DISRUPTION OF THE ACTIN CYTOSKELETON OF MAMMALIAN-CELLS BY THE CAPPING COMPLEX ACTIN-FRAGMIN IS INHIBITED BY ACTIN PHOSPHORYLATION AND REGULATED BY CA2+ IONS

Fragmin from Physarum polycephalum is a gelsolin-like actin-binding pr otein and interferes with the growth of actin filaments in vitro by se vering actin filaments and capping their barbed ends through formation of an actin-fragmin dimer in a Ca2+-dependent manner. The actin-fragm in dimer is phosphorylated in vivo and in vitro on the actin subunit b y the actin-fragmin kinase, We have studied the properties of these ca pping proteins and their regulation by actin phosphorylation and Ca2ions in living PtK2, CV1 and NIH3T3 cultured cells by microinjection o r by expression in conjunction with immunostaining and fluorescence mi croscopy. Microinjection of the actin-fragmin dimer disintegrated the actin cytoskeleton and altered cell morphology. This in vivo effect co uld be blocked by phosphorylation of the actin subunit by the actin-fr agmin kinase in low Ca2+ conditions, and the capping activity could be recovered by high Ca2+ concentration, probably through activation of the second actin-binding site in fragmin, This suggests that in Physar um microplasmodia, actin polymerization can be controlled in a Ca2+-de pendent manner through the phosphorylation of actin. Microinjected or overexpressed recombinant fragmin did not affect the actin-based cytos keleton or cell morphology of resting cells, unless the cytosolic free Ca2+ concentration was increased by microinjection of a Ca2+-containi ng buffer. The cells were able to revert to their normal phenotype whi ch indicates that endogenous regulatory mechanisms counteracted fragmi n activity, probably by uncapping fragmin from the barbed ends of fila ments. Fragmin also antagonized formation of stress fibers induced by lysophosphatidic acid. Our findings demonstrate that the interactions between actin and fragmin are tightly regulated by the cytosolic Ca2concentration and this provides a basis for a more general mechanism i n higher organisms to regulate microfilament organization.