ACTIN ASSEMBLY BY CADMIUM IONS

Cadmium is a highly toxic metal entering cells by a variety of mechani sms. Its toxic action is far from being completely understood, althoug h specific interaction with the cellular calcium metabolism has been i ndicated. Metal ions that influence intracellular Ca2+ concentrations or compete with Ca2+ for protein binding sites may exert an effect on actin filaments, whose assembly and disassembly are both regulated by a number of calcium-dependent factors. Cadmium is such a metal. Much e vidence demonstrates that cadmium interferes with the dynamics of acti n filaments in various types of cells. Here we show that, at high (0.8 -1.0 mM) concentrations, CdCl2 causes actin denaturation. At such Cd2 concentrations, actin precipitates (really actin, as shown by SDS-PAG E, see Fig. 1B) in the form of irregular, disordered clots, clearly ap preciable by electron microscopy. Denaturation seems to be reversible since, after Cd2+ removal by dialysis, the polymerizability of sedimen ted actin is restored almost completely. On the other hand, at concent rations ranging from 0.25 to 0.6 mM, CdCl2 is more effective as an act in polymerizing agent than both MgCl2 and CaCl2. The Cd-related increa se in the actin assembly rate is ascribable to an enhanced nucleation rather than to an increased monomer addition to filament growing ends. The latter, in contrast, appears quite slow. Critical concentration m easurements revealed that the extent of polymerization of both Mg- and Cd-assembled actin are very close (C-c ranges from 0.25 to 0.5 mu M), while Ca-polymerized actin shows a polymerization extent markedly low er (C-c = 4.0 mu M). By both the fluorescent Ca2+ chelator Quin-2 assa y and limited proteolysis of actin by trypsin and cr-chymotrypsin, the real substitution of G-actin-bound Ca2+ by Cd2+ has been appreciated. The increase in Quin-2 fluorescence after addition of excess CdCl2 in dicates that, in our experimental conditions, Ca2+ tightly-bound to ac tin is partially (60-70%) replaced by Cd2+, forming cd-actin. Electrop horetic patterns after limited proteolysis reveal that the trypsin cle avage sites in the segment 61-69 of the actin polypeptide chain are le ss accessible in Cd-actin than in Ca-actin, although the cation-depend ent effect is less pronounced in Cd-actin than in Mg-actin. Our result s are consistent with some of the consequences on microfilament organi zation observed in Cd2+-treated cells; however, considering the positi ve effect of Cd2+ on actin polymerization in solution we have noticed that this was never observed in vivo. A different indirect effect of C d2+ on some cellular event(s) influencing cytoplasmic actin polymeriza tion appears to be reasonable. (C) 1997 Elsevier Science B.V.