Photoactivated localization microscopy combined with image spatial correlation analysis to study of the distribution and diffusion of Angiotensin II type 1 Receptor in the plasma membrane.

Abstract

Super resolution fluorescence microscopy, (SRFM) techniques which overcome the spatial diffraction-limit has enabled us to visualize nanoscale cellular events with high temporal and spatial resolution in living cells, but in order to gain reliable mechanistic insights, a quantitative analysis is essential 1–3. In this study we used SRFM combined with novel image analysis as a powerful tool to investigate the mechanisms by which Angiotensin II type 1 Receptors (AT1Rs), are distributed and diffuse in the plasma membrane. This mechanism remains still unclear, despite its crucial role in cardiovascular homeostasis in the cell4,5. The aim of this study is to obtain quantitative information about AT1R dynamics and organization, and their corresponding receptor-ligand complexes in living cells. This was achieved by using photoactivated localization microscopy (PALM) combined with image spatial-temporal correlation analysis. To study nanoscale receptor distribution, a combination of expansion microscopy (ExM) and PALM was used. This study showed that AT1R lateral diffusion increased after binding to the agonist, Angiotensin II (Ang II) while no change for either the biased agonist, SII-Ang II or the antagonist, Losartan, was observed. The receptor diffusion was found to be transiently confined in the PM. In addition, ExM revealed that AT1Rs form nano clusters at the PM, and the cluster size significantly decreased after Ang II treatment. Taking these results together suggests that Ang II binding and activation cause reorganization and changes in the dynamics of AT1Rs at the PM. Our work contributes to a better comprehension of AT1R dynamic in PM and makes for a better understanding of signalling and receptor mechanisms.