Involvement of the cohesin Rad21 and SCP3 in monopolar attachment of sister kinetochores during mouse meiosis I

Mara Teresa Parra 2 Alberto Viera 2 Roco Gmez 2 Jess Page 2 Ricardo Benavente 1 Juan Luis Santos 0 Julio S. Rufas 2 Jos A. Suja ) 2 0 Departamento de Genetica, Facultad de Biologia, Universidad Complutense , 28040 Madrid , Spain 1 Department of Cell and Developmental Biology, Theodor Boveri Institute, University of Wurzburg , 97074 Wurzburg , Germany 2 Departamento de Biologia, Edificio de Biologicas, Universidad Autonoma de Madrid , 28049 Madrid , Spain - SCP3 is a meiosis-specific structural protein appearing at axial elements and lateral elements of the synaptonemal complex. We have analysed the behaviour of SCP3 and the cohesin subunit Rad21 in mouse spermatocytes by means of a squashing technique. Our results demonstrate that both proteins colocalize and are partially released from chromosome arms during late prophase I stages, although they persist at the interchromatid domain of metaphase I bivalents. Thus, Rad21 cannot be considered a mitoticspecific variant, but coexists with Rec8. During late prophase I SCP3 and Rad21 accumulate at centromeres, and together with the chromosomal passenger proteins INCENP and aurora-B kinase, show a complex double cornet-like distribution at the inner domain of metaphase During meiosis two successive cellular divisions take place without an intervening DNA replication step thus resulting in the generation of haploid germ cells. During meiotic prophase I homologues pair, synapse and recombine. These processes are essential to ensure a correct chromosome segregation during meiosis I, and are thought to be mediated by a meiosisspecific structure, the synaptonemal complex (SC). The SC is a proteinaceous structure found between synapsed homologues during the pachytene stage of prophase I (for review, see Zickler and Kleckner, 1999). Ultrastructurally, the SC appears as a tripartite structure composed by two parallel lateral elements (LEs) and a central element. The LEs and the central element are held together by fine fibres called transverse filaments. Axial chromosome structures called axial elements (AEs) appear during leptotene, associated with the two sister chromatids of each homologue, and are the precursors of the LEs found in zygotene and pachytene. During diplotene and diakinesis, homologues desynapse and the SC disassembles with the concomitant separation of LEs and disappearance of the central element. Two AE/LE components have been characterised in mammals: SC proteins SCP2 and SCP3. The localization of rat/mouse SCP3 (Moens et al., 1987; Lammers et al., 1994), also called COR1 in hamster (Dobson et al., 1994), has been I centromeres beneath the associated sister kinetochores. We have observed that Rad21 and SCP3 are displaced from centromeres during telophase I when sister kinetochores separate, and are not present at metaphase II centromeres. Thus, we hypothesise that Rad21, and the superimposed SCP3 and SCP2, are involved in the monopolar attachment of sister kinetochores during meiosis I, and are not responsible for the maintenance of sister-chromatid centromere cohesion during meiosis II as previously suggested. mainly studied in surface-spread rodent spermatocytes. SCP3 first appears at AEs in leptotene spermatocytes, and is found at LEs during pairing and synapsis during zygotene and pachytene, respectively. In diplotene spermatocytes, SCP3 is present at desynapsed LEs and still persists between sister chromatids until the metaphase I/anaphase I transition (Dobson et al., 1994; Moens and Spyropoulos, 1995). SCP2 shows a similar distribution pattern during prophase I (Schalk et al., 1998). Interestingly, it has been reported that SCP3 persists at centromeres up to anaphase II. Consequently, it has been proposed that SCP3 may have a role in sister-chromatid arm cohesion during meiosis I and in centromere cohesion during meiosis II (Moens and Spyropoulos, 1995). It has been demonstrated that SCP3 is essential for both formation and maintenance of AEs/LEs since in its absence no AEs/LEs are found (Yuan et al., 2000; Pelttari et al., 2001). During meiosis sister-chromatid cohesion is released in two steps (Suja et al., 1992; Miyazaki and Orr-Weaver, 1994; Petronczki et al., 2003). Arm cohesion is lost during the metaphase I/anaphase I transition to allow the segregation of recombined homologues. This reductional segregation is possible since sister kinetochores remain closely associated and then bivalents reach an accurate biorientation. Recent studies have proposed that in budding yeast the monopolar attachment of sister kinetochores is facilitated by the monopolin complex (Toth et al., 2000; Rabitsch et al., 2003), while in fission yeast this is ensured by the cohesin subunit Rec8 (Watanabe and Nurse, 1999; Yokobayashi et al., 2003). Centromere cohesion is released during the metaphase II/anaphase II transition to allow chromatid segregation. It has been demonstrated that arm and centromere cohesion are maintained by a cohesin complex in both mitotic and meiotic chromosomes (for reviews, see Nasmyth, 2001; Nasmyth, 2002). Recently, several subunits of the cohesin complex have been found to be associated with mammalian AEs/LEs (for reviews, see Lee and Orr-Weaver, 2001; Nasmyth, 2001; Petronczki et al., 2003). Thus, it has been reported that the cohesin subunits belonging to the structural maintenance of chromosome proteins SMC1a , SMC1b and SMC3 (Eijpe et al., 2000; Revenkova et al., 2001), and the non-SMC cohesin subunits STAG3 (Pezzi et al., 2000; Prieto et al., 2001; Pelttari et al., 2001), STAG2 and Rad21 (Prieto et al., 2002), and Rec8, a meiosis-specific variant of Rad21 (Eijpe et al., 2003; Lee et al., 2003), are present at AEs/LEs from leptotene up to diplotene. After the SC disassembly, STAG3 remains at the interchromatid domain of metaphase I chromosomes, but is lost during the metaphase I/anaphase I transition (Prieto et al., 2001). Accordingly, it has been proposed that STAG3 is mainly involved in maintaining sister-chromatid arm cohesion. By contrast, it has been reported that SMC1b SMC3 and Rec8 persist at centromeres from metaphase I up to anaphase II thus regulating sister-chromatid centromere cohesion (Revenkova et al., 2001; Eijpe et al., 2003; Lee et al., 2003). During recent years it has been suggested that INCENP and aurora-B kinase, two chromosomal passenger proteins present at the inner centromere during mitotic metaphase, and redistributing to the spindle midzone during anaphase, are implicated, not only in coordinating chromosome segregation with cytokinesis, but also in centromere cohesion (Adams et al., 2001; Vagnarelli and Earnshaw, 2001). Interestingly, INCENP distributes abnormally at centromeres in chicken mutant cells for the cohesin subunit Rad21 (Sonoda et al., 2001), and its dynamics is affected in Drosophila cells depleted of Rad21 (Vass et al., 2003). Additionally, aurora-B phosphorylates the cohesin subunit Rec8, and therefore promotes the release of sister-chromatid c (...truncated)