Biology subjects

Chavez-Abiega, S.

Publications and source records attributed to Chavez-Abiega, S..

3 recordsLinked to original sources

bioRxiv2025

Multiplexed imaging of G-proteins and ERK activity upon activation of CaSR

We have previously shown that the calcium sensing receptor (CaSR) activates different G proteins and second messengers in single cells, and that GPCR and G protein-mediated ERK activity can be highly dynamic and heterogeneous at the single cell level. Here, we attempted to investigate the CaSR-Gi-ERK signaling pathway in single cells with previously characterized biosensors. We developed a strategy to simultaneously track G-protein and ERK activities in a single cell, using FRET-based and translocation sensors, a novel large-Stokes-shift green fluorescent protein, and an imaging setup with a single detector and a single wavelength for excitation. Extensive characterization and optimization to unmix the signals showed that the differences in dynamic range and type of read-out obtained from these biosensors made it very challenging to obtain robust FRET-measurements in the developed setup. When focusing solely on ERK, we found signs of possible ERK activity in response to calcium stimulation via CaSR, as well as milder changes in the absence of calcium treatment. Further optimization of the model, probes, and image processing will be necessary to develop a setup to robustly measure FRET and translocation simultaneously in single cells. We present the challenges we encountered and discuss future developments that can overcome them.

cell biology↗

bioRxiv2024

Biosensor imaging of G protein mediated Signaling Pathways Activated Immediately Downstream of CaSR

Genetically encoded biosensors allow us to study events in single cells in real time. Here, we used biosensors with a read-out based on Forster resonance energy transfer (FRET) or intensity to investigate the main signaling pathways activated immediately downstream of the calcium sensing receptor (CaSR) in HEK-293 cells. CaSR responds to its endogenous ligand, calcium, to activate Gq and Gi protein dependent pathways. We demonstrate that CaSR activates Gq to promote intracellular calcium mobilization, and we monitored these two events simultaneously in the same cells. We also observed that CaSR activates the 3 Gi subtypes and decreases cAMP levels in a Gi-dependent manner. Moreover, the use of negative allosteric modulator NPS-2143 can inhibit these signaling events and also rapidly disrupts them if added after receptor activation by calcium stimulation. In addition, the increases in calcium and cAMP levels were respectively enhanced when activation of Gi or Gq proteins was prevented. Finally, we provide evidence that CaSR does not couple to G13 proteins, and that activation of RhoA by CaSR is solely dependent on Gq/G11 activity.

cell biology↗

bioRxiv2021

Heterogeneity and dynamics of ERK and Akt activation by G protein-coupled receptors depend on the activated heterotrimeric G proteins

Kinases are fundamental regulators of cellular functions and play key roles in GPCR-mediated signaling pathways. Kinase activities are generally inferred from cell lysates, hiding the heterogeneity of the individual cellular responses to extracellular stimuli. Here, we study the dynamics and heterogeneity of ERK and Akt in genetically identical cells in response to activation of endogenously expressed GPCRs. We use kinase translocation reporters, high-content imaging, automated segmentation and clustering methods to assess cell-to-cell signaling heterogeneity. We observed ligand-concentration dependent response kinetics to histamine, a2-adrenergic, and S1P receptor stimulation that varied between cells. By using G protein inhibitors, we observed that Gq mediated the ERK and Akt responses to histamine. In contrast, Gi was necessary for ERK and Akt activation in response to 2-adrenergic receptor activation. ERK and Akt were also strongly activated by S1P, showing high heterogeneity at the single cell level, especially for ERK. In all cases, the cellular heterogeneity was not explained by distinct pre-stimulation levels or saturation of the measured response. Cluster analysis of time-series derived from 68,000 cells obtained under the different conditions revealed several distinct populations of cells that display similar response dynamics. The single-cell ERK responses to histamine and brimonidine showed remarkably similar dynamics, despite the activation of different heterotrimeric G proteins. In contrast, the ERK response dynamics to S1P showed high heterogeneity, which was reduced by the inhibition of Gi. To conclude, we have set up an imaging and analysis strategy that reveals substantial cell-to-cell heterogeneity in kinase activity driven by GPCRs. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

cell biology↗