University of Twente Student Theses


Polarization microscopy for visualizing FRET between identical fluorophores : monitoring receptor clustering in live cells

Krabbendam, Rick (2012) Polarization microscopy for visualizing FRET between identical fluorophores : monitoring receptor clustering in live cells.

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Abstract:Clustering behavior of plasma membrane receptors such as the epidermal growth factor receptor (EGFR) is intensively investigated. Learning about this clustering behavior provides information about the biological role and function of the EGF-receptor in general and possibly about its role in the development of cancer. We investigated the clustering behavior of EGFR using fluorescent probes. Förster resonance energy transfer between identical fluorescent probes (homo-FRET) in an EGFR cluster can be visualized by its effect on the fluorescence anisotropy. In order to do so a fluorescence polarization microscope was implemented in a wide-field fluorescence microscope. The possibility to observe homo-FRET in live cells by a decrease in anisotropy using this microscope was verified with a model protein consisting of two closely spaced fluorescent proteins connected by an amino acid linker. In this model protein, homo-FRET can occur. It was also verified that homo-FRET cannot be observed by a decrease in the fluorescence lifetime of the donor. The effect of homo-FRET on the fluorescence anisotropy is simulated based on equations describing photophysical properties of fluorescent molecules. Results from these simulations show that the maximum decrease in anisotropy due to homo-FRET between a single donor and a single acceptor is 0.20 for randomly orientated non-rotating fluorophores. When there is a small angle between the absorption and emission transition moment in a single fluorophore, the anisotropy decrease due to homo-FRET is smaller. Then the polarization microscope was used to obtain anisotropy images of EGF receptor and model systems. The clustering behavior of the receptor was altered by addition of its ligand EGF or the addition of antibodies against EGFR which may induce dimerization. This experiment uses MCF-7 and NIH-3T3 cells expressing EGFR protein fusions consisting of EGFR and a fluorescent protein and protein fusions consisting of EGFR and a fluorescently labeled SNAP-tag. No change in anisotropy was found after the addition of EGF or antibodies compared to before. The anisotropy did also not depend on the intensity. Also in cells expressing SNAP-TMD-Fv, a model system that can be dimerized upon addition of a dimerizer molecule (AP20187), no significant change in anisotropy was found within five minutes after addition of this dimerizer molecule. In literature can be found that TMD-Fv protein domains and EGFR protein domains dimerize within five minutes after addition of AP20187 and EGF respectively, therefore we conclude that the used fluorescent probes are too far away from each other for homo-FRET to occur.
Item Type:Essay (Master)
Faculty:TNW: Science and Technology
Subject:33 physics
Programme:Applied Physics MSc (60436)
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