RRC ID 39521
著者 Sugimoto K, Fukuda R, Himeno M.
タイトル Centromere/kinetochore localization of human centromere protein A (CENP-A) exogenously expressed as a fusion to green fluorescent protein.
ジャーナル Cell Struct Funct
Abstract Three human centromere proteins, CENP-A, CENP-B and CENP-C, are a set of autoantigens specifically recognized by anticentromere antibodies often produced by patients with scleroderma. Microscopic observation has indicated that CENP-A and CENP-C localize to the inner plate of metaphase kinetochore, while CENP-B localizes to the centromere heterochromatin beneath the kinetochore. The antigenic structure, called "prekinetochore", is also present in interphase nuclei, but little is known about its molecular organization and the relative position of these antigens. Here, to visualize prekinetochore in living cells, we first obtained a stable human cell line, MDA-AF8-A2, in which human CENP-A is exogenously expressed as a fusion to a green fluorescent protein of Aequorea victoria. Simultaneous staining with anti-CENP-B and anti-CENP-C antibodies showed that the recombinant CENP-A colocalized with the endogenous CENP-C and constituted small discrete dots attaching to larger amorphous mass of CENP-B heterochromatin. When the cell growth was arrested in G1/ S phase with hydroxyurea, CENP-B heterochromatin was sometimes highly extended, while the relative location between GFP-fused CENP-A and the endogenous CENP-C was not affected. These results indicated that the fluorescent CENP-A faithfully localizes to the centromere/kinetochore throughout the cell cycle. We then obtained several mammalian cell lines where the same GFP-fused human CENP-A construct was stably expressed and their centromere/kinetochore is fluorescent throughout the cell cycle. These cell lines will further be used for visualizing the prekinetochore locus in interphase nuclei as well as analyzing kinetochore dynamics in the living cells.
巻・号 25(4)
ページ 253-61
公開日 2000-8-1
DOI 10.1247/csf.25.253
PMID 11129795
MeSH Animals Autoantigens / biosynthesis* Autoantigens / genetics Cattle Cell Line Cell Nucleus / genetics Centromere / genetics Centromere / metabolism* Centromere Protein A Centromere Protein B Chromosomal Proteins, Non-Histone / biosynthesis* Chromosomal Proteins, Non-Histone / genetics Chromosomal Proteins, Non-Histone / metabolism DNA-Binding Proteins* Green Fluorescent Proteins Heterochromatin / genetics Heterochromatin / metabolism Humans Interphase / genetics Kinetochores / metabolism* Luminescent Proteins / biosynthesis* Luminescent Proteins / genetics* Mice Recombinant Fusion Proteins / biosynthesis* Scyphozoa Swine Tumor Cells, Cultured
IF 2.429
引用数 26
WOS 分野 CELL BIOLOGY
リソース情報
ヒト・動物細胞 MDBK(RCB0180) PK15(RCB0534)