Kinetochore localisation and phosphorylation of the mitotic checkpoint components Bub1 and BubR1 are differentially regulated by spindle events in human cells

2001 ◽  
Vol 114 (24) ◽  
pp. 4385-4395 ◽  
Author(s):  
Stephen S. Taylor ◽  
Deema Hussein ◽  
Yunmei Wang ◽  
Sarah Elderkin ◽  
Christopher J. Morrow
Keyword(s):  
Cell Cycle ◽  
Protein Kinases ◽  
Budding Yeast ◽  
Spindle Assembly ◽  
Mitotic Checkpoint ◽  
Spindle Pole ◽  
Cell Cycle Regulators ◽  
Yeast Gene ◽  
Microtubule Attachment ◽  
Spindle Dynamics

BUB1 is a budding yeast gene required to ensure that progression through mitosis is coupled to correct spindle assembly. Two related human protein kinases, Bub1 and BubR1, both localise to kinetochores during mitosis, suggesting that they play a role in delaying anaphase until all chromosomes achieve correct, bipolar attachment to the spindle. However, how the activities of Bub1 and BubR1 are regulated by spindle events and how their activities regulate downstream cell cycle events is not known.To investigate how spindle events regulate Bub1 and BubR1, we characterised their relative localisations during mitosis in the presence and absence of microtubule toxins. In prometaphase cells, both kinases colocalise to the same domain of the kinetochore. However, whereas the localisation of BubR1 at sister kinetochores is symmetrical, localisation of Bub1 is often asymmetrical. This asymmetry is dependent on microtubule attachment, and the kinetochore exhibiting weaker Bub1 staining is typically closer to the nearest spindle pole. In addition, a 30 minute nocodazole treatment dramatically increases the amount of Bub1 localising to kinetochores but has little effect on BubR1. Furthermore, Bub1 levels increase at metaphase kinetochores following loss of tension caused by taxol treatment. Thus, these observations suggest that Bub1 localisation is sensitive to changes in both tension and microtubule attachment.Consistent with this, we also show that Bub1 is rapidly phosphorylated following brief treatments with nocodazole or taxol. In contrast, BubR1 is phosphorylated in the absence of microtubule toxins, and spindle damage has little additional effect. Although these observations indicate that Bub1 and BubR1 respond differently to spindle dynamics, they are part of a common complex during mitosis. We suggest therefore that Bub1 and BubR1 may integrate different ‘spindle assembly signals’ into a single signal which can then be interpreted by downstream cell cycle regulators.

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.

The novel human protein serine/threonine phosphatase 6 is a functional homologue of budding yeast Sit4p and fission yeast ppe1, which are involved in cell cycle regulation

1996 ◽  
Vol 109 (12) ◽  
pp. 2865-2874 ◽  
Author(s):  
H. Bastians ◽  
H. Ponstingl
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Shape Control ◽  
Cell Cycle Regulation ◽  
Budding Yeast ◽  
Mitotic Checkpoint ◽  
Human Protein ◽  
Predicted Amino Acid Sequence ◽  
Temperature Sensitive ◽  
Cycle Regulation

We identified a novel human protein serine/threonine phosphatase cDNA, designated protein phosphatase 6 (PP6) by using a homology-based polymerase chain reaction. The predicted amino acid sequence indicates a 35 kDa protein showing high homology to other protein phosphatases including human PP2A (57%), human PP4 (59%), rat PPV (98%), Drosophila PPV (74%), Schizosaccharomyces pombe ppe1 (68%) and Saccharomyces cerevisiae Sit4p (61%). In human cells, three forms of PP6 mRNA were found with highest levels of expression in testis, heart and skeletal muscle. The PP6 protein was detected in lysates of human heart muscle and in bull testis. Complementation studies using a temperature sensitive mutant strain of S. cerevisiae SIT4, which is required for the G1 to S transition of the cell cycle, showed that PP6 can rescue the mutant growth arrest. In addition, a loss of function mutant of S. pombe ppe1, described as a gene interacting with the pim1/spi1 mitotic checkpoint and involved in cell shape control, can be complemented by expression of human PP6. These data indicate that human PP6 is a functional homologue of budding yeast Sit4p and fission yeast ppe1, implying a function of PP6 in cell cycle regulation.

scholarly journals Spc98p Directs the Yeast γ-Tubulin Complex into the Nucleus and Is Subject to Cell Cycle-dependent Phosphorylation on the Nuclear Side of the Spindle Pole Body

1998 ◽  
Vol 9 (4) ◽  
pp. 775-793 ◽  
Author(s):  
Gislene Pereira ◽  
Michael Knop ◽  
Elmar Schiebel
Keyword(s):  
Cell Cycle ◽  
Spindle Pole Body ◽  
Mitotic Checkpoint ◽  
Spindle Pole ◽  
Nuclear Localization Sequence ◽  
Checkpoint Control ◽  
Yeast Saccharomyces Cerevisiae ◽  
Pole Body ◽  
Cycle Dependent ◽  
Cytoplasmic Side

In the yeast Saccharomyces cerevisiae, microtubules are organized by the spindle pole body (SPB), which is embedded in the nuclear envelope. Microtubule organization requires the γ-tubulin complex containing the γ-tubulin Tub4p, Spc98p, and Spc97p. The Tub4p complex is associated with cytoplasmic and nuclear substructures of the SPB, which organize the cytoplasmic and nuclear microtubules. Here we present evidence that the Tub4p complex assembles in the cytoplasm and then either binds to the cytoplasmic side of the SPB or is imported into the nucleus followed by binding to the nuclear side of the SPB. Nuclear import of the Tub4p complex is mediated by the essential nuclear localization sequence of Spc98p. Our studies also indicate that Spc98p in the Tub4p complex is phosphorylated at the nuclear, but not at the cytoplasmic, side of the SPB. This phosphorylation is cell cycle dependent and occurs after SPB duplication and nucleation of microtubules by the new SPB and therefore may have a role in mitotic spindle function. In addition, activation of the mitotic checkpoint stimulates Spc98p phosphorylation. The kinase Mps1p, which functions in SPB duplication and mitotic checkpoint control, seems to be involved in Spc98p phosphorylation. Our results also suggest that the nuclear and cytoplasmic Tub4p complexes are regulated differently.

scholarly journals Augmin accumulation on long-lived microtubules drives amplification and kinetochore-directed growth

2019 ◽  
Vol 218 (7) ◽  
pp. 2150-2168 ◽  
Author(s):  
Ana F. David ◽  
Philippe Roudot ◽  
Wesley R. Legant ◽  
Eric Betzig ◽  
Gaudenz Danuser ◽  
...  
Keyword(s):  
Spindle Assembly ◽  
Light Sheet ◽  
Spindle Pole ◽  
Directional Bias ◽  
Fiber Assembly ◽  
Light Sheet Microscopy ◽  
Sister Chromatids ◽  
Spindle Dynamics ◽  
Dividing Cells ◽  
Microtubule Growth

Dividing cells reorganize their microtubule cytoskeleton into a bipolar spindle, which moves one set of sister chromatids to each nascent daughter cell. Early spindle assembly models postulated that spindle pole–derived microtubules search the cytoplasmic space until they randomly encounter a kinetochore to form a stable attachment. More recent work uncovered several additional, centrosome-independent microtubule generation pathways, but the contributions of each pathway to spindle assembly have remained unclear. Here, we combined live microscopy and mathematical modeling to show that most microtubules nucleate at noncentrosomal regions in dividing human cells. Using a live-cell probe that selectively labels aged microtubule lattices, we demonstrate that the distribution of growing microtubule plus ends can be almost entirely explained by Augmin-dependent amplification of long-lived microtubule lattices. By ultrafast 3D lattice light-sheet microscopy, we observed that this mechanism results in a strong directional bias of microtubule growth toward individual kinetochores. Our systematic quantification of spindle dynamics reveals highly coordinated microtubule growth during kinetochore fiber assembly.

scholarly journals Regulation of spindle pole body assembly and cytokinesis by the centrin-binding protein Sfi1 in fission yeast

2014 ◽  
Vol 25 (18) ◽  
pp. 2735-2749 ◽  
Author(s):  
I-Ju Lee ◽  
Ning Wang ◽  
Wen Hu ◽  
Kersey Schott ◽  
Jürg Bähler ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Fission Yeast ◽  
Budding Yeast ◽  
Binding Protein ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Centrosome Duplication ◽  
Pole Body ◽  
The Individual

Centrosomes play critical roles in the cell division cycle and ciliogenesis. Sfi1 is a centrin-binding protein conserved from yeast to humans. Budding yeast Sfi1 is essential for the initiation of spindle pole body (SPB; yeast centrosome) duplication. However, the recruitment and partitioning of Sfi1 to centrosomal structures have never been fully investigated in any organism, and the presumed importance of the conserved tryptophans in the internal repeats of Sfi1 remains untested. Here we report that in fission yeast, instead of doubling abruptly at the initiation of SPB duplication and remaining at a constant level thereafter, Sfi1 is gradually recruited to SPBs throughout the cell cycle. Like an sfi1Δ mutant, a Trp-to-Arg mutant (sfi1-M46) forms monopolar spindles and exhibits mitosis and cytokinesis defects. Sfi1-M46 protein associates preferentially with one of the two daughter SPBs during mitosis, resulting in a failure of new SPB assembly in the SPB receiving insufficient Sfi1. Although all five conserved tryptophans tested are involved in Sfi1 partitioning, the importance of the individual repeats in Sfi1 differs. In summary, our results reveal a link between the conserved tryptophans and Sfi1 partitioning and suggest a revision of the model for SPB assembly.

scholarly journals The Maize Homologue of the Cell Cycle Checkpoint Protein MAD2 Reveals Kinetochore Substructure and Contrasting Mitotic and Meiotic Localization Patterns

1999 ◽  
Vol 145 (3) ◽  
pp. 425-435 ◽  
Author(s):  
Hong-Guo Yu ◽  
Michael G. Muszynski ◽  
R. Kelly Dawe
Keyword(s):  
Cell Cycle ◽  
Spindle Assembly ◽  
Cell Cycle Checkpoint ◽  
Checkpoint Protein ◽  
Microtubule Attachment ◽  
Checkpoint Pathway ◽  
Outer Domain ◽  
Cycle Checkpoint ◽  
Mitosis And Meiosis ◽  
Meiosis I

We have identified a maize homologue of yeast MAD2, an essential component in the spindle checkpoint pathway that ensures metaphase is complete before anaphase begins. Combined immunolocalization of MAD2 and a recently cloned maize CENPC homologue indicates that MAD2 localizes to an outer domain of the prometaphase kinetochore. MAD2 staining was primarily observed on mitotic kinetochores that lacked attached microtubules; i.e., at prometaphase or when the microtubules were depolymerized with oryzalin. In contrast, the loss of MAD2 staining in meiosis was not correlated with initial microtubule attachment but was correlated with a measure of tension: the distance between homologous or sister kinetochores (in meiosis I and II, respectively). Further, the tension-sensitive 3F3/2 phosphoepitope colocalized, and was lost concomitantly, with MAD2 staining at the meiotic kinetochore. The mechanism of spindle assembly (discussed here with respect to maize mitosis and meiosis) is likely to affect the relative contributions of attachment and tension. We support the idea that MAD2 is attachment-sensitive and that tension stabilizes microtubule attachments.

scholarly journals Cdc14 phosphatase directs centrosome re-duplication at the meiosis I to meiosis II transition in budding yeast

2017 ◽  
Vol 2 ◽  
pp. 2 ◽  
Author(s):  
Colette Fox ◽  
Juan Zou ◽  
Juri Rappsilber ◽  
Adele L. Marston
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Chromosome Segregation ◽  
Budding Yeast ◽  
Spindle Pole Body ◽  
Initial Step ◽  
Mitotic Cell ◽  
Spindle Pole ◽  
Fluorescence And Electron Microscopy ◽  
Meiosis I

Background Gametes are generated through a specialized cell division called meiosis, in which ploidy is reduced by half because two consecutive rounds of chromosome segregation, meiosis I and meiosis II, occur without intervening DNA replication. This contrasts with the mitotic cell cycle where DNA replication and chromosome segregation alternate to maintain the same ploidy. At the end of mitosis, CDKs are inactivated. This low CDK state in late mitosis/G1 allows for critical preparatory events for DNA replication and centrosome/spindle pole body (SPB) duplication. However, their execution is inhibited until S phase, where further preparatory events are also prevented. This “licensing” ensures that both the chromosomes and the centrosomes/SPBs replicate exactly once per cell cycle, thereby maintaining constant ploidy. Crucially, between meiosis I and meiosis II, centrosomes/SPBs must be re-licensed, but DNA re-replication must be avoided. In budding yeast, the Cdc14 protein phosphatase triggers CDK down regulation to promote exit from mitosis. Cdc14 also regulates the meiosis I to meiosis II transition, though its mode of action has remained unclear. Methods Fluorescence and electron microscopy was combined with proteomics to probe SPB duplication in cells with inactive or hyperactive Cdc14. Results We demonstrate that Cdc14 ensures two successive nuclear divisions by re-licensing SPBs at the meiosis I to meiosis II transition. We show that Cdc14 is asymmetrically enriched on a single SPB during anaphase I and provide evidence that this enrichment promotes SPB re-duplication. Cells with impaired Cdc14 activity fail to promote extension of the SPB half-bridge, the initial step in morphogenesis of a new SPB. Conversely, cells with hyper-active Cdc14 duplicate SPBs, but fail to induce their separation. Conclusion Our findings implicate reversal of key CDK-dependent phosphorylations in the differential licensing of cyclical events at the meiosis I to meiosis I transition.

scholarly journals Identifying Novel Cell Cycle Regulators in Budding Yeast

2009 ◽  
Vol 23 (S1) ◽  
Author(s):  
Tesia Stephenson ◽  
Paula Fearon ◽  
Orna Cohen‐Fix
Keyword(s):  
Cell Cycle ◽  
Budding Yeast ◽  
Cell Cycle Regulators

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.

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