Spatial signals link exit from mitosis to spindle position.
ABSTRACT: In budding yeast, if the spindle becomes mispositioned, cells prevent exit from mitosis by inhibiting the mitotic exit network (MEN). The MEN is a signaling cascade that localizes to spindle pole bodies (SPBs) and activates the phosphatase Cdc14. There are two competing models that explain MEN regulation by spindle position. In the 'zone model', exit from mitosis occurs when a MEN-bearing SPB enters the bud. The 'cMT-bud neck model' posits that cytoplasmic microtubule (cMT)-bud neck interactions prevent MEN activity. Here we find that 1) eliminating cMT- bud neck interactions does not trigger exit from mitosis and 2) loss of these interactions does not precede Cdc14 activation. Furthermore, using binucleate cells, we show that exit from mitosis occurs when one SPB enters the bud despite the presence of a mispositioned spindle. We conclude that exit from mitosis is triggered by a correctly positioned spindle rather than inhibited by improper spindle position.
Project description:How spatial information is translated into a chemical signal is a fundamental problem in all organisms. The spindle position checkpoint is a prime example of this problem. This checkpoint senses spindle position and, in budding yeast, inhibits the mitotic exit network (MEN), a signaling pathway that promotes exit from mitosis. We find that spindle position is sensed by a system composed of MEN-inhibitory and -activating zones and a sensor that moves between them. The MEN inhibitory zone is located in the mother cell, the MEN-activating zone in the bud, and the spindle pole body (SPB), where the components of the MEN reside, functions as the sensor. Only when an SPB escapes the MEN inhibitor Kin4 in the mother cell and moves into the bud where the MEN activator Lte1 resides can exit from mitosis occur. In this manner, spatial information is sensed and translated into a chemical signal.
Project description:The Mitotic Exit Network (MEN), a budding yeast Ras-like signal transduction cascade, translates nuclear position into a signal to exit from mitosis. Here we describe how scaffolding the MEN onto spindle pole bodies (SPB-centrosome equivalent) allows the MEN to couple the final stages of mitosis to spindle position. Through the quantitative analysis of the localization of MEN components, we determined the relative importance of MEN signaling from the SPB that is delivered into the daughter cell (dSPB) during anaphase and the SPB that remains in the mother cell. Movement of half of the nucleus into the bud during anaphase causes the active form of the MEN GTPase Tem1 to accumulate at the dSPB. In response to Tem1's activity at the dSPB, the MEN kinase cascade, which functions downstream of Tem1, accumulates at both SPBs. This localization to both SPBs serves an important role in promoting efficient exit from mitosis. Cells that harbor only one SPB delay exit from mitosis. We propose that MEN signaling is initiated by Tem1 at the dSPB and that association of the downstream MEN kinases with both SPBs serves to amplify MEN signaling, enabling the timely exit from mitosis.
Project description:For a daughter cell to receive a complete genomic complement, it is essential that the mitotic spindle be positioned accurately within the cell. In budding yeast, a signaling system known as the spindle position checkpoint (SPOC) monitors spindle position and regulates the activity of the mitotic exit network (MEN), a GTPase signaling pathway that promotes exit from mitosis. The protein kinase Kin4 is a central component of the spindle position checkpoint. Kin4 primarily localizes to the mother cell and associates with spindle pole bodies (SPBs) located in the mother cell to inhibit MEN signaling. In contrast, the kinase does not associate with the SPB in the bud. Thus, only when a MEN bearing SPB leaves the mother cell and the spindle is accurately positioned along the mother-bud axis can MEN signaling occur and cell division proceed. Here, we describe a mechanism ensuring that Kin4 only associates with mother cell-located SPBs. The bud-localized MEN regulator Lte1, whose molecular function has long been unclear, prevents Kin4 that escapes into the bud from associating with SPBs in the daughter cell.
Project description:Components of the mitotic exit network (MEN), a signaling pathway that triggers exit from mitosis, localize to the spindle pole body (SPB) that migrates into the daughter cell during anaphase but are largely absent from the SPB that remains in the mother cell. Through the analysis of one of the determinants of this asymmetry, Bfa1, we find that the machinery responsible for establishing cell polarity and cytoplasmic microtubules collaborate to establish MEN asymmetry. In cells defective in the Cdc42 signaling pathway or the formin Bni1, Bfa1 localizes to both SPBs. The quantitative analysis of Bfa1 localization further shows that Bfa1 can associate with both SPBs in a transient and highly dynamic fashion, but the protein is stabilized on the SPB that migrates into the daughter cell during anaphase through microtubule-bud cortex interactions. Our results indicate that mother-daughter cell asymmetry determinants establish MEN signaling asymmetry through microtubule-bud cortex interactions.
Project description:Background Gametes are generated through a specialized cell division called meiosis, in which ploidy is reduced by half because two consecutive rounds of chromosome segregation, meiosis I and meiosis II, occur without intervening DNA replication. This contrasts with the mitotic cell cycle where DNA replication and chromosome segregation alternate to maintain the same ploidy. At the end of mitosis, CDKs are inactivated. This low CDK state in late mitosis/G1 allows for critical preparatory events for DNA replication and centrosome/spindle pole body (SPB) duplication. However, their execution is inhibited until S phase, where further preparatory events are also prevented. This "licensing" ensures that both the chromosomes and the centrosomes/SPBs replicate exactly once per cell cycle, thereby maintaining constant ploidy. Crucially, between meiosis I and meiosis II, centrosomes/SPBs must be re-licensed, but DNA re-replication must be avoided. In budding yeast, the Cdc14 protein phosphatase triggers CDK down regulation to promote exit from mitosis. Cdc14 also regulates the meiosis I to meiosis II transition, though its mode of action has remained unclear. Methods Fluorescence and electron microscopy was combined with proteomics to probe SPB duplication in cells with inactive or hyperactive Cdc14. Results We demonstrate that Cdc14 ensures two successive nuclear divisions by re-licensing SPBs at the meiosis I to meiosis II transition. We show that Cdc14 is asymmetrically enriched on a single SPB during anaphase I and provide evidence that this enrichment promotes SPB re-duplication. Cells with impaired Cdc14 activity fail to promote extension of the SPB half-bridge, the initial step in morphogenesis of a new SPB. Conversely, cells with hyper-active Cdc14 duplicate SPBs, but fail to induce their separation. Conclusion Our findings implicate reversal of key CDK-dependent phosphorylations in the differential licensing of cyclical events at the meiosis I to meiosis I transition.
Project description:The budding yeast Polo-like kinase Cdc5 is a key regulator of many mitotic events. Cdc5 coordinates its functions spatially and temporally by changing its localization during the cell cycle: Cdc5 is imported into the nucleus in G2 phase and released to the cytoplasm in anaphase, where it accumulates at the bud neck. Cdc5 also localizes to the spindle pole bodies (SPBs) from S phase until the end of mitosis. Whether Cdc5 changes its SPB population during the cell cycle is not known. We find that Cdc5 localizes to distinct SPB subpopulations, depending on the mitotic stage. Cdc5 localizes to the nuclear side of the SPBs during metaphase and early anaphase and to the cytoplasmic surface of the SPBs during late anaphase. Cdc14 is necessary to relocalize Cdc5 from the nuclear SPB plaque. Accumulation of Cdc5 at the daughter SPB in late anaphase is controlled by Bfa1. We also show that Cdc5 and Bfa1 are found in spatially distinct locations at the SPBs during G2/M arrest after DNA damage. Collectively our data reveal that Cdc5 is a dynamic component of the SPBs during mitosis and provide new insight into its regulation during both late mitotic events and DNA damage-induced G2/M arrest.
Project description:Cytoplasmic microtubules (cMT) control mitotic spindle positioning in many organisms, and are therefore pivotal for successful cell division. Despite its importance, the temporal control of cMT formation remains poorly understood. Here we show that unlike the best-studied yeast <i>Saccharomyces cerevisiae</i>, position of pre-anaphase nucleus is not strongly biased toward bud neck in <i>Ogataea polymorpha</i> and the regulation of spindle positioning becomes active only shortly before anaphase. This is likely due to the unstable property of cMTs compared to those in <i>S. cerevisiae</i>. Furthermore, we show that cMT nucleation/anchoring is restricted at the level of recruitment of the ?-tubulin complex receptor, Spc72, to spindle pole body (SPB), which is regulated by the polo-like kinase Cdc5. Additionally, electron microscopy revealed that the cytoplasmic side of SPB is structurally different between G1 and anaphase. Thus, polo-like kinase dependent recruitment of ?-tubulin receptor to SPBs determines the timing of spindle orientation in <i>O. polymorpha.</i>
Project description:The mitotic exit network (MEN) is a spindle pole body (SPB)-associated, GTPase-driven signaling cascade that controls mitotic exit. The inhibitory Bfa1-Bub2 GTPase-activating protein (GAP) only associates with the daughter SPB (dSPB), raising the question as to how the MEN is regulated on the mother SPB (mSPB). Here, we show mutual regulation of cyclin-dependent kinase 1 (Cdk1) and the MEN. In early anaphase Cdk1 becomes recruited to the mSPB depending on the activity of the MEN kinase Cdc15. Conversely, Cdk1 negatively regulates binding of Cdc15 to the mSPB. In addition, Cdk1 phosphorylates the Mob1 protein to inhibit the activity of Dbf2-Mob1 kinase that regulates Cdc14 phosphatase. Our data revise the understanding of the spatial regulation of the MEN. Although MEN activity in the daughter cells is controlled by Bfa1-Bub2, Cdk1 inhibits MEN activity at the mSPB. Consistent with this model, only triple mutants that lack BUB2 and the Cdk1 phosphorylation sites in Mob1 and Cdc15 show mitotic exit defects.
Project description:Cytokinesis, which leads to the physical separation of two dividing cells, is normally restrained until after nuclear division. In Saccharomyces cerevisiae, chitin synthase 2 (Chs2), which lays down the primary septum at the mother-daughter neck, also ensures proper actomyosin ring constriction during cytokinesis. During the metaphase-to-anaphase transition, phosphorylation of Chs2 by the mitotic cyclin-dependent kinase (Cdk1) retains Chs2 at the endoplasmic reticulum (ER), thereby preventing its translocation to the neck. Upon Cdk1 inactivation at the end of mitosis, Chs2 is exported from the ER and targeted to the neck. The mechanism for triggering Chs2 ER export thus far is unknown. We show here that Chs2 ER export requires the direct reversal of the inhibitory Cdk1 phosphorylation sites by Cdc14 phosphatase, the ultimate effector of the mitotic exit network (MEN). We further show that only Cdc14 liberated by the MEN after completion of chromosome segregation, and not Cdc14 released in early anaphase by the Cdc fourteen early anaphase release pathway, triggers Chs2 ER exit. Presumably, the reduced Cdk1 activity in late mitosis further favors dephosphorylation of Chs2 by Cdc14. Thus, by requiring declining Cdk1 activity and Cdc14 nuclear release for Chs2 ER export, cells ensure that septum formation is contingent upon chromosome separation and exit from mitosis.
Project description:To function in diverse cellular processes, the dynamic behavior of microtubules (MTs) must be differentially regulated within the cell. In budding yeast, the spindle position checkpoint (SPOC) inhibits mitotic exit in response to mispositioned spindles. To maintain SPOC-mediated anaphase arrest, astral MTs must maintain persistent interactions with and/or extend through the bud neck. However, the molecular mechanisms that ensure the stability of these interactions are not known.The presence of an MT extending through and/or interacting with the bud neck is maintained by spatial control of catastrophe and rescue, which extends MT lifetime >25-fold and controls the length of dynamic MTs within the bud compartment. Moreover, the single kinesin-8 motor Kip3 alternately mediates both catastrophe and rescue of the bud MT. Kip3 accumulates in a length-dependent manner along the lattice of MTs within the bud, yet induces catastrophe spatially near the bud tip. Rather, this accumulation of Kip3 facilitates its association with depolymerizing MT plus ends, where Kip3 promotes rescue before MTs exit the bud. MT rescue within the bud requires the tail domain of Kip3, whereas the motor domain mediates catastrophe at the bud tip. In vitro, Kip3 exerts both stabilizing and destabilizing effects on reconstituted yeast MTs.The kinesin-8 Kip3 is a multifunctional regulator that differentially stabilizes and destabilizes specific MTs. Control over MT catastrophe and rescue by Kip3 defines the length and lifetime of MTs within the bud compartment of cells with mispositioned spindles. This subcellular regulation of MT dynamics is critical to maintaining mitotic arrest in response to mispositioned spindles.