Coupling spindle position with mitotic exit in budding yeast: The multifaceted role of the small GTPase Tem1

In budding yeast, the spindle position checkpoint (SPC) delays mitotic exit until the mitotic spindle moves into the neck between the mother and bud. This checkpoint works by inhibiting the mitotic exit network (MEN), a signaling cascade initiated and controlled by Tem1, a small GTPase. Tem1 is regulated by a putative guanine exchange factor, Lte1, but the function and regulation of Lte1 remains poorly understood. Here, we identify novel components of the checkpoint that operate upstream of Lte1. We present genetic evidence in agreement with existing biochemical evidence for the molecular mechanism of a pathway that links microtubule-cortex interactions with Lte1 and mitotic exit. Each component of this pathway is required for the spindle position checkpoint to delay mitotic exit until the spindle is positioned correctly.

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Computational modelling of mitotic exit in budding yeast: the role of separase and Cdc14 endocycles

Journal of The Royal Society Interface ◽

10.1098/rsif.2010.0649 ◽

2011 ◽

Vol 8 (61) ◽

pp. 1128-1141 ◽

Cited By ~ 20

Author(s):

P. K. Vinod ◽

Paula Freire ◽

Ahmed Rattani ◽

Andrea Ciliberto ◽

Frank Uhlmann ◽

...

Keyword(s):

Regulatory Networks ◽

Budding Yeast ◽

Cell Cycle Control ◽

Computational Modelling ◽

Eukaryotic Cell ◽

Mitotic Exit ◽

Intuitive Reasoning ◽

Systematic Analysis ◽

Systems View

The operating principles of complex regulatory networks are best understood with the help of mathematical modelling rather than by intuitive reasoning. Hereby, we study the dynamics of the mitotic exit (ME) control system in budding yeast by further developing the Queralt's model. A comprehensive systems view of the network regulating ME is provided based on classical experiments in the literature. In this picture, Cdc20–APC is a critical node controlling both cyclin (Clb2 and Clb5) and phosphatase (Cdc14) branches of the regulatory network. On the basis of experimental situations ranging from single to quintuple mutants, the kinetic parameters of the network are estimated. Numerical analysis of the model quantifies the dependence of ME control on the proteolytic and non-proteolytic functions of separase. We show that the requirement of the non-proteolytic function of separase for ME depends on cyclin-dependent kinase activity. The model is also used for the systematic analysis of the recently discovered Cdc14 endocycles. The significance of Cdc14 endocycles in eukaryotic cell cycle control is discussed as well.

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A Mathematical Model of Mitotic Exit in Budding Yeast: The Role of Polo Kinase

PLoS ONE ◽

10.1371/journal.pone.0030810 ◽

2012 ◽

Vol 7 (2) ◽

pp. e30810 ◽

Cited By ~ 7

Author(s):

Baris Hancioglu ◽

John J. Tyson

Keyword(s):

Mathematical Model ◽

Budding Yeast ◽

Mitotic Exit ◽

Polo Kinase

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Inhibition of Cdc42 during mitotic exit is required for cytokinesis

The Journal of Cell Biology ◽

10.1083/jcb.201301090 ◽

2013 ◽

Vol 202 (2) ◽

pp. 231-240 ◽

Cited By ~ 55

Author(s):

Benjamin D. Atkins ◽

Satoshi Yoshida ◽

Koji Saito ◽

Chi-Fang Wu ◽

Daniel J. Lew ◽

...

Keyword(s):

Cell Cycle ◽

General Feature ◽

Budding Yeast ◽

Mitotic Exit ◽

Division Site ◽

Polo Kinase ◽

Cell Cycle Regulator ◽

P21 Activated Kinase

The role of Cdc42 and its regulation during cytokinesis is not well understood. Using biochemical and imaging approaches in budding yeast, we demonstrate that Cdc42 activation peaks during the G1/S transition and during anaphase but drops during mitotic exit and cytokinesis. Cdc5/Polo kinase is an important upstream cell cycle regulator that suppresses Cdc42 activity. Failure to down-regulate Cdc42 during mitotic exit impairs the normal localization of key cytokinesis regulators—Iqg1 and Inn1—at the division site, and results in an abnormal septum. The effects of Cdc42 hyperactivation are largely mediated by the Cdc42 effector p21-activated kinase Ste20. Inhibition of Cdc42 and related Rho guanosine triphosphatases may be a general feature of cytokinesis in eukaryotes.

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BFA1 has multiple positive roles in directing late mitotic events in S. cerevisiae

10.1101/352427 ◽

2018 ◽

Author(s):

J Whalen ◽

C Sniffen ◽

S Gartland ◽

M Vannini ◽

A Seshan

Keyword(s):

Budding Yeast ◽

Spindle Pole Body ◽

Biological Significance ◽

Spindle Pole ◽

Mitotic Exit ◽

Genome Integrity ◽

Mitotic Exit Network ◽

M Phase ◽

Regulation Of Cell Cycle ◽

Spindle Position

ABSTRACTThe proper regulation of cell cycle transitions is paramount to the maintenance of cellular genome integrity. In budding yeast, the mitotic exit network (MEN) is a Ras-like signaling cascade that effects the transition from M phase to G1 during the cell division cycle in budding yeast. MEN activation is tightly regulated. It occurs during anaphase and is coupled to mitotic spindle position by the spindle position checkpoint (SPoC). Bfa1 is a key component of the SPoC and functions as part of a two-component GAP complex along with Bub2. The GAP activity of Bfa1-Bub2 keeps the MEN GTPase Tem1 inactive in cells with mispositioned spindles, thereby preventing inappropriate mitotic exit and preserving genome integrity. Interestingly, a GAP-independent role for Bfa1 in mitotic exit regulation has been previously identified. However the nature of this Bub2-independent role and its biological significance are not understood. Here we show that Bfa1 also activates the MEN by promoting the localization of Tem1 primarily to the daughter spindle pole body (dSPB). We demonstrate that the overexpression of BFA1 is lethal due to defects in Tem1 localization, which is required for its activity. In addition, our studies demonstrate a Tem1-independent role for Bfa1 in promoting proper cytokinesis. Cells lacking TEM1, in which the essential mitotic exit function is bypassed, exhibit cytokinesis defects. These defects are suppressed by the overexpression of BFA1. We conclude that Bfa1 functions to both inhibit and activate late mitotic events.

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

Cells ◽

10.3390/cells11010046 ◽

2021 ◽

Vol 11 (1) ◽

pp. 46

Author(s):

Michael Vannini ◽

Victoria R. Mingione ◽

Ashleigh Meyer ◽

Courtney Sniffen ◽

Jenna Whalen ◽

...

Keyword(s):

Cell Cycle ◽

Budding Yeast ◽

Signal Transduction Pathway ◽

Spindle Pole ◽

Mitotic Exit ◽

Independent Manner ◽

Spindle Pole Bodies ◽

Mitotic Exit Network ◽

Cell Cycle Transition ◽

Spindle Position

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.

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Different Levels of Bfa1/Bub2 GAP Activity Are Required to Prevent Mitotic Exit of Budding Yeast Depending on the Type of Perturbations

Molecular Biology of the Cell ◽

10.1091/mbc.e08-02-0149 ◽

2008 ◽

Vol 19 (10) ◽

pp. 4328-4340 ◽

Cited By ~ 11

Author(s):

Junwon Kim ◽

Selma Sun Jang ◽

Kiwon Song

Keyword(s):

Budding Yeast ◽

Mitotic Exit ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Surveillance Mechanism ◽

Spindle Position ◽

First Time ◽

Different Levels

In budding yeast, Tem1 is a key regulator of mitotic exit. Bfa1/Bub2 stimulates Tem1 GTPase activity as a GTPase-activating protein (GAP). Lte1 possesses a guanine-nucleotide exchange factor (GEF) domain likely for Tem1. However, recent observations showed that cells may control mitotic exit without either Lte1 or Bfa1/Bub2 GAP activity, obscuring how Tem1 is regulated. Here, we assayed BFA1 mutants with varying GAP activities for Tem1, showing for the first time that Bfa1/Bub2 GAP activity inhibits Tem1 in vivo. A decrease in GAP activity allowed cells to bypass mitotic exit defects. Interestingly, different levels of GAP activity were required to prevent mitotic exit depending on the type of perturbation. Although essential, more Bfa1/Bub2 GAP activity was needed for spindle damage than for DNA damage to fully activate the checkpoint. Conversely, Bfa1/Bub2 GAP activity was insufficient to delay mitotic exit in cells with misoriented spindles. Instead, decreased interaction of Bfa1 with Kin4 was observed in BFA1 mutant cells with a defective spindle position checkpoint. These findings demonstrate that there is a GAP-independent surveillance mechanism of Bfa1/Bub2, which, together with the GTP/GDP switch of Tem1, may be required for the genomic stability of cells with misaligned spindles.

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

10.1101/2020.08.30.273946 ◽

2020 ◽

Author(s):

Dilara Kocakaplan ◽

Hüseyin Karabürk ◽

Cansu Dilege ◽

Idil Kirdok ◽

Şeyma Nur Erkan ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Mitotic Spindle ◽

Protein Phosphatase ◽

Budding Yeast ◽

Mitotic Exit ◽

Protein Phosphatase 1 ◽

Inhibitory Function ◽

Surveillance Mechanism ◽

Spindle Position

AbstractSaccharomyces 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.

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Checkpoint control of mitotic exit—do budding yeast mind the GAP?

The Journal of Cell Biology ◽

10.1083/jcb.200512153 ◽

2006 ◽

Vol 172 (3) ◽

pp. 331-333 ◽

Cited By ~ 5

Author(s):

John A. Cooper ◽

Scott A. Nelson

Keyword(s):

Budding Yeast ◽

Spindle Pole ◽

Small Gtpase ◽

Mitotic Exit ◽

Cell Cycle Checkpoints ◽

Nucleotide Exchange ◽

Checkpoint Control ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Spindle Pole Bodies

Cell cycle checkpoints can delay mitotic exit in budding yeast. The master controller is the small GTPase Tem1, with inputs from a proposed guanine nucleotide exchange factor (GEF), Lte1, and a GTPase-activating protein (GAP), Bub2/Bfa1. In this issue, Fraschini et al. (p. 335) show that GAP activity of Bub2/Bfa1 appears to be dispensable for inactivation of Tem1 in cells. Their results call into question the GTP/GDP switch model for Tem1 activity, as have other results in the past. The paper also focuses attention on the two spindle pole bodies as potential sites for regulation of Tem1.

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Functional Characterization of Dma1 and Dma2, the Budding Yeast Homologues of Schizosaccharomyces pombe Dma1 and Human Chfr

Molecular Biology of the Cell ◽

10.1091/mbc.e04-02-0094 ◽

2004 ◽

Vol 15 (8) ◽

pp. 3796-3810 ◽

Cited By ~ 35

Author(s):

Roberta Fraschini ◽

Denis Bilotta ◽

Giovanna Lucchini ◽

Simonetta Piatti

Keyword(s):

Schizosaccharomyces Pombe ◽

Budding Yeast ◽

Functional Characterization ◽

Ectopic Expression ◽

Mitotic Exit ◽

Spindle Positioning ◽

Septin Ring ◽

Daughter Cells ◽

Spindle Position

Proper transmission of genetic information requires correct assembly and positioning of the mitotic spindle, responsible for driving each set of sister chromatids to the two daughter cells, followed by cytokinesis. In case of altered spindle orientation, the spindle position checkpoint inhibits Tem1-dependent activation of the mitotic exit network (MEN), thus delaying mitotic exit and cytokinesis until errors are corrected. We report a functional analysis of two previously uncharacterized budding yeast proteins, Dma1 and Dma2, 58% identical to each other and homologous to human Chfr and Schizosaccharomyces pombe Dma1, both of which have been previously implicated in mitotic checkpoints. We show that Dma1 and Dma2 are involved in proper spindle positioning, likely regulating septin ring deposition at the bud neck. DMA2 overexpression causes defects in septin ring disassembly at the end of mitosis and in cytokinesis. The latter defects can be rescued by either eliminating the spindle position checkpoint protein Bub2 or overproducing its target, Tem1, both leading to MEN hyperactivation. In addition, dma1Δ dma2Δ cells fail to activate the spindle position checkpoint in response to the lack of dynein, whereas ectopic expression of DMA2 prevents unscheduled mitotic exit of spindle checkpoint mutants treated with microtubule-depolymerizing drugs. Although their primary functions remain to be defined, our data suggest that Dma1 and Dma2 might be required to ensure timely MEN activation in telophase.

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