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.