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Intracellular localization and biochemical function of variant beta-actin, which inhibits metastasis of B16 melanoma
H Sadano1, R Shimokawa-Kuroki, S Taniguchi
1Department of Molecular and Cellular Biology, Kyushu University, Fukuoka.
Abstract:
We analyzed the biochemical nature of beta m-actin protein found in mouse 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 not 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 immunoprecipitate contained beta m-, beta- and gamma-actin. This indicated that the beta m-actin was incorporated into an actin filament together with beta- and gamma-actin in vitro, and this phenomenon was consistently suggested by cellular double immunostaining with anti-beta m-actin and common anti-actin antibody. When the actin fraction containing beta m-actin under a regular depolymerizing condition was subjected to immuno-adsorption assay using anti-beta m antibody and protein-A Sepharose, the immunoadsorbed aggregates contained beta m-, beta- and gamma-actin. This indicates that the actin fraction was not completely depolymerized and contained beta m-actin-containing oligomers, which were too small to be precipitated with anti-beta m-actin antibody alone. The incomplete 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 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 beta m-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.