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 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 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 The cortical protein Lte1 promotes mitotic exit by inhibiting the spindle position checkpoint kinase Kin4

2011 ◽  
Vol 193 (6) ◽  
pp. 1033-1048 ◽  
Author(s):  
Daniela Trinca Bertazzi ◽  
Bahtiyar Kurtulmus ◽  
Gislene Pereira
Keyword(s):  
Kinase Activity ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Mitotic Exit ◽  
Cell Protein ◽  
Cell Axis ◽  
Pole Body ◽  
Catalytically Active ◽  
Surveillance Mechanism ◽  
Spindle Position

The spindle position checkpoint (SPOC) is an essential surveillance mechanism that allows mitotic exit only when the spindle is correctly oriented along the cell axis. Key SPOC components are the kinase Kin4 and the Bub2–Bfa1 GAP complex that inhibit the mitotic exit–promoting GTPase Tem1. During an unperturbed cell cycle, Kin4 associates with the mother spindle pole body (mSPB), whereas Bub2–Bfa1 is at the daughter SPB (dSPB). When the spindle is mispositioned, Bub2–Bfa1 and Kin4 bind to both SPBs, which enables Kin4 to phosphorylate Bfa1 and thereby block mitotic exit. Here, we show that the daughter cell protein Lte1 physically interacts with Kin4 and inhibits Kin4 kinase activity. Specifically, Lte1 binds to catalytically active Kin4 and promotes Kin4 hyperphosphorylation, which restricts Kin4 binding to the mSPB. This Lte1-mediated exclusion of Kin4 from the dSPB is essential for proper mitotic exit of cells with a correctly aligned spindle. Therefore, Lte1 promotes mitotic exit by inhibiting Kin4 activity at the dSPB.

scholarly journals The yeast centrosome translates the positional information of the anaphase spindle into a cell cycle signal

2007 ◽  
Vol 179 (3) ◽  
pp. 423-436 ◽  
Author(s):  
Hiromi Maekawa ◽  
Claire Priest ◽  
Johannes Lechner ◽  
Gislene Pereira ◽  
Elmar Schiebel
Keyword(s):  
Spindle Pole Body ◽  
Coiled Coil ◽  
Positional Information ◽  
Spindle Pole ◽  
Mitotic Exit ◽  
Spindle Orientation ◽  
Additional Function ◽  
A Cell ◽  
Cytoplasmic Microtubules ◽  
Guanosine Triphosphatase

The spindle orientation checkpoint (SPOC) of budding yeast delays mitotic exit when cytoplasmic microtubules (MTs) are defective, causing the spindle to become misaligned. Delay is achieved by maintaining the activity of the Bfa1–Bub2 guanosine triphosphatase–activating protein complex, an inhibitor of mitotic exit. In this study, we show that the spindle pole body (SPB) component Spc72, a transforming acidic coiled coil–like molecule that interacts with the γ-tubulin complex, recruits Kin4 kinase to both SPBs when cytoplasmic MTs are defective. This allows Kin4 to phosphorylate the SPB-associated Bfa1, rendering it resistant to inactivation by Cdc5 polo kinase. Consistently, forced targeting of Kin4 to both SPBs delays mitotic exit even when the anaphase spindle is correctly aligned. Moreover, we present evidence that Spc72 has an additional function in SPOC regulation that is independent of the recruitment of Kin4. Thus, Spc72 provides a missing link between cytoplasmic MT function and components of the SPOC.

scholarly journals Control of Mitotic Spindle Position by the Saccharomyces cerevisiae Formin Bni1p

1999 ◽  
Vol 144 (5) ◽  
pp. 947-961 ◽  
Author(s):  
Laifong Lee ◽  
Saskia K. Klee ◽  
Marie Evangelista ◽  
Charles Boone ◽  
David Pellman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Spindle ◽  
Cortical Microtubule ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Spindle Orientation ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Bud Site ◽  
Spindle Position

Alignment of the mitotic spindle with the axis of cell division is an essential process in Saccharomyces cerevisiae that is mediated by interactions between cytoplasmic microtubules and the cell cortex. We found that a cortical protein, the yeast formin Bni1p, was required for spindle orientation. Two striking abnormalities were observed in bni1Δ cells. First, the initial movement of the spindle pole body (SPB) toward the emerging bud was defective. This phenotype is similar to that previously observed in cells lacking the kinesin Kip3p and, in fact, BNI1 and KIP3 were found to be in the same genetic pathway. Second, abnormal pulling interactions between microtubules and the cortex appeared to cause preanaphase spindles in bni1Δ cells to transit back and forth between the mother and the bud. We therefore propose that Bni1p may localize or alter the function of cortical microtubule-binding sites in the bud. Additionally, we present evidence that other bipolar bud site determinants together with cortical actin are also required for spindle orientation.

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 Saccharomyces cerevisiae Mob1p Is Required for Cytokinesis and Mitotic Exit

2001 ◽  
Vol 21 (20) ◽  
pp. 6972-6983 ◽  
Author(s):  
Francis C. Luca ◽  
Manali Mody ◽  
Cornelia Kurischko ◽  
David M. Roof ◽  
Thomas H. Giddings ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Mitotic Exit ◽  
Live Cells ◽  
Ring Assembly ◽  
Spindle Poles ◽  
Spindle Pole Bodies ◽  
Bud Neck ◽  
Mitotic Cyclin

ABSTRACT The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved set of genes that mediate the transition from mitosis to G1 by regulating mitotic cyclin degradation and the inactivation of cyclin-dependent kinase (CDK). Here, we demonstrate that, in addition to mitotic exit, S. cerevisiae MEN gene MOB1 is required for cytokinesis and cell separation. The cytokinesis defect was evident in mob1mutants under conditions in which there was no mitotic-exit defect. Observation of live cells showed that yeast myosin II, Myo1p, was present in the contractile ring at the bud neck but that the ring failed to contract and disassemble. The cytokinesis defect persisted for several mitotic cycles, resulting in chains of cells with correctly segregated nuclei but with uncontracted actomyosin rings. The cytokinesis proteins Cdc3p (a septin), actin, and Iqg1p/ Cyk1p (an IQGAP-like protein) appeared to correctly localize inmob1 mutants, suggesting that MOB1functions subsequent to actomyosin ring assembly. We also examined the subcellular distribution of Mob1p during the cell cycle and found that Mob1p first localized to the spindle pole bodies during mid-anaphase and then localized to a ring at the bud neck just before and during cytokinesis. Localization of Mob1p to the bud neck requiredCDC3, MEN genes CDC5,CDC14, CDC15, and DBF2, and spindle pole body gene NUD1 but was independent ofMYO1. The localization of Mob1p to both spindle poles was abolished in cdc15 and nud1 mutants and was perturbed in cdc5 and cdc14mutants. These results suggest that the MEN functions during the mitosis-to-G1 transition to control cyclin-CDK inactivation and cytokinesis.

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 Dynamic Localization of the Swe1 Regulator Hsl7 During theSaccharomyces cerevisiae Cell Cycle

2001 ◽  
Vol 12 (6) ◽  
pp. 1645-1669 ◽  
Author(s):  
Victor J. Cid ◽  
Mark J. Shulewitz ◽  
Kent L. McDonald ◽  
Jeremy Thorner
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Fine Tuning ◽  
Dependent Manner ◽  
Binding Assays ◽  
Bud Neck

In Saccharomyces cerevisiae, entry into mitosis requires activation of the cyclin-dependent kinase Cdc28 in its cyclin B (Clb)-associated form. Clb-bound Cdc28 is susceptible to inhibitory tyrosine phosphorylation by Swe1 protein kinase. Swe1 is itself negatively regulated by Hsl1, a Nim1-related protein kinase, and by Hsl7, a presumptive protein-arginine methyltransferase. In vivo all three proteins localize to the bud neck in a septin-dependent manner, consistent with our previous proposal that formation of Hsl1-Hsl7-Swe1 complexes constitutes a checkpoint that monitors septin assembly. We show here that Hsl7 is phosphorylated by Hsl1 in immune-complex kinase assays and can physically associate in vitro with either Hsl1 or Swe1 in the absence of any other yeast proteins. With the use of both the two-hybrid method and in vitro binding assays, we found that Hsl7 contains distinct binding sites for Hsl1 and Swe1. A differential interaction trap approach was used to isolate four single-site substitution mutations in Hsl7, which cluster within a discrete region of its N-terminal domain, that are specifically defective in binding Hsl1. When expressed in hsl7Δ cells, each of these Hsl7 point mutants is unable to localize at the bud neck and cannot mediate down-regulation of Swe1, but retains other functions of Hsl7, including oligomerization and association with Swe1. GFP-fusions of these Hsl1-binding defective Hsl7 proteins localize as a bright perinuclear dot, but never localize to the bud neck; likewise, inhsl1Δ cells, a GFP-fusion to wild-type Hsl7 or native Hsl7 localizes to this dot. Cell synchronization studies showed that, normally, Hsl7 localizes to the dot, but only in cells in the G1 phase of the cell cycle. Immunofluorescence analysis and immunoelectron microscopy established that the dot corresponds to the outer plaque of the spindle pole body (SPB). These data demonstrate that association between Hsl1 and Hsl7 at the bud neck is required to alleviate Swe1-imposed G2-M delay. Hsl7 localization at the SPB during G1 may play some additional role in fine-tuning the coordination between nuclear and cortical events before mitosis.

scholarly journals A FRET-based study reveals site-specific regulation of spindle position checkpoint proteins at yeast centrosomes

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Yuliya Gryaznova ◽  
Ayse Koca Caydasi ◽  
Gabriele Malengo ◽  
Victor Sourjik ◽  
Gislene Pereira
Keyword(s):  
Spindle Pole Body ◽  
Spindle Pole ◽  
Mitotic Exit ◽  
Site Specific ◽  
Checkpoint Proteins ◽  
Pole Body ◽  
Family Protein ◽  
Specific Regulation ◽  
Surveillance Mechanism ◽  
Spindle Position

The spindle position checkpoint (SPOC) is a spindle pole body (SPB, equivalent of mammalian centrosome) associated surveillance mechanism that halts mitotic exit upon spindle mis-orientation. Here, we monitored the interaction between SPB proteins and the SPOC component Bfa1 by FRET microscopy. We show that Bfa1 binds to the scaffold-protein Nud1 and the γ-tubulin receptor Spc72. Spindle misalignment specifically disrupts Bfa1-Spc72 interaction by a mechanism that requires the 14-3-3-family protein Bmh1 and the MARK/PAR-kinase Kin4. Dissociation of Bfa1 from Spc72 prevents the inhibitory phosphorylation of Bfa1 by the polo-like kinase Cdc5. We propose Spc72 as a regulatory hub that coordinates the activity of Kin4 and Cdc5 towards Bfa1. In addition, analysis of spc72∆ cells shows that a mitotic-exit-promoting dominant signal, which is triggered upon elongation of the spindle into the bud, overrides the SPOC. Our data reinforce the importance of daughter-cell-associated factors and centrosome-based regulations in mitotic exit and SPOC control.

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