Image mean square displacement to study the lateral mobility of Angiotensin II type 1 and Endothelin 1 type A receptors on living cells

• Published in: Microscopy research and technique Vol. 83; no. 4; pp. 381 - 392
• Main Authors: Planes, Nadir, Vanderheyden, Patrick P.M.L., Gratton, Enrico, Caballero‐George, Catherina
• Format: Journal Article
• Language: English
• Published: Hoboken, USA John Wiley & Sons, Inc 01.04.2020; Wiley Subscription Services, Inc
• Subjects: Angiotensin AT1 receptors; Angiotensin II; Angiotensin II type 1 receptor; Animals; Binding; Biological Transport; Cells (biology); CHO Cells; Coordination compounds; Cricetulus; Diffusion; diffusion coefficient; Diffusivity; Endothelin 1; Endothelin 1 type A receptor; Endothelins; Fluorescence; Fluorescence microscopy; G‐protein‐coupled receptor; image mean square displacement; Image Processing, Computer-Assisted - methods; Information processing; Ligands; Membrane fluidity; Membranes; Microscopy; Microscopy, Fluorescence; Mobility; Molecular interactions; Protein Binding; Proteins; Qualitative analysis; Receptor mechanisms; Receptor, Angiotensin, Type 1 - metabolism; Receptor, Endothelin A - metabolism; Receptors; Reflection; total internal reflection fluorescence microscopy; total internal reflection fluorescence microscopy; Angiotensin II type 1 receptor; image mean square displacement; Endothelin 1 type A receptor; G-protein-coupled receptor; diffusion coefficient
• ISSN: 1059-910X; 1097-0029
• DOI: 10.1002/jemt.23425

The lateral mobility of membrane receptors provides insights into the molecular interactions of protein binding and the complex dynamic plasma membrane. The image mean square displacement (iMSD) analysis is a method used to extract qualitative and quantitative information of the protein diffusion law and infers how diffusion dynamic processes may change when the cellular environment is modified. The aim of the study was to describe the membrane diffusing properties of two G‐protein‐coupled receptors namely Angiotensin II type 1 (AT1) and Endothelin 1 type A (ETA) receptors and their corresponding receptor–ligand complexes in living cells using total internal reflection fluorescent microscopy and iMSD analysis. This study showed that both AT1 and ETA receptors displayed a mix of three modes of diffusion: free, confined, and partially confined. The confined mode was the predominant at the plasma membrane of living cells and was not affected by ligand binding. However, the local diffusivity and the confinement zone of AT1 receptors were reduced by the binding of its antagonist losartan, and the long‐range diffusion with the local diffusivity coefficient of ETA receptors was reduced upon exposure to its antagonist BQ123. To the best of our knowledge, this is the first study addressing the protein diffusion laws of these two receptors on living cells using total internal reflection fluorescence microscopy and iMSD. Membrane receptor lateral mobility has been a subject of many studies using single particle tracking (SPT). However, SPT by definition only tracks a particle at a time. To detect obstacles to particle mobility and particle confinement regions, a large number of tracks are needed. The need of high statistics does not allow simultaneous exploration of a large part of the membrane. The time changes of the confinement zones are also difficult to obtain and also the condition of single particle requires labeling receptors using large particles conjugated to antibodies. The image mean square displacement (iMSD) method provides the location of confinement zones and the complete map of the membrane where the motion is characterized by free, confined, and partially confined diffusion and the time evolution (in the second timescale) of these regions. Given the sensitivity of the iMSD method, it can be performed with receptors labeled with a single fluorescent molecule. The iMSD method could become the method of choice for the determination of membrane dynamics and overcome the technical issues related to the SPT technique.