Evaluation of a ligand-directed labeling molecule targeting integrin alphaVbeta5
department or school: Arts and Sciences, Irving K. Barber School of (Okanagan)
data provider: DSpace
degree campus: UBCO
creator: Slattery, Wyatt Thomas
issued: 2018-12-13T18:28:16Z
issued: 2018
related degree: Master of Science - MSc
degree grantor: University of British Columbia
description: Integrins are transmembrane receptors that mediate cellular adhesion to the extracellular matrix and initiate signaling cascades that may be relevant to synaptic elimination events occurring in neurodegenerative disease. To study the role of integrins in synaptic elimination, a novel ligand-directed labeling molecule has been created, comprised of the Pacific Blue™ (PB) fluorophore and the high-affinity integrin inhibitor, cilengitide. It was hypothesized that the function of integrin αVβ5 could be studied in live cells by using this probe to track subcellular integrin localization through fluorescence microscopy. This study evaluated the pharmacodynamic properties of the PB-probe, finding that its fluorescence excitation and emission properties are similar to PB and highly photostable over the time period of at least 2 h. Fluorescence enhancement and anisotropy experiments revealed that a derivative of the probe binds integrin αVβ5 with a dissociation constant of 0.11 ± 0.07 μM. Binding was competitively inhibited by cilengitide, suggesting that both molecules compete for the integrin binding domain with similar affinities. Application to BV-2 murine microglia in vitro confirmed that the PB-probe is a robust and rapidly applicable labeling method for the fluorescence detection of endogenous integrins. These experiments demonstrated that PB-probe labeling is specific, amenable to a variety of sample preparation methods, and compatible with simultaneous immunocytochemistry techniques. Separately, the PB-probe was found to inhibit integrin-mediated signaling cascades with equal efficacy as cilengitide. The results of this study confirm that the PB-probe retains both similar fluorescent properties as PB and the integrin-binding properties of cilengitide. These findings establish the PB-probe as an effective means of tracking endogenous integrin αVβ5 in live-cell systems, representing a critical first step in understanding the role of integrin signaling in synaptic elim…
aggregated CHO: https://circle.library.ubc.ca/rest/handle/2429/68069
note: Evaluation of a ligand-directed labeling molecule targeting integrin alphaVbeta5 by Wyatt Thomas Slattery A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science in The College of Graduate Studies (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) December 2018 © Wyatt Thomas Slattery, 2018 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis/dissertation entitled: Evaluation of a ligand-directed labeling molecule targeting integrin alphaVbeta5 Submitted by Wyatt Thomas Slattery in partial fulfillment of the requirements of the degree of Master of Science. Dr. Frederic Menard, Irving K. Barber School of Arts and Sciences Supervisor Dr. Andis Klegeris, Irving K. Barber School of Arts and Sciences Supervisory Committee Member Dr. Kirsten Wolthers, Irving K. Barber School of Arts and Sciences Supervisory Committee Member Dr. Michael Deyholos, Irving K. Barber School of Arts and Sciences University Examiner iii Abstract Integrins are transmembrane receptors that mediate cellular adhesion to the extracellular matrix and initiate signaling cascades that may be relevant to synaptic elimination events occurring in neurodegenerative disease. To study the role of integrins in synaptic elimination, a novel ligand-directed labeling molecule has been created, comprised of the Pacific BlueÔ (PB) fluorophore and the high-affinity integrin inhibitor, cilengitide. It was hypothesized that the function of integrin αVβ5 could be studied in live cells by using this probe to track subcellular integrin localization through fluorescence microscopy. This study evaluated the pharmacodynamic properties of the PB-probe, finding that its fluorescence excitation and emission properties are similar to PB and highly photostable over the time period of at least 2 h. Fluorescence enhancement and anisotropy experiments revealed that a derivative of …
has type: Thesis/Dissertation
date issued: 2019-02
is shown at: 10.14288/1.0375728
language: eng
degree discipline: Biochemistry and Molecular Biology
provider: Vancouver : University of British Columbia Library
publisher: University of British Columbia
rights: Attribution-NonCommercial-NoDerivatives 4.0 International
rights URI: http://creativecommons.org/licenses/by-nc-nd/4.0/
scholar level: Graduate
type: Text
identifier URI: http://hdl.handle.net/2429/68069