Intracellular Localization and Biochemical Function of Variant β-Actin, which Inhibits Metastasis of B16 Melanoma

We analyzed the biochemical nature of βm-actin protein found in mouse B16 melanoma. When we carried out immunostaining with the antibody specific to βm-actin, filamentous immunofluorescence was observed in B16-F1, a low-metastatic cell line expressing βm-actin, but not in highly metastatic B16-F10 that did not express βm-actin. When a purified actin fraction containing βm-actin was polymerized and immunoprecipitated with anti-βm-actin antibody, the immunoprecipitate contained βm-, β- and γ-actin. This indicated that the βm-actin was incorporated into an actin filament together with β- and γ-actin in vitro , and this phenomenon was consistently suggested by cellular double immunostaining with anti-βm-actin and common anti-actin antibody. When the actin fraction containing βm-actin under a regular depolymerizing condition was subjected to immuno-adsorption assay using anti-βm antibody and protein-A Sepharose, the immunoadsorbed aggregates contained βm-, β-and γ-actin. This indicates that the actin fraction was not completely depolymerized and contained βm-actin-containing oligomers, which were too small to be precipitated with anti-βm-actin antibody alone. The incomplete depolymerization of the βm-actin-containing fraction was also suggested by the much lower DNase 1 inhibition activity of the βm-actin-containing fraction than that of β- and γ-actin fraction. Furthermore, a DNase 1 binding assay showed that cytoplasmic supernatant prepared from B16-F1 under a low-ionic condition contained less monomeric actin than the cytoplasmic preparation from B16-F10. These results suggested that βm-actin protein in B16 melanoma probably inhibits the dynamic conversion between the monomeric and polymerized forms of actin, leading to a decrease in cell motility and consequently the suppression of invasiveness and metastasis.