Quantification of epidermal growth factor receptor dynamics and interactions in living cells by fluorescent correlation and cross correlation spectroscopy

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. The combination of diffusion coefficient analysis and FCS diffusion law studies provides insights about the influence of lipid rafts, cytoskeleton, and receptor activation on the mobility of EGFR. Chapter concludes and presents an outlook for future FCS and FCCS developments for a better understanding of biological systems. ix Kolin, D. L., D. Ronis, et al. (2006). "k-Space image correlation spectroscopy: a method for accurate transport measurements independent of fluorophore photophysics." Biophys J 91(8): 3061-3075. Kolin, D. L. and P. W. Wiseman (2007). "Advances in image correlation spectroscopy: measuring number densities, aggregation states, and dynamics of fluorescently labeled macromolecules in cells." Cell Biochem Biophys 49(3): 141-164. Kozer, N., M. P. Kelly, et al. (2011). "Differential and synergistic effects of epidermal growth factor receptor antibodies on unliganded ErbB dimers and oligomers." Biochemistry 50(18): 3581-3590. Kuan, C. T., C. J. Wikstrand, et al. (2001). "EGF mutant receptor vIII as a molecular target in cancer therapy." Endocr Relat Cancer 8(2): 83-96. Kwik, J., S. Boyle, et al. (2003). "Membrane cholesterol, lateral mobility, and the phosphatidylinositol 4,5-bisphosphate-dependent organization of cell actin." Proc Natl Acad Sci U S A 100(24): 13964-13969. Lajoie, P., E. A. Partridge, et al. (2007). "Plasma membrane domain organization regulates EGFR signaling in tumor cells." J Cell Biol 179(2): 341-356. Lamb, D. C., B. K. Muller, et al. (2005). "Enhancing the sensitivity of fluorescence correlation spectroscopy by using time-correlated single photon counting." Curr Pharm Biotechnol 6(5): 405-414. Lambert, S., D. Vind-Kezunovic, et al. (2006). "Ligand-independent activation of the EGFR by lipid raft disruption." J Invest Dermatol 126(5): 954-962. Lee, A. G., N. J. Birdsall, et al. (1974). "Clusters in lipid bilayers and the interpretation of thermal effects in biological membranes." Biochemistry 13(18): 3699-3705. Lee, N. Y., T. L. Hazlett, et al. (2006). "Structure and dynamics of the epidermal growth factor receptor C-terminal phosphorylation domain." Protein Sci 15(5): 1142-1152. Lee, W., Y. I. Lee, et al. (2010). "Cross-talk-free dual-color fluorescence cross-correlation spectroscopy for the study of enzyme activity." Anal Chem 82(4): 1401-1410. Lemmon, M. A. (2009). "Ligand-induced ErbB receptor dimerization." Exp Cell Res 315(4): 638-648. Lemoine, N. R., D. M. Barnes, et al. (1992). "Expression of the ERBB3 gene product in breast cancer." Br J Cancer 66(6): 1116-1121. 156 Lenne, P. F., L. Wawrezinieck, et al. (2006). "Dynamic molecular confinement in the plasma membrane by microdomains and the cytoskeleton meshwork." EMBO J 25(14): 3245-3256. Leu, T. H. and M. C. Maa (2003). "Functional implication of the interaction between EGF receptor and c-Src." Front Biosci 8: s28-38. Levi-Montalcini, R. and S. Cohen (1960). "Effects of the extract of the mouse submaxillary salivary glands on the sympathetic system of mammals." Ann N Y Acad Sci 85: 324-341. Levitzki, A. and A. Gazit (1995). "Tyrosine kinase inhibition: an approach to drug development." Science 267(5205): 1782-1788. Lill, N. L. and N. I. Sever (2012). "Where EGF receptors transmit their signals." Sci Signal 5(243): pe41. Lim, K. I. and J. Yin (2005). "Localization of receptors in lipid rafts can inhibit signal transduction." Biotechnol Bioeng 90(6): 694-702. Lin, S. Y., K. Makino, et al. (2001). "Nuclear localization of EGF receptor and its potential new role as a transcription factor." Nat Cell Biol 3(9): 802-808. Litherland, G. J., M. S. Elias, et al. (2010). "Protein kinase C isoforms zeta and iota mediate collagenase expression and cartilage destruction via STAT3- and ERKdependent c-fos induction." J Biol Chem 285(29): 22414-22425. Liu, P., T. Sudhaharan, et al. (2007). "Investigation of the dimerization of proteins from the epidermal growth factor receptor family by single wavelength fluorescence crosscorrelation spectroscopy." Biophys J 93(2): 684-698. Lo, H. W., S. C. Hsu, et al. (2005). "Nuclear interaction of EGFR and STAT3 in the activation of the iNOS/NO pathway." Cancer Cell 7(6): 575-589. Loman, A., I. Gregor, et al. (2010). "Measuring rotational diffusion of macromolecules by fluorescence correlation spectroscopy." Photochem Photobiol Sci 9(5): 627-636. Lui, V. W. and J. R. Grandis (2002). "EGFR-mediated cell cycle regulation." Anticancer Res 22(1A): 1-11. Lund, M. J., K. F. Trivers, et al. (2009). "Race and triple negative threats to breast cancer survival: a population-based study in Atlanta, GA." Breast Cancer Res Treat 113(2): 357370. Lundmark, R. and S. R. Carlsson (2010). "Driving membrane curvature in clathrindependent and clathrin-independent endocytosis." Semin Cell Dev Biol 21(4): 363-370. 157 Lurje, G. and H. J. Lenz (2009). "EGFR signaling and drug discovery." Oncology 77(6): 400-410. Ma, X., S. Ahmed, et al. (2011). "EGFR activation monitored by SW-FCCS in live cells." Front Biosci (Elite Ed) 3: 22-32. Maemondo, M., A. Inoue, et al. (2010). "Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR." N Engl J Med 362(25): 2380-2388. Magde, D., E. Elson, et al. (1972). "Thermodynamic Fluctuations in a Reacting System— Measurement by Fluorescence Correlation Spectroscopy." Physical Review Letters 29(11): 705-708. Magde, D., W. W. Webb, et al. (1978). "Fluorescence correlation spectroscopy. III. Uniform translation and laminar flow." Biopolymers 17(2): 361-376. Malchus, N. and M. Weiss (2010). "Elucidating anomalous protein diffusion in living cells with fluorescence correlation spectroscopy-facts and pitfalls." J Fluoresc 20(1): 19-26. Marti, U., S. J. Burwen, et al. (1991). "Localization of epidermal growth factor receptor in hepatocyte nuclei." Hepatology 13(1): 15-20. Mays, R. W., K. A. Siemers, et al. (1995). "Hierarchy of mechanisms involved in generating Na/K-ATPase polarity in MDCK epithelial cells." J Cell Biol 130(5): 1105-1115. Meguid, R. A., C. M. Hooker, et al. (2010). "Long-term survival outcomes by smoking status in surgical and nonsurgical patients with non-small cell lung cancer: comparing never smokers and current smokers." Chest 138(3): 500-509. Meseth, U., T. Wohland, et al. (1999). "Resolution of fluorescence correlation measurements." Biophys J 76(3): 1619-1631. Mets, Ü. and R. Rigler (1994). "Submillisecond detection of single rhodamine molecules in water." J Fluoresc 4(3): 259-264. Milon, S., R. Hovius, et al. (2003). "Factors influencing fluorescence correlation spectroscopy measurements on membranes: simulations and experiments." Chem Phys 288(2): 171-186. Mineo, C., G. L. James, et al. (1996). "Localization of epidermal growth factor-stimulated Ras/Raf-1 interaction to caveolae membrane." J Biol Chem 271(20): 11930-11935. Mitsudomi, T. and Y. Yatabe (2010). "Epidermal growth factor receptor in relation to tumor development: EGFR gene and cancer." FEBS J 277(2): 301-308. 158 Miyagi, H. and I. N. Maruyama (2010). "Analysis of Ligand-Receptor Interaction on the Surface of Living Cells by Fluorescence Correlation Spectroscopy." Open Spectrosc J 4(1): 28-31. Moriki, T., H. Maruyama, et al. (2001). "Activation of preformed EGF receptor dimers by ligand-induced rotation of the transmembrane domain." J Mol Biol 311(5): 1011-1026. Mounier, N. and A. P. Arrigo (2002). "Actin cytoskeleton and small heat shock proteins: how they interact?" Cell Stress Chaperones 7(2): 167-176. Mueller, V., C. Ringemann, et al. (2011). "STED nanoscopy reveals molecular details of cholesterol- and cytoskeleton-modulated lipid interactions in living cells." Biophys J 101(7): 1651-1660. Mukherjee, S., T. T. Soe, et al. (1999). "Endocytic sorting of lipid analogues differing solely in the chemistry of their hydrophobic tails." J Cell Biol 144(6): 1271-1284. Müller, B. K., E. Zaychikov, et al. (2005). "Pulsed interleaved excitation." Biophys J 89(5): 3508-3522. Muraoka-Cook, R. S., M. A. Sandahl, et al. (2009). "ErbB4 splice variants Cyt1 and Cyt2 differ by 16 amino acids and exert opposing effects on the mammary epithelium in vivo." Mol Cell Biol 29(18): 4935-4948. Murphy, L. O. and J. Blenis (2006). "MAPK signal specificity: the right place at the right time." Trends Biochem Sci 31(5): 268-275. Nagy, P., J. Claus, et al. (2010). "Distribution of resting and ligand-bound ErbB1 and ErbB2 receptor tyrosine kinases in living cells using number and brightness analysis." Proc Natl Acad Sci U S A 107(38): 16524-16529. Nicholson, R. I., J. M. Gee, et al. (2001). "EGFR and cancer prognosis." Eur J Cancer 37 Suppl 4: S9-15. Nie, Z., D. S. Hirsch, et al. (2006). "A BAR domain in the N terminus of the Arf GAP ASAP1 affects membrane structure and trafficking of epidermal growth factor receptor." Curr Biol 16(2): 130-139. Nogales, E. (2000). "Structural insights into microtubule function." Annu Rev Biochem 69: 277-302. Nogales, E., K. H. Downing, et al. (1998). "Tubulin and FtsZ form a distinct family of GTPases." Nat Struct Biol 5(6): 451-458. 159 Octobre, G., C. Lemercier, et al. (2005). "Monitoring the interaction between DNA and a transcription factor (MEF2A) using fluorescence correlation spectroscopy." C R Biol 328(12): 1033-1040. Offterdinger, M., V. Georget, et al. (2004). "Imaging phosphorylation dynamics of the epidermal growth factor receptor." J Biol Chem 279(35): 36972-36981. Ogiso, H., R. Ishitani, et al. (2002). "Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains." Cell 110(6): 775-787. Oh, H. Y., J. Leem, et al. (2010). "Lipid raft cholesterol and genistein inhibit the cell viability of prostate cancer cells via the partial contribution of EGFR-Akt/p70S6k pathway and down-regulation of androgen receptor." Biochem Biophys Res Commun 393(2): 319-324. Omary, M. B., P. A. Coulombe, et al. (2004). "Intermediate Filament Proteins and Their Associated Diseases." N Engl J Med 351(20): 2087-2100. Onishi-Haraikawa, Y., M. Funaki, et al. (2001). "Unique phosphorylation mechanism of Gab1 using PI 3-kinase as an adaptor protein." Biochem Biophys Res Commun 288(2): 476-482. Orr, G., D. Hu, et al. (2005). "Cholesterol dictates the freedom of EGF receptors and HER2 in the plane of the membrane." Biophys J 89(2): 1362-1373. Orth, J. D., E. W. Krueger, et al. (2006). "A novel endocytic mechanism of epidermal growth factor receptor sequestration and internalization." Cancer Res 66(7): 3603-3610. Pao, W., V. A. Miller, et al. (2005). "Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain." PLoS Med 2(3): e73. Pennock, S. and Z. Wang (2008). "A tale of two Cbls: interplay of c-Cbl and Cbl-b in epidermal growth factor receptor downregulation." Mol Cell Biol 28(9): 3020-3037. Peres, C., A. Yart, et al. (2003). "Modulation of phosphoinositide 3-kinase activation by cholesterol level suggests a novel positive role for lipid rafts in lysophosphatidic acid signalling." FEBS Lett 534(1-3): 164-168. Petersen, N. O., P. L. Hoddelius, et al. (1993). "Quantitation of membrane receptor distributions by image correlation spectroscopy: concept and application." Biophys J 65(3): 1135-1146. Petrasek, Z. and P. Schwille (2008). "Precise measurement of diffusion coefficients using scanning fluorescence correlation spectroscopy." Biophys J 94(4): 1437-1448. 160 Petrelli, F. and S. Barni (2012). "Anti-EGFR-targeting agents in recurrent or metastatic head and neck carcinoma: a meta-analysis." Head Neck 34(11): 1657-1664. Piccart-Gebhart, M. J., M. Procter, et al. (2005). "Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer." N Engl J Med 353(16): 1659-1672. Pike, L. J. (2006). "Rafts defined: a report on the Keystone Symposium on Lipid Rafts and Cell Function." J Lipid Res 47(7): 1597-1598. Pike, L. J. (2009). "The challenge of lipid rafts." J Lipid Res 50 Suppl: S323-328. Pike, L. J. and L. Casey (2002). "Cholesterol levels modulate EGF receptor-mediated signaling by altering receptor function and trafficking." Biochemistry 41(32): 1031510322. Pike, L. J., X. Han, et al. (2002). "Lipid rafts are enriched in arachidonic acid and plasmenylethanolamine and their composition is independent of caveolin-1 expression: a quantitative electrospray ionization/mass spectrometric analysis." Biochemistry 41(6): 2075-2088. Pike, L. J., X. Han, et al. (2005). "Epidermal growth factor receptors are localized to lipid rafts that contain a balance of inner and outer leaflet lipids: a shotgun lipidomics study." J Biol Chem 280(29): 26796-26804. Posner, I., M. Engel, et al. (1992). "Kinetic model of the epidermal growth factor (EGF) receptor tyrosine kinase and a possible mechanism of its activation by EGF." J Biol Chem 267(29): 20638-20647. Press, M. F. and H. J. Lenz (2007). "EGFR, HER2 and VEGF pathways: validated targets for cancer treatment." Drugs 67(14): 2045-2075. Prickett, T. D., N. S. Agrawal, et al. (2009). "Analysis of the tyrosine kinome in melanoma reveals recurrent mutations in ERBB4." Nat Genet 41(10): 1127-1132. Przybylo, M., J. Sykora, et al. (2006). "Lipid diffusion in giant unilamellar vesicles is more than times faster than in supported phospholipid bilayers under identical conditions." Langmuir 22(22): 9096-9099. Quesnelle, K. M., A. L. Boehm, et al. (2007). "STAT-mediated EGFR signaling in cancer." J Cell Biochem 102(2): 311-319. Rappoport, J. Z. and S. M. Simon (2009). "Endocytic trafficking of activated EGFR is AP-2 dependent and occurs through preformed clathrin spots." J Cell Sci 122(Pt 9): 13011305. 161 Reck-Peterson, S. L., A. Yildiz, et al. (2006). "Single-molecule analysis of dynein processivity and stepping behavior." Cell 126(2): 335-348. Red Brewer, M., S. H. Choi, et al. (2009). "The juxtamembrane region of the EGF receptor functions as an activation domain." Mol Cell 34(6): 641-651. Resat, H., M. N. Costa, et al. (2011). "Spatial aspects in biological system simulations." Methods Enzymol 487: 485-511. Resat, H., J. A. Ewald, et al. (2003). "An integrated model of epidermal growth factor receptor trafficking and signal transduction." Biophys J 85(2): 730-743. Rička, J. and T. Binkert (1989). "Direct measurement of a distinct correlation function by fluorescence cross correlation." Phys Rev A 39(5): 2646-2652. Ridgeway, W. K., D. P. Millar, et al. (2012). "Quantitation of ten 30S ribosomal assembly intermediates using fluorescence triple correlation spectroscopy." Proc Natl Acad Sci U S A 109(34): 13614-13619. Ries, J., S. Chiantia, et al. (2009). "Accurate determination of membrane dynamics with line-scan FCS." Biophys J 96(5): 1999-2008. Ries, J. and P. Schwille (2006). "Studying slow membrane dynamics with continuous wave scanning fluorescence correlation spectroscopy." Biophys J 91(5): 1915-1924. Rigler, R., Ü. Mets, et al. (1993). "Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion." Eur Biophys J 22(3): 169175. Ringerike, T., F. D. Blystad, et al. (2002). "Cholesterol is important in control of EGF receptor kinase activity but EGF receptors are not concentrated in caveolae." J Cell Sci 115(Pt 6): 1331-1340. Rio, C., J. D. Buxbaum, et al. (2000). "Tumor necrosis factor-alpha-converting enzyme is required for cleavage of erbB4/HER4." J Biol Chem 275(14): 10379-10387. Ritchie, K., X. Y. Shan, et al. (2005). "Detection of non-Brownian diffusion in the cell membrane in single molecule tracking." Biophys J 88(3): 2266-2277. Roepstorff, K., P. Thomsen, et al. (2002). "Sequestration of epidermal growth factor receptors in non-caveolar lipid rafts inhibits ligand binding." J Biol Chem 277(21): 1895418960. Romberg, L. and P. A. Levin (2003). "Assembly dynamics of the bacterial cell division protein FTSZ: poised at the edge of stability." Annu Rev Microbiol 57: 125-154. 162 Rosenzweig, S. A. (2012). "Acquired resistance to drugs targeting receptor tyrosine kinases." Biochem Pharmacol 83(8): 1041-1048. Ross, J. S., E. A. Slodkowska, et al. (2009). "The HER-2 receptor and breast cancer: ten years of targeted anti-HER-2 therapy and personalized medicine." Oncologist 14(4): 320368. Rossow, M., W. W. Mantulin, et al. (2009). "Spatiotemporal image correlation spectroscopy measurements of flow demonstrated in microfluidic channels." J Biomed Opt 14(2): 024014. Rossow, M. J., J. M. Sasaki, et al. (2010). "Raster image correlation spectroscopy in live cells." Nat Protoc 5(11): 1761-1774. Ruttinger, S., V. Buschmann, et al. (2008). "Comparison and accuracy of methods to determine the confocal volume for quantitative fluorescence correlation spectroscopy." J Microsc 232(2): 343-352. Saffarian, S., Y. Li, et al. (2007). "Oligomerization of the EGF receptor investigated by live cell fluorescence intensity distribution analysis." Biophys J 93(3): 1021-1031. Saffman, P. G. and M. Delbruck (1975). "Brownian motion in biological membranes." Proc Natl Acad Sci U S A 72(8): 3111-3113. Saka, S. and S. O. Rizzoli (2012). "Super-resolution imaging prompts re-thinking of cell biology mechanisms: selected cases using stimulated emission depletion microscopy." Bioessays 34(5): 386-395. Sankaran, J., N. Bag, et al. (2013). "Accuracy and precision in camera-based fluorescence correlation spectroscopy measurements." Anal Chem 85(8): 3948-3954. Sankaran, J., M. Manna, et al. (2009). "Diffusion, transport, and cell membrane organization investigated by imaging fluorescence cross-correlation spectroscopy." Biophys J 97(9): 2630-2639. Sankaran, J., X. Shi, et al. (2010). "ImFCS: a software for imaging FCS data analysis and visualization." Opt Express 18(25): 25468-25481. Schaefer, G., L. Haber, et al. (2011). "A two-in-one antibody against HER3 and EGFR has superior inhibitory activity compared with monospecific antibodies." Cancer Cell 20(4): 472-486. Schaetzel, K. and R. Peters (1991). "Noise on multiple-tau photon correlation data." 109115. 163 Schindler, T., F. Sicheri, et al. (1999). "Crystal structure of Hck in complex with a Src family-selective tyrosine kinase inhibitor." Mol Cell 3(5): 639-648. Schlieper, D., M. A. Oliva, et al. (2005). "Structure of bacterial tubulin BtubA/B: evidence for horizontal gene transfer." Proc Natl Acad Sci U S A 102(26): 9170-9175. Schoeberl, B., E. A. Pace, et al. (2009). "Therapeutically targeting ErbB3: a key node in ligand-induced activation of the ErbB receptor-PI3K axis." Sci Signal 2(77): ra31. Schuck, S., M. Honsho, et al. (2003). "Resistance of cell membranes to different detergents." Proc Natl Acad Sci U S A 100(10): 5795-5800. Schwille, P., F. J. Meyer-Almes, et al. (1997). "Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution." Biophys J 72(4): 1878-1886. Schwille, P., F. Oehlenschlager, et al. (1996). "Quantitative hybridization kinetics of DNA probes to RNA in solution followed by diffusional fluorescence correlation analysis." Biochemistry 35(31): 10182-10193. Sengupta, P., E. Bosis, et al. (2009). "EGFR juxtamembrane domain, membranes, and calmodulin: kinetics of their interaction." Biophys J 96(12): 4887-4895. Sergeev, M., J. L. Swift, et al. (2012). "Ligand-induced clustering of EGF receptors: a quantitative study by fluorescence image moment analysis." Biophys Chem 161: 50-53. Sergina, N. V., M. Rausch, et al. (2007). "Escape from HER-family tyrosine kinase inhibitor therapy by the kinase-inactive HER3." Nature 445(7126): 437-441. Seshacharyulu, P., M. P. Ponnusamy, et al. (2012). "Targeting the EGFR signaling pathway in cancer therapy." Expert Opin Ther Targets 16(1): 15-31. Sharma, S. V. and J. Settleman (2009). "ErbBs in lung cancer." Exp Cell Res 315(4): 557571. Shenolikar, S. and A. C. Nairn (1991). "Protein phosphatases: recent progress." Adv Second Messenger Phosphoprotein Res 23: 1-121. Shepherd, F. A., J. Rodrigues Pereira, et al. (2005). "Erlotinib in previously treated nonsmall-cell lung cancer." N Engl J Med 353(2): 123-132. Shi, X., Y. H. Foo, et al. (2009). "Determination of dissociation constants in living zebrafish embryos with single wavelength fluorescence cross-correlation spectroscopy." Biophys J 97(2): 678-686. Shih, J. Y., C. H. Gow, et al. (2005). "EGFR mutation conferring primary resistance to gefitinib in non-small-cell lung cancer." N Engl J Med 353(2): 207-208. 164 Shih, Y. L. and L. Rothfield (2006). "The bacterial cytoskeleton." Microbiol Mol Biol Rev 70(3): 729-754. Sigismund, S., E. Argenzio, et al. (2008). "Clathrin-mediated internalization is essential for sustained EGFR signaling but dispensable for degradation." Dev Cell 15(2): 209-219. Sigismund, S., T. Woelk, et al. (2005). "Clathrin-independent endocytosis of ubiquitinated cargos." Proc Natl Acad Sci U S A 102(8): 2760-2765. Simons, K. and D. Toomre (2000). "Lipid rafts and signal transduction." Nat Rev Mol Cell Biol 1(1): 31-39. Simons, K. and G. van Meer (1988). "Lipid sorting in epithelial cells." Biochemistry 27(17): 6197-6202. Singer, S. J. and G. L. Nicolson (1972). "The fluid mosaic model of the structure of cell membranes." Science 175(4023): 720-731. Singh, A. P., J. W. Krieger, et al. (2013). "The performance of 2D array detectors for light sheet based fluorescence correlation spectroscopy." Opt Express 21(7): 8652-8668. Song, W., H. Xuan, et al. (2008). "Epidermal growth factor induces changes of interaction between epidermal growth factor receptor and actin in intact cells." Acta Biochim Biophys Sin (Shanghai) 40(8): 754-760. Sorkin, A. and L. K. Goh (2009). "Endocytosis and intracellular trafficking of ErbBs." Exp Cell Res 315(4): 683-696. Starr, A., J. Greif, et al. (2006). "ErbB4 increases the proliferation potential of human lung cancer cells and its blockage can be used as a target for anti-cancer therapy." Int J Cancer 119(2): 269-274. Staubach, S. and F. G. Hanisch (2011). "Lipid rafts: signaling and sorting platforms of cells and their roles in cancer." Expert Rev Proteomics 8(2): 263-277. Subramanian, S., E. T. Boder, et al. (2007). "Phylogenetic divergence of CD47 interactions with human signal regulatory protein alpha reveals locus of species specificity. Implications for the binding site." J Biol Chem 282(3): 1805-1818. Sungkaworn, T., Y. Lenbury, et al. (2011). "Oxidative stress increases angiotensin receptor type I responsiveness by increasing receptor degree of aggregation using image correlation spectroscopy." Biochim Biophys Acta 1808(10): 2496-2500. Swift, J. L., M. C. Burger, et al. (2007). "Two-photon excitation fluorescence crosscorrelation assay for ligand-receptor binding: cell membrane nanopatches containing the human micro-opioid receptor." Anal Chem 79(17): 6783-6791. 165 Thews, E., M. Gerken, et al. (2005). "Cross talk free fluorescence cross correlation spectroscopy in live cells." Biophys J 89(3): 2069-2076. Tian, A. and T. Baumgart (2009). "Sorting of lipids and proteins in membrane curvature gradients." Biophys J 96(7): 2676-2688. Toomre, D. and D. J. Manstein (2001). "Lighting up the cell surface with evanescent wave microscopy." Trends Cell Biol 11(7): 298-303. Torres, R., J. R. Genzen, et al. (2012). "Clinical measurement of von Willebrand factor by fluorescence correlation spectroscopy." Clin Chem 58(6): 1010-1018. Treanor, B., D. Depoil, et al. (2010). "The membrane skeleton controls diffusion dynamics and signaling through the B cell receptor." Immunity 32(2): 187-199. Triffo, S. B., H. H. Huang, et al. (2012). "Monitoring lipid anchor organization in cell membranes by PIE-FCCS." J Am Chem Soc 134(26): 10833-10842. Tsai, M. Y., S. Wang, et al. (2006). "A mitotic lamin B matrix induced by RanGTP required for spindle assembly." Science 311(5769): 1887-1893. Tudor, C., J. N. Feige, et al. (2007). "Association with coregulators is the major determinant governing peroxisome proliferator-activated receptor mobility in living cells." J Biol Chem 282(7): 4417-4426. Ueda, S., S. Ogata, et al. (2004). "The correlation between cytoplasmic overexpression of epidermal growth factor receptor and tumor aggressiveness: poor prognosis in patients with pancreatic ductal adenocarcinoma." Pancreas 29(1): e1-8. Ullrich, A., L. Coussens, et al. (1984). "Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells." Nature 309(5967): 418-425. van den Ent, F., L. A. Amos, et al. (2001). "Prokaryotic origin of the actin cytoskeleton." Nature 413(6851): 39-44. van den Ent, F., J. Moller-Jensen, et al. (2002). "F-actin-like filaments formed by plasmid segregation protein ParM." EMBO J 21(24): 6935-6943. Vanhaesebroeck, B. and D. R. Alessi (2000). "The PI3K-PDK1 connection: more than just a road to PKB." Biochem J 346 Pt 3: 561-576. Vermorken, J. B., R. Mesia, et al. (2008). "Platinum-based chemotherapy plus cetuximab in head and neck cancer." N Engl J Med 359(11): 1116-1127. 166 Vexler, A., G. Lidawi, et al. (2008). "Anti-ERBb4 targeted therapy combined with radiation therapy in prostate cancer. Results of in vitro and in vivo studies." Cancer Biol Ther 7(7): 1090-1094. Vivanco, I. and C. L. Sawyers (2002). "The phosphatidylinositol 3-Kinase AKT pathway in human cancer." Nat Rev Cancer 2(7): 489-501. Vrljic, M., S. Y. Nishimura, et al. (2002). "Translational diffusion of individual class II MHC membrane proteins in cells." Biophys J 83(5): 2681-2692. Vrljic, M., S. Y. Nishimura, et al. (2005). "Cholesterol depletion suppresses the translational diffusion of class II major histocompatibility complex proteins in the plasma membrane." Biophys J 88(1): 334-347. Wang, L., H. C. Chiang, et al. (2012). "Epidermal growth factor receptor is a preferred target for treating Amyloid-beta-induced memory loss." Proc Natl Acad Sci U S A 109(41): 16743-16748. Wang, S. C. and M. C. Hung (2009). "Nuclear translocation of the epidermal growth factor receptor family membrane tyrosine kinase receptors." Clin Cancer Res 15(21): 6484-6489. Wang, Y., J. Wu, et al. (2006). "Akt binds to and phosphorylates phospholipase Cgamma1 in response to epidermal growth factor." Mol Biol Cell 17(5): 2267-2277. Wang, Y. N., H. Yamaguchi, et al. (2010). "Nuclear trafficking of the epidermal growth factor receptor family membrane proteins." Oncogene 29(28): 3997-4006. Ward, C. W., P. A. Hoyne, et al. (1995). "Insulin and epidermal growth factor receptors contain the cysteine repeat motif found in the tumor necrosis factor receptor." Proteins 22(2): 141-153. Watanabe, N. and T. J. Mitchison (2002). "Single-molecule speckle analysis of actin filament turnover in lamellipodia." Science 295(5557): 1083-1086. Wawrezinieck, L., H. Rigneault, et al. (2005). "Fluorescence correlation spectroscopy diffusion laws to probe the submicron cell membrane organization." Biophys J 89(6): 4029-4042. Weidemann, T., R. Worch, et al. (2011). "Single cell analysis of ligand binding and complex formation of interleukin-4 receptor subunits." Biophys J 101(10): 2360-2369. Weiss, M., H. Hashimoto, et al. (2003). "Anomalous protein diffusion in living cells as seen by fluorescence correlation spectroscopy." Biophys J 84(6): 4043-4052. 167 Wells, A., J. B. Welsh, et al. (1990). "Ligand-induced transformation by a noninternalizing epidermal growth factor receptor." Science 247(4945): 962-964. Wenger, J., F. Conchonaud, et al. (2007). "Diffusion analysis within single nanometric apertures reveals the ultrafine cell membrane organization." Biophys J 92(3): 913-919. Widengren, J., U. Mets, et al. (1995). "Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study." J Phys Chem 99(36): 1336813379. Widengren, J., R. Rigler, et al. (1994). "Triplet-state monitoring by fluorescence correlation spectroscopy." J Fluoresc 4(3): 255-258. Worch, R., C. Bokel, et al. (2010). "Focus on composition and interaction potential of single-pass transmembrane domains." Proteomics 10(23): 4196-4208. Xia, W., C. M. Gerard, et al. (2005). "Combining lapatinib (GW572016), a small molecule inhibitor of ErbB1 and ErbB2 tyrosine kinases, with therapeutic anti-ErbB2 antibodies enhances apoptosis of ErbB2-overexpressing breast cancer cells." Oncogene 24(41): 6213-6221. Yarden, Y. (2001). "The EGFR family and its ligands in human cancer. signalling mechanisms and therapeutic opportunities." Eur J Cancer 37 Suppl 4: S3-8. Yarden, Y. and J. Schlessinger (1987). "Self-phosphorylation of epidermal growth factor receptor: evidence for a model of intermolecular allosteric activation." Biochemistry 26(5): 1434-1442. Yarden, Y. and M. X. Sliwkowski (2001). "Untangling the ErbB signalling network." Nat Rev Mol Cell Biol 2(2): 127-137. Yi, E. S., D. Harclerode, et al. (1997). "High c-erbB-3 protein expression is associated with shorter survival in advanced non-small cell lung carcinomas." Mod Pathol 10(2): 142148. Yildiz, A., M. Tomishige, et al. (2004). "Kinesin walks hand-over-hand." Science 303(5658): 676-678. You, L., S. C. Cowin, et al. (2001). "A model for strain amplification in the actin cytoskeleton of osteocytes due to fluid drag on pericellular matrix." J Biomech 34(11): 1375-1386. Yu, C., J. Hale, et al. (2009). "Receptor overexpression or inhibition alters cell surface dynamics of EGF-EGFR interaction: new insights from real-time single molecule analysis." Biochem Biophys Res Commun 378(3): 376-382. 168 Zhang, X., J. Gureasko, et al. (2006). "An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor." Cell 125(6): 1137-1149. Zhou, M.-M., J. E. Harlan, et al. (1996). "Binding Affinities of Tyrosine-phosphorylated Peptides to the COOH-terminal SH2 and NH-terminal Phosphotyrosine Binding Domains of Shc." J Biol Chem 270(52): 31119-31123. Zhou, M. M., K. S. Ravichandran, et al. (1995). "Structure and ligand recognition of the phosphotyrosine binding domain of Shc." Nature 378(6557): 584-592. Zhou, S. W., Y. Y. Huang, et al. (2012). "No survival benefit from adding cetuximab or panitumumab to oxaliplatin-based chemotherapy in the first-line treatment of metastatic colorectal cancer in KRAS wild type patients: a meta-analysis." PLoS One 7(11): e50925. Zhuang, L., J. Lin, et al. (2002). "Cholesterol-rich lipid rafts mediate akt-regulated survival in prostate cancer cells." Cancer Res 62(8): 2227-2231. Zwang, Y. and Y. Yarden (2009). "Systems biology of growth factor-induced receptor endocytosis." 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