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  • The molecular mechanisms underlying spindle orientation are

    2022-04-13

    The molecular mechanisms underlying spindle orientation are still largely unknown. Budding yeast has been used to study spindle positioning and asymmetric cell division. Indeed, after cytokinesis, daughter cells have different sizes. The cell division plane is established early in the cell cycle and before assembly of the mitotic spindle (Etienne-Manneville, 2004, Fraschini et al., 2008, Segal and Bloom, 2001); therefore, a surveillance mechanism called the spindle position checkpoint (SPOC) oversees spindle positioning and delays mitotic exit and cytokinesis in case of errors (Bardin et al., 2000, Lew and Burke, 2003, Nelson and Cooper, 2007, Pereira et al., 2000). A similar checkpoint has recently been described in Drosophila (Cheng et al., 2011), suggesting that these mechanisms exist in various multicellular eukaryotes. Haspin is an atypical protein kinase, highly conserved throughout evolution (Higgins, 2001). The UM171 synthesis yeast genome codes for two haspin paralogs, Alk1 and Alk2, whose protein levels peak in mitosis and late S/G2, respectively, and are modulated by the anaphase-promoting complex. Both proteins are phosphorylated in mitosis, albeit the physiological significance of this modification is still unclear. Finally, overexpression of ALK2 prevents mitotic progression, suggesting that a precise regulation of haspin is critical for cell division (Nespoli et al., 2006). In mammalian cells, haspin knockdown arrests cells in mitosis and prevents proper chromosome congression at the metaphase plate (Dai et al., 2005, Dai et al., 2006); moreover, haspin is responsible for histone H3Thr3 phosphorylation, which is involved in the recruitment of the chromosomal passenger complex (Wang et al., 2010, Kelly et al., 2010, Yamagishi et al., 2010). In Arabidopsis thaliana, haspin is also involved in plant development; indeed, it contributes to embryonic patterning (Ashtiyani et al., 2011). However, the precise function of haspin is still not fully understood and it is likely that more kinase targets exist, particularly in budding yeast, where phosphorylation of H3Thr3 has never been observed. Here we show that in the absence of haspin, budding yeast cells fail to properly recruit polarity factors that are important for the establishment of a balance in the forces acting on spindle positioning. If mitosis is prolonged in these cells, the spindle is pulled within the daughter cell and nuclear division generates an anucleated mother and a binucleated daughter. Haspin is thus essential to tolerate a transient mitotic arrest and for the maintenance of the coupling between polarization and cell-cycle progression. Our findings may help explain the developmental defects observed in Arabidopsis haspin mutants, and its evolutionary conservation suggests that haspin may be important for the proper positioning of polarity factors also in other eukaryotic cells and may be a key player in the control of asymmetric cell division.
    Results
    Discussion Haspin is an atypical protein kinase that has been conserved throughout eukaryotic evolution; it is also present in the Encephalitozoon cuniculi genome, which contains just 2,000 genes and is only ∼2.9 Mb long (Katinka et al., 2001). This extreme conservation suggests a critical function for haspin. In human, A. thaliana, and fission yeast cells, haspin has been reported to be responsible for the phosphorylation of H3Thr3. This phosphosite is a docking site for the chromosome passenger complex, including Aurora B kinase, that is critical for proper chromosomal alignment at the metaphase plate and for chromosomal movement (Kelly et al., 2010, Wang et al., 2010, Yamagishi et al., 2010, Ashtiyani et al., 2011). S. cerevisiae contains two haspin paralogs, coded by ALK1 and ALK2, but H3Thr3 does not seem to be phosphorylated. This suggests that budding yeast may be a good model for identifying other processes where haspin kinase is involved. We report that yeast cells lacking ALK1 and ALK2 are exquisitely sensitive to microtubule-depolymerizing drugs (i.e., benomyl, nocodazole). Recent work has suggested that chemical inhibition of haspin may impair the activation of SAC (De Antoni et al., 2012, Wang et al., 2012); however, haspin downregulation via siRNA does not seem to produce the same SAC defect (Wang et al., 2012). By monitoring the stabilization of the securin Pds1 and the separation of sister chromatids, prevented by the SAC, after exposure to nocodazole or with conditional mutations affecting kinetochore functionality, we show that yeast cells completely lacking haspin properly activate the SAC. This excludes that a SAC defect could explain nocodazole-induced cell death. After release from nocodazole treatment, the kinetics of reassembly of the mitotic spindle is normal in alk1Δalk2Δ cells, but surprisingly, mutant cells exhibit a highly penetrant spindle positioning defect. Indeed, after a transient nocodazole treatment, cells lacking haspin position the mitotic spindle entirely within the bud, where it elongates and drives nuclear division, producing an anucleated mother and a binucleated daughter cell. To our knowledge, although nuclear mispositioning in the bud has been reported and linked to a FEAR defect (Ross and Cohen-Fix, 2004), this is the first observation reporting exit from mitosis where spindle elongation and nuclear division are restricted within the bud. The SPOC has been shown to be triggered when the mispositioned spindle is within the mother cell (Fraschini et al., 2008, Bertazzi et al., 2011); in our mutant, the SPOC is functional (data not shown) but it is not activated, likely because the spindle is mispositioned within the bud. All these data suggest that haspin loss does not interfere with microtubule dynamics, and it does not affect the Kar9 and Dyn1 pathways; instead, haspin function becomes essential after transient mitotic arrest. Indeed, all the phenotypes described above can be observed by genetically inducing a mitotic UM171 synthesis delay in alk1Δalk2Δ cells. Our results indicate that yeast haspin has a role in allowing cells to tolerate the mitotic delay induced by SAC activation and reestablish proper spindle positioning.