Elucidating Calcium-Sensing Receptor signaling pathways in single cells with fluorescence-based biosensors

The aim of this thesis was to gain a better understanding of the signaling dynamics of GPCRs and G proteins in single cells using fluorescence-based biosensors, with a special focus on the calcium sensing receptor (CaSR). In chapter 1, I reviewed extensively the structure and function this GPCR, fundamental for calcium homeostasis regulation. CaSR is expressed in various tissues and has multiple known endogenous ligands, which can bind to different sites to activate preferentially specific downstream signaling pathways. This ligand-biased signaling makes CaSR a promising pharmacological target to treat disease as it offers varied modulation possibilities. In chapter 2, I provided an overview of how biosensors and imaging technologies are used to dissect the complex physical biology of GPCR signaling dynamics. I highlighted developments to investigate receptor mobility and localization, conformational changes upon ligand binding, subsequent activation and downstream signaling, and resulting negative feedback mechanisms. I also discussed the need for approaches that capture the dynamics of multiple components simultaneously, given the enormous complexity of GPCR signaling networks. In chapter 3, I employed some of these tools to investigate G protein mediated signaling pathways activated by CaSR. Through transient transfection with various FRET-based and an intensity based biosensor in HEK-293 cells stably expressing CaSR, I evaluated the activity of signaling components downstream of CaSR and their dependency to G protein activation. I found well-established associations in literature, and also evidence for RhoA being exclusively activated by Gq/G11 proteins, contrary to the hypothesis that it is mediated by G13. In chapter 4, I used translocation reporters to measure the activities of ERK and Akt in single HeLa cells in response to activation of various GPCRs. By screening multiple generated stable cell lines containing an engineered optimized plasmid, I obtained a model suitable for high-content imaging and automated image analysis that yielded readily large numbers of robust single cell measurements. I stimulated endogenous histamine, a2-adrenergic, and S1P receptors with multiple ligand concentrations, and found that ERK and Akt activities were largely correlated. ERK responses between ligands and concentrations were heterogeneous in amplitude and kinetics, and displayed novel dynamics upon inhibition of Gq and Gi protein families. Due to the observed diversity in the ERK responses, I decided to investigate how the single cell trajectories differed between the tested conditions. In chapter 5, I evaluated the performance of multiple clustering methods to classify the observed ERK and Akt trajectories across all conditions. ERK responses upon activation of histamine and a2-adrenergic receptors, although mediated by different G proteins, showed similar transient dynamics. Responses to S1P, on the other hand, displayed multiple kinetics pattern, which were dependent mainly on Gi but also on Gq proteins. Given these encouraging results to generate large amounts of robust single cell data, in chapter 6 I attempted to combine
the ERK KTR with a FRET-based G protein sensor in the previously characterized CaSR-expressing HEK-293 cell line. The strategy included an imaging setup with a single detector and a single wavelength for excitation, technically feasible by including large-Stokes-shift fluorescent proteins for the KTR and the nuclear marker to complement the Gi1 FRET sensor. Despite consistent expression of the four signals in the generated cell line, and extensive efforts to characterize the imaging setup, I could not unmix these signals sufficiently to measure clear FRET changes. In addition, the measured ERK responses upon calcium stimulation were limited, and less robust than those I observed after stimulating endogenous receptors in HeLa cells. After presenting the encountered challenges and limitations, I discussed possible approaches to overcome them. Finally, in the last part I discuss the most relevant results from the previous chapters.