the surface of osteoclasts, and its receptor Plexin-B1, expressed by osteoblasts, to be critical for the induction of contact inhibition of locomotion. I developed an optogenetic tool to initiate Plexin-B1 signaling using blue light, named optoPlexin. Localized stimulation of optoPlexin in subcellular regions elucidated spatial and temporal dynamics of signaling pathways involved in mediating contact inhibition of locomotion. In addition, I used optoPlexin to develop a novel protein-protein interaction assay in live cells, which revealed a previously unknown role of the RGS-like domain of PDZ-RhoGEF in mediating recruitment by Plexin-B1. This thesis provides new understanding of the interactions between osteoclasts and osteoblasts during bone remodeling, illustrates the impact of localized Semaphorin 4D-Plexin-B1 stimulation in migrating osteoblasts, and provides new information on the molecular mechanism of Plexin-B1 signaling. The observations on spatial regulation of signaling pathways in cells undergoing contact inhibition of locomotion are pertinent to understanding the molecular pathways mediating cell repolarization. Furthermore, the novel optogenetic approach utilized to locally stimulate Plexin-B1 may be extended to other plexins, and potentially to other repulsive-guidance molecules. Thus, this work presents a significant advance not only in the field of bone biology and semaphorin-plexin signaling, but cell biology in general.

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