Quantification of membrane protein diffusion on endothelial cell surfaces under shear stress using a photochromic fluorescent protei

The fluidity of endothelial cell (EC) membranes, consisting of a lipid bilayer and heterogeneous multicom-ponent, undergoes alterations in response to shear stress. Although localized variations in membrane fluidity are assumed to induce region-specific signal transduction by modulating membrane protein dynamics on almost planar bilayers, membrane protein diffusion under shear stress remains uncertain. Hence, this study aimed to quantify membrane protein diffusion on ECs under fluid shear stress. We used the photochromic fluorescent protein Dronpa to tag a glycosylphosphatidylinositol-anchored protein (GPI-AP), which diffuses across the outer membranes, and quantified its surface diffusion based on the spatiotemporal distribution of Dronpa-Green-labeled GPI-AP (DGGPI-AP) on EC membranes. We developed an experimental platform to meas-ure the GPI-AP surface diffusion under fluid shear stress and quantified the diffusion coefficient of GPI-AP in two distinct membrane regions: upstream and downstream relative to the direction of fluid flow. Our experimental results showed that there were not statistically significant differ-ences in GPI-AP diffusion on EC membranes between the time points or between the upstream and downstream regions of ECs for at least 15 minutes under shear stress. Our developed methodology and experimental results will be useful to understand a relationship between the membrane protein diffusion and shear-induced cellular processes.