A Novel Hyperactive Nud1 Mitotic Exit Network Scaffold Causes Spindle Position Checkpoint Bypass in Budding Yeast

Mitotic exit is a critical cell cycle transition that requires the careful coordination of nuclear positioning and cyclin B destruction in budding yeast for the maintenance of genome integrity. The mitotic exit network (MEN) is a Ras-like signal transduction pathway that promotes this process during anaphase. A crucial step in MEN activation occurs when the Dbf2-Mob1 protein kinase complex associates with the Nud1 scaffold protein at the yeast spindle pole bodies (SPBs; centrosome equivalents) and thereby becomes activated. This requires prior priming phosphorylation of Nud1 by Cdc15 at SPBs. Cdc15 activation, in turn, requires both the Tem1 GTPase and the Polo kinase Cdc5, but how Cdc15 associates with SPBs is not well understood. We have identified a hyperactive allele of NUD1, nud1-A308T, that recruits Cdc15 to SPBs in all stages of the cell cycle in a CDC5-independent manner. This allele leads to early recruitment of Dbf2-Mob1 during metaphase and requires known Cdc15 phospho-sites on Nud1. The presence of nud1-A308T leads to loss of coupling between nuclear position and mitotic exit in cells with mispositioned spindles. Our findings highlight the importance of scaffold regulation in signaling pathways to prevent improper activation.

Combined effect of cell geometry and polarity domains determines the orientation of unequal division

Cell division orientation is thought to result from a competition between cell geometry and polarity domains controlling the position of the mitotic spindle during mitosis. Depending on the level of cell shape anisotropy or the strength of the polarity domain, one dominates the other and determines the orientation of the spindle. Whether and how such competition is also at work to determine unequal cell division (UCD), producing daughter cells of different size, remains unclear. Here, we show that cell geometry and polarity domains cooperate, rather than compete, in positioning the cleavage plane during UCDs in early ascidian embryos. We found that the UCDs and their orientation at the ascidian third cleavage rely on the spindle tilting in an anisotropic cell shape, and cortical polarity domains exerting different effects on spindle astral microtubules. By systematically varying mitotic cell shape, we could modulate the effect of attractive and repulsive polarity domains and consequently generate predicted daughter cell size asymmetries and position. We therefore propose that the spindle position during UCD is set by the combined activities of cell geometry and polarity domains, where cell geometry modulates the effect of cortical polarity domain(s).

Protein Phosphatase 1 in association with Bud14 inhibits mitotic exit in Saccharomyces cerevisiae

Mitotic exit in budding yeast is dependent on correct orientation of the mitotic spindle along the cell polarity axis. When accurate positioning of the spindle fails, a surveillance mechanism named the Spindle Position Checkpoint (SPOC) prevents cells from exiting mitosis. Mutants with a defective SPOC become multinucleated and lose their genomic integrity. Yet, a comprehensive understanding of the SPOC mechanism is missing. In this study, we identified the type 1 protein phosphatase, Glc7, in association with its regulatory protein Bud14 as a novel checkpoint component. We further showed that Glc7-Bud14 promotes dephosphorylation of the SPOC effector protein Bfa1. Our results suggest a model in which two mechanisms act in parallel for a robust checkpoint response: first, the SPOC kinase Kin4 isolates Bfa1 away from the inhibitory kinase Cdc5 and second, Glc7-Bud14 dephosphorylates Bfa1 to fully activate the checkpoint effector.

Human Microcephaly Protein RTTN Is Required for Proper Mitotic Progression and Correct Spindle Position

Autosomal recessive primary microcephaly (MCPH) is a complex neurodevelopmental disorder characterized by a small brain size with mild to moderate intellectual disability. We previously demonstrated that human microcephaly RTTN played an important role in regulating centriole duplication during interphase, but the role of RTTN in mitosis is not fully understood. Here, we show that RTTN is required for normal mitotic progression and correct spindle position. The depletion of RTTN induces the dispersion of the pericentriolar protein γ-tubulin and multiple mitotic abnormalities, including monopolar, abnormal bipolar, and multipolar spindles. Importantly, the loss of RTTN altered NuMA/p150Glued congression to the spindle poles, perturbed NuMA cortical localization, and reduced the number and the length of astral microtubules. Together, our results provide a new insight into how RTTN functions in mitosis.

Unifying the mechanism of mitotic exit control in a spatiotemporal logical model

The transition from mitosis into the first gap phase of the cell cycle in budding yeast is controlled by the Mitotic Exit Network (MEN). The network interprets spatiotemporal cues about the progression of mitosis and ensures that release of Cdc14 phosphatase occurs only after completion of key mitotic events. The MEN has been studied intensively; however, a unified understanding of how localisation and protein activity function together as a system is lacking. In this paper, we present a compartmental, logical model of the MEN that is capable of representing spatial aspects of regulation in parallel to control of enzymatic activity. We show that our model is capable of correctly predicting the phenotype of the majority of mutants we tested, including mutants that cause proteins to mislocalise. We use a continuous time implementation of the model to demonstrate that Cdc14 Early Anaphase Release (FEAR) ensures robust timing of anaphase, and we verify our findings in living cells. Furthermore, we show that our model can represent measured cell–cell variation in Spindle Position Checkpoint (SPoC) mutants. This work suggests a general approach to incorporate spatial effects into logical models. We anticipate that the model itself will be an important resource to experimental researchers, providing a rigorous platform to test hypotheses about regulation of mitotic exit.

Protein Phosphatase 1 in association with Bud14 inhibits mitotic exit in Saccharomyces cerevisiae

Saccharomyces cerevisiae, also known as the budding yeast, orients and elongates its mitotic spindle along its polarity axis in order to segregate one copy of its genomic DNA to the daughter cell. When accurate positioning of the mitotic spindle fails, a surveillance mechanism, named the Spindle Position Checkpoint (SPOC), prevents cells from exiting mitosis unless the spindle orientation is corrected. Mutants with a defective SPOC loss their genomic integrity, become multiploid and aneuploid. Thus, SPOC is a crucial checkpoint for the budding yeast. Yet, a comprehensive understanding of how the SPOC mechanism works is missing. In this study, we identified Bud14 as a novel checkpoint protein. We showed that the mitotic exit inhibitory function of Bud14 requires its association with the type 1 protein phosphatase, Glc7. Our data indicate that Glc7-Bud14 promotes dephosphorylation of the SPOC effector protein Bfa1. Our results support a model in which Glc7-Bud14 works parallel to the SPOC kinase Kin4 in inhibiting mitotic exit.

Spindle position dictates division site during asymmetric cell division in moss

Asymmetric cell division (ACD) underlies the development of multicellular organisms. The division site in plant cells is predetermined prior to mitosis and the localization of the mitotic spindle is considered static, unlike in animal ACD, where the cell division site is defined by active spindle-positioning mechanisms. Here, we isolated a novel mutant of the microtubule-associated protein TPX2 in the moss Physcomitrella patens and observed abnormal spindle motility, which led to inverted asymmetric division during organ development. This phenotype was rescued by restoring endogenous TPX2 function and, unexpectedly, by depolymerizing actin filaments. Thus, we identify an active spindle-positioning mechanism involving microtubules and actin filaments that sets the division site in plants, which is reminiscent of the acentrosomal ACD in animals, and suggests the existence of a common ancestral mechanism.

Theoretical and experimental investigations of different area ratios of a supersonic ejector driven by compressed air

Ejectors have some advantages such as being simple, reliable and no moving parts. They can be used in air-conditioning and refrigeration applications. This paper presents a comparison of ejector performance, primary pressure (Pp), back pressure (Pb) and area ratios of ejectors (A2/At) predictions by an analytical model and a computational fluid dynamics model for different operating conditions. Six different area ratios of ejector using air as working fluid in this study were proposed and tested experimentally. The variable area ratios of ejectors (A2/At) were used with a range from 10.68 to 30.62. Two sets of ejectors (A and C) are studied and examined depend on the kind of nozzle. The aim of this study was to investigate these ejectors under variation of primary pressure (Pp) (1.5–6.0 bar) and adjustable spindle position (0 to −25 mm). Two groups of ejectors (A and C) were categorized based on the type of nozzle. The experimental results validate the solutions of the main parameters of ejectors using air as working fluid. The results show that group A is more appropriate for higher values of back pressure, while group C is more suitable for high performance of the ejector. Finally, the main parameters were carried out on six different ejectors to find the best combination based on various nozzles and constant area sections.

SWR1 Chromatin Remodeling Complex Prevents Mitotic Slippage during Spindle Position Checkpoint Arrest

ABSTRACTFaithful chromosome segregation in budding yeast requires correct positioning of the mitotic spindle along the mother to daughter cell polarity axis. When the anaphase spindle is not correctly positioned, a surveillance mechanism, named as the spindle position checkpoint (SPOC), prevents the progression out of mitosis until correct spindle positioning is achieved. How SPOC works on a molecular level is not well-understood. Here, we performed a genome-wide genetic screen to search for components required for SPOC. We identified the SWR1 chromatin-remodeling complex (SWR1-C) among the several novel factors that are essential for SPOC integrity. Cells lacking SWR1-C were able to activate SPOC upon spindle misorientation but underwent mitotic slippage upon prolonged SPOC arrest. This mitotic slippage required the Cdc14-early anaphase release pathway and other factors including the SAGA histone acetyltransferase complex, proteasome components, the mitotic cyclin-dependent kinase inhibitor Sic1 and the mitogen-activated protein kinase Slt2/Mpk1. Together, our data establish a novel link between chromatin remodeling and robust checkpoint arrest in late anaphase.AUTHORS SUMMARYBefore it physically divides into two, the cell must duplicate its genetic material and separate the duplicated copies to the opposite poles of the cell with the help of the spindle machinery. The direction along which the genetic material is separated has different consequences on cell division, especially when the opposite poles of the cell differ from each other, as is the case of asymmetric cell division. Every cell division in budding yeast is asymmetric. The new (daughter) cell grows on the old (mother) cell and pinches of from this location at the end of the cell division, giving rise to a new and an old cell. The daughter and mother cells differ in size and composition, thus the cell division is asymmetric. In order for the daughter cell to receive one copy of the duplicated genetic material, budding yeast has to separate the copies of its genetic material along the mother to daughter cell direction, which is possible by placing the spindle apparatus along this direction.A surveillance mechanism named the Spindle Position Checkpoint (SPOC) in budding yeast monitors the position of the mitotic spindle and prevents cells from dividing if the spindle fails to align in the mother to daughter direction. The cell can resume cell division only after correcting the position of the spindle followed by inactivation of SPOC. This way SPOC prevents multi-nucleation and enucleation, and hence it is a crucial mechanism to maintain the correct ploidy. It has been known that about five proteins play a role in positively supporting the SPOC. Yet, how SPOC works on a molecular level remains ill understood.In this study, we aimed to find out novel components of SPOC. Through an unbiased genome-wide genetic screen, we successfully identified several new components of the SPOC machinery. Among several other novel proteins identified, we investigated the role of the SWR1 chromatin remodeling complex (SWR1-C) in more detail. We show that the SWR1-C has a function in preventing cells with mis-positioned spindles from resuming cell division when the spindle stays mis-positioned for a prolonged time (mitotic slippage). Our data indicated that SWR1-C is not required to start the immediate SPOC response, rather it is important to keep the prolonged SPOC arrest.