Shugoshin Prevents Dissociation of Cohesin from Centromeres During Mitosis in Vertebrate Cells

Cohesion between sister chromatids is essential for their bi-orientation on mitotic spindles. It is mediated by a multisubunit complex called cohesin. In yeast, proteolytic cleavage of cohesin's α kleisin subunit at the onset of anaphase removes cohesin from both centromeres and chromosome arms and thus triggers sister chromatid separation. In animal cells, most cohesin is removed from chromosome arms during prophase via a separase-independent pathway involving phosphorylation of its Scc3-SA1/2 subunits. Cohesin at centromeres is refractory to this process and persists until metaphase, whereupon its α kleisin subunit is cleaved by separase, which is thought to trigger anaphase. What protects centromeric cohesin from the prophase pathway? Potential candidates are proteins, known as shugoshins, that are homologous to Drosophila MEI-S332 and yeast Sgo1 proteins, which prevent removal of meiotic cohesin complexes from centromeres at the first meiotic division. A vertebrate shugoshin-like protein associates with centromeres during prophase and disappears at the onset of anaphase. Its depletion by RNA interference causes HeLa cells to arrest in mitosis. Most chromosomes bi-orient on a metaphase plate, but precocious loss of centromeric cohesin from chromosomes is accompanied by loss of all sister chromatid cohesion, the departure of individual chromatids from the metaphase plate, and a permanent cell cycle arrest, presumably due to activation of the spindle checkpoint. Remarkably, expression of a version of Scc3-SA2 whose mitotic phosphorylation sites have been mutated to alanine alleviates the precocious loss of sister chromatid cohesion and the mitotic arrest of cells lacking shugoshin. These data suggest that shugoshin prevents phosphorylation of cohesin's Scc3-SA2 subunit at centromeres during mitosis. This ensures that cohesin persists at centromeres until activation of separase causes cleavage of its α kleisin subunit. Centromeric cohesion is one of the hallmarks of mitotic chromosomes. Our results imply that it is not an intrinsically stable property, because it can easily be destroyed by mitotic kinases, which are kept in check by shugoshin.

姐妹染色单体（sister chromatids）之间的黏连对于其在有丝分裂纺锤体（mitotic spindles）上的双向定向排布至关重要，该黏连由一种名为黏连蛋白（cohesin）的多亚基复合物所介导。在酵母中，于分裂后期（anaphase）起始时对黏连蛋白的α kleisin亚基（α kleisin subunit）进行蛋白水解切割（proteolytic cleavage），可将黏连蛋白从着丝粒（centromeres）与染色体臂（chromosome arms）上移除，进而触发姐妹染色单体分离。在动物细胞中，绝大多数黏连蛋白会在分裂前期（prophase）通过一条不依赖分离酶的通路（separase-independent pathway）从染色体臂上被清除，该通路涉及黏连蛋白Scc3-SA1/2亚基（Scc3-SA1/2 subunits）的磷酸化修饰（phosphorylation）。着丝粒处的黏连蛋白对该过程具有耐受性，会一直存留至分裂中期（metaphase），直至其α kleisin亚基被分离酶切割，而这一过程被认为是触发分裂后期的关键事件。那么，究竟是什么保护着丝粒处的黏连蛋白免受前期清除通路的影响呢？潜在的候选蛋白是一类被称为保护素（shugoshins）的蛋白质，它们与果蝇MEI-S332（Drosophila MEI-S332）及酵母Sgo1（yeast Sgo1）蛋白同源，能够在第一次减数分裂时阻止减数分裂黏连蛋白复合物（meiotic cohesin complexes）从着丝粒处被移除。一种脊椎动物来源的保护素样蛋白（vertebrate shugoshin-like protein）会在分裂前期与着丝粒结合，并于分裂后期起始时消失。通过RNA干扰（RNA interference）技术将其敲低后，海拉细胞（HeLa cells）会停滞在有丝分裂阶段。大多数染色体能够在赤道板（metaphase plate）上完成双向定向排布，但染色体上着丝粒处黏连蛋白的过早丢失（precocious loss），会伴随所有姐妹染色单体黏连的丧失、单个染色单体从赤道板脱离，以及永久性的细胞周期阻滞，这一现象推测是由纺锤体检验点（spindle checkpoint）的激活所导致的。值得注意的是，将Scc3-SA2的有丝分裂磷酸化位点突变为丙氨酸后所表达的突变体，能够缓解因保护素缺失所导致的姐妹染色单体黏连过早丢失与细胞有丝分裂阻滞。上述实验结果表明，保护素可在有丝分裂过程中阻止着丝粒处黏连蛋白的Scc3-SA2亚基发生磷酸化。这一机制确保了黏连蛋白能够稳定存留于着丝粒处，直至分离酶被激活并切割其α kleisin亚基。着丝粒黏连是有丝分裂染色体的标志性特征之一。我们的研究结果暗示，着丝粒黏连并非一种内在稳定的特性，因为它可轻易被有丝分裂激酶（mitotic kinases）破坏，而这类激酶的活性受到保护素的抑制。