Magnesium Cations Modulate Actin Bundles Formed by α-Actinin

α-Actinin is an actin-crosslinking protein that influences cytoskeletal organization by forming actin bundles or networks. Divalent cations, such as magnesium cations (Mg²⁺), promote bundling through counterion condensation. While α-actinin and Mg²⁺ individually promote actin bundle assembly, their combined effects on actin bundling remain unclear. Here, we determine how Mg²⁺ affects α-actinin's binding to filaments and its subsequent bundling activities through biophysical analysis. We show that increasing Mg²⁺ concentrations enhance the binding of α-actinin to actin filaments, producing more compact bundles instead of loose networks. Molecular dynamics simulation indicates that Mg²⁺ modulates the interaction between filaments and the calponin homology 1 domain of α-actinin. We propose a mechanism by which divalent cations and α-actinin crosslinking act simultaneously to drive actin bundle formation.