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  • Title: Actin assembly by cadmium ions.
    Author: DalleDonne I, Milzani A, Colombo R.
    Journal: Biochim Biophys Acta; 1997 Jun 05; 1357(1):5-17. PubMed ID: 9202170.
    Abstract:
    Cadmium is a highly toxic metal entering cells by a variety of mechanisms. Its toxic action is far from being completely understood, although specific interaction with the cellular calcium metabolism has been indicated. 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 evidence demonstrates that cadmium interferes with the dynamics of actin 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-PAGE, see Fig. 1B) in the form of irregular, disordered clots, clearly appreciable by electron microscopy. Denaturation seems to be reversible since, after Cd2+ removal by dialysis, the polymerizability of sedimented actin is restored almost completely. On the other hand, at concentrations ranging from 0.25 to 0.6 mM, CdCl2 is more effective as an actin polymerizing agent than both MgCl2 and CaCl2. The Cd-related increase 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 measurements 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 microM), while Ca-polymerized actin shows a polymerization extent markedly lower (C(c) = 4.0 microM). By both the fluorescent Ca2+ chelator Quin-2 assay and limited proteolysis of actin by trypsin and alpha-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 indicates that, in our experimental conditions, Ca2+ tightly-bound to actin is partially (60-70%) replaced by Cd2+, forming Cd-actin. Electrophoretic patterns after limited proteolysis reveal that the trypsin cleavage sites in the segment 61-69 of the actin polypeptide chain are less accessible in Cd-actin than in Ca-actin, although the cation-dependent effect is less pronounced in Cd-actin than in Mg-actin. Our results are consistent with some of the consequences on microfilament organization observed in Cd2(+)-treated cells; however, considering the positive effect of Cd2+ on actin polymerization in solution we have noticed that this was never observed in vivo. A different indirect effect of Cd2+ on some cellular event(s) influencing cytoplasmic actin polymerization appears to be reasonable.
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