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The objective of my Ph. D. study is to develop novel genetically encoded fluorescent biosensors to image and dissect biological signaling pathways in the context of live cells. I utilized protein engineering techniques to convert fluorescent proteins into fluorescent biosensors that can actively respond to specific, spatiotemporally organized cellular changes.
One of the major challenges of modern biology and medicine consists in finding means to visualize biomolecules in their natural environment with the greatest level of accuracy, so as to gain insight into their properties and behaviour in a physiological and pathological setting. This has been achieved thanks to the design of novel imaging agents, in particular to fluorescent biosensors. Fluorescence Biosensors comprise a large set of tools which are useful for fundamental purposes as well as for applications in biomedicine, drug discovery and biotechnology. These tools have been designed and engineered thanks to the combined efforts of chemists and biologists over the last decade, and developed hand in hand together with imaging technologies. This volume will convey the many exciting developments the field of fluorescent biosensors and reporters has witnessed over the recent years, from concepts to applications, including chapters on the chemistry of fluorescent probes, on technologies for monitoring protein/protein interactions and technologies for imaging biosensors in cultured cells and in vivo. Other chapters are devoted to specific examples of genetically-encoded reporters, or to protein and peptide biosensors, together with examples illustrating their application to cellular and in vivo imaging, biomedical applications, drug discovery and high throughput screening. Contributions from leading authorities Informs and updates on all the latest developments in the field
Fluorescent proteins (FPs) are essential tools of biochemical research. Traditionally, FPs have been utilized as markers of gene expression, protein localization, and organelle structure. In recent years, however, FPs have gained in popularity as active biosensors of cellular activity, in which the fluorescence intensity or colour of a FP chromophore is modulated in response to a change in its environment. Current methods for converting FPs into active biosensors of live cell biochemistry remain few in number and are technically challenging. In addition, a growing demand has emerged for spectrally orthogonal biosensors for monitoring multiple biological parameters in a single live cell. A challenge in realizing this goal is that the broad spectral profiles of FPs have limited the number of biosensors that can be used together. In this thesis I describe the engineering of new FP-based biosensors and further validating them via live cell multiparameter imaging. The first class of FP-based biosensors addressed in this thesis is Förster resonance energy transfer (FRET) biosensors. I described our efforts to use optimized spectrally distinct FRET-based biosensors to image Ca2+ dynamics in two distinct subcellular compartments as well as Ca2+ and caspase-3 activity in the same subcellular compartment. Although the inherently ratiometric response of FRET biosensor permits quantitative measurements, the fact that two FPs are involved in each FRET pair present a challenge with respect to their application in multiparameter imaging. To overcome this issue, we engineered intensiometric biosensors based on the recently introduced dimerization-dependent fluorescent protein (ddFP) technology. I demonstrate that ddFP-based protease biosensors enable the reporting of protease activity either by the intensiometric loss of the initially bright fluorescence or with dramatic green-to-red and red-to-green colour switches and translocation from the cytoplasm to the nucleus. In addition to detection of caspase activities, we also achieved specific highlighting of the mitochondria-associated membrane (MAM) using ddFP technology. Attempts to detect polyADP-ribose and polyubiquitin using ddFP technology were ultimately unsuccessful. Overall, this work serves to expand the scope of current FRET and ddFP-based biosensor designs and applications.
In Fluorescent Protein-Based Biosensors: Methods and Protocols, experts in the field have assembled a series of protocols describing several methods in which fluorescent protein-based reporters can be used to gain unique insights into the regulation of cellular signal transduction. Genetically encodable fluorescent biosensors have allowed researchers to observe biochemical processes within the endogenous cellular environment with unprecedented spatiotemporal resolution. As the number and diversity of available biosensors grows, it is increasingly important to equip researchers with an understanding of the key concepts underlying the design and application of genetically encodable fluorescent biosensors to live cell imaging. Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Fluorescent Protein-Based Biosensors: Methods and Protocols promises to be a valuable resource for researchers interested in applying current biosensors to the study of biochemical processes in living cells as well as those interested in developing novel biosensors to visualize other cellular phenomena.
Since the discovery of the gene for green fluorescent protein (GFP), derived from jellyfish, this protein that emits a green glow has initiated a revolution in molecular biosciences. With this tool, it is now possible to visualize nearly any protein of interest in any cell or tissue of any species. Since the publication of the first edition, there have been tremendously significant technological advances, including development of new mutant variants. Proteins are now available in yellow and blue, and Novel Fluorescent Proteins (NFPs) have expanded their utility in developing biosensors, biological markers, and other biological applications. This updated, expanded new edition places emphasis on the rise of NFPs, including new chapters on NFP properties with detailed protocols, applications of GFPs and NFPs in industry research, and biosensors. This book provides a solid theoretical framework, along with detailed, practical guidance on use of GFPs and NFPs with discussion of potential pitfalls. The expert contributors provide real examples in showing how to tailor GFP/NFP to specific systems, maximize expression, and enhance detection.
Chloride is the most abundant anion in our body and is essential for all forms of life. The transport of chloride is linked to cellular functions including cell volume, pH regulation, cell division, muscle contraction, and neuroexcitation. However, dysregulation of cellular chloride transport has been implicated in human diseases such as cystic fibrosis, pancreatitis, and epilepsy suggesting that chloride could be a signal of cellular status. Moreover, we lack a clear molecular-level picture of what chloride is doing. In this thesis, I will discuss the various approaches researchers have used to study chloride in biological systems. Chapter 1 will review the different types of fluorescent proteins that have been used to develop sensors for chloride. To expand on the current state of the art, we have revealed the first examples of standalone turn-on fluorescent protein-based sensors for chloride using the naturally occurring yellow fluorescent protein from the jellyfish Phialidium sp., which is ratiometric and undergoes an excited state proton transfer in the presence of chloride (Chapter 2), and the engineered mNeonGreen protein from the cephalochordate Branchiostoma lanceloatum (Chapter 3). Given that the mNeonGreen sensor operates best at pH 4.5, Chapter 4 will describe how we designed and carried out double site-saturation mutagenesis of noncoordinating residues in the mNeonGreen chloride binding pocket. This protein engineering effort not only improved the chloride sensing properties of mNeonGreen at physiological pH but also generated sensors with the largest turn-on fluorescence responses to chloride thus far (ChlorON). Fluorescence imaging experiments in mammalian cells expressing a ChlorON sensor demonstrate how the advantage of using turn-on fluorescent sensors to provide spatial and temporal resolution for mapping chloride dynamics. Lastly, Chapter 5 will highlight an alternative de novo strategy to convert the membrane-bound, proton-pumping rhodopsin from the cyanobacterium Gloeobacter violaceus (GR) into a non-pumping, red-shifted, and turn-on fluorescent sensor for chloride that can be used to image chloride in bacteria. In this study, we identified how a single point mutation of a key residue in the proton transport pathway can create a new chloride binding site in GR while also altering the protein function and spectroscopic properties to sense chloride. Taken together, this body of work lays the foundation of building protein-based hosts for chloride recognition and illustrates how we can use and adapt naturally occurring proteins to expand the turn-on fluorescent imaging toolkit for chloride that can enable the discovery of new roles for chloride in biology.
This new edition of Fluorescent Proteins presents current applications of autofluorescent proteins in cell and molecular biology authored by researchers from many of the key laboratories in the field. Starting from a current review of the broad palette of fluorescent proteins available, several chapters focus on key autofluorescent protein variants, including spectral variants, photodynamic variants as well as chimeric FP approaches. Molecular applications are addressed in chapters that detail work with single molecules, approaches to generating protein fusions and biosensors as well as analysis of protein-protein interactions in vivo by FRET, fluorescence polarization and fluorescence cross correlation techniques. A number of approaches to in vivo dynamics are presented, including FRAP, photoactivation, and 4-dimensional microscopy. Behavior of spindle components, membrane proteins, mRNA trafficking as well as analysis of cell types in tissues and in development are detailed and provide models for a wide variety of experimental approaches. In addition, several chapters deal directly with the computational issues involved in processing multidimensional image data and using fluorescent imaging to probe cellular behavior with quantitative modeling. This volume brings together the latest perspective and techniques on fluorescent proteins and will be an invaluable reference in a wide range of laboratories.
Advances in fluorescent proteins, live-cell imaging, and superresolution instrumentation have ushered in a new era of investigations in cell biology, medicine, and physiology. From the identification of the green fluorescent protein in the jellyfish Aequorea victoria to the engineering of novel fluorescent proteins, The Fluorescent Protein Revoluti
This volume brings together cutting-edge laboratory protocols to characterize the novel fluorescent proteins (FPs) and approaches based on fluorescent proteins that aim to answer some of the key cell biological questions. The book covers topics ranging from the database of fluorescent proteins to their characterization and adaptation to a wide range of biological systems. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Fluorescent Proteins: Methods and Protocols serves as an ideal guide for students and academicians enthusiastic about the recent progress in the practical application of fluorescent protein technology.
Cells execute specific responses to diverse environmental cues by encoding information in distinctly compartmentalized biochemical signaling reactions. Genetically encoded fluorescent biosensors enable the spatial and temporal monitoring of signaling events in live cells. Chapter 1 of this dissertation is a reprint of work submitted for publication, that introduces the concepts of compartmentalized signaling, genetically encoded fluorescent biosensors, and temporal and spatiotemporal deterministic computational modeling and then demonstrates the power of applying computational models to interpret biosensor experiments in complex biochemical networks in two case studies. Chapter 2 of this dissertation is a reprint of published work in which we demonstrate that a mutation on a solvent-exposed residue of a red fluorescent protein (RFP) leads to a brighter, more stably fluorescent RFP. We also show that this RFP enhances green-red FRET biosensors and can be incorporated into a spectrally orthogonal FRET biosensor multiplexing scheme to investigate the crosstalk between Src, Akt, and Extracellular Regulated Kinase (ERK) kinases in single cells. Chapter 3 is a reprint of published work wherein we demonstrate that a platform combing an open-source liquid handler with a fluorescent microscope can automate and enhance throughput of fluorescent biosensor imaging experiments in response to multiple compound stimulations. We demonstrate this platform using protein kinase A (PKA) and 3',5'-cyclic adenosine monophosphate (cAMP) FRET biosensors to measure the signaling dynamics in response to varying concentrations of a single G-protein coupled receptor (GPCR) agonist, to a panel of GPCR agonists and antagonists, or to pH media exchanges. Finally, Chapter 4 is a conclusion where we summarize the findings and discuss future extensions of the work. This work describes three different methods that increase the utility of fluorescent biosensors experiments to investigate cell signaling phenomenon: quantitatively evaluating biosensor experimental data using computational models, developing a new red fluorescent protein and a three FRET biosensor multiplexing scheme, and engineering a platform that automates and increases throughput of fluorescent biosensor imaging experiments.