Curiously, in cell division the proper separation of chromosomes into daughter cells needs set periods when they are stuck together. So how do they come apart at the right time and place? Their ‘guardian spirits’ intercede.
To avoid cell death or genetic diseases such as cancer, chromosomes must be transmitted to progeny cells with high fidelity. During cell division, chromosomes are duplicated to form sister chromatids, which must then be divided into two equal groups and separated into daughter cells (a process termed segregation). To prevent random segregation, sister chromatids are held together along their lengths by a ring-shaped protein complex called cohesin1. At the cell-cycle stage known as anaphase, cohesins are cleaved and cohesion between sister chromatids dissolves, triggering their segregation. So the chromosomal association and dissociation of cohesin must be tightly regulated for proper segregation. In this issue, Kitajima et al. (page 46)2 and Riedel et al. (page 53)3 describe how proteins known as shugoshins — Japanese for ‘guardian spirits’ — and an associated regulatory enzyme temporally and spatially control the removal of cohesins from chromosomes.
The association of cohesins with the chromosome is particularly robust near the centromere4. This specialized region of the chromosome mediates assembly of the kinetochore complex that attaches the chromosome to the ‘spindle’, the cellular structure that pulls sister chromatids apart once cohesion has dissolved. Strong cohesion flanking the kinetochores is crucial, because it promotes the attachment of the two kinetochores on each chromatid pair to spindle fibres that originate from opposite sides of the dividing cell. It also opposes the splitting forces exerted by spindle fibres, preventing premature separation of sister chromatids.
There are two types of cell division — meiosis, a specialized process that generates reproductive cells, and mitosis, which produces all other cells. Each has a pathway to deal with cohesin removal. In vertebrate mitosis, cohesins are detached from the chromosome arms first, in prophase (early mitosis), but the cohesins around the centromere are retained until the onset of anaphase (late mitosis) (Fig. 1). At anaphase, an enzyme called separase cleaves the cohesin subunit known as Mcd1/Scc1, causing cohesins to dissociate from the centromeres5. How cohesins are removed from the chromosomal arms during prophase is less clear, other than it does not require separase and it is triggered by the phosphorylation (addition of a phosphate group) of a cohesin subunit called Scc3/SA (ref. 6).
In meiosis, DNA replication is followed by two successive rounds of chromosome segregation and cell division, producing four egg or sperm cells with half the chromosomal content of the other cells in the body (so that the fusion of two cells at fertilization gives the correct genetic complement). Maternally and paternally derived copies of chromosomes — referred to as homologues — are segregated in the first round, and sister chromatids are segregated in the second round. Before the first meiotic division, homologues usually undergo ‘recombination’, the reciprocal exchange of chromosomal segments. Recombination produces crossovers, which are physical links between homologues that can only be untangled by the release of sister-chromatid cohesion (Fig. 1). However, complete removal of cohesins at this stage would lead to random segregation of sister chromatids during the second division, so cohesin association persists in centromeric regions until the second division. At the first division, cohesins are removed in prophase by a pathway that is probably similar to that operating in mitosis7, and by the separase-mediated cleavage of Rec8, a meiosis-specific variant of Mcd1. Cohesin removal by these pathways seems to be enhanced by phosphorylation of cohesin subunits7,8. As in mitosis, the removal of centromeric cohesins is dependent on separase.
How cohesin binding is stabilized specifically in centromeric regions is unclear. But hints came from the fruitfly protein MEI-S332, which, when inactivated, leads to defective centromeric cohesion9. MEI-S332 is the founding member of a family of proteins called shugoshins, whose localization to the kinetochore is essential for the persistence of centromeric cohesin10,11,12. Shugoshin family members exist in organisms from yeast to humans, implying that there is an evolutionarily conserved mechanism to protect centromeric cohesin.
To determine how shugoshins protect centromeric cohesin, Kitajima et al.2, Riedel et al.3 and Tang et al.13 set out to identify proteins that interact with shugoshin in mitotic and meiotic cells. Each group identified an enzyme called protein phosphatase 2A (PP2A) that dephosphorylates certain protein residues. PP2A is composed of catalytic, scaffolding and regulatory (B) subunits, and although several subfamilies of PP2A regulatory subunits exist, the shugoshins interacted only with the subset of PP2A that contained B′ regulatory subunits2,3. Moreover, despite the fact that PP2A is found in many places in the cell, Kitajima et al.2 and Riedel et al.3 show that the B′-containing PP2A forms are found preferentially in centromeric regions, where they co-localize with shugoshins.
Given that phosphorylation of cohesin subunits triggers cohesin removal6, all three research groups2,3,13 propose that shugoshins protect centromeric cohesion in both mitotic and meiotic cells by recruiting PP2A to centromeres, where it stabilizes centromeric cohesin by dephosphorylating cohesin subunits. Consistent with this model was the dramatic increase in premature chromatid separation and chromosome mis-segregation that was observed when either PP2A activity was eliminated in yeast, or PP2A protein levels were reduced in human cells2,3,13. Furthermore, Kitajima et al.2 showed that in cells during late mitosis, chromosome-bound cohesins (which are mostly centromeric) were phosphorylated to a lesser extent than were free cohesins (presumably those formerly associated with chromosome arms). So it seems likely that PP2A protects centromeric cohesin by reducing cohesin-subunit phosphorylation in centromeric regions.
But is getting PP2A to the centromeres sufficient to prevent cohesin removal? Kitajima et al.2 and Riedel et al.3 artificially tethered PP2A to chromosomes independently of shugoshins. Under these conditions, cohesins were retained on the chromosomes and the cells either failed to undergo nuclear divisions or aberrantly segregated their chromosomes. This supports the idea that the main function of shugoshins is to recruit PP2A to the centromere2,3. However, the depletion of one shugoshin protein in cells expressing a second family member induced premature chromatid separation even though PP2A was tethered to the centromere, suggesting that shugoshins may have additional roles in protecting centromeric cohesion2.
In a related study published online in Nature, Brar et al.14 investigated whether the phosphorylation of the meiotic cohesin subunit Rec8 is involved in the stepwise loss of cohesins during meiosis. Previous results showed that depletion of the enzyme thought to phosphorylate Rec8 leads to defects in Rec8 cleavage and entry into anaphase8. And consistent with this, Brar et al. found that cells with a mutant version of Rec8 in which phosphorylation was reduced had difficulties initiating the first meiotic division because of a defect in the separase-mediated cleavage of Rec8. The requirement for Rec8 phosphorylation was abolished, however, if there was no recombination between homologues. Without recombination-mediated crossovers, the removal of cohesins from chromosome arms was not required to separate homologues, and cohesins persisted on chromosomes until the second meiotic division.
So the two meiotic divisions have different requirements for Rec8 phosphorylation in triggering the separase-mediated cleavage of Rec8 — Rec8 phosphorylation is important for cleavage in the first meiotic division, but less so in the second division. Surprisingly, it seems that recombination itself might contribute to the ordered removal of cohesins from chromosome arms. In addition, separase-mediated cleavage of the mutant Rec8 did occur when shugoshin was depleted, leading Brar et al.14 to conclude, as have Kitajima et al.2, that shugoshins may have other duties in addition to protecting centromeric Rec8 from phosphorylation.
These four studies have made significant progress in elucidating the role of shugoshins in protecting centromeric cohesin, but they also raise questions. For example, several organisms, including humans, have multiple shugoshin family members that have different expression patterns during mitotic and meiotic cell divisions, raising the likelihood that specific family members have different roles in protecting centromeric cohesin. Determining precisely how shugoshins function as a chromosome segregation nexus, coordinating both centromeric cohesion and other potential roles, will be fundamental to our understanding of the molecular mechanisms of cell division.
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Celestial Mechanics and Dynamical Astronomy (2011)