FRET-BASED BIOSENSORS TO ELUCIDATE EXTRACELLULAR SIGNAL- REGULATED KINASE (ERK) DYNAMICS By Archer R. Hamidzadeh A thesis submitted to Johns Hopkins University in conformity with the requirements for the degree of Master of Science in Engineering Baltimore, MD May, 2015 Abstract Proper cell functions are dependent on the precise regulation of transduction events to relay external environmental cues into the cell for processing. The propagation and regulation of the signal involves a diverse array of enzymes and molecules with distinct functions and differences in spatio-temporal regulation. Further understanding cell signaling dynamics will continue to provide a knowledge basis for developing disease therapies. While an active field of research, a great deal of information has remained elusive with the previously used tools to monitor such pathways either through discrete snapshots or analyses of single components of signaling pathways outside of their natural biological context. To address these limitations, FRET-based biosensors provide a powerful means to dissect the complexities of signaling pathways in a real-time manner in live cells. The first objective is to optimize the previously developed ERK Activity Reporter, EKAR, by examining the dependence of its dynamic range on fluorophore orientation. To alter relative fluorophore orientations, variations of EKAR were developed with reversed orders of fluorescent proteins. Reversing the order of a previously optimized yellow-cyan EKAR led to a cyan-yellow EKAR with an improved dynamic range. Moreover, a closer examination of the previously optimized EeVee- linker, which was believed to render FRET-based biosensors solely distance-dependent, reveals that the FRET efficiency is likely dependent on FP orientation in EeVee-linker based biosensors. This conclusion for EKAR can likely be extended for further improvements of other FRET-based biosensors with the EeVee linker. ii The second objective is to expand the versatility of the EKAR biosensor for co- imaging capabilities, which allow for more precise correlations between multiple activity readouts on a single-cell level. Utilizing EKAR color variants, a co-imaging strategy was employed to allow for imaging two sensors with spectrally distinct FRET-donor fluorescent proteins (FP) and a common FRET-acceptor FP. This strategy allowed for simultaneously tracking ERK activity in multiple subcellular compartments, monitoring both ERK and Protein kinase A (PKA) on a single-cell level, and for the development of a novel single-chain biosensor capable of monitoring ERK and Ras activity in the plasma membrane. Continued investigations will provide powerful tools to elucidate signaling dynamics with stronger precision. Thesis advisors and readers: Professors Jin Zhang, Andre Levchenko, and Kevin Yarema iii Preface Foremost, I would like to express my immense gratitude to my advisors, Professor Jin Zhang and Professor Andre Levchenko, for their incredible guidance and support during my undergraduate and graduate studies. I greatly appreciate their motivation, enthusiasm, and vast knowledge in the area of cellular signaling, all of which have allowed me to grow towards becoming an independent researcher. I owe my great enthusiasm to continue studying biological signaling to both of them. In addition, I would like to express my immense gratitude to Professor Kevin Yarema for his invaluable comments and guidance for this thesis. In addition, I would like to thank the other members of both labs, who have provided excellent environments to conduct these research projects, collaborate, and learn. I greatly appreciate the guidance and mentoring from everyone in these two labs. A great deal of appreciation is due to my research mentor, and good friend, Dr. Ambhi Ganesan. He has truly been an excellent guide and friend throughout my studies. I would also like to thank my earliest research and academic advisers at Hopkins, Dr. Vincent Hilser and Dr. Ludwig Brand, who guided me towards the right path - quantitatively understanding biological phenomena. Last but not least, I am indebted to my father, Dr. Hamid Hamidzadeh, my mother, Azar, and my brother, Cyrus, for their unconditional compassion and support over the years. iv Table of Contents Title Page..................................................................................................................i Abstract...................................................................................................................ii Preface....................................................................................................................iii Table of Contents...................................................................................................iv List of Figures..........................................................................................................v Introduction.......................................................................................................1-15 Chapter 1 Optimization of FRET-based Biosensor for Investigation of Extracellular Signal-Regulated Kinase Dynamics......................................................16-33 ERK-MAPK signaling...............................................................................17 Regulation of ERK activity and specificity................................................20 Optimization of EKAR...............................................................................22 Methods......................................................................................................30 References..................................................................................................32 Chapter 2 Expansion of the Application of EKAR for Investigation of Extracellular Signal Regulated Kinase Dynamics......................................................34-54 Development and characterization of EKAR color variants......................35 Visualization of ERK activity in the cytoplasm.........................................42 ERK activity in the plasma membrane and nucleus...................................44 Co-Imaging of subcellular-targeted EKAR................................................48 Methods......................................................................................................51 References..................................................................................................53 Chapter 3 Investigation of Crosstalk and Pathway dynamics of Extracellular Signal- Regulated Kinase..................................................................................55-76 Biological crosstalk: cAMP-PKA and ERK-MAPK pathways.................56 EKAR and AKAR co-imaging...................................................................57 Role of calcium in the ERK-MAPK pathway............................................65 Simultaneously monitoring the activities of Ras and ERK........................66 Methods......................................................................................................74 References..................................................................................................76 Curriculum Vitae..................................................................................................77 v List of Figures Figure 1.1 ERK-MAPK cascade...................................................................................18 Figure 1.2 Average EGF-induced responses of cyan-yellow EKARs..........................24 Figure 1.3 Average dynamic ranges of cytoplasmic-targeted cyan-yellow EKARs....25 Figure 1.4 Effects of EeVee linker length on EKAR dynamic range...........................27 Figure 1.5 Effects of EeVee linker length on EKAR basal FRET values.....................29 Figure 2.1 Design and characterization of diffusible EKAR color variants.................38 Figure 2.2 Representative characterization experiments of EKAR color variants.......39 Figure 2.3 Average dynamic ranges of diffusible EKAR color variants......................41 Figure 2.4 Cytoplasmic targeting of EKAR..................................................................43 Figure 2.5 Plasma membrane and nuclear targeting of EKAR.....................................46 Figure 2.6 Targeted EKAR Co-Imaging.......................................................................50 Figure 3.1 EKAR and AKAR co-imaging....................................................................59 Figure 3.2 EKAR and AKAR co-imaging controls......................................................62 Figure 3.3 EKAR control under co-imaging stimulus conditions.................................64 Figure 3.4 SABER1.0...................................................................................................68 Figure 3.5 SABER1.0: single cell results - Oscillatory Ras Response.........................70 Figure 3.6 SABER1.0: single cell results - Sustained Ras Response...........................71 Figure 3.7 SABER1.0: mutant control..........................................................................72 vi Intended to be blank vii Introduction 1 Cellular Signaling With the realization that cells do not live in isolation, cellular communication is an inherent process that drives functional responses of a cell to its surrounding environment. This process of exchanging messages between a cell and its environment is present in all cells. Single-celled prokaryotes sense and navigate toward nutrients sources. Eukaryotic microorganisms, such as yeasts and protozoans, utilize secreted pheromones to coordinate aggregation and mating. Lastly, multicellular eukaryotes use a plethora of signaling molecules and mechanisms to coordinate the developmental, regulatory, and metabolic activities of the organism as a whole. Signal transduction cascades are molecular circuits responsible for sensing, amplifying, and integrating various external environmental stimuli into intracellular responses, such as changes in gene expression and enzyme activity. A signaling cascade is generally triggered by the binding of a ligand to a specific protein receptor. The subsequent conformational change results in the initiation of one or more pathways that transmit the stimulus to the interior of the cell. These cascades, which involve a complex network of interwoven molecules, enzymes, and transcription factors, control cellular responses such as: proliferation, differentiation, and apoptosis. Normal cell function is dependent on the precise regulation of transduction events to relay the external environmental cue into an intracellular outcome. The propagation and regulation of the signal involves a diverse array of enzymes with distinct functions. In particular, post-translational modifications (PTMs) of cellular proteins are important mechanisms for propagating the signal. One of the more common PTMs, phosphorylation, has a governing role in many signaling events. Protein kinases 2 enzymatically transfer the γ-phosphate from adenosine triphosphate (ATP) to serine, threonine, or tyrosine residues of a target protein, resulting in changes in the protein's activity, localization, or interactions (1). The human genome is reported to contain 518 protein kinase genes, about 2% of all known human genes (2). While protein kinases share a conserved catalytic core, various structural components dictate their activation, regulation, target protein preferences, and localization within the cell (3). These kinases modify several downstream targets, which comprise 30% of all human proteins. The dynamics of kinase levels, activities, and localization influence the regulation of major processes in the cell. Dysregulation of kinase function or localization is a hallmark of numerous diseases including neurodegenerative, autoimmune, and immunological disorders and most human cancers. Due to their roles in various cellular activities and pathologies, kinases have been prime targets for therapies. Over the past two decades, a small number of small- molecule kinase inhibitors have been identified and approved for clinical use (4). Conventional methods of investigating kinase activity involve quantification of the phosphoryl transfer using enzyme-based and lysis-based radiometric or phospho- specific antibody assays in vitro. Traditional radioisotope filtration binding assays utilize radioisotope labeled γ-ATP reactions and cumbersome binding and washing steps for the ultimate detection of kinase activity (5). Moreover, later assays were directed to detect kinase activity using phospho-specific antibodies or fluorescent signals (6). While these methods have been successful in detecting kinase activity and function, a great deal of information is lost when monitoring the activity of these enzymes outside of the cell or in discrete snapshots through Western blots and immunostaining. Further understanding 3