Probing the mechanoreceptor protein Piezo1 on red blood cell membrane using atomic force microscopy

Details

Supervisors

Alsteens, David

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries

Abstract

Mechanotransduction is a vital process for all cell type that triggers cellular response according to an external stimulus applied on their plasma membrane. Piezo1 protein is a mechanosensitive ion channel linked with the passage of calcium ion into the intracellular compartment. Dysfunction of the protein or mutation in the gene of the Piezo1 cause pathologies such as anemias. Embedded on the plasma membrane of red blood cell, gain-of function mutation of Piezo1 lead to hereditary xerocytosis, a pathology marked by dehydrated erythrocytes that will be removed by hemolysis. The structure of Piezo1 channel allows it to sense change in curvature of the membrane generate by external forces and undergo reversible deformation, opening its central pore and causing calcium ion influx. Piezo1 is also known to be surrounded by specific lipids and proteins, enhancing its ability to detect stimulus, and forming clusters that may be characterized by distinct biophysical properties. This work focuses on the detection of Piezo1, the nature of its interactions with its antibody and its distribution along the plasma membrane. FD curved-based AFM is used to understand the nature of the interaction and its quantitative parameters. With its nanoscale resolution, this technique allows to work under physiological conditions at unprecedented pace. The interactions between the mechanoreceptor and its antibody is probed on model surface and fixed cells, extracting thermodynamic parameters of the bond. Confocal imaging was also used to visualize Piezo1 clusters and their distribution over the plasma membrane of red blood cells. Our investigations showed the specificity of the antibody used for the Piezo1 protein and the double interactions, of hydrogen bond nature, between the pair. A result expected as the antibody use dis an immunoglobulin G which possesses two antigen binding sites. We also showed that the interaction was not impaired by the fixation of the cell as the same values from the thermodynamic parameters were found. AFM and confocal images both show the existence of Piezo1 clusters along the plasma membrane. Multiparametric AFM allows the correlation of regions displaying high adhesion frequency, hence the presence of Piezo1, with different biophysical features than the rest of the membrane. These clusters, under fixative condition, are marked by an increased roughness and a stiffer behavior than the rest of the surface. While an increase roughness can be explain by the presence of ganglioside GM1 and proteins in the cluster, the stiffer behavior of these regions can be imputed to their high content of cholesterol and the presence of Piezo1 itself ,cross-linked under fixed conditions. Confocal imaging on the other hand allowed us to determine that cells fixed overnight gave the optimal fluorescent response for the identification of Piezo1 clusters. Confocal analysis also served to count the number of clusters encountered on each hemi-erythrocyte with this procedure. With 2.5 cluster per hemi-erythrocytes we find the similar results as given by the literature. However, in fixative conditions the distribution of Piezo1 clusters is contrary to expected results. This observation might underline the fact that Piezo1 is recruited by GM1 clusters which cannot diffuse in the plasma membrane under fixed conditions.