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.
|Qualification||Doctor of Philosophy|
|Award date||10 May 2010|
|Place of Publication||Eindhoven|
|Publication status||Published - 2010|