RESUMEN
The asymmetrically dividing yeast S. cerevisiae assembles a bipolar spindle well after establishing the future site of cell division (i.e., the bud neck) and the division axis (i.e., the mother-bud axis). A surveillance mechanism called spindle position checkpoint (SPOC) delays mitotic exit and cytokinesis until the spindle is properly positioned relative to the mother-bud axis, thereby ensuring the correct ploidy of the progeny. SPOC relies on the heterodimeric GTPase-activating protein Bub2/Bfa1 that inhibits the small GTPase Tem1, in turn essential for activating the mitotic exit network (MEN) kinase cascade and cytokinesis. The Bub2/Bfa1 GAP and the Tem1 GTPase form a complex at spindle poles that undergoes a remarkable asymmetry during mitosis when the spindle is properly positioned, with the complex accumulating on the bud-directed old spindle pole. In contrast, the complex remains symmetrically localized on both poles of misaligned spindles. The mechanism driving asymmetry of Bub2/Bfa1/Tem1 in mitosis is unclear. Furthermore, whether asymmetry is involved in timely mitotic exit is controversial. We investigated the mechanism by which the GAP Bub2/Bfa1 controls GTP hydrolysis on Tem1 and generated a series of mutants leading to constitutive Tem1 activation. These mutants are SPOC-defective and invariably lead to symmetrical localization of Bub2/Bfa1/Tem1 at spindle poles, indicating that GTP hydrolysis is essential for asymmetry. Constitutive tethering of Bub2 or Bfa1 to both spindle poles impairs SPOC response but does not impair mitotic exit. Rather, it facilitates mitotic exit of MEN mutants, likely by increasing the residence time of Tem1 at spindle poles where it gets active. Surprisingly, all mutant or chimeric proteins leading to symmetrical localization of Bub2/Bfa1/Tem1 lead to increased symmetry at spindle poles of the Kar9 protein that mediates spindle positioning and cause spindle misalignment. Thus, asymmetry of the Bub2/Bfa1/Tem1 complex is crucial to control Kar9 distribution and spindle positioning during mitosis.
Asunto(s)
Citocinesis/genética , Mitosis/genética , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Saccharomyces cerevisiae/genética , Polos del Huso/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Polaridad Celular/genética , Proteínas del Citoesqueleto/genética , GTP Fosfohidrolasas/genética , Regulación Fúngica de la Expresión Génica , Glutamina/genética , Glutamina/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Budding yeast spindle position checkpoint is engaged by misoriented spindles and prevents mitotic exit by inhibiting the G protein Tem1 through the GTPase-activating protein (GAP) Bub2/Bfa1. Bub2 and Bfa1 are found on both duplicated spindle pole bodies until anaphase onset, when they disappear from the mother-bound spindle pole under unperturbed conditions. In contrast, when spindles are misoriented they remain symmetrically localized at both SPBs. Thus, symmetric localization of Bub2/Bfa1 might lead to inhibition of Tem1, which is also present at SPBs. Consistent with this hypothesis, we show that a Bub2 version symmetrically localized on both SPBs throughout the cell cycle prevents mitotic exit in mutant backgrounds that partially impair it. This effect is Bfa1 dependent and can be suppressed by high Tem1 levels. Bub2 removal from the mother-bound SPB requires its GAP activity, which in contrast appears to be dispensable for Tem1 inhibition. Moreover, it correlates with the passage of one spindle pole through the bud neck because it needs septin ring formation and bud neck kinases.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Proteínas del Citoesqueleto/fisiología , Mitosis/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/fisiología , Huso Acromático/fisiología , Alelos , Anafase/genética , Proteína Quinasa CDC2/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ciclina B/metabolismo , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/genética , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Regulación Fúngica de la Expresión Génica/genética , Genes Dominantes/genética , Factores de Intercambio de Guanina Nucleótido/genética , Metionina/farmacología , Proteínas de Microtúbulos/genética , Mitosis/efectos de los fármacos , Modelos Genéticos , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Mutación/genética , Feromonas/farmacología , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Quinasas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Huso Acromático/metabolismoRESUMEN
During asymmetric cell division, spindle positioning is critical to ensure the unequal segregation of polarity factors and generate daughter cells with different sizes or fates. In budding yeast the boundary between mother and daughter cell resides at the bud neck, where cytokinesis takes place at the end of the cell cycle. Since budding and bud neck formation occur much earlier than bipolar spindle formation, spindle positioning is a finely regulated process. A surveillance device called the SPOC (spindle position checkpoint) oversees this process and delays mitotic exit and cytokinesis until the spindle is properly oriented along the division axis, thus ensuring genome stability.
Asunto(s)
División Celular , Saccharomycetales/citología , Huso Acromático/metabolismo , Centro Organizador de los Microtúbulos/metabolismoRESUMEN
Mitotic exit and cytokinesis must be tightly coupled to nuclear division both in time and space in order to preserve genome stability and to ensure that daughter cells inherit the right set of chromosomes after cell division. This is achieved in budding yeast through control over a signal transduction cascade, the mitotic exit network (MEN), which is required for mitotic CDK inactivation in telophase and for cytokinesis. Current models of MEN activation emphasize on the bud as the place where most control is exerted. This review focuses on recent data that instead point to the mother cell as being the residence of key regulators of late mitotic events.