Calcium-dependent human erythrocyte cytoskeleton stability analysis through atomic force microscopy

Erythrocytes affected by age and diseases such as sickle cell anemia, hypertension, diabetes, etc., exhibit abnormally high intracellular Ca ²⁺ ion levels, and appear to have altered cytoskeleton properties. It has been proposed that extra binding of Ca²⁺ to membrane-associated calmodulin attenuates the spectrin-ankyrin-Band 3 tether of the cytoskeleton to the cytoplasmic membrane and might change the cytoskeleton structure. Due to the close apposition of the network, direct observation of such a structural change in vivo is restricted. In this study, atomic force microscopy and quantitative image analysis were applied to investigate the structural change of young healthy erythrocyte cytoskeletons upon extra Ca²⁺ binding to the cytoplasmic membrane in vitro. The results show that extra Ca²⁺ binding increased the cytoskeleton rigidity and prevented spectrin aggregation during sample preparation. The cytoskeleton morphology observed in Ca ²⁺-incubated healthy young cell were similar to the glutaraldehyde-fixed healthy young cells.