Distinct Roles of RZZ and Bub1-KNL1 in Mitotic Checkpoint Signaling and Kinetochore Expansion.

The Mad1-Mad2 heterodimer is the catalytic hub of the spindle assembly checkpoint (SAC), which controls M phase progression through a multi-subunit anaphase inhibitor, the mitotic checkpoint complex (MCC) [1, 2]. During interphase, Mad1-Mad2 generates MCC at nuclear pores [3]. After nuclear envelope breakdown (NEBD), kinetochore-associated Mad1-Mad2 catalyzes MCC assembly until all chromosomes achieve bipolar attachment [1, 2]. Mad1-Mad2 and other factors are also incorporated into the fibrous corona, a phospho-dependent expansion of the outer kinetochore that precedes microtubule attachment [4-6]. The factor(s) involved in targeting Mad1-Mad2 to kinetochores in higher eukaryotes remain controversial [7-12], and the specific phosphorylation event(s) that trigger corona formation remain elusive [5, 13]. We used genome editing to eliminate Bub1, KNL1, and the Rod-Zw10-Zwilch (RZZ) complex in human cells. We show that RZZ's sole role in SAC activation is to tether Mad1-Mad2 to kinetochores. Separately, Mps1 kinase triggers fibrous corona formation by phosphorylating two N-terminal sites on Rod. In contrast, Bub1 and KNL1 activate kinetochore-bound Mad1-Mad2 to produce a "wait anaphase" signal but are not required for corona formation. We also show that clonal lines isolated after BUB1 disruption recover Bub1 expression and SAC function through nonsense-associated alternative splicing (NAS). Our study reveals a fundamental division of labor in the mammalian SAC and highlights a transcriptional response to nonsense mutations that can reduce or eliminate penetrance in genome editing experiments.

Mps1 Regulates Kinetochore-Microtubule Attachment Stability via the Ska Complex to Ensure Error-Free Chromosome Segregation.

The spindle assembly checkpoint kinase Mps1 not only inhibits anaphase but also corrects erroneous attachments that could lead to missegregation and aneuploidy. However, Mps1's error correction-relevant substrates are unknown. Using a chemically tuned kinetochore-targeting assay, we show that Mps1 destabilizes microtubule attachments (K fibers) epistatically to Aurora B, the other major error-correcting kinase. Through quantitative proteomics, we identify multiple sites of Mps1-regulated phosphorylation at the outer kinetochore. Substrate modification was microtubule sensitive and opposed by PP2A-B56 phosphatases that stabilize chromosome-spindle attachment. Consistently, Mps1 inhibition rescued K-fiber stability after depleting PP2A-B56. We also identify the Ska complex as a key effector of Mps1 at the kinetochore-microtubule interface, as mutations that mimic constitutive phosphorylation destabilized K fibers in vivo and reduced the efficiency of the Ska complex's conversion from lattice diffusion to end-coupled microtubule binding in vitro. Our results reveal how Mps1 dynamically modifies kinetochores to correct improper attachments and ensure faithful chromosome segregation.

A New Methodology for the Quantification of In Vivo Cdc14 Phosphatase Activity.

The phosphatase Cdc14 has a pivotal function in the mitotic exit of Saccharomyces cerevisiae. During interphase, Cdc14 remains inactive in the nucleolus bound to the inhibitor Net1. Cdc14 activation occurs in the metaphase to anaphase transition and it is promoted by at least two signaling pathways called FEAR (CdcFourteen Early Anaphase Release) and MEN (Mitotic Exit Network). These two pathways act in parallel and target the phosphorylation of Net1, thus decreasing Net1 affinity for Cdc14. The activity of Cdc14 can be used as a readout to assay functional interactions of different components of the mitotic exit signaling pathways.

Mitotic Exit Function of Polo-like Kinase Cdc5 Is Dependent on Sequential Activation by Cdk1.

To complete mitosis, Saccharomyces cerevisiae needs to activate the mitotic phosphatase Cdc14. Two pathways contribute to Cdc14 regulation: FEAR (Cdc14 early anaphase release) and MEN (mitotic exit network). Cdc5 polo-like kinase was found to be an important mitotic exit component. However, its specific role in mitotic exit regulation and its involvement in Cdc14 release remain unclear. Here, we provide insight into the mechanism by which Cdc5 contributes to the timely release of Cdc14. Our genetic and biochemical data indicate that Cdc5 acts in parallel with MEN during anaphase. This MEN-independent Cdc5 function requires active separase and activation by Cdk1-dependent phosphorylation. Cdk1 first phosphorylates Cdc5 to activate it in early anaphase, and then, in late anaphase, further phosphorylation of Cdc5 by Cdk1 is needed to promote its MEN-related functions.

Dual Regulation of the mitotic exit network (MEN) by PP2A-Cdc55 phosphatase.

Exit from mitosis in budding yeast is triggered by activation of the key mitotic phosphatase Cdc14. At anaphase onset, the protease separase and Zds1 promote the downregulation of PP2A(Cdc55) phosphatase, which facilitates Cdk1-dependent phosphorylation of Net1 and provides the first wave of Cdc14 activity. Once Cdk1 activity starts to decline, the mitotic exit network (MEN) is activated to achieve full Cdc14 activation. Here we describe how the PP2A(Cdc55) phosphatase could act as a functional link between FEAR and MEN due to its action on Bfa1 and Mob1. We demonstrate that PP2A(Cdc55) regulates MEN activation by facilitating Cdc5- and Cdk1-dependent phosphorylation of Bfa1 and Mob1, respectively. Downregulation of PP2A(Cdc55) initiates MEN activity up to Cdc15 by Bfa1 inactivation. Surprisingly, the premature Bfa1 inactivation observed does not entail premature MEN activation, since an additional Cdk1-Clb2 inhibitory signal acting towards Dbf2-Mob1 activity restrains MEN activity until anaphase. In conclusion, we propose a clear picture of how PP2A(Cdc55) functions affect the regulation of various MEN components, contributing to mitotic exit.

Zds1 regulates PP2A(Cdc55) activity and Cdc14 activation during mitotic exit through its Zds_C motif.

At anaphase onset, highly active mitotic cyclin-dependent kinase (Cdk) is inactivated to promote exit from mitosis and completion of cytokinesis. The budding yeast Cdc14p phosphatase is a key mitotic regulator that counteracts cyclin-dependent kinase (Cdk) activity during mitotic exit. Separase, together with Zds1p, promotes the downregulation of the protein phosphatase 2A in conjunction with its Cdc55p regulatory subunit (PP2A(Cdc55)) in early anaphase, enabling accumulation of phosphorylated forms of Net1p and release of Cdc14p from the nucleolus. Here we show that the C-terminal domain of Zds1p, called the Zds_C motif, is required for Zds1-induced release of Cdc14p, and the N-terminal domain of the protein might be involved in regulating this activity. More interestingly, Zds1p physically interacts with Cdc55p, and regulates its localization through the Zds_C motif. Nevertheless, expression of the Zds_C motif at endogenous levels cannot induce timely release of Cdc14p from the nucleolus, despite the proper (nucleolar) localization of Cdc55p. Our results suggest that the activity of PP2A(Cdc55) cannot be modulated solely through regulation of its localization, and that an additional regulatory step is probably required. These results suggest that Zds1p recruits PP2A(Cdc55) to the nucleolus and induces its inactivation by an unknown mechanism.

Susceptibilidad antibiótica y capacidad de producción de la Beta-lactamasa / Antibiotic susceptibility and beta-lactamasa outpout capacity

Se estudiaron 100 muestras de exudados nasales obtenidos de estudiantes de segundo año de Medicina del Instituto Superior de Ciencias Médicas de Villa Clara, de enero a julio de 1987. El objectivo es conocer el índice de portadores de estafilococos por la implicación que tiene en su futura actividad hospitalaria a partir del tercer año, y para investigar la sensibilidad y resistencia de las cepas a los diferentes antimicrobianos. Se aislaron 86 cepas de estafilococos y se les realizó la prueba coagulasa, de las que 48 resultaron positivas. Se comprobó que la novobiocina era el agente antimicrobiano más efectivo. Se verificó la alta resistencia de los estafilococos a la penicilina y se pudo comprobar que un gran por ciento de las cepas son productoras de Beta-lactamasas

Susceptibilidad antibiótica y capacidad de producción de la Beta-lactamasa / Antibiotic susceptibility and beta-lactamasa outpout capacity

Se estudiaron 100 muestras de exudados nasales obtenidos de estudiantes de segundo año de Medicina del Instituto Superior de Ciencias Médicas de Villa Clara, de enero a julio de 1987. El objectivo es conocer el índice de portadores de estafilococos por la implicación que tiene en su futura actividad hospitalaria a partir del tercer año, y para investigar la sensibilidad y resistencia de las cepas a los diferentes antimicrobianos. Se aislaron 86 cepas de estafilococos y se les realizó la prueba coagulasa, de las que 48 resultaron positivas. Se comprobó que la novobiocina era el agente antimicrobiano más efectivo. Se verificó la alta resistencia de los estafilococos a la penicilina y se pudo comprobar que un gran por ciento de las cepas son productoras de Beta-lactamasas

Estudio de la incidencia de portadores de meningococo en un colectivo de estudiantes universitarios / Study of the incidence of meningococus host in a group of university students

Se seleccionó, dentro de un colectivo de estudiantes universitarios, una muestra de 284 alumnos, cifras que representa el 50.9 porciento. Se realizó 3 exudados de orofaringe en busca de portadores de meningococo, siendo sembrados en medio de Thayer Martin y mediando entre cada toma de muestra una semana. De los 284 alumnos, 83 resultaron positivos, para un 29.2 porciento de positividad. Después de 5 meses de habérsele tomado las muestras a estos casos y no habiendo recibido tratamiento, se les realizó 3 exudados más, descubriéndose que de los 83 portadores, continuaban siéndolo 38; mientras un 54.2 porciento de los mismos dejó de serlo. Las diferentes cepas encontradas se clasificaron serológicamente predominando el serogrupo B. Se realizó prueba de sensibilidad a la Rifampicina y Metaciclina por el método de dilución, detectándose una menor sensibilidad de las cepas estudiadas a la Rifampicina que a la Metaciclina. En el presente trabajo se pone de manifiesto que el número de portadores aumenta al utilizarse tres exudados seriados en lugar de uno

Estudio de la incidencia de portadores de meningococo en un colectivo de estudiantes universitarios / Study of the incidence of meningococus host in a group of university students

Se seleccionó, dentro de un colectivo de estudiantes universitarios, una muestra de 284 alumnos, cifras que representa el 50.9 porciento. Se realizó 3 exudados de orofaringe en busca de portadores de meningococo, siendo sembrados en medio de Thayer Martin y mediando entre cada toma de muestra una semana. De los 284 alumnos, 83 resultaron positivos, para un 29.2 porciento de positividad. Después de 5 meses de habérsele tomado las muestras a estos casos y no habiendo recibido tratamiento, se les realizó 3 exudados más, descubriéndose que de los 83 portadores, continuaban siéndolo 38; mientras un 54.2 porciento de los mismos dejó de serlo. Las diferentes cepas encontradas se clasificaron serológicamente predominando el serogrupo B. Se realizó prueba de sensibilidad a la Rifampicina y Metaciclina por el método de dilución, detectándose una menor sensibilidad de las cepas estudiadas a la Rifampicina que a la Metaciclina. En el presente trabajo se pone de manifiesto que el número de portadores aumenta al utilizarse tres exudados seriados en lugar de uno(AU)