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

Cells ◽  
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.

scholarly journals Disappearance of the budding yeast Bub2–Bfa1 complex from the mother-bound spindle pole contributes to mitotic exit

2006 ◽  
Vol 172 (3) ◽  
pp. 335-346 ◽  
Author(s):  
Roberta Fraschini ◽  
Claudio D'Ambrosio ◽  
Marianna Venturetti ◽  
Giovanna Lucchini ◽  
Simonetta Piatti
Keyword(s):  
Cell Cycle ◽  
Budding Yeast ◽  
Spindle Pole ◽  
Mitotic Exit ◽  
Gtpase Activating Protein ◽  
Septin Ring ◽  
Anaphase Onset ◽  
Spindle Pole Bodies ◽  
Bud Neck ◽  
Spindle Position

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.

scholarly journals Spatial signals link exit from mitosis to spindle position

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Jill Elaine Falk ◽  
Dai Tsuchiya ◽  
Jolien Verdaasdonk ◽  
Soni Lacefield ◽  
Kerry Bloom ◽  
...  
Keyword(s):  
Spindle Pole ◽  
Mitotic Exit ◽  
Zone Model ◽  
Cytoplasmic Microtubule ◽  
Binucleate Cells ◽  
Spindle Pole Bodies ◽  
Mitotic Exit Network ◽  
Bud Neck ◽  
Competing Models ◽  
Spindle Position

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.

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

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.

scholarly journals LTE1 promotes exit from mitosis by multiple mechanisms

2016 ◽  
Vol 27 (25) ◽  
pp. 3991-4001 ◽  
Author(s):  
Jill E. Falk ◽  
Ian W. Campbell ◽  
Kelsey Joyce ◽  
Jenna Whalen ◽  
Anupama Seshan ◽  
...  
Keyword(s):  
Mother Cell ◽  
Regulatory Elements ◽  
Spindle Pole ◽  
Mitotic Exit ◽  
Genome Integrity ◽  
Cell Compartment ◽  
Activating Function ◽  
Spindle Pole Bodies ◽  
Mitotic Exit Network ◽  
Spindle Position

In budding yeast, alignment of the anaphase spindle along the mother–bud axis is crucial for maintaining genome integrity. If the anaphase spindle becomes misaligned in the mother cell compartment, cells arrest in anaphase because the mitotic exit network (MEN), an essential Ras-like GTPase signaling cascade, is inhibited by the spindle position checkpoint (SPoC). Distinct localization patterns of MEN and SPoC components mediate MEN inhibition. Most components of the MEN localize to spindle pole bodies. If the spindle becomes mispositioned in the mother cell compartment, cells arrest in anaphase due to inhibition of the MEN by the mother cell–restricted SPoC kinase Kin4. Here we show that a bud-localized activating signal is necessary for full MEN activation. We identify Lte1 as this signal and show that Lte1 activates the MEN in at least two ways. It inhibits small amounts of Kin4 that are present in the bud via its central domain. An additional MEN-activating function of Lte1 is mediated by its N- and C-terminal GEF domains, which, we propose, directly activate the MEN GTPase Tem1. We conclude that control of the MEN by spindle position is exerted by both negative and positive regulatory elements that control the pathway’s GTPase activity.

scholarly journals Control of the mitotic exit network during meiosis

2012 ◽  
Vol 23 (16) ◽  
pp. 3122-3132 ◽  
Author(s):  
Michelle A. Attner ◽  
Angelika Amon
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Signaling Pathway ◽  
Vegetative Growth ◽  
Spindle Pole ◽  
Mitotic Exit ◽  
Regulatory Pathways ◽  
Spindle Pole Bodies ◽  
Mitotic Exit Network ◽  
Meiosis I

The mitotic exit network (MEN) is an essential GTPase signaling pathway that triggers exit from mitosis in budding yeast. We show here that during meiosis, the MEN is dispensable for exit from meiosis I but contributes to the timely exit from meiosis II. Consistent with a role for the MEN during meiosis II, we find that the signaling pathway is active only during meiosis II. Our analysis further shows that MEN signaling is modulated during meiosis in several key ways. Whereas binding of MEN components to spindle pole bodies (SPBs) is necessary for MEN signaling during mitosis, during meiosis MEN signaling occurs off SPBs and does not require the SPB recruitment factor Nud1. Furthermore, unlike during mitosis, MEN signaling is controlled through the regulated interaction between the MEN kinase Dbf20 and its activating subunit Mob1. Our data lead to the conclusion that a pathway essential for vegetative growth is largely dispensable for the specialized meiotic divisions and provide insights into how cell cycle regulatory pathways are modulated to accommodate different modes of cell division.

scholarly journals Budding Yeast Bub2 Is Localized at Spindle Pole Bodies and Activates the Mitotic Checkpoint via a Different Pathway from Mad2

1999 ◽  
Vol 145 (5) ◽  
pp. 979-991 ◽  
Author(s):  
Roberta Fraschini ◽  
Elisa Formenti ◽  
Giovanna Lucchini ◽  
Simonetta Piatti
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Cell Cycle Progression ◽  
Budding Yeast ◽  
Spindle Pole Body ◽  
Mitotic Checkpoint ◽  
Spindle Pole ◽  
Cycle Progression ◽  
Spindle Pole Bodies

The mitotic checkpoint blocks cell cycle progression before anaphase in case of mistakes in the alignment of chromosomes on the mitotic spindle. In budding yeast, the Mad1, 2, 3, and Bub1, 2, 3 proteins mediate this arrest. Vertebrate homologues of Mad1, 2, 3, and Bub1, 3 bind to unattached kinetochores and prevent progression through mitosis by inhibiting Cdc20/APC-mediated proteolysis of anaphase inhibitors, like Pds1 and B-type cyclins. We investigated the role of Bub2 in budding yeast mitotic checkpoint. The following observations indicate that Bub2 and Mad1, 2 probably activate the checkpoint via different pathways: (a) unlike the other Mad and Bub proteins, Bub2 localizes at the spindle pole body (SPB) throughout the cell cycle; (b) the effect of concomitant lack of Mad1 or Mad2 and Bub2 is additive, since nocodazole-treated mad1 bub2 and mad2 bub2 double mutants rereplicate DNA more rapidly and efficiently than either single mutant; (c) cell cycle progression of bub2 cells in the presence of nocodazole requires the Cdc26 APC subunit, which, conversely, is not required for mad2 cells in the same conditions. Altogether, our data suggest that activation of the mitotic checkpoint blocks progression through mitosis by independent and partially redundant mechanisms.

scholarly journals The Differential Roles of Budding Yeast Tem1p, Cdc15p, and Bub2p Protein Dynamics in Mitotic Exit

2004 ◽  
Vol 15 (4) ◽  
pp. 1519-1532 ◽  
Author(s):  
Jeffrey N. Molk ◽  
Scott C. Schuyler ◽  
Jenny Y. Liu ◽  
James G. Evans ◽  
E. D. Salmon ◽  
...  
Keyword(s):  
Budding Yeast ◽  
Protein Localization ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Mitotic Exit ◽  
Yeast Saccharomyces Cerevisiae ◽  
Late Anaphase ◽  
Gfp Fluorescence ◽  
Mitotic Exit Network

In the budding yeast Saccharomyces cerevisiae the mitotic spindle must be positioned along the mother-bud axis to activate the mitotic exit network (MEN) in anaphase. To examine MEN proteins during mitotic exit, we imaged the MEN activators Tem1p and Cdc15p and the MEN regulator Bub2p in vivo. Quantitative live cell fluorescence microscopy demonstrated the spindle pole body that segregated into the daughter cell (dSPB) signaled mitotic exit upon penetration into the bud. Activation of mitotic exit was associated with an increased abundance of Tem1p-GFP and the localization of Cdc15p-GFP on the dSPB. In contrast, Bub2p-GFP fluorescence intensity decreased in mid-to-late anaphase on the dSPB. Therefore, MEN protein localization fluctuates to switch from Bub2p inhibition of mitotic exit to Cdc15p activation of mitotic exit. The mechanism that elevates Tem1p-GFP abundance in anaphase is specific to dSPB penetration into the bud and Dhc1p and Lte1p promote Tem1p-GFP localization. Finally, fluorescence recovery after photobleaching (FRAP) measurements revealed Tem1p-GFP is dynamic at the dSPB in late anaphase. These data suggest spindle pole penetration into the bud activates mitotic exit, resulting in Tem1p and Cdc15p persistence at the dSPB to initiate the MEN signal cascade.

scholarly journals Spindle Pole Bodies Exploit the Mitotic Exit Network in Metaphase to Drive Their Age-Dependent Segregation

Cell ◽  
2012 ◽  
Vol 148 (5) ◽  
pp. 958-972 ◽  
Author(s):  
Manuel Hotz ◽  
Christian Leisner ◽  
Daici Chen ◽  
Cristina Manatschal ◽  
Thomas Wegleiter ◽  
...  
Keyword(s):  
Spindle Pole ◽  
Mitotic Exit ◽  
Spindle Pole Bodies ◽  
Mitotic Exit Network ◽  
Age Dependent

scholarly journals Downregulation of the Tem1 GTPase by Amn1 after cytokinesis involves both nuclear import and SCF-mediated degradation

2021 ◽  
Vol 134 (19) ◽  
Author(s):  
Alain Devault ◽  
Simonetta Piatti
Keyword(s):  
Cell Cycle ◽  
Daughter Cell ◽  
Spindle Pole ◽  
Mitotic Exit ◽  
Spindle Pole Bodies ◽  
Origin Licensing ◽  
Kinase Cascade ◽  
Scf Ubiquitin Ligase ◽  
Dual Mechanism ◽  
Dependent Processes

ABSTRACT At mitotic exit the cell cycle engine is reset to allow crucial processes, such as cytokinesis and replication origin licensing, to take place before a new cell cycle begins. In budding yeast, the cell cycle clock is reset by a Hippo-like kinase cascade called the mitotic exit network (MEN), whose activation is triggered at spindle pole bodies (SPBs) by the Tem1 GTPase. Yet, MEN activity must be extinguished once MEN-dependent processes have been accomplished. One factor contributing to switching off the MEN is the Amn1 protein, which binds Tem1 and inhibits it through an unknown mechanism. Here, we show that Amn1 downregulates Tem1 through a dual mode of action. On one side, it evicts Tem1 from SPBs and escorts it into the nucleus. On the other, it promotes Tem1 degradation as part of a Skp, Cullin and F-box-containing (SCF) ubiquitin ligase. Tem1 inhibition by Amn1 takes place after cytokinesis in the bud-derived daughter cell, consistent with its asymmetric appearance in the daughter cell versus the mother cell. This dual mechanism of Tem1 inhibition by Amn1 may contribute to the rapid extinguishing of MEN activity once it has fulfilled its functions.

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