INTRACELLULAR-LOCALIZATION AND BIOCHEMICAL FUNCTION OF VARIANT BETA-ACTIN, WHICH INHIBITS METASTASIS OF B16 MELANOMA

We analyzed the biochemical nature of beta m-actin protein found in mo use B16 melanoma. When we carried out immunostaining with the antibody specific to beta m-actin, filamentous immunofluorescence was observed in B16-F1, a low-metastatic cell line expressing beta m-actin, but no t in highly metastatic B16-F10 that did not express beta m-actin. When a purified actin fraction containing beta m-actin was polymerized and immunoprecipitated with anti-beta m-actin antibody, the immunoprecipi tate contained beta m-, beta- and gamma-actin. This indicated that the beta m-actin was incorporated into an actin filament together with be ta- and gamma-actin in vitro, and this phenomenon was consistently sug gested by cellular double immunostaining with anti-beta m-actin and co mmon anti-actin antibody. When the actin fraction containing beta m-ac tin under a regular depolymerizing condition was subjected to immuno-a dsorption assay using anti-beta m antibody and protein-A Sepharose, th e immunoadsorbed aggregates contained beta m-, beta- and gamma-actin. This indicates that the actin fraction was not completely depolymerize d and contained beta m-actin-containing oligomers, which were too smal l to be precipitated with anti-beta m-actin antibody alone. The incomp lete depolymerization of the beta m-actin-containing fraction was also suggested by the much lower DNase 1 inhibition activity of the beta m -actin-containing fraction than that of beta- and gamma-actin fraction . Furthermore, a DNase 1 binding assay showed that cytoplasmic superna tant prepared from B16-F1 under a low-ionic condition contained less m onomeric actin than the cytoplasmic preparation from B16-F10. These re sults suggested that beta m-actin protein in B16 melanoma probably inh ibits the dynamic conversion between the monomeric and polymerized for ms of actin, leading to a decrease in cell motility and consequently t he suppression of invasiveness and metastasis.