Angiotensin II Treatment Induces Reorganization and Changes in the Lateral Dynamics of Angiotensin II Type 1 Receptor in the Plasma Membrane Elucidated by Photoactivated Localization Microscopy Combined with Image Spatial Correlation Analysis.

The mechanisms by which angiotensin II type 1 receptor is distributed and the diffusional pattern in the plasma membrane (PM) remain unclear, despite their crucial role in cardiovascular homeostasis. In this work, we obtained quantitative information of angiotensin II type 1 receptor (AT1R) lateral dynamics as well as changes in the diffusion properties after stimulation with ligands in living cells using photoactivated localization microscopy (PALM) combined with image spatial-temporal correlation analysis. To study the organization of the receptor at the nanoscale, expansion microscopy (ExM) combined with PALM was performed. This study revealed that AT1R lateral diffusion increased after binding to angiotensin II (Ang II) and the receptor diffusion was transiently confined in the PM. In addition, ExM revealed that AT1R formed nanoclusters at the PM and the cluster size significantly decreased after Ang II treatment. Taking these results together suggest that Ang II binding and activation cause reorganization and changes in the dynamics of AT1R at the PM.

Using kICS to Reveal Changed Membrane Diffusion of AQP-9 Treated with Drugs.

The formation of nanodomains in the plasma membrane are thought to be part of membrane proteins regulation and signaling. Plasma membrane proteins are often investigated by analyzing the lateral mobility. k-space ICS (kICS) is a powerful image correlation spectroscopy (ICS) technique and a valuable supplement to fluorescence correlation spectroscopy (FCS). Here, we study the diffusion of aquaporin-9 (AQP9) in the plasma membrane, and the effect of different membrane and cytoskeleton affecting drugs, and therefore nanodomain perturbing, using kICS. We measured the diffusion coefficient of AQP9 after addition of these drugs using live cell Total Internal Reflection Fluorescence imaging on HEK-293 cells. The actin polymerization inhibitors Cytochalasin D and Latrunculin A do not affect the diffusion coefficient of AQP9. Methyl-ß-Cyclodextrin decreases GFP-AQP9 diffusion coefficient in the plasma membrane. Human epidermal growth factor led to an increase in the diffusion coefficient of AQP9. These findings led to the conclusion that kICS can be used to measure diffusion AQP9, and suggests that the AQP9 is not part of nanodomains.

Defining the Diffusion in Model Membranes Using Line Fluorescence Recovery after Photobleaching.

In this study, we explore the use of line FRAP to detect diffusion in synthetic lipid membranes. The study of the dynamics of these membrane lipids can, however, be challenging. The diffusion in two different synthetic membranes consisting of the lipid mixtures 1:1 DOPC:DPPC and 2:2:1 DOPC:DPPC:Cholesterol was studied with line FRAP. A correlation between diffusion coefficient and temperature was found to be dependent on the morphology of the membrane. We suggest line FRAP as a promising accessible and simple technique to study diffusion in plasma membranes.

Revealing Plasma Membrane Nano-Domains with Diffusion Analysis Methods.

Nano-domains are sub-light-diffraction-sized heterogeneous areas in the plasma membrane of cells, which are involved in cell signalling and membrane trafficking. Throughout the last thirty years, these nano-domains have been researched extensively and have been the subject of multiple theories and models: the lipid raft theory, the fence model, and the protein oligomerization theory. Strong evidence exists for all of these, and consequently they were combined into a hierarchal model. Measurements of protein and lipid diffusion coefficients and patterns have been instrumental in plasma membrane research and by extension in nano-domain research. This has led to the development of multiple methodologies that can measure diffusion and confinement parameters including single particle tracking, fluorescence correlation spectroscopy, image correlation spectroscopy and fluorescence recovery after photobleaching. Here we review the performance and strengths of these methods in the context of their use in identification and characterization of plasma membrane nano-domains.