Degree Type

Dissertation

Date of Award

2016

Degree Name

Doctor of Philosophy

Department

Chemistry

Major

Analytical Chemistry

First Advisor

Emily A. Smith

Abstract

Systematic spatial and temporal fluctuations are a fundamental part of any biological process. For example, lateral diffusion of membrane proteins is one of the key mechanisms in their cellular function. Lateral diffusion governs how membrane proteins interact with intracellular, transmembrane, and extracellular components to achieve their function. Herein, fluorescence-based techniques are used to elucidate the dynamics of receptor for advanced glycation end-products (RAGE) and integrin membrane proteins.

RAGE is a transmembrane protein that is being used as a biomarker for various diseases. RAGE dependent signaling in numerous pathological conditions is well studied. However, RAGE lateral diffusion in the cell membrane is poorly understood. For this purpose, effect of cholesterol, cytoskeleton dynamics, and presence of ligand on RAGE lateral diffusion is investigated. RAGE diffusion in the cell membrane is probed with fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT). The ensemble diffusion measurement of RAGE with FRAP indicated that RAGE diffuses freely in the cell membrane with a large mobile fraction. Also, a decrease in the mobile fraction is observed when the actin cytoskeleton dynamics are altered with cytoskeletal drugs cytochalasin-D and Jasplakinolide. Further, RAGE signaling is observed to be dependent on an intact actin cytoskeleton in cells. On the other hand, confined diffusion of RAGE is measured when the lateral diffusion is probed one protein at a time with SPT. Methylglyoxal modified-bovine serum albumin (MGO-BSA) is used as a RAGE ligand to study how the presence of ligand affects RAGE lateral diffusion. RAGE’s affinity for ligand increases linearly with the net negative surface charge on the MGO-BSA. Although incubation of MGO-BSA with different percent primary amine modification changes the diffusion properties of RAGE, no correlation is measured in the magnitude of the change in the diffusion properties with the ligand binding affinity and the net negative surface charge. Ligand induced changes are not present when cholesterol is depleted from the cell membrane, indicating that cholesterol plays a role in ligand induced changes on RAGE lateral diffusion. The work advances our understanding of factors that affect RAGE lateral diffusion and signaling and also the connection between RAGE lateral diffusion and signaling.

The effect of a highly conserved membrane proximal cysteine (Cys1368) residue on the diffusion properties of integrins in Drosophila S2 cells is elucidated using FRAP and SPT. Mutating Cys1368 to Val1368 altered the lateral diffusion properties of this receptor. Both FRAP and SPT indicate larger mobile population for the Val1368 integrins compared to wild-type integrins. Tandem mass spectrometry results and protein sequence analysis show that Cys1368 is a potential palmitoylation or redox post-translational modification site.

Finally, a method for imaging protein distribution and for probing local environment around fluorophore in cells at sub-diffraction spatial resolution is discussed. Stimulated emission depletion coupled with fluorescence lifetime microscopy (STED-FLIM) is used to image the actin cytoskeleton with 40-nm spatial resolution at ambient conditions. The quality of the fluorescence lifetime data is evaluated by time binning the photon data to increase the number of photons in the peak channel of the fluorescence decay curve. STED-FLIM can be used to image the distribution of protein in the cell membrane or STED-FLIM can be coupled with complementary techniques such as fluorescence correlation spectroscopy to obtain the diffusion properties of membrane proteins.

Copyright Owner

Aleem Syed

Language

en

File Format

application/pdf

File Size

233 pages

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