Functional and dynamic studies GPCRs and other membrane proteins using ultrasensitive fluorescence spectroscopy

Purpose and goal

The implementation of recently developed fluorescence microscopy techniques to biological questions is not straightforward but often leads to original results and shifts in knowledge about the studied system. In this project, we aimed to apply superresolution, correlative methods and dark state fluorescence spectroscopy to study GPCRs, the largest family of membrane receptors, on the surface of living cells. This project also aimed to generate and reinforce collaboration and to create synergies between local and international research institutions.

Expected results and effects

The main goal was to study GPCRs membrane organization with advanced fluorescence microscopy methods. The results of this work led to novel techniques development in microscopy, including data analysis, and protein labelling. The collaboration with EPFL, Lausanne gave the opportunity to combine expertise in biology, biochemistry and applied physics. The project demonstrated precise measurement of how GPCRs are organized in membrane and, furthermore, how to measure receptors activity by looking at their organization.

Planned approach and implementation

All the steps of the project involved cutting edge microscopy methods. The first part of the project was dedicated to familiarization of the researcher with the instrumentation. It was also necessary to address the outcome and limitations of the labelling strategies. During the next steps, experiments were performed in parallel on living and artificial systems to obtain reliable quantitative values. We had to design a software to analyze the large amount of data generated by STED-FCS. The last step allowed to obtain significant biological knowledge on GPCRs organization.