Nanoscale Tools to Expand the Biophysical Understanding of Epidermal Growth Factor Receptor Function Öffentlichkeit
Stabley, Daniel Ryan (2014)
Abstract
Modern techniques in biochemistry and fluorescence imaging have done much to
reveal the vast chemical signaling networks associated with cell surface receptors,
however many aspects of receptor function and regulation remain obscure. This is
especially evident in the case of the epidermal growth factor receptor (EGFR), a longstudied
receptor tyrosine kinase. While the signaling networks of the EGFR are largely
known, there are still critical elements of receptor function that have not been elucidated.
For example, the EGFR has long been shown to self-assemble during the process of
activation, but the precise functional role of this oligomerization is not well defined.
Additionally, mechanical forces are thought to play a role during receptor endocytosis,
but direct evidence has not been obtained.
This dissertation describes the development of novel methods to answer longstanding
questions about cell receptor function that are difficult to answer with current
techniques. Chapter 1 gives a brief history of the EGFR as well as an overview of the
known aspects of its signaling networks and corresponding regulatory elements. Chapter
2 describes the development of a fluorescence force sensor that can directly measure
forces exerted by the cell through receptor-ligand interactions. This sensor was used to
visualize the force exerted on the EGFR during endocytosis, and remains as a facile and
broadly applicable method for the characterization of mechanotransduction events in
living cells. In chapter 3 we apply recently developed supported lipid bilayer
nanopatterning techniques to determine whether the formation of micron-sized EGFR
oligomers impacts biochemical signaling output. Supported lipid bilayers were formed on
surfaces modified with diffusion barriers and were subsequently functionalized with the
epidermal growth factor (EGF), a ligand for the EGFR. By controlling the size of the
diffusion barriers oligomer size could be controlled, and pairing this approach with
standard immunofluorescence staining revealed a correlation between the cluster size and
signaling output. Chapter 4 details the development of simultaneous two-wavelength
axial ratiometry (STAR), a new fluorescence microscopy technique based on total
internal reflection fluorescence microscopy (TIRF-M) that allows nanoscale imaging
along the optical axis with millisecond time resolution. This technique was used to
investigate the dynamics of EGFR internalization and stands as a turn-key method to
study cell membrane processes with high spatial and temporal resolution. The final
chapter gives a summary of these techniques as they have been implemented to date, and
also describes future directions for their continued application. These approaches serve to
expand the modern biochemical toolkit, allowing access to previously unknown aspects
of long-studied biological systems.
Table of Contents
Chapter 1: The EGFR Signaling Pathway
1.1 Introduction...2
1.1.1 Historical Perspectives...2
1.1.2 Impact on Human Health...5
1.2 Current Models of EGFR Signaling...6
1.2.1 Critical Events in EGFR Signaling...6
1.2.2 EGFR Endocytosis...10
1.3 The Aims and Scope of This Dissertation...13
1.4 References...16
Chapter 2: Visualizing Mechanical Tension Across Membrane Receptors with a
Fluorescent Sensor
2.1 Introduction...21
2.2 Sensor Design and Characterization...22
2.3 Imaging Force in Live Cells...27
2.4 Correlating Force to Endocytosis...31
2.5 Quantification of Force Signals...33
2.6 Conclusion...35
2.7 Author Contributions and Acknowledgments...37
2.8 Methods...38
2.9 References...48
Chapter 3: Manipulating the Lateral Diffusion of Surface-Anchored EGF
Demonstrates that Receptor Clustering Modulates Phosphorylation Levels
3.1 Introduction...51
3.2 Experimental Platforms to Study EGFR Clustering...54
3.3 The Formation of Clusters Affects Signaling...56
3.4 Cluster Size Modulates Receptor Phosphorylation...60
3.5 Investigating the Mechanisms of EGFR Cluster Formation...66
3.6 Conclusion...69
3.7 Acknowledgements...71
3.8 Methods...71
3.9 References...8
Chapter 4: Simultaneous Two-Wavelength Axial Ratiometry (STAR) for Imaging
Protein Dynamics with sub-15 nm Accuracy in Living Cells
4.1 Introduction...83
4.2 Total Internal Reflection Fluorescence (TIRF)...83
4.3 Theory...86
4.4 The Effects of FRET on STAR Imaging...88
4.5 Proof of Concept...93
4.6 Mapping Microtubule z Positions with STAR...96
4.7 Real-time Imaging of EGFR Internalization...97
4.8 Conclusion...102
4.9 Methods...103
4.10 References...108
Chapter 5: Conclusion
5.1 Perspectives...110
5.2 The Role of force in EGFR endocytosis...110
5.3 The effect of clustering on EGFR signaling...115
5.4 Pushing the boundaries of optical microscopy along the z axis...117
5.5 Final Comments...119
5.6 References...120
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