STUDY OF BIOMOLECULAR INTERACTIONS IN VIVO BY MULTICOLOUR FLUORESCENCE SPECTROSCOPY FOO YONG HWEE (B.Sc.(Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2011 This work was performed in the Department of Chemistry at the National University of Singapore under the supervision of Associate Professor Thorsten Wohland, and in the Institute of Medical Biology, Agency for Science, Technology and Research, under the co-supervision of Associate Professor Sohail Ahmed, between August 2006 to July 2011. The translocation project of p21 and PCNA was done in collaboration with Dr Carsten Schultz in the European Molecular Biology Laboratory (Heidelberg), between October 2009 to January 2010. Part of the PIEFCCS measurements was performed in Ludwig-Maximilians-Universität München in collaboration with Professor Don C. Lamb in December 2009. The results have been partly published in  Y. H. Foo, V. Korzh, and T. Wohland. Fluorescence Correlation and CrossCorrelation Spectroscopy Using Fluorescent Proteins for Measurements of Biomolecular Processes in Living Organisms. 2011. Springer Series on Fluorescence, Online FirstTM, 31 March 2011.  Shi, X., Y. H. Foo, T. Sudhaharan, S. W. Chong, V. Korzh, S. Ahmed, and T. Wohland. 2009. Determination of dissociation constants in living zebrafish embryos with single wavelength fluorescence cross-correlation spectroscopy. Biophys J 97:678-686.  Sudhaharan, T., P. Liu, Y. H. Foo, W. Bu, K. B. Lim, T. Wohland, and S. Ahmed. 2009. Determination of in vivo dissociation constant, KD, of Cdc42-effector complexes in live mammalian cells using single wavelength fluorescence crosscorrelation spectroscopy. J Biol Chem 284:13602-13609. Foo Yong Hwee 17/08/2011 i Acknowledgements I started off with only an analytical chemistry background. Thus, this thesis in the field of biophysics would not be possible without the help of many individuals. I would like to express my special thanks to my supervisor Associate Professor Thorsten Wohland for the opportunities to work in these projects. The support, guidance, patience and freedom he has given me are greatly appreciated. I have gained a lot from his passion for research and his eye for details. I am very grateful to my co-supervisor Associate Professor Sohail Ahmed for making me a part of his lab and showing me the ropes of molecular cell biology. As I have spent most of my time in his lab, I am lucky to have his support, patience and guidance. I have learned a great deal about molecular biology and imaging from the numerous discussions with him. I am greatly thankful to Professor Ernst H. K. Stelzer for hosting me during my short-term EMBO fellowship in the European Molecular Biology Laboratory (EMBL). I would like to thank Dr Carsten Schultz and the technical assistance given by Dr Alan Piljic and Dr Malte Wachsmuth in the EMBL for the project during the stay. I am grateful for the collaboration and support given by Professor Don C. Lamb of Ludwig-Maximilians-Universität München during my short stay in his lab. Special thanks to Nikolaus Naredi-Rainer and Dr Matthias Höller, for setting up the instruments during that period. I wish to thank all the colleagues from the Biophysical Fluorescence Laboratory in the National University of Singapore and from the Neural Stem Cells group in the Institute of Medical Biology for their discussions, guidance, patience and friendship. In particular, Dr Liu Ping for the guidance in SW-FCCS; Dr Shi Xianke for the zebrafish embryo measurements; Dr Thankiah Sudhaharan and Dr Eric Lam Chen Sok for the guidance and discussion in molecular cell biology. Last but not least, I would like to thank my parents for their love, support and understanding. ii Table of Contents Acknowledgements . ii Summary vii List of Tables ix List of Figures .x Lists of Symbols and Acronyms xii Introduction .1 Theory and Instrumental Setup 11 2.1. Fluorescence Correlation Spectroscopy (FCS) .13 2.1.1. Fluorescence fluctuations . 13 2.1.2. The Autocorrelation function . 14 2.1.3. Theoretical ACF models 15 2.2. Fluorescence Cross-Correlation Spectroscopy (FCCS) 23 2.2.1. The Cross-Correlation Function 24 2.3. Applying FCS and FCCS in vivo 27 2.3.1. Background corrections . 27 2.3.2. Crosstalk 30 2.3.3. Optimizing measurement conditions . 31 2.4. Instrument Setup and SW-FCCS 31 iii Quantitation Using Cross-Correlation Ratios: A Simulation Study 34 3.1. Introduction .34 3.2. Materials and Method .36 3.3. Cross-correlation ratios for 1:1 binding 37 3.4. Quantitation for a dimerization system .41 3.5. Summary .43 Determination of Dissociation Constants in Living Cells 45 4.1. Interaction of Cdc42 with IQGAP1 in living cells and zebrafish embryo. 47 4.1.1 Theory . 47 4.1.2. Cell culture . 49 4.1.3. Plasmids . 49 4.1.4. Instrumentation and data analysis 50 4.1.5. FCCS Calibration . 50 4.1.6. Controls 52 4.1.7. Interaction of Cdc42T17N with IQGAP1 . 53 4.1.8. Interaction of Cdc42G12V with IQGAP1 . 54 4.1.9. Comparison of the results in CHO cells and zebrafish embryos . 56 4.1.10 Conclusion 58 4.2. Interaction of p21 with PCNA in Living Cells with FCCS and Translocation 59 iv 4.2.1 Introduction . 59 4.2.2 FCCS Instrumentation 60 4.2.3. Plasmids and cell culture . 60 4.2.4. Translocation 61 4.2.5. FCCS measurements 62 4.2.6 Conclusion 63 Factors Affecting Fluorescence Cross-Correlation Spectroscopy 65 5.1. Introduction .65 5.2. Theory .66 5.2.1. Cross-correlation volume . 66 5.2.2. CCF ratios 68 5.2.3. Pulsed interleave excitation-FCCS and FRET . 69 5.3. Materials and Methods 71 5.3.1. Plasmids and cell cultures 71 5.3.2. Cycloheximide chase experiment 72 5.3.3. SW-FCCS Instrumentation 72 5.3.4. Obtaining the brightnesses of GFP dimers 72 5.3.5. Pulsed interleave excitation-FCCS Instrumentation 73 5.4. Results and Discussions 74 5.4.1. Calibration of SW-FCCS observation volumes with a single dye.74 v 5.4.2. Photophysical properties of tandem FPs change the auto- and cross-correlation amplitudes 75 5.4.3. Non-fluorescent FPs and their influence on the FCCS data 79 5.4.4. Influence of FRET on the amplitudes 83 5.4.5. Determination of effective observation volumes and correction parameters 85 5.4.6. Influence of non-fluorescent labels on binding experiments . 87 5.4.7. Influence of endogenous labels on binding experiments . 90 5.4.8. Experimental Kd (mRFP against mCherry) . 93 5.4.9. Experimental Kd with endogenous proteins . 94 5.5. Conclusion 95 Conclusion and Outlook .97 Bibliography .102 vi Summary Fluorescence correlation spectroscopy (FCS) and its modality fluorescence crosscorrelation spectroscopy (FCCS) are single molecule sensitive optical tools to study mobility, concentrations and interactions. Due to their non-invasive nature, they are gaining popularity in studying molecular processes in vivo. The aim of this thesis is to apply and develop single-wavelength-FCCS (SW-FCCS), a variant of FCCS, to study protein-protein interactions in vivo. The thesis is organized into the following chapters: Chapter starts with discussing the importance of green florescent proteins (GFP) in modern cell biology. The advent of GFP led to the development of many optical tools to study molecular interactions and dynamics in vivo. A review of the different modalities of FCS/FCCS is presented and what are the different types of GFP mutants that are commonly used in FCCS. Chapter introduces the principles and instrumental setup of FCS and FCCS. It discusses the additional corrections and conditions when applied in vivo. Chapter discusses the cross-correlation ratios, which is commonly used to quantitate binding. It was shown, using a series of simulations, that these crosscorrelation ratios are dependent on the Kd of the binding, concentration range and the relative amount of red to green labeled molecules in the system. Chapter applies SW-FCCS to quantitate protein-protein interactions. It is divided into two parts. In the first part, the binding between a small GTPases protein vii Cdc42, and one of its effectors, IQGAP1, is investigated. The Kd of the binding in cell culture was compared with that in zebrafish embryo. In the second part, SW-FCCS is applied to study interaction between two proteins, p21 and PCNA, which are involved in DNA replication and DNA damage repair. Chapter addresses issues which constantly surface during measurements in vivo but are not studied extensively. These issues include mismatch in effective volumes, non-fluorescent fluorescent labels, FRET, photobleaching and endogenous proteins. All these factors influence the quality of the determined Kd. Major findings include quantitating the fraction of non-fluorescent red fluorescent proteins (mRFP and mCherry) and investigating the relationship of Kd with non-fluorescent labels both by simulations and experiments. Chapter concludes and presents outlook for future FCS and FCCS research. viii List of Tables 4.1 Summary of FCCS study in zebrafish muscle fiber and CHO cells 57 5.1 SW-FCCS measurements of different tandem fluorescent proteins 78 ix Chapter Despite being able to determine the Kds of interactions, discrepancies were observed. In theory, the tandem FPs should result in a cross-correlation ratio of 1. However, measurements to date by us and others achieved only a ratio of ~0.5. In chapter 5, it was demonstrated that this non-ideal ratio is due to imperfect overlap of effective volumes and can be corrected. From measurements of different tandem FPs, it was shown that the ratio is also dependent on the type of FPs and FRET. This leads to the finding that a large fraction of mRFP (78 %) and mCherry (60%) was found to be non-fluorescent hence affecting the quantitation of the amount of red labeled molecules. Due to this issue, the quality of the Kd was compromised. Endogenous proteins also act as a form of non-detected molecule which competes for binding. The effect of these was studied using Kd simulations followed by experiments using either mRFP or mCherry labeled Cdc42 and in the presence or absence of unlabeled Cdc42 (to mimic endogenous proteins). As the amount of fluorescent mRFP and mCherry are very different, the Kds determined for mRFP/mCherry-Cdc42 binding to EGFPIQGAP1 showed very different results. However, when the fraction of nonfluorescent FPs is taken into account, the Kds for both experiments becomes similar, indicating that non-fluorescent labels can be accounted for. In summary, chapter showed that the quantity of FCCS measurements is dependent on photobleaching, selection of the FPs, FRET and overlap of the effective volumes, and that these factors can be accounted for. Only by understanding these issues can the technique be developed further. 99 Chapter 6.2 Outlook In this thesis, the capabilities and limitations of FCCS were shown. It is a powerful tool to determine molecular interaction in live cells and organisms. However, the technique remains largely inaccessible to most biological laboratories. Although there are commercial systems available, they are not as ubiquitously available as other biophysical techniques. Commercial systems only provide basic data treatment and are mainly based on confocal FCS/FCCS. The more advanced applications or systems, such as scanning or camera based FCS/FCCS, are not yet commercialized or require special modification to the commercial system which is only possible in specialized laboratories. This is because many modalities of FCS/FCCS are still being developed and are usually used by only a few groups around the world. These new FCS modalities often require self-written software packages, require newly developed data treatment algorithms, and need handling and maintenance by specialists. In addition, the mathematical concepts involved in FCS/FCCS are not trivial for new comers who wish to use the technique. Hence, very often, collaborations between biological laboratories and laboratories specialized in biophysical techniques are required. FCCS is an extension of FCS. Future developments in FCS will impact the way FCCS can be applied. Some new FCS modalities such as the combination of stimulated emission depletion (STED) with FCS (STED-FCS) can reduce the diffraction limited volume allowing higher concentration and smaller region to be measured [166]. FCS has also been used with nearfield scanning optical microscopy [167], nano-apertures [168] and supercritical angle illumination [169] to manipulate the observation volume. Fluorescence lifetime correlation spectroscopy (FLCS), 100 Chapter where contributions due to different fluorescent species can be separated based on their lifetime and not emission wavelengths, is able to generate ACF free from unwanted background fluorescence or individual ACFs from spectrally similar fluorescent species as long as their fluorescence lifetime can be resolved [106, 108]. It can also separate particles with similar diffusion time, something which the conventional FCS cannot achieve. FLCS has been applied to living cells very recently [170]. Optical fiber-based FCS has also been developed [171-174]. Although these studies were performed in solution, optical fiber based FCS has the potential of measuring in remote areas of an organism. The initial motivation of applying FCS or single molecule techniques in live cells or organisms is its physiological relevance. Techniques which induce high amount of laser radiation thus inducing photodamage and phototoxicity, or require extensive physical manipulation of the organism to the point that they are no longer physiological relevant should be avoided. Ideally, the measurement should be as noninvasive and use the lowest amount of radiation per photon detected as possible. Light-sheet based illumination as used in SPIM-FCS, greatly enhances the multiplexing capabilities of FCS, allowing the measurements of whole areas in an organism simultaneously with greatly reduced phototoxicity [82, 175]. This allows capturing many more measurements per organism and makes it possible to establish FCS/FCCS images in which each pixel reports not on the fluorescence intensity but on molecular parameters, including molecular mobility, concentration, and degree of biomolecular interactions. A similar technique which uses image correlation concept is raster image correlation spectroscopy (RICS) [176, 177]. RICS uses the images acquired from a confocal laser scanning microscope to perform correlations instead of using a separate 101 Chapter FCS module. The fluorescence intensity in one of the pixels is considered to be a single point measurement which can be spatially and/or temporally correlated. RICS exploits the time structure hidden during the acquisition of an image to allow one to monitor processes in the microseconds to seconds timescale. Looking at the way how single-molecule experiments are done nowadays, if properly designed, the photons collected from one single experiment can actually be used by a number of techniques to extract information. Apart from FCS/FCCS, one can perform single-molecule FRET [160], FLCS [106, 108], FLIM-FRET [170, 178] or photon counting histograms [161, 162, 179] on a single instrument and experiment. This will maximize the amount of information acquired per experiment. As GFPs and its variants are probably going to be the preferred choice of reporters for biological studies in the future, understanding their photophysical properties will be helpful not only in FCS but in many other single molecule sensitive techniques. Although many new FPs have been generated in recent years [180, 181], the wait continues for a brighter and more stable RFP than mCherry. 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Chemphyschem, 2009. 10(9-10): p. 1369-79. 114 [...]... 1 In summary, FCCS is still in the stages of development In vivo application and interpretation of the data still require attention and improvement Therefore the aim of this thesis is the application and development of FCCS to quantitate protein-protein interactions in vivo The thesis contains six chapters and is structured into the following sections: Chapter 2 introduces the basic principles and instrumentation... co-immunoprecipitation involves lysing the cell before using anti-bodies to pull down the target protein complexes Another technique, the yeast-two-hybrid system detects protein-protein 2 Chapter 1 interactions in vivo but it requires the proteins to be expressed in yeast which is not the native environment for the protein of interest (unless it is a yeast protein) Therefore there is a need to monitor protein-protein interactions. .. strength of interaction In special cases such as the combination of fluorescence lifetime imaging microscopy to FRET (FLIM-FRET), one can determine the amount of donor molecules in complex which gives an estimate of the binding strength [14] Binding strength is typically represented by dissociation constant Kd A technique which allows the determination of Kd is fluorescence cross-correlation spectroscopy. .. role in a cell system As a cell functions through a network of protein-protein interactions, it is vital to study these interactions as it allows one to understand the role of a particular protein and its place in the whole network Many techniques, which are mainly biochemical in nature, are available to detected protein-protein interactions However, they are either in vitro methods or qualitative in. .. FCCS investigates the synchronized fluorescence fluctuations of two different fluorophores in order to detect biomolecular interactions When the movements of two molecules are synchronized, they are most likely to be interacting FCCS is an extension of fluorescence correlation spectroscopy (FCS) The basic principles of FCS is based on extracting statistical information that is embedded within the fluorescence. .. imaging and spectroscopy are important techniques in the area of modern biology Today, fluorescent tagging of biomolecules allows researchers to monitor even single molecules of interest in organisms This advance has been possible because of the advent of green fluorescence protein (GFP), which allowed genetic labeling of proteins within cell cultures or in vivo in a selective and specific manner GFP was... interactions using non-invasive methods in the native cell environment and this is where the optical imaging and spectroscopy tools fill the gap Fluorescence microscopy is most commonly used to monitor the expression of proteins in cells Due to its diffraction limited resolution of ~250 nm, it is not possible to detect protein-protein interactions even if two proteins are localized in the same pixel of an image... Shimomura, Martin Chalfie and Roger Y Tsien for the discovery and development of GFP [10] The advent of GFP leads to the development and applications of many optical imaging and spectroscopy tools in biology These tools have been helpful in discovering molecular interactions, molecular dynamics and localization of molecules Among these many different processes in a cell, protein-protein interactions. .. affect FCCS studies in vivo but is usually overlooked These issues include mismatch in effective volumes, nonfluorescent fluorescent labels, FRET, photobleaching and endogenous proteins All 9 Chapter 1 these factors influence the quality of the determined Kd Major findings include quantitating the fraction of non-fluorescent RFPs (mRFP and mCherry) and investigating the relationship of Kd with non-fluorescent... fitting algorithms [101] Despite the technological advancements, performing single molecule sensitive measurements such as FCS/FCCS in vivo is a challenge The background autofluorescence of other molecules in a cell sometimes interfere with the measurement On top of this, the commonly used GFP mutants are low in brightness and less photostable when compared to organic dyes resulting in less photons being . processes in a cell, protein-protein interactions play an important role in a cell system. As a cell functions through a network of protein-protein interactions, it is vital to study these interactions. variant of FCCS, to study protein-protein interactions in vivo. The thesis is organized into the following chapters: Chapter 1 starts with discussing the importance of green florescent proteins. STUDY OF BIOMOLECULAR INTERACTIONS IN VIVO BY MULTICOLOUR FLUORESCENCE SPECTROSCOPY FOO YONG HWEE (B.Sc.(Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY