The organization and dynamics of the spectrin-actin membrane cytoskeleton play a crucial role in determining the mechanical properties of red blood cells (RBC). RBC are subjected to various forces that induce deformation during blood microcirculation. Such forces also regulate membrane tension, leading to Piezo1 channel activation, which is functionally linked to RBC dehydration through calcium influx and subsequent activation of Gardos channels, ultimately resulting in variations in RBC volume. In this study, we investigated how actin instability affects Piezo1 channel gating, in relation to RBC deformation and mechanical properties, using micropipette aspiration and optical tweezers. Actin instability, induced by 0.5 μM Cytochalasin-D (Cyt-D), led to a 22% reduction in the activation pressure. Additionally, we observed a decreasing trend in Young's modulus, membrane tension, and viscosity. By measuring the time required for cell shape recovery after deformation in an optical trap, we found that Cyt-D-treated RBC took approximately 14% longer to recover compared to untreated cells. The bimodal imaging feature of our experimental approach allowed us to simultaneously measure and correlate activation pressure with mechanical properties at the single-cell level. A significant correlation was found between these parameters in both treated and untreated RBC. Our findings demonstrate the influence of actin instability on both Piezo1 activation and RBC mechanics. These results offer new insights into the interplay between F-actin and Piezo1 in RBC mechanobiology.
