QUANTIFICATION OF EPIDERMAL GROWTH FACTOR RECEPTOR DYNAMICS AND INTERACTIONS IN LIVING CELLS BY FLUORESCENCE CORRELATION AND CROSSCORRELATION SPECTROSCOPY MA XIAOXIAO (B.Sc.(Hons.) Beijing Normal University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2014 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. MA Xiaoxiao i Acknowledgements For this thesis, there are many people to whom I would like to express my deep appreciation. I would like to thank my supervisor Professor Dr. Thorsten Wohland for his guidance during my PhD project. Thorsten is one of the few people I've ever met who really love their work. I hope that in my life I could be as lively, enthusiastic and energetic as he is. I will always think fondly of my time as a student in his lab. I also thank my co-supervisor Dr. Sohail Ahmed for his advice for my PhD projects. Sohail has given me the freedom to pursue various projects without objection, even when projects were beyond his focus. I will never forget the support from both of them. I also thank the members of my PhD committee, Professors Rachel Susan Kraut, Qing-hua Xu, and Ganesh Srinivasan Anand for their helpful advice and suggestions in general. I will forever be grateful to Dr Yong Hwee Foo for his scientific advice and knowledge and many insightful discussions and suggestions. He was my primary resource for getting my science questions answered for many years and was extremely helpful in helping produce this thesis. I will also be thankful to all my colleagues in both Thorsten's and Sohail's lab for their warm friendship, especially Dr. Jagadish Sankaran, Ms Xi Wang, Ms Guangyu Sun, Mr. Nirmalya Bag, and Ms Shuangru Huang for their various support and discussion, I couldn't have finished the projects smoothly without them. I was lucky to know them and the time I spent with all of them was happy and will be unforgettable through all my life. I also thank people who were not part of the labs but supported me including my parents and friends (too many to list here). My parents, Mr Baochao Ma and Ms Runhua Cui, always encourage me to pursue the life and career I want to have. They are my couragegenerator all the time whenever I was sad or lost. Yi Zhu has been so helpful before and right after I firstly arrived to NUS. She was instrumental in helping me through my candidacy and I am deeply grateful to her. Last but not least, I want to sincerely thank my best friend, Juan Cheng, for her consistent positive attitude despite the situation and numerous times of helping me out. I know that when we are old, Juan will still be there as a supportive and caring friend. ii Publication list  Ma X, Foo YH, Wohland T. "Fluorescence Cross-Correlation Spectroscopy (FCCS) in Living Cells", Methods Mol Biol. 2014;1076:557-73.  Kay JG, Koivusalo M, Ma X, Wohland T, Grinstein S. "Phosphatidylserine dynamics in cellular membranes.", Mol Biol Cell. 2012 Jun;23(11):2198-212. Epub 2012 Apr 11.  Ma X, Ahmed S, Wohland T. "EGFR activation monitored by SW-FCCS in live cells", Front Biosci (Elite Ed). 2011 Jan 1;3:22-32. iii Table of Contents DECLARATION . i Acknowledgements . ii Publication list . iii Table of Contents . iv Summary viii List of Tables x List of Figures xi List of Symbols and Abbreviations xiii Chapter 1. Introduction . 1.1 Epidermal Growth Factor Receptor (EGFR) 1.1.1 The importance and clinical trials 1.1.2 The structure of EGFR . 1.1.3 The cycle of EGFR in a cell . 1.1.4 Interaction of EGFR upon activation (signalling pathway map) . 10 1.1.5 EGFR in the nucleus 13 1.1.6 Dimerization of EGFR . 14 1.1.7 EGFR and lipid raft 16 1.1.8 EGFR and the cytoskeleton 22 1.2 Other members of ErbB family . 25 1.2.1 ErbB2 . 25 1.2.2 ErbB3 . 26 1.2.3 ErbB4 . 27 Chapter 2. FCS theory . 29 2.1 Fluorescence Correlation Spectroscopy (FCS) . 29 2.2. Derivatives of FCS . 31 2.3. Principle of confocal FCS and SW-FCCS 32 2.3.1. Theory of FCS . 32 2.3.2. Theory of FCCS 35 2.3.3. Calibration for FCCS 39 2.3.4 Instrumentation of SW-FCCS 39 2.4. Imaging Total Internal Reflection-FCS (ITIR-FCS) . 40 iv 2.4.1 Total Internal Reflection (TIR) illumination 40 2.4.2 ITIR-FCS . 42 2.4.3 Instrumentation of ITIR-FCS . 48 2.5. FCS diffusion law 50 2.5.1 Theory 51 2.5.2 Diffusion law in confocal and TIRF FCS setup . 52 2.6 Objectives and significance of the study . 53 Chapter 3. Materials and methods . 54 3.1 Construction of PTB-EGFP 54 3.2 Cell sample preparation 54 3.3 Drug treatments . 55 3.4 lipid-mimetic dialkylindocarbocyanine (DiI) analogues 56 3.5 Cholesterol concentration determination 58 Appendix . 58 1. EGFR 58 1.1 Sequence of EGFR 58 1.2 Map of vectors containing EGFR sequence and the vector in re-constructed plasmids 61 PMT (Plasma membrane targeting sequence, negative controls) 64 2.1 The sequence of PMT . 64 2.2 The map of the vectors 64 PTB 65 3.1. The PTB sequence . 65 3.2 Map of the vector of the plasmid 66 4. Map of mRFP-EGFR-EGFP plasmid (positive control) . 67 Chapter 4. Quantitative study of dimerization of EGFR 68 4.1 Result 68 4.1.1. Dimer% value consistency 68 4.1.2. Determination of receptor dimer fractions 73 4.1.3. EGF induced an increase in the dimer% and induced strong EGFR clustering in some cells 74 4.1.4. The fraction of slow component is related to the receptors' response to EGF stimulation 80 4.1.5. Influence of dimer formation on molecular brightness . 81 v 4.2 Discussion . 84 Chapter 5. Interaction of EGFR and PTB . 87 5.1. Introduction of PTB . 87 5.2. Results 88 5.2.1 z-scan 88 5.2.2 Controls and brightness parameters . 91 5.2.3 Interaction between EGFR and PTB stimulated by EGF 92 5.2.4 PTB domain translocation induced by EGF stimulation 97 5.2.5 Inhibition of EGFR-PTB interaction by inhibiting phosphorylation . 99 5.3. Discussion 100 Chapter 6. Study of proteins by imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) 103 6.1 System testing . 103 6.1.1 Determination of measurement parameters . 103 6.1.2. Bleach correction method determination 105 6.1.3. Comparison of the results from confocal FCS and ITIR-FCS 107 6.2 The mobility of EGFR and PTB on the bottom membrane of CHO cells recorded and analyzed by ITIR-FCS . 110 6.2.1 The mobility of EGFR . 110 6.2.2 The mobility of PTB 112 6.3. ITIR-FCS as a tool to study large cluster diffusion on cell membrane 113 6.3.1 The cluster observed in resting cells 113 6.3.2 EGFR cluster formation regulated by EGF 114 6.4. Discussion 116 Chapter 7. The study of factors affecting the diffusion of EGFR . 119 7.1. Diffusion law plots in ITIR-FCS . 119 7. 2. ITIR-FCS diffusion law study on EGFR 121 7.2.1. The heterogeneity of EGFR diffusion revealed by ITIR-FCS diffusion law 121 7.2.2. The effect of cholesterol depletion by mβCD on the mobility of EGFR 122 7.2.3. Cytoskeletal effects are negligible on EGFR partitioning and diffusion 129 7.2.4. EGF stimulation caused the reorganization of EGFR-contained rafts in certain cells . 131 7.3. Discussion 136 Chapter 8. Conclusion and Outlook 138 vi 8.1 Conclusion 138 8.2 Outlook . 140 Bibliography . 144 Appendices 170 vii Summary Fluorescence correlation spectroscopy (FCS) and its modality fluorescence crosscorrelation spectroscopy (FCCS) as well as imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) are single molecule sensitive optical tools to study mobility, concentrations and/or interactions. These methods are gaining popularity in the past few years due to their non-invasive nature for in vivo biological systems. The aim of this thesis is to apply and develop single-wavelength-FCCS (SW-FCCS), a variant of FCCS, to quantitate the protein-protein interactions in vivo, and to apply ITIR-FCS to quantitatively study the mobility modes of membrane molecules. The thesis is organized into the following chapters: Chapter gives an introduction of the epidermal growth factor receptor (EGFR). The introduction starts from a history of EGFR study, and focuses on the function and clinical importance of EGFR, its structure, the cycling within the cells, important signalling pathways triggered by EGFR, EGFR in the nucleus, and special topics such as dimerization of EGFR as well as its relationship with membrane domains and the cytoskeleton. Chapter introduces the principles of FCS, FCCS and ITIR-FCS, followed by instrumental setup of SW-FCCS and ITIR-FCS, respectively. It further introduces the FCS diffusion law applied using our ITIR-FCS setup. Chapter presents materials and methods besides FCS used in this thesis. Chapter discusses the pre-formed dimers of EGFR measured by SW-FCCS in living cells. This chapter firstly confirms the data consistency with the previous results, and then presents the discoveries about the dimer% changes upon EGF stimulation. Chapter reports the measurement of an activation and time dependent interaction between a cytosolic and a membrane bound protein by SW-FCCS in live cells. This chapter demonstrates the activation of the receptor through detecting the phosphorylation dependent binding of a phosphotyrosine binding (PTB) domain. Chapter describes the application of ITIR-FCS in studying the diffusion of EGFR. The first part discusses the calibration of important parameters which should be determined viii before the method is applied in our setting. Then the results from confocal FCS and ITIRFCS are compared to confirm the consistency of the data. The last part of this chapter introduces the information of EGFR and PTB which can be extracted from ITIR-FCS measurements. Chapter focus on the factors affecting the diffusion mode of EGFR. 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Traffic 10(4): 349-363. 169 Appendices Permission of citations 170 171 [...]... 2006), which serves as the docking sites for the receptors' downstream proteins or domains including SH2 and PTB (Ferguson 2008) Besides, in the 2 -receptor- 2-ligand stoichiometry, binding of the first ligand shows negative cooperativity, leading to reduced affinity for binding of the second ligand and the existence of "high-affinity" and "low-affinity" classes (Alvarado, Klein et al 2010) However, it has... spot and block the activation induced by the ligands -receptor binding Besides, cetuximab can induce EGFR internalization and down-regulation to overcome the overexpression 2) TK inhibitors (TKI), such as competitors of ATP (erlotinib, efitinib, and Gifitinib, for example), target to the ATP-binding pockets within the EGFR tyrosine kinase (TK) domain and block the phosphorylation and the following signalling... acid EGFR Epidermal growth factor receptor EGF Epidermal growth factor FCCS Fluorescence cross -correlation spectroscopy FCS Fluorescence correlation spectroscopy FP Fluorescent protein 𝐺(0) fL 𝐺(𝜏) Femtolitres Amplitude of the correlation function Correlation function xiii 𝐺 𝑔 (0) 𝐺 𝑔𝑟 (0) 𝐺 𝑟 (0) Amplitude of the autocorrelation function of the signal in the green channel Amplitude of the cross -correlation. .. folds, and shares 37% amino acid identity CR1 and CR2, as indicated by the names, are rich in cysteine CR1 domain forms disulfide bonds with CR1 domain of the other receptor in an activated dimer The crystal structure revealed two distinct conformations of EGFR ectodomains: in the inactive conformation, CR1 and CR2 interact with each other and 6 prevent interaction with their ligand; while in the active... events, which causes inconsistent results between artificial membrane and in vivo studies, e.g the introduction of cholesterol in DOPC vesicles led to an increase in the affinity between EGFR and its ligand (den Hartigh, van Bergen en Henegouwen et al 1993) but in living cells, depletion of cholesterol enhanced the binding of EGFR to EGF, whereas cholesterol loading lowers the binding (Pike and Casey 2002;... hydrocarbon chains in raft-related lipids such as sphingolipids (Simons and Toomre 2000) Therefore, one of the most important experimental methods for studying rafts is interrupting rafts by changing the amount of cholesterol in cells, which includes increasing its amount by adding in extra cholesterol and extracting it by drug treatments Recent lipidomics studies have shown a good picture of the lipid... acquisition of functions (Jorissen, Walker et al 2003) In the following parts of this chapter, the functions and clinical importance of EGFR will be introduced in section 1.1.1, followed by the structure in section 1.1.2, and the cycling of EGFR within the cells will be summarized in section 1.1.3 Next, the signalling pathways triggered by EGFR, EGFR in the nucleus, 1 dimerization of EGFR and EGFR's... domains and cytoskeleton will be in section 1.1.4 - 1.1.8, respectively 1.1.1 The importance and clinical trials There are 7 ligands known to bind to EGFR, including EGF, transforming growth factor (TGF- α), amphiregulin (AR), epigen (EPN), betacellulin(BTC), heparin-binding EGF (HBEGF) and epiregulin (EPR) (Mitsudomi and Yatabe 2010) EGFR transfers the signals from extracellular space into the cells. .. However, EGFR was reported to lack a putative DNA binding domain, so it is presumed to function by interacting with DNA binding transcription factors such as STAT3 and E2F1, and the bindings were found to be associated with overexpression of Cyclin-D1 (a regulator of cyclin-dependent kinase), iNOS (inducible nitric oxide synthase), and B-Myb (Myb-related protein B) (Lo, Hsu et al 2005; Hanada, Lo et al 2006;... by dwellings within nanodomains, that is, rafts (Orr, Hu et al 2005) Localization of EGFR is important because it exerts a special effect on the functions of EGFR such as binding with ligands and subsequent activation Interestingly, again, opposing effects were observed in different settings of experiments In artificial 20 membranes, the binding of EGF-EGFR was observed to be enhanced by adding cholesterol . QUANTIFICATION OF EPIDERMAL GROWTH FACTOR RECEPTOR DYNAMICS AND INTERACTIONS IN LIVING CELLS BY FLUORESCENCE CORRELATION AND CROSS- CORRELATION SPECTROSCOPY MA XIAOXIAO. importance and clinical trials There are 7 ligands known to bind to EGFR, including EGF, transforming growth factor- α (TGF- α), amphiregulin (AR), epigen (EPN), betacellulin(BTC), heparin-binding. pockets within the EGFR tyrosine kinase (TK) domain and block the phosphorylation and the following signalling. 3) chemopreventive agents, including Genistain, Curcumin, and Caspacin, aim at