Fluorescent sensor proteins for intracellular metal imaging

Metal ions such as Ca2+, Mg2+, Zn2+ and Fe2+ are essential for proper functioning of the human body. Current knowledge regarding the intracellular homeostasis of these metal ions is mostly based on the characterization of individual metal binding proteins or on clinical studies involving patients suffering from (putative) metal related diseases. To obtain a more detailed understanding of the molecular mechanisms involved in metal homeostasis, tools are required that enable intracellular detection of these metal ions in living cells with high spatiotemporal resolution. Pioneered by the Nobel laureate Roger Tsien in the field of Ca2+-imaging, fluorescent protein-based sensors have shown great benefits as tools for intracellular metal imaging. Such sensors are non-invasive and their concentration and localization can be carefully controlled. In this thesis, the design, characterization and application in living cells of various genetically encoded metal sensors is described, with the main focus on sensors for the transition metal ion Zn2+. In Chapter 1, the various roles of Zn2+ in the human body are described together with the proteins involved in the homeostasis of this metal ion. The available synthetic dyes to monitor intracellular Zn2+ levels are also discussed, together with their strengths and limitations. Finally, an overview is given of the protein-based sensors that have been created to measure Zn2+ concentrations in the cell. Chapter 2 describes a rational attempt to improve an existing genetically encoded Zn2+ sensor called ZinCh-9, which displayed a large signal change but also bound Zn2+ with a relatively weak affinity. Mutant variants that contain a (Cys)4 metal binding site were created, but unfortunately none of these variants displayed an improved affinity for Zn2+. To test whether the pocket was slightly too large to accommodate Zn2+, Cd2+ titrations were performed, leading to the serendipitous discovery of a protein-based Cd2+ sensor with a 2500-fold specificity over Zn2+. In Chapter 3, an ECFP-linker-EYFP construct was used as a model system to test whether redesign of the dimerization interface of these fluorescent domains could be used to improve the signal change of protein-based FRET sensors. The hydrophobic mutations S208F and V224L were introduced on ECFP and EYFP, leading to intramolecular complex formation between these fluorescent domains and thus high energy transfer. Proteolytic cleavage of the flexible peptide linker resulted in dissociation of ECFP and EYFP and consequently a large decrease in energy transfer. The enhancement of the imaging properties and intracellular application of CALWY, an existing protein-based Zn2+ sensor, is described in Chapter 4. CALWY displayed a femtomolar affinity for Zn2+, but also only a 15% signal change upon binding of this metal ion. This small signal change has been attributed to the presence of a distribution of conformations in absence of Zn2+ that reduced the average change in distance between ECFP and EYFP upon binding of Zn2+. Introduction of the S208F and V224L mutations promoted complex formation of the fluorescent domains of CALWY in absence of Zn2+, yielding high energy transfer. Binding of Zn2+ to enhanced CALWY (eCALWY-1) resulted in a 2-fold signal change. By systematically further decreasing the affinity of eCALWY-1, a toolbox of sensors was created. These sensors were used to reliably determine the cytosolic free Zn2+ concentration in HEK293 cells, which was found to be 400 pM. In Chapter 5, the application of the eCALWY variants in pancreatic ¿-cells is described. In these cells, Zn2+ plays an important role in the storage and secretion of insulin. Determination of the free Zn2+ concentration in ¿-cells revealed similar Zn2+ levels as observed in HEK293 cells. In addition, insulin secretion and simultaneous release of Zn2+ did not affect cytosolic Zn2+ levels. The sensors were also targeted to the insulin-containing granules of these cells. In these vesicles, the free Zn2+ concentration was found to be orders of magnitude higher compared to the cytosol. Chapter 6 describes the development of different genetically encoded sensors for Mg2+. Introduction of Mg2+ binding residues at the dimerization interface of improved variants of ECFP and EYFP did not result in a Mg2+-sensitive equivalent of ZinCh-9. The second approach involved flanking of a native Mg2+ binding domain with improved variants of ECFP and EYFP, resembling a more classical FRET sensor design. Two sensors were created via this approach and both were able to detect changes in intracellular Mg2+ levels. In addition, the sensors showed intracellular specificity for Mg2+ over Ca2+, even during Ca2+ signaling. The final Chapter discusses future applications of the sensors described in this thesis. These applications include the extension of the protein-based sensor platform to other (transition) metals or to use the available sensors to simultaneously image multiple metals in different locations of the cell. In addition, different strategies are described in which the ‘sticky’ fluorescent domains are used to improve the signal change of genetically encoded FRET sensors

Fluorescent imaging of transition metal homeostasis using genetically encoded sensors

The ability to image the concentration of transition metals in living cells in real time is important for understanding transition metal (TM) homeostasis and its involvement in diseases. Genetically encoded fluorescent sensor proteins are attractive because they do not require cell-invasive procedures, can be targeted to different locations in the cell, and allow ratiometric detection. Important progress in the development of Zn2+ sensors has allowed sensitive detection of the very low free concentrations of Zn2+ in single cells, both in the cytosol and various organelles. Together with other recent advances in chemical biology, these tools seem particularly useful to interrogate the dynamics and compartmentation of TM homeostasis

Reengineering of a fluorescent zinc sensor yields the first genetically encoded cadnium probe

Introduction of a (Cys)4 metal binding site at the dimerization interface of two fluorescent protein domains yields a chelating FRET sensor protein that shows a 2500-fold selectivity for Cd2+ over Zn2+ by taking advantage of their different ionic radii

Reengineering of a fluorescent zinc sensor yields the first genetically encoded cadnium probe

Introduction of a (Cys)4 metal binding site at the dimerization interface of two fluorescent protein domains yields a chelating FRET sensor protein that shows a 2500-fold selectivity for Cd2+ over Zn2+ by taking advantage of their different ionic radii

Rational design of FRET sensor proteins based on mutually exclusive domain interactions

Proteins that switch between distinct conformational states are ideal to monitor and control molecular processes within the complexity of biological systems. Inspired by the modular architecture of natural signalling proteins, our group explores generic design strategies for the construction of FRET-based sensor proteins and other protein switches. In the present article, I show that designing FRET sensors based on mutually exclusive domain interactions provides a robust method to engineer sensors with predictable properties and an inherently large change in emission ratio. The modularity of this approach should make it easily transferable to other applications of protein switches in fields ranging from synthetic biology, optogenetics and molecular diagnostics

Engineering protein switches : sensors, regulators, and spare parts for biology and biotechnology

Switch protein switch! Proteins that switch between distinct conformational states are ideal for monitoring and controlling molecular processes in biological systems. We discuss new engineering concepts for the construction of protein switches that have the potential to be generally applicable and discuss them according to their mechanism of action

Enhanced sensitivity of FRET-based protease sensors by redesign of the GFP dimerization interface

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Genetically encoded FRET sensors to monitor intracellular Zn2+ homeostasis

We developed genetically encoded fluorescence resonance energy transfer (FRET)-based sensors that display a large ratiometric change upon Zn2+ binding, have affinities that span the pico- to nanomolar range and can readily be targeted to subcellular organelles. Using this sensor toolbox we found that cytosolic Zn2+ was buffered at 0.4 nM in pancreatic ß cells, and we found substantially higher Zn2+ concentrations in insulin-containing secretory vesicles

Dynamic imaging of cytosolic zinc in Arabidopsis roots combining FRET sensors and RootChip technology

Zinc plays a central role in all living cells as a cofactor for enzymes and as a structural element enabling the adequate folding of proteins. In eukaryotic cells, metals are highly compartmentalized and chelated. Although essential to characterize the mechanisms of Zn(2+) homeostasis, the measurement of free metal concentrations in living cells has proved challenging and the dynamics are difficult to determine. Our work combines the use of genetically encoded Förster resonance energy transfer (FRET) sensors and a novel microfluidic technology, the RootChip, to monitor the dynamics of cytosolic Zn(2+) concentrations in Arabidopsis root cells. Our experiments provide estimates of cytosolic free Zn(2+) concentrations in Arabidopsis root cells grown under sufficient (0.4 nM) and excess (2 nM) Zn(2+) supply. In addition, monitoring the dynamics of cytosolic [Zn(2+) ] in response to external supply suggests the involvement of high- and low-affinity uptake systems as well as release from internal stores. In this study, we demonstrate that the combination of genetically encoded FRET sensors and microfluidics provides an attractive tool to monitor the dynamics of cellular metal ion concentrations over a wide concentration range in root cells