Sugimoto K, Fukuda R, Himeno M
Division of Applied Biochemistry, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, Sakai, Japan.
Cell Struct Funct. 2000 Aug;25(4):253-61. doi: 10.1247/csf.25.253.
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.
三种人类着丝粒蛋白，即着丝粒蛋白A（CENP-A）、着丝粒蛋白B（CENP-B）和着丝粒蛋白C（CENP-C），是一组自身抗原，通常由硬皮病患者产生的抗着丝粒抗体特异性识别。显微镜观察表明，CENP-A和CENP-C定位于中期动粒的内板，而CENP-B定位于动粒下方的着丝粒异染色质。这种被称为“前动粒”的抗原结构在间期核中也存在，但对其分子组织以及这些抗原的相对位置了解甚少。在这里，为了在活细胞中可视化前动粒，我们首先获得了一个稳定的人类细胞系MDA-AF8-A2，其中人类CENP-A作为与维多利亚水母绿色荧光蛋白的融合蛋白被外源表达。用抗CENP-B和抗CENP-C抗体同时染色表明，重组CENP-A与内源性CENP-C共定位，并形成附着在CENP-B异染色质较大无定形团块上的小离散点。当用羟基脲使细胞生长停滞在G1/S期时，CENP-B异染色质有时会高度伸展，而绿色荧光蛋白融合的CENP-A与内源性CENP-C之间的相对位置不受影响。这些结果表明，荧光CENP-A在整个细胞周期中忠实地定位于着丝粒/动粒。然后，我们获得了几个哺乳动物细胞系，其中相同的绿色荧光蛋白融合的人类CENP-A构建体稳定表达，并且它们的着丝粒/动粒在整个细胞周期中都是荧光的。这些细胞系将进一步用于可视化间期核中的前动粒位点以及分析活细胞中的动粒动力学。
