Nanoscale Tools to Expand the Biophysical Understanding of Epidermal Growth Factor Receptor Function Open Access

Stabley, Daniel Ryan (2014)

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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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