Degree Type

Dissertation

Date of Award

2018

Degree Name

Doctor of Philosophy

Department

Chemistry

Major

Analytical Chemistry

First Advisor

Emily A. Smith

Abstract

Receptors on the cell membrane initiate cell signaling upon stimulation with extracellular molecules; this results in signal transduction pathways. Receptors are redistributed as clusters upon extracellular stimuli with the change of receptor dynamics. The lateral diffusion of receptors plays a role in how receptors interface with other membrane proteins, extracellular ligands, intracellular proteins, and how receptors function. In this dissertation, the lateral diffusion and clusters of receptors for advanced glycation end-products (RAGE) at both a single molecule and the ensemble level are discussed. The effects of extracellular ligands, cholesterol levels, and actin polymerization on RAGE diffusion are considered.

RAGE is a member of the immunoglobulin superfamily of membrane proteins that are involved in numerous pathological conditions. Ligand binding to the extracellular domain of RAGE drives RAGE clusters and initiates the downstream signal transduction. The signal transduction also consists of the interaction between diaphanous-1 (Diaph1) with the cytoplasmic tail of RAGE. Diaph1 affects the nanoscale clustering and diffusion of RAGE as measured by super-resolution stochastic optical reconstruction microscopy (STORM) and single particle tracking (SPT). A reduced expression level of Diaph1 or a disrupted interaction between Diaph1 and RAGE results in a decreased size and number of RAGE clusters. RAGE diffusion is increased after reduced Diaph1 expression. In contrast, when the interaction site between RAGE and Diaph1 is mutated, RAGE diffusion is slowed.

The effect of the ligand on the lateral diffusion of the receptor for advanced glycation endproducts involved in numerous pathological conditions. A methylglyoxal-modified bovine serum albumin (MGO-BSA) RAGE ligand is prepared and characterized. The effect of MGO-BSA on the lateral diffusion of RAGE is measured by SPT. Ligand incubation affects RAGE diffusion and the phosphorylation of extracellular signal-regulated kinases. However, there is no correlation between MGO-BSA ligand binding affinity and the change in RAGE diffusion. Moreover, the mechanism for the ligand-induced change in RAGE diffusion is dependent on cholesterol.

The actin cytoskeleton plays a crucial role in RAGE functions. The effect of the actin cytoskeleton on RAGE diffusion is measured by fluorescence recovery after photobleaching at the ensemble-level. When depolymerization of the actin cytoskeleton is inhibited, RAGE diffusion and mobile fraction are decreased. Also, when the actin cytoskeleton is disrupted, the phosphorylation of extracellular signal-regulated kinase is reduced.

Copyright Owner

Qiaochu Zhu

Language

en

File Format

application/pdf

File Size

144 pages

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