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 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.
Sadano
H
H
Department of Molecular and Cellular Biology, Kyushu University, Fukuoka.
Shimokawa-Kuroki
R
R
Taniguchi
S
S
D000199:Actins / Q000032:analysis* / Q000276:immunology / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D003850:Deoxyribonuclease I / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D007163:Immunosorbent Techniques; D008546:Melanoma, Experimental / Q000473:pathology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009362:Neoplasm Metastasis; D014407:Tumor Cells, Cultured
D000199:Actins; D000906:Antibodies; D005456:Fluorescent Dyes; D003850:Deoxyribonuclease I; D016650:Fluorescein-5-isothiocyanate