Date of Award

Spring 5-15-2021

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Computational & Molecular Biophysics)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Glucagon is secreted from pancreatic islet alpha-cells in response to hypoglycemia. The regulation of this secretion likely involves multiple interacting molecular pathways. There are three general types of proposed models for glucose-dependent regulation of glucagon secretion: direct regulation by glucose mediated modulation of cell electrophysiology, paracrine regulation by other endocrine cell types within the islets of Langerhans, and juxtacrine regulation by surface protein interactions from neighboring beta-cells. This work is focused on one pathway of juxtacrine regulation that occurs through signaling from EphA4 receptors on the surface of α-cells upon interaction with ephrin-A5 ligands on the surface of beta-cells. In this pathway, stimulation of EphA4 leads to a dense F-actin network that inhibits the secretion of glucagon vesicles. In this work, we present evidence that RhoA, a Rho family GTPase, links EphA4 stimulation to F-actin polymerization and is required for the formation of cortical actin networks. Pharmacological inhibition of RhoA, but not Cdc42 or Rac1, resulted in the disruption of proper glucose-inhibition of glucagon secretion. We showed that EphA4 stimulation increase RhoA activity through direct visualization and quantification using a FRET biosensor reporting on RhoA activity. Using a fluorescent nanobody for F-actin, we also showed that RhoA activity and F-actin formation are correlated. Further, quantitative imaging of intracellular Ca2+ using Ca2+ biosensors showed that RhoA activity modulates intracellular Ca2+ activity. These results implicate RhoA as the key regulator of F-actin in the EphA/Ephrin-A juxtacrine regulation pathway and suggest a larger role of RhoA as a signaling hub facilitating crosstalk between the different regulatory pathways.

Language

English (en)

Chair and Committee

David W. Piston Kathleen Hall

Committee Members

Thomas J. Baranski, Anders E. Carlsson, Maria S. Remedi,

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