scholarly journals The budding yeast Dbf2 protein kinase localises to the centrosome and moves to the bud neck in late mitosis

2000 ◽  
Vol 113 (19) ◽  
pp. 3399-3408 ◽  
Author(s):  
L.M. Frenz ◽  
S.E. Lee ◽  
D. Fesquet ◽  
L.H. Johnston
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Preliminary Investigation ◽  
Spindle Pole ◽  
Mitotic Exit ◽  
Temperature Sensitive ◽  
Multifunctional Protein ◽  
Double Ring ◽  
Bud Neck ◽  
Late Mitosis

Dbf2 is a multifunctional protein kinase in Saccharomyces cerevisiae that functions in transcription, the stress response and as part of a network of genes in exit from mitosis. By analogy with fission yeast it seemed likely that these mitotic exit genes would be involved in cytokinesis. As a preliminary investigation of this we have used Dbf2 tagged with GFP to examine intracellular localisation of the protein in living cells. Dbf2 is found on the centrosomes/spindle pole bodies (SPBs) and also at the bud neck where it forms a double ring. The localisation of Dbf2 is cell cycle regulated. It is on the SPBs for much of the cell cycle and migrates from there to the bud neck in late mitosis, consistent with a role in cytokinesis. Dbf2 partly co-localises with septins at the bud neck. A temperature-sensitive mutant of dbf2 also blocks progression of cytokinesis at 37 degrees C. Following cytokinesis some Dbf2 moves into the nascent bud. Localisation to the bud neck depends upon the septins and also the mitotic exit network proteins Mob1, Cdc5, Cdc14 and Cdc15. The above data are consistent with Dbf2 acting downstream in a pathway controlling cytokinesis.

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 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 Genetic and Biochemical Evaluation of the Importance of Cdc6 in Regulating Mitotic Exit

2003 ◽  
Vol 14 (11) ◽  
pp. 4592-4604 ◽  
Author(s):  
Vincent Archambault ◽  
Caihong X. Li ◽  
Alan J. Tackett ◽  
Ralph Wäsch ◽  
Brian T. Chait ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mitotic Exit ◽  
Terminal Region ◽  
Cyclin Dependent Kinase ◽  
Replication Control ◽  
Mitotic Cyclin ◽  
Biochemical Evaluation ◽  
Inhibitory Domain ◽  
Late Mitosis ◽  
Control Protein

We evaluated the hypothesis that the N-terminal region of the replication control protein Cdc6 acts as an inhibitor of cyclin-dependent kinase (Cdk) activity, promoting mitotic exit. Cdc6 accumulation is restricted to the period from mid-cell cycle until the succeeding G1, due to proteolytic control that requires the Cdc6 N-terminal region. During late mitosis, Cdc6 is present at levels comparable with Sic1 and binds specifically to the mitotic cyclin Clb2. Moderate overexpression of Cdc6 promotes viability of CLB2Δdb strains, which otherwise arrest at mitotic exit, and rescue is dependent on the N-terminal putative Cdk-inhibitory domain. These observations support the potential for Cdc6 to inhibit Clb2-Cdk, thus promoting mitotic exit. Consistent with this idea, we observed a cytokinesis defect in cdh1Δ sic1Δ cdc6Δ2–49 triple mutants. However, we were able to construct viable strains, in three different backgrounds, containing neither SIC1 nor the Cdc6 Cdk-inhibitory domain, in contradiction to previous work. We conclude, therefore, that although both Cdc6 and Sic1 have the potential to facilitate mitotic exit by inhibiting Clb2-Cdk, mitotic exit nevertheless does not require any identified stoichiometric inhibitor of Cdk activity.

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.

Yeast myosin light chain, Mlc1p, interacts with both IQGAP and class II myosin to effect cytokinesis

2000 ◽  
Vol 113 (24) ◽  
pp. 4533-4543 ◽  
Author(s):  
J.R. Boyne ◽  
H.M. Yosuf ◽  
P. Bieganowski ◽  
C. Brenner ◽  
C. Price
Keyword(s):  
Cell Cycle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Direct Interaction ◽  
Class Ii ◽  
Dominant Negative ◽  
Temperature Sensitive ◽  
Gene Encoding ◽  
Bud Neck ◽  
Bud Site

MLC1 (myosin light chain) acts as a dosage suppressor of a temperature sensitive mutation in the gene encoding the S. cerevisiae IQGAP protein. Both proteins localize to the bud neck in mitosis although Mlc1p localisation precedes Iqg1p. Mlc1p is also found at the incipient bud site in G(1) and the growing bud tip during S and G(2) phases of the cell cycle. A dominant negative GST-Mlc1p fusion protein specifically blocks cytokinesis and prevents Iqg1p localisation to the bud neck, as does depletion of Mlc1p. These data support a direct interaction between the two proteins and immunoprecipitation experiments confirm this prediction. Mlc1p is also shown to interact with the class II conventional myosin (Myo1p). All three proteins form a complex, however, the interaction between Mlc1p and Iqg1p can be separated from the Mlc1p/Myo1p interaction. Mlc1p localisation and maintenance at the bud neck is independent of actin, Myo1p and Iqg1p. It is proposed that Mlc1p therefore functions to recruit Iqg1p and in turn actin to the actomyosin ring and that it is also required for Myo1p function during ring contraction.

Parkinson´s related protein DJ-1 is involved in aberrant cell cycle re-entry through Cdk5

2020 ◽  
Author(s):  
Mª José López-Grueso ◽  
Carmen Alicia Padilla ◽  
José Antonio Bárcena ◽  
Raquel Requejo-Aguilar
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Cortical Neurons ◽  
Cell Cycle Progression ◽  
Cell Cycle Checkpoints ◽  
Mitogen Activated Protein Kinase ◽  
Signalling Pathways ◽  
Multifunctional Protein ◽  
Mitogen Activated Protein ◽  
Phosphoinositide 3 Kinase

Abstract DJ-1 is a multifunctional protein involved in Parkinson disease (PD) that can act as antioxidant, molecular chaperone, protease, glyoxalase and transcriptional regulator. However, the exact mechanism by which DJ-1 dysfunction contributes to development of Parkinson´s disease remains elusive. Here, using a comparative proteomic analysis between normal cortical neurons and neurons lacking DJ-1, we show that this protein is involved in cell cycle checkpoints disruption as a consequence of increased amount of p-Tau and a-synuclein proteins, altered signalling pathways, as the phosphoinositide-3-kinase/protein kinase B (PI3K/AKT) and mitogen-activated protein kinase (MAPK), and deregulation of cyclin-dependent kinase 5 (Cdk5). Cdk5 is normally involved in dendritic growth, axon formation and the establishment of synapses, but can also contribute to cell cycle progression, as in our case, in pathological conditions. In addition, we observed a decrease in proteasomal activity, probably due to Tau phosphorylation that can also lead to activation of mitogenic signalling pathways. Taken together, our findings indicate, for the first time, that aborted cell cycle re-entry could be at the onset of DJ-1 associated PD. Thereby, new approaches targeting cell cycle re-entry can be envisaged to improve current therapeutic strategies.

scholarly journals Cell cycle regulation of the yeast Cdc7 protein kinase by association with the Dbf4 protein.

1993 ◽  
Vol 13 (5) ◽  
pp. 2899-2908 ◽  
Author(s):  
A L Jackson ◽  
P M Pahl ◽  
K Harrison ◽  
J Rosamond ◽  
R A Sclafani
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Protein Interactions ◽  
Kinase Activity ◽  
Mitotic Cell ◽  
Common Point ◽  
Temperature Sensitive ◽  
Cdc7 Kinase

Yeast Cdc7 protein kinase and Dbf4 protein are both required for the initiation of DNA replication at the G1/S phase boundary of the mitotic cell cycle. Cdc7 kinase function is stage-specific in the cell cycle, but total Cdc7 protein levels remained unchanged. Therefore, regulation of Cdc7 function appears to be the result of posttranslational modification. In this study, we have attempted to elucidate the mechanism responsible for achieving this specific execution point of Cdc7. Cdc7 kinase activity was shown to be maximal at the G1/S boundary by using either cultures synchronized with alpha factor or Cdc- mutants or with inhibitors of DNA synthesis or mitosis. Therefore, Cdc7 kinase is regulated by a posttranslational mechanism that ensures maximal Cdc7 activity at the G1/S boundary, which is consistent with Cdc7 function in the cell cycle. This cell cycle-dependent regulation could be the result of association with the Dbf4 protein. In this study, the Dbf4 protein was shown to be required for Cdc7 kinase activity in that Cdc7 kinase activity is thermolabile in vitro when extracts prepared from a temperature-sensitive dbf4 mutant grown under permissive conditions are used. In vitro reconstitution assays, in addition to employment of the two-hybrid system for protein-protein interactions, have demonstrated that the Cdc7 and Dbf4 proteins interact both in vitro and in vivo. A suppressor mutation, bob1-1, which can bypass deletion mutations in both cdc7 and dbf4 was isolated. However, the bob1-1 mutation cannot bypass all events in G1 phase because it fails to suppress temperature-sensitive cdc4 or cdc28 mutations. This indicates that the Cdc7 and Dbf4 proteins act at a common point in the cell cycle. Therefore, because of the common point of function for the two proteins and the fact that the Dbf4 protein is essential for Cdc7 function, we propose that Dbf4 may represent a cyclin-like molecule specific for the activation of Cdc7 kinase.

scholarly journals MIF2 is required for mitotic spindle integrity during anaphase spindle elongation in Saccharomyces cerevisiae.

1993 ◽  
Vol 123 (2) ◽  
pp. 387-403 ◽  
Author(s):  
M T Brown ◽  
L Goetsch ◽  
L H Hartwell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Structural Integrity ◽  
Spindle Pole ◽  
Chromosome Loss ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
In Vitro Mutagenesis ◽  
Spindle Elongation

The function of the essential MIF2 gene in the Saccharomyces cerevisiae cell cycle was examined by overepressing or creating a deficit of MIF2 gene product. When MIF2 was overexpressed, chromosomes missegregated during mitosis and cells accumulated in the G2 and M phases of the cell cycle. Temperature sensitive mutants isolated by in vitro mutagenesis delayed cell cycle progression when grown at the restrictive temperature, accumulated as large budded cells that had completed DNA replication but not chromosome segregation, and lost viability as they passed through mitosis. Mutant cells also showed increased levels of mitotic chromosome loss, supersensitivity to the microtubule destabilizing drug MBC, and morphologically aberrant spindles. mif2 mutant spindles arrested development immediately before anaphase spindle elongation, and then frequently broke apart into two disconnected short half spindles with misoriented spindle pole bodies. These findings indicate that MIF2 is required for structural integrity of the spindle during anaphase spindle elongation. The deduced Mif2 protein sequence shared no extensive homologies with previously identified proteins but did contain a short region of homology to a motif involved in binding AT rich DNA by the Drosophila D1 and mammalian HMGI chromosomal proteins.

scholarly journals A Temperature-Sensitive dcw1 Mutant of Saccharomyces cerevisiae Is Cell Cycle Arrested with Small Buds Which Have Aberrant Cell Walls

2004 ◽  
Vol 3 (5) ◽  
pp. 1297-1306 ◽  
Author(s):  
Hiroshi Kitagaki ◽  
Kiyoshi Ito ◽  
Hitoshi Shimoi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Wall ◽  
Cell Walls ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Mrna Levels ◽  
Homologous Proteins ◽  
Aberrant Cell ◽  
Cell Wall Biogenesis

ABSTRACT Dcw1p and Dfg5p in Saccharomyces cerevisiae are homologous proteins that were previously shown to be involved in cell wall biogenesis and to be essential for growth. Dcw1p was found to be a glycosylphosphatidylinositol-anchored membrane protein. To investigate the roles of these proteins in cell wall biogenesis and cell growth, we constructed mutant alleles of DCW1 by random mutagenesis, introduced them into a Δdcw1 Δdfg5 background, and isolated a temperature-sensitive mutant, DC61 (dcw1-3 Δdfg5). When DC61 cells were incubated at 37°C, most cells had small buds, with areas less than 20% of those of the mother cells. This result indicates that DC61 cells arrest growth with small buds at 37°C. At 37°C, fewer DC61 cells had 1N DNA content and most of them still had a single nucleus located apart from the bud neck. In addition, in DC61 cells incubated at 37°C, bipolar spindles were not formed. These results indicate that DC61 cells, when incubated at 37°C, are cell cycle arrested after DNA replication and prior to the separation of spindle pole bodies. The small buds of DC61 accumulated chitin in the bud cortex, and some of them were lysed, which indicates that they had aberrant cell walls. A temperature-sensitive dfg5 mutant, DF66 (Δdcw1 dfg5-29), showed similar phenotypes. DCW1 and DFG5 mRNA levels peaked in the G1 and S phases, respectively. These results indicate that Dcw1p and Dfg5p are involved in bud formation through their involvement in biogenesis of the bud cell wall.

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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