The study of interactions of transmembrane receptors and intracellular signaling proteins in live cells by fluorescence correlation and cross correlation spectroscopy

THE STUDY OF INTERACTIONS OF TRANSMEMBRANE RECEPTORS AND INTRACELLULAR SIGNALING PROTEINS IN LIVE CELLS BY FLUORESCENCE CORRELATION AND CROSS-CORRELATION SPECTROSCOPY LIU PING NATIONAL UNIVERSITY OF SINGAPORE 2007 THE STUDY OF INTERACTIONS OF TRANSMEMBRANE RECEPTORS AND INTRACELLULAR SIGNALING PROTEINS IN LIVE CELLS BY FLUORESCENCE CORRELATION AND CROSS-CORRELATION SPECTROSCOPY LIU PING (M.Sc), CAS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2007 Acknowledgements This thesis is a cross-disciplinary work in which I have been accompanied and supported by people from both physics and biology The completion of this work would not be possible without their expertise and contribution I would like to express my deepest gratitude to the following people: Asst Prof Thorsten Wohland, my supervisor, for his enthusiastic guidance, incredible patience and endless support all through the work Dr Ichiro Maruyama, my co-supervisor, for his attentive guidance in biological experiments, strong encouragement and great support throughout the time Assoc Prof Sohail Ahmed, for his warm help and support Prof Stuart J Edelstein, for his constructive advice on experiments and detailed guidance in scientific writing Dr Ling Chin Hwang provided great support in alignment of the optical system and helped in mathematic analysis Dr Sudhaharan Thankiah and Miss Rosita M L Koh helped greatly in biological sample preparation Dr Haihe Wang enthusiastically shared his invaluable research experience without reservation and provided helpful recommendations whenever I met problems in biology All my colleagues in TW lab (NUS), IM lab (GIS) and SA lab (IMB), for their friendship, companionship and all the help Finally, I would like to thank my husband Mr Zang Jianfeng, for his love, continuous encouragement and support in my pursuing for MSc and PhD i Table of Contents Acknowledgements i Table of contents ii Summary v List of Tables viii List of Figures ix List of Symbols xi Chapter Introduction 1.1 Methods to detect protein-protein interactions 1.1.1 Biochemical methods and library-based methods 1.1.2 FCS, FCCS and other biophysical approaches 1.2 Cell signal input by ErbB receptor tyrosine kinases 1.2.1 Signal initiation: activation of the ErbB receptor tyrosine kinases 1.2.2 Activation mechanisms of the ErbB receptors 1.3 Intracellular signal processing by Cdc42 1.3.1 Signal transduction: Cdc42 as a signaling node on intracellular signaling networks 1.3.2 Interactions between Cdc42 and its effectors 7 12 12 14 1.4 Objectives and significance of the study 17 Chapter FCS/FCCS theory, application and experimental setup 2.1 FCS theory and applications 23 24 2.1.1 The theory of FCS 24 2.1.2 Applications of FCS 29 2.2 FCCS Theory and applications 34 2.2.1 The theory of SW-FCCS 34 2.2.2 FCCS applications 41 2.3 FCS and SW-FCCS setups 43 2.3.1 FCS setup 43 2.3.2 SW-FCCS setup 46 ii Chapter Biological sample preparations 48 3.1 FP-fusion plasmid constructions 48 3.1.1 Construction of EGFR-FP fusions 48 3.1.2 Construction of ErbB2-EGFP and ErbB2-mRFP 51 3.1.3 Construction of mRFP-EGFR and mRFP-EGFR-EGFP 52 3.1.4 Construction of PMT-FPs 54 3.1.5 Summary 56 3.2 Sample preparations for imaging, FCS/FCCS and phosphorylation assay 3.2.1 Cell culture and transfection 58 58 3.2.2 Imaging and FCS/FCCS on FP-fusion proteins in live cells 60 3.2.3 Phosphorylation assays of ErbB receptors chimera with FP 61 3.3 Sample preparation for the determination of ErbB2 expression level in CHO-K1 cells by FACS 62 Chapter FCS study on fluorescent proteins and fluorescent protein-fusion proteins in live cells 64 4.1 FCS on EGFP and EGFP-fusion proteins in live cells 64 4.1.1 Characterization of the photodynamic properties of EGFP in CHO cells 4.1.2 Characteristics of the photophysical dynamics of PMT-EGFP and EGFR-EGFP in CHO cells 4.2 FCS on EYFP, mRFP and their fusion-proteins in live cells 64 67 72 4.3 Quantification of the expression level of endogenous ErbB2 in CHO cells 76 Chapter FCCS study on homo- and heterodimerization of EGFR/ErbB2 80 5.1 System calibration 81 5.1.1 cps of each FP in the two channels 81 5.1.2 Positive and negative controls 83 5.2 SW-FCCS measurements on EGFR/ErbB2 87 5.3 Quantitative analysis to determine the dimer percentages of ErbB receptors on the cell surface 88 5.4 SW-FCCS investigation on EGFR dimerization using EYFP/mRFP pair 95 5.5 Summary 98 iii Chapter Activation of ErbB receptors using EGF stimulation 6.1 Phosphorylation assay 99 99 6.2 Imaging of FP-fusion EGFR internalization after EGF stimulation 102 6.3 FCS/SW-FCCS observations on the activation of FP-fusion EGFR by using EGF stimulation 103 6.3.1 FCS observations on EGFR-EGFP activation 103 6.3.2 SW-FCCS observations on FP-fusion EGFRs on cell surface after EGF stimulation 108 6.4 Discussion 111 Chapter 113 SW-FCCS studies on Cdc42-related signaling complexes 7.1 SW-FCCS investigation on the interactions between Cdc42 and Its effectors 113 7.1.1 Positive and negative controls 113 7.1.2 Interactions between Cdc42 and its effectors 116 7.1.3 Plot of the equilibrium dissociation constant K D 7.2 Competition effect on the dimerization of IRSp53 125 7.3 Discussion 129 Chapter Conclusion and outlook 132 8.1 Conclusion 132 8.2 Outlook 136 References 141 Publications 158 118 iv Summary The objective of this study was to apply biophysical fluorescence techniques, i.e FCS and FCCS, to quantitatively study protein-protein interactions in live cells Although both methods have been established, a large portion of FCS/FCCS work were done in vitro, and the applications of FCS/FCCS on studies of biomolecular interactions in live cells are limited In particular, single wavelength fluorescence cross-correlation spectroscopy (SW-FCCS) had not been applied to study molecular interactions in live cell systems In this thesis, we further developed SW-FCCS to study protein-protein interactions in vivo The biological systems studied here are two groups of signaling proteins: the ErbB receptor family and Cdc42-related signaling complexes Chapter first provides a brief review on both biochemical and biophysical methods that are applied to detect protein-protein interactions The primary focus of this chapter is the introduction of the biological backgrounds of the systems that were studied in this thesis: the physiological functions of ErbB receptors and Cdc42 together with its effectors, and their related topics that were studied by using FCS/SW-FCCS Chapter introduces the theory of FCS and FCCS, together with their relevant applications The focal points in this chapter are the related mathematic models that were utilized in the quantification of complex (dimer) percentages of interacting proteins and the determination of the equilibrium dissociation v constants of binding proteins The experimental setups of FCS/SW-FCCS are introduced in the last section of this chapter Chapter introduces the procedures of biological sample preparations, including plasmids construction, tissue culture, transfection methods and sample preparation for imaging, FCS/FCCS, phosphorylation assays and quantitative flow cytometry Chapter describes the characterization of the photodynamic properties of three commonly-used fluorescent proteins (FPs), EGFP, EYFP and mRFP, by performing FCS on cells expressing these proteins and their fusion proteins The results provided the basic information on the mobility, brightness and photodynamic characteristics of FPs and fusion FPs Chapters and focus on the interactions between ErbB receptors Chapter describes the study of the interactions between ErbB receptors before ligand stimulation by performing SW-FCCS The dimer percentages between EGFR/EGFR, EGFR/ErbB2, and ErbB2/ErbB2 were determined through quantitative data analysis The results suggest that the majority of ErbB receptors preform dimeric structures on the cell surface before ligand binding Chapter describes observations of slower diffusion and irregular fluorescence fluctuations with high intensity, which indicates aggregates or oligomerization of the receptors vi after EGF activation The results of the two chapters shed light on the activation mechanism of ErbB receptors Chapter describes further applications of SW-FCCS to investigate the intracellular interactions between Cdc42 and its effectors The concentrations of bound complexes ( cGR ) and unbound EGFP- and mRFP-fusion proteins ( cG and c R ) were determined by SW-FCCS The equilibrium dissociation constants K D were obtained through plotting cG × c R vs cGR The K D values for effectors containing different Cdc42 binding domains indicate SW-FCCS may be applied to distinguish the binding strength between interacting molecules in vivo In conclusion, this thesis reveals SW-FCCS as a novel tool to quantitatively study biomolecular interactions in live cells vii List of Tables Table 4.1 Characteristic parameters 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C-terminus domain, to expose binding sites for Src-homology-2 (SH2) domain-containing proteins and phosphotyrosine-binding (PTB) domain-containing proteins Adaptor proteins with a SH2-binding domain... ligands bind to the extracellular domains of two receptor subunits and form a 2:2 complex (42), resulting in activation of the intracellular kinases of the receptors Upon ligand binding, the