Appelgren Henrik, Kniola Barbara, Ekwall Karl
Karolinska Institute, Department of Biosciences, University College Sodertorn, Sweden.
J Cell Sci. 2003 Oct 1;116(Pt 19):4035-42. doi: 10.1242/jcs.00707. Epub 2003 Aug 19.
Fission yeast (Saccharomyces pombe) centromere DNA is organized in a central core region flanked on either side by a region of outer repeat (otr) sequences. The otr region is known to be heterochromatic and bound by the Swi6 protein whereas the central core region contains an unusual chromatin structure involving the histone H3 variant Cnp1 (S. pombe CENP-A). The central core is the base for formation of the kinetochore structure whereas the flanking region is important for sister centromere cohesion. We have previously shown that the ultrastructural domain structure of S. pombe centromeres in interphase is similar to that of human centromeres. Here we demonstrate that S. pombe centromeres are organized in cytologically distinct domains even in mitosis. Fluorescence in situ hybridization of fixed metaphase cells revealed that the otr regions of the centromere were still held together by cohesion even after the sister kinetochores had separated. In live cells, the central cores and kinetochores of sister chromosomes could be distinguished from one another when they were subjected to mitotic tension. The function of the different centromeric domains was addressed. Transacting mutations affecting the kinetochore (nuf2) central core domain (mis6) and the heterochromatin domain (rik1) were analyzed in live cells. In interphase, both nuf2 and mis6 caused declustering of centromeres from the spindle pole body whereas centromere clustering was normal in rik1 despite an apparent decondensation defect. The declustering of centromeres in mis6 cells correlated with loss the Ndc80 kinetochore marker protein from the centromeres. Interestingly the declustered centromeres were still restricted to the nuclear periphery thus revealing a kinetochore-independent peripheral localization mechanism for heterochromatin. Time-lapse microscopy of live mis6 and nuf2-1 mutant cells in mitosis showed similar severe misaggregation phenotypes whereas the rik1 mutants showed a mild cohesion defect. Thus, S. pombe centromeres have two distinguishable domains even during mitosis, and our functional analyses support the previous observations that the kinetochore/central core and the heterochromatin domains have distinct functions both in interphase and mitosis.
裂殖酵母（粟酒裂殖酵母）的着丝粒DNA组织在一个中央核心区域，两侧各有一个外侧重复序列（otr）区域。已知otr区域是异染色质的，且与Swi6蛋白结合，而中央核心区域包含一种不寻常的染色质结构，涉及组蛋白H3变体Cnp1（粟酒裂殖酵母的CENP - A）。中央核心是动粒结构形成的基础，而侧翼区域对姐妹着丝粒的黏连很重要。我们之前已经表明，间期粟酒裂殖酵母着丝粒的超微结构域结构与人类着丝粒的相似。在此我们证明，即使在有丝分裂中，粟酒裂殖酵母着丝粒也组织成细胞学上不同的结构域。对固定的中期细胞进行荧光原位杂交显示，即使姐妹动粒分离后，着丝粒的otr区域仍通过黏连结合在一起。在活细胞中，当姐妹染色体受到有丝分裂张力时，其中央核心和动粒可以相互区分。研究了不同着丝粒结构域的功能。在活细胞中分析了影响动粒（nuf2）、中央核心结构域（mis6）和异染色质结构域（rik1）的反式作用突变。在间期，nuf2和mis6都导致着丝粒从纺锤极体解聚，而尽管rik1存在明显的解聚缺陷，但着丝粒的聚集正常。mis6细胞中着丝粒的解聚与着丝粒上Ndc80动粒标记蛋白的丢失相关。有趣的是，解聚的着丝粒仍局限于核周边，从而揭示了一种不依赖动粒的异染色质周边定位机制。对活的mis6和nuf2 - 1突变体细胞进行的延时显微镜观察显示出类似的严重错聚表型，而rik1突变体表现出轻微的黏连缺陷。因此，即使在有丝分裂期间，粟酒裂殖酵母着丝粒也有两个可区分的结构域，我们的功能分析支持了之前的观察结果，即动粒/中央核心和异染色质结构域在间期和有丝分裂中都具有不同的功能。
