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 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 SIN-like pathway kinases regulate the end of mitosis in the methylotrophic yeast Ogataea polymorpha

2021 ◽  
Author(s):  
Shen Jiangyan ◽  
Kaoru Takegawa ◽  
Gislene Pereira ◽  
Hiromi Maekawa
Keyword(s):  
Budding Yeast ◽  
Methylotrophic Yeast ◽  
Growth Conditions ◽  
Mitotic Exit ◽  
Signalling Pathway ◽  
Significant Delay ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mitotic Exit Network ◽  
Septation Initiation Network ◽  
Ogataea Polymorpha

The Mitotic exit network (MEN) is a conserved signalling pathway essential for termination of mitosis in the budding yeast Saccharomyces cerevisiae. All MEN components are highly conserved in the methylotrophic budding yeast Ogataea polymorpha, except for Cdc15 kinase. Amongst O. polymorpha protein kinases that have some similarity to ScCdc15, only two had no other obvious homologues in S. cerevisiae and these were named OpHCD1 and OpHCD2 for homologue candidate of ScCdc15. A search in other yeast species revealed that OpHcd2 has an armadillo type fold in the C-terminal region as found in SpCdc7 kinases of the fission yeast Schizosaccharomyces pombe, which are homologues of ScCdc15; while OpHcd1 is homologous to SpSid1 kinase, a component of the Septation Initiation Network (SIN) of S. pombe not present in the MEN. Since the deletion of either OpHCD1 or OpHCD2 resulted in lethality under standard growth conditions, conditional mutants were constructed by introducing an ATP analog sensitive mutation. For OpHCD2, we constructed and used new genetic tools for O. polymorpha that combined the Tet promoter and the improved auxin-degron systems. Conditional mutants for OpHCD1 and OpHCD2 exhibited significant delay in late anaphase and defective cell separation, suggesting that both genes have roles in mitotic exit and cytokinesis. These results suggest a SIN-like signalling pathway regulates termination of mitosis in O. polymorpha and that the loss of Sid1/Hcd1 kinase in the MEN occurred relatively recently during the evolution of budding yeast.

scholarly journals A non-canonical Hippo pathway regulates spindle disassembly and cytokinesis during meiosis in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Scott M. Paulissen ◽  
Cindy A. Hunt ◽  
Christian J. Slubowski ◽  
Yao Yu ◽  
Dang Truong ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Hippo Pathway ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Mitotic Exit ◽  
Prospore Membrane ◽  
Mitotic Exit Network ◽  
Similar Phenotype ◽  
Spindle Disassembly ◽  
Ndr Kinase

ABSTRACTMeiosis in the budding yeast Saccharomyces cerevisiae is used to create haploid yeast spores from a diploid mother cell. During meiosis II, cytokinesis occurs by closure of the prospore membrane, a membrane that initiates at the spindle pole body and grows to surround each of the haploid meiotic products. Timely prospore membrane closure requires SPS1, which encodes a STE20-family GCKIII kinase. To identify genes that may activate SPS1, we utilized a histone phosphorylation defect of sps1 mutants to screen for genes with a similar phenotype and found that cdc15 shared this phenotype. CDC15 encodes a Hippo-like kinase that is part of the mitotic exit network. We find that Sps1 complexes with Cdc15, that Sps1 phosphorylation requires Cdc15, and that CDC15 is also required for timely prospore membrane closure. We also find that SPS1, like CDC15, is required for meiosis II spindle disassembly and sustained anaphase II release of Cdc14 in meiosis. However, the NDR-kinase complex encoded by DBF2/DBF20 MOB1 which functions downstream of CDC15 in mitotic cells, does not appear to play a role in spindle disassembly, timely prospore membrane closure, or sustained anaphase II Cdc14 release. Taken together, our results suggest that the mitotic exit network is rewired for exit from meiosis II, such that SPS1 replaces the NDR-kinase complex downstream of CDC15.

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 A lysine deacetylase Hos3 is targeted to the bud neck and involved in the spindle position checkpoint

2014 ◽  
Vol 25 (18) ◽  
pp. 2720-2734 ◽  
Author(s):  
Mengqiao Wang ◽  
Ruth N. Collins
Keyword(s):  
Spindle Pole Body ◽  
Neck Region ◽  
Spindle Pole ◽  
Mitotic Exit ◽  
Spindle Orientation ◽  
Lysine Deacetylase ◽  
Bud Neck ◽  
Morphogenesis Checkpoint ◽  
Spindle Position

An increasing number of cellular activities can be regulated by reversible lysine acetylation. Targeting the enzymes responsible for such posttranslational modifications is instrumental in defining their substrates and functions in vivo. Here we show that a Saccharomyces cerevisiae lysine deacetylase, Hos3, is asymmetrically targeted to the daughter side of the bud neck and to the daughter spindle pole body (SPB). The morphogenesis checkpoint member Hsl7 recruits Hos3 to the neck region. Cells with a defect in spindle orientation trigger Hos3 to load onto both SPBs. When associated symmetrically with both SPBs, Hos3 functions as a spindle position checkpoint (SPOC) component to inhibit mitotic exit. Neck localization of Hos3 is essential for its symmetric association with SPBs in cells with misaligned spindles. Our data suggest that Hos3 facilitates cross-talk between the morphogenesis checkpoint and the SPOC as a component of the intricate monitoring of spindle orientation after mitotic entry and before commitment to mitotic exit.

scholarly journals A Noncanonical Hippo Pathway Regulates Spindle Disassembly and Cytokinesis During Meiosis in Saccharomyces cerevisiae

Genetics ◽  
2020 ◽  
Vol 216 (2) ◽  
pp. 447-462
Author(s):  
Scott M. Paulissen ◽  
Cindy A. Hunt ◽  
Brian C. Seitz ◽  
Christian J. Slubowski ◽  
Yao Yu ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Hippo Pathway ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Mitotic Exit ◽  
Link Type ◽  
Prospore Membrane ◽  
Mitotic Exit Network ◽  
Spindle Disassembly ◽  
Ndr Kinase

Meiosis in the budding yeast Saccharomyces cerevisiae is used to create haploid yeast spores from a diploid mother cell. During meiosis II, cytokinesis occurs by closure of the prospore membrane, a membrane that initiates at the spindle pole body and grows to surround each of the haploid meiotic products. Timely prospore membrane closure requires SPS1, which encodes an STE20 family GCKIII kinase. To identify genes that may activate SPS1, we utilized a histone phosphorylation defect of sps1 mutants to screen for genes with a similar phenotype and found that cdc15 shared this phenotype. CDC15 encodes a Hippo-like kinase that is part of the mitotic exit network. We find that Sps1 complexes with Cdc15, that Sps1 phosphorylation requires Cdc15, and that CDC15 is also required for timely prospore membrane closure. We also find that SPS1, like CDC15, is required for meiosis II spindle disassembly and sustained anaphase II release of Cdc14 in meiosis. However, the NDR-kinase complex encoded by DBF2/DBF20MOB1 which functions downstream of CDC15 in mitotic cells, does not appear to play a role in spindle disassembly, timely prospore membrane closure, or sustained anaphase II Cdc14 release. Taken together, our results suggest that the mitotic exit network is rewired for exit from meiosis II, such that SPS1 replaces the NDR-kinase complex downstream of CDC15.

scholarly journals Novel Functional Dissection of the Localization-Specific Roles of Budding Yeast Polo Kinase Cdc5p

2004 ◽  
Vol 24 (22) ◽  
pp. 9873-9886 ◽  
Author(s):  
Jung-Eun Park ◽  
Chong J. Park ◽  
Krisada Sakchaisri ◽  
Tatiana Karpova ◽  
Satoshi Asano ◽  
...  
Keyword(s):  
Budding Yeast ◽  
Spindle Pole Body ◽  
Kinase Domain ◽  
Spindle Pole ◽  
Mitotic Exit ◽  
Negative Regulator ◽  
Triple Mutant ◽  
Polo Kinase ◽  
Bud Neck ◽  
M Phase

ABSTRACT Budding yeast polo kinase Cdc5p localizes to the spindle pole body (SPB) and to the bud-neck and plays multiple roles during M-phase progression. To dissect localization-specific mitotic functions of Cdc5p, we tethered a localization-defective N-terminal kinase domain of Cdc5p (Cdc5pΔC) to the SPB or to the bud-neck with components specifically localizing to one of these sites and characterized these mutants in a cdc5Δ background. Characterization of a viable, SPB-localizing, CDC5ΔC-CNM67 mutant revealed that it is defective in timely degradation of Swe1p, a negative regulator of Cdc28p. Loss of BFA1, a negative regulator of mitotic exit, rescued the lethality of a neck-localizing CDC5ΔC-CDC12 or CDC5ΔC-CDC3 mutant but yielded severe defects in cytokinesis. These data suggest that the SPB-associated Cdc5p activity is critical for both mitotic exit and cytokinesis, whereas the bud neck-localized Cdc5p is required for proper Swe1p regulation. Interestingly, a cdc5Δ bfa1Δ swe1Δ triple mutant is viable but grows slowly, whereas cdc5Δ cells bearing both CDC5ΔC-CNM67 and CDC5ΔC-CDC12 grow well with only a mild cell cycle delay. Thus, SPB- and the bud-neck-localized Cdc5p control most of the critical Cdc5p functions and downregulation of Bfa1p and Swe1p at the respective locations are two critical factors that require Cdc5p.

scholarly journals The microtubule polymerase Stu2 promotes oligomerization of the γ-TuSC for cytoplasmic microtubule nucleation

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Judith Gunzelmann ◽  
Diana Rüthnick ◽  
Tien-chen Lin ◽  
Wanlu Zhang ◽  
Annett Neuner ◽  
...  
Keyword(s):  
Budding Yeast ◽  
Spindle Pole Body ◽  
Family Members ◽  
Spindle Pole ◽  
Microtubule Nucleation ◽  
Cytoplasmic Microtubule ◽  
Pole Body ◽  
Cytoplasmic Microtubules

Stu2/XMAP215/ZYG-9/Dis1/Alp14/Msps/ch-TOG family members in association with with γ-tubulin complexes nucleate microtubules, but we know little about the interplay of these nucleation factors. Here, we show that the budding yeast Stu2 in complex with the γ-tubulin receptor Spc72 nucleates microtubules in vitro without the small γ-tubulin complex (γ-TuSC). Upon γ-TuSC addition, Stu2 facilitates Spc72–γ-TuSC interaction by binding to Spc72 and γ-TuSC. Stu2 together with Spc72–γ-TuSC increases microtubule nucleation in a process that is dependent on the TOG domains of Stu2. Importantly, these activities are also important for microtubule nucleation in vivo. Stu2 stabilizes Spc72–γ-TuSC at the minus end of cytoplasmic microtubules (cMTs) and an in vivo assay indicates that cMT nucleation requires the TOG domains of Stu2. Upon γ-tubulin depletion, we observed efficient cMT nucleation away from the spindle pole body (SPB), which was dependent on Stu2. Thus, γ-TuSC restricts cMT assembly to the SPB whereas Stu2 nucleates cMTs together with γ-TuSC and stabilizes γ-TuSC at the cMT minus end.

scholarly journals Inactivation of Mitotic Kinase Triggers Translocation of MEN Components to Mother-Daughter Neck in Yeast

2003 ◽  
Vol 14 (11) ◽  
pp. 4734-4743 ◽  
Author(s):  
Hong Hwa Lim ◽  
Foong May Yeong ◽  
Uttam Surana
Keyword(s):  
Chromosome Segregation ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Mitotic Exit ◽  
Mitotic Kinase ◽  
Division Site ◽  
Pole Body ◽  
Spindle Pole Bodies ◽  
Mitotic Exit Network ◽  
Mutant Cells

Chromosome segregation, mitotic exit, and cytokinesis are executed in this order during mitosis. Although a scheme coordinating sister chromatid separation and initiation of mitotic exit has been proposed, the mechanism that temporally links the onset of cytokinesis to mitotic exit is not known. Exit from mitosis is regulated by the mitotic exit network (MEN), which includes a GTPase (Tem1) and various kinases (Cdc15, Cdc5, Dbf2, and Dbf20). Here, we show that Dbf2 and Dbf20 functions are necessary for the execution of cytokinesis. Relocalization of these proteins from spindle pole bodies to mother daughter neck seems to be necessary for this role because cdc15-2 mutant cells, though capable of exiting mitosis at semipermissive temperature, are unable to localize Dbf2 (and Dbf20) to the “neck” and fail to undergo cytokinesis. These cells can assemble and constrict the actomyosin ring normally but are incapable of forming a septum, suggesting that MEN components are critical for the initiation of septum formation. Interestingly, the spindle pole body to neck translocation of Dbf2 and Dbf20 is triggered by the inactivation of mitotic kinase. The requirement of kinase inactivation for translocation of MEN components to the division site thus provides a mechanism that renders mitotic exit a prerequisite for cytokinesis.

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