Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Mad1p, a phosphoprotein component of the spindle assembly checkpoint in budding yeast.

ABSTRACT: The spindle assembly checkpoint prevents cells from initiating anaphase until the spindle has been fully assembled. We previously isolated mitotic arrest deficient (mad) mutants that inactivate this checkpoint and thus increase the sensitivity of cells to benomyl, a drug that interferes with mitotic spindle assembly by depolymerizing microtubules. We have cloned the MAD1 gene and show that when it is disrupted yeast cells have the same phenotype as the previously isolated mad1 mutants: they fail to delay the metaphase to anaphase transition in response to microtubule depolymerization. MAD1 is predicted to encode a 90-kD coiled-coil protein. Anti-Mad1p antibodies give a novel punctate nuclear staining pattern and cell fractionation reveals that the bulk of Mad1p is soluble. Mad1p becomes hyperphosphorylated when wild-type cells are arrested in mitosis by benomyl treatment, or by placing a cold sensitive tubulin mutant at the restrictive temperature. This modification does not occur in G1-arrested cells treated with benomyl or in cells arrested in mitosis by defects in the mitotic cyclin proteolysis machinery, suggesting that Mad1p hyperphosphorylation is a step in the activation of the spindle assembly checkpoint. Analysis of Mad1p phosphorylation in other spindle assembly checkpoint mutants reveals that this response to microtubule-disrupting agents is defective in some (mad2, bub1, and bub3) but not all (mad3, bub2) mutant strains. We discuss the possible functions of Mad1p at this cell cycle checkpoint.

SUBMITTER: Hardwick KG

PROVIDER: S-EPMC2120625 | biostudies-other | 1995 Nov

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Cdc14 inhibition by the spindle assembly checkpoint prevents unscheduled centrosome separation in budding yeast.

Project description:The spindle assembly checkpoint (SAC) is an evolutionarily conserved surveillance mechanism that delays anaphase onset and mitotic exit in response to the lack of kinetochore attachment. The target of the SAC is the E3 ubiquitin ligase anaphase-promoting complex (APC) bound to its Cdc20 activator. The Cdc20/APC complex is in turn required for sister chromatid separation and mitotic exit through ubiquitin-mediated proteolysis of securin, thus relieving inhibition of separase that unties sister chromatids. Separase is also involved in the Cdc-fourteen early anaphase release (FEAR) pathway of nucleolar release and activation of the Cdc14 phosphatase, which regulates several microtubule-linked processes at the metaphase/anaphase transition and also drives mitotic exit. Here, we report that the SAC prevents separation of microtubule-organizing centers (spindle pole bodies [SPBs]) when spindle assembly is defective. Under these circumstances, failure of SAC activation causes unscheduled SPB separation, which requires Cdc20/APC, the FEAR pathway, cytoplasmic dynein, and the actin cytoskeleton. We propose that, besides inhibiting sister chromatid separation, the SAC preserves the accurate transmission of chromosomes also by preventing SPBs to migrate far apart until the conditions to assemble a bipolar spindle are satisfied.

| S-EPMC2682603 | biostudies-literature

Dual mechanisms regulate the recruitment of spindle assembly checkpoint proteins to the budding yeast kinetochore.

Project description:Recruitment of spindle assembly checkpoint (SAC) proteins by an unattached kinetochore leads to SAC activation. This recruitment is licensed by the Mps1 kinase, which phosphorylates the kinetochore protein Spc105 at one or more of its six MELT repeats. Spc105 then recruits the Bub3-Bub1 and Mad1-Mad2 complexes, which produce the inhibitory signal that arrests cell division. The strength of this signal depends, in part, on the number of Bub3-Bub1 and Mad1-Mad2 molecules that Spc105 recruits. Therefore regulation of this recruitment will influence SAC signaling. To understand this regulation, we established the physiological binding curves that describe the binding of Bub3-Bub1 and Mad1-Mad2 to the budding yeast kinetochore. We find that the binding of both follows the mass action law. Mps1 likely phosphorylates all six MELT repeats of Spc105. However, two mechanisms prevent Spc105 from recruiting six Bub3-Bub1 molecules: low Bub1 abundance and hindrance in the binding of more than one Bub3-Bub1 molecule to the same Spc105. Surprisingly, the kinetochore recruits two Mad1-Mad2 heterotetramers for every Bub3-Bub1 molecule. Finally, at least three MELT repeats per Spc105 are needed for accurate chromosome segregation. These data reveal that kinetochore-intrinsic and -extrinsic mechanisms influence the physiological operation of SAC signaling, potentially to maximize chromosome segregation accuracy.

| S-EPMC5221577 | biostudies-literature

The MEN mediates the effects of the spindle assembly checkpoint on Kar9-dependent spindle pole body inheritance in budding yeast.

Project description:Many asymmetrically dividing cells segregate the poles of the mitotic spindle non-randomly between their two daughters. In budding yeast, the protein Kar9 localizes almost exclusively to the astral microtubules emanating from the old spindle pole body (SPB) and promotes its movement toward the bud. Thereby, Kar9 orients the spindle relative to the division axis. Here, we show that beyond perturbing Kar9 distribution, activation of the spindle assembly checkpoint (SAC) randomizes SPB inheritance. Inactivation of the B-type cyclin Clb5 led to a SAC-dependent defect in Kar9 orientation and SPB segregation. Furthermore, unlike the Clb4-dependent pathway, the Clb5- and SAC-dependent pathways functioned genetically upstream of the mitotic exit network (MEN) in SPB specification and Kar9-dependent SPB inheritance. Together, our study indicates that Clb5 functions in spindle assembly and that the SAC controls the specification and inheritance of yeast SPBs through inhibition of the MEN.

| S-EPMC3442921 | biostudies-literature

Protein phosphatase 1 regulates exit from the spindle checkpoint in budding yeast.

Project description:Accurate chromosome segregation depends on sister kinetochores coming under tension when they make bioriented attachments to microtubules from opposite poles. The spindle checkpoint halts the cell cycle in response to defects in generating proper attachments or tension on kinetochores, although the precise signal that triggers the checkpoint is unclear because tension and attachment are coupled. The target of the checkpoint is the Cdc20 protein, which initiates the anaphase-promoting complex (APC)-dependent degradation of the anaphase inhibitor Pds1/securin. Although the molecular details of spindle checkpoint activation are still being elucidated, phosphorylation by at least four kinases is a crucial requirement. However, less is known about the mechanisms that silence the checkpoint after kinetochores biorient. Here, we show that the catalytic subunit of the budding yeast protein phosphatase 1 (PP1) homolog, Glc7, regulates exit from the checkpoint. Glc7 overexpression prevents spindle checkpoint activation in response to both tension and attachment defects. Although glc7 mutant cells are able to efficiently release from a non-checkpoint-mediated metaphase arrest, they are uniquely sensitive to transient spindle checkpoint activation as a result of a failure in spindle checkpoint exit. We therefore propose that PP1 activity silences the checkpoint by reversing key phosphorylation events.

| S-EPMC2731492 | biostudies-literature

B-cyclin/CDKs regulate mitotic spindle assembly by phosphorylating kinesins-5 in budding yeast.

Project description:Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle and entry into mitosis, the full complement of relevant CDK targets has not been identified. It has previously been shown in a variety of model systems that B-type cyclin/CDK complexes, kinesin-5 motors, and the SCF(Cdc4) ubiquitin ligase are required for the separation of spindle poles and assembly of a bipolar spindle. It has been suggested that, in budding yeast, B-type cyclin/CDK (Clb/Cdc28) complexes promote spindle pole separation by inhibiting the degradation of the kinesins-5 Kip1 and Cin8 by the anaphase-promoting complex (APC(Cdh1)). We have determined, however, that the Kip1 and Cin8 proteins are present at wild-type levels in the absence of Clb/Cdc28 kinase activity. Here, we show that Kip1 and Cin8 are in vitro targets of Clb2/Cdc28 and that the mutation of conserved CDK phosphorylation sites on Kip1 inhibits spindle pole separation without affecting the protein's in vivo localization or abundance. Mass spectrometry analysis confirms that two CDK sites in the tail domain of Kip1 are phosphorylated in vivo. In addition, we have determined that Sic1, a Clb/Cdc28-specific inhibitor, is the SCF(Cdc4) target that inhibits spindle pole separation in cells lacking functional Cdc4. Based on these findings, we propose that Clb/Cdc28 drives spindle pole separation by direct phosphorylation of kinesin-5 motors.

| S-EPMC2865516 | biostudies-literature

The fission yeast gamma-tubulin complex is required in G(1) phase and is a component of the spindle assembly checkpoint.

Project description:Microtubule polymerization is initiated from the microtubule organizing centre (MTOC), which contains the gamma-tubulin complex. We have identified fission yeast Alp4 and Alp6, which are homologues of the gamma-tubulin-interacting proteins Sc.Spc97/Hs.Gcp2 and Sc. Spc98/Hs.Gcp3, respectively. The size of the fission yeast gamma-tubulin complex is large (>2000 kDa), comparable to that in metazoans. Both Alp4 and Alp6 localize to the spindle pole body (SPB) and also to the equatorial MTOC. Temperature-sensitive (ts) alp4 and alp6 mutants show two types of microtubular defects. First, monopolar mitotic spindles form. Secondly, abnormally long cytoplasmic microtubules appear that do not stop at the cell tips and are still associated with the SPB. Alp4 function is required in G(1) phase and ts mutants become lethal before S-phase. alp4 and alp6 mutants are hypersensitive to the microtubule- destabilizing drug thiabendazole (TBZ) and show a lethal 'cut' phenotype in its presence. Furthermore, alp4mad2 double mutants show an exaggerated multiple septation phenotype in TBZ. These results indicate that Alp4 and Alp6 may play a crucial role in the spindle pole-mediated checkpoint pathway.

| S-EPMC305819 | biostudies-literature

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

Project description: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.

| S-EPMC8750578 | biostudies-literature

The spindle assembly checkpoint and speciation.

Project description:A mechanism is proposed by which speciation may occur without the need to postulate geographical isolation of the diverging populations. Closely related species that occupy overlapping or adjacent ecological niches often have an almost identical genome but differ by chromosomal rearrangements that result in reproductive isolation. The mitotic spindle assembly checkpoint normally functions to prevent gametes with non-identical karyotypes from forming viable zygotes. Unless gametes from two individuals happen to undergo the same chromosomal rearrangement at the same place and time, a most improbable situation, there has been no satisfactory explanation of how such rearrangements can propagate. Consideration of the dynamics of the spindle assembly checkpoint suggest that chromosomal fission or fusion events may occur that allow formation of viable heterozygotes between the rearranged and parental karyotypes, albeit with decreased fertility. Evolutionary dynamics calculations suggest that if the resulting heterozygous organisms have a selective advantage in an adjoining or overlapping ecological niche from that of the parental strain, despite the reproductive disadvantage of the population carrying the altered karyotype, it may accumulate sufficiently that homozygotes begin to emerge. At this point the reproductive disadvantage of the rearranged karyotype disappears, and a single population has been replaced by two populations that are partially reproductively isolated. This definition of species as populations that differ from other, closely related, species by karyotypic changes is consistent with the classical definition of a species as a population that is capable of interbreeding to produce fertile progeny. Even modest degrees of reproductive impairment of heterozygotes between two related populations may lead to speciation by this mechanism, and geographical isolation is not necessary for the process.

| S-EPMC7224227 | biostudies-literature

The fission yeast dma1 gene is a component of the spindle assembly checkpoint, required to prevent septum formation and premature exit from mitosis if spindle function is compromised.

Project description:Premature initiation of cytokinesis can lead to loss of chromosomes, and 'cutting' of the nucleus. Therefore, the proper spatial and temporal co-ordination of mitosis and cytokinesis is essential for maintaining the integrity of the genome. The fission yeast cdc16 gene is implicated both in the spindle assembly checkpoint and control of septum formation. To identify other proteins involved in these controls, we have isolated multicopy suppressors of the cdc16-116 mutation, and the characterization of one of these, dma1 (defective in mitotic arrest), is presented here. dma1 is not an essential gene, but in a dma1 null background (dma1-D1) the function of the spindle assembly checkpoint is compromised. If assembly of the spindle is prevented, dma1-D1 cells do not arrest, the activity of cdc2 kinase decays and cells form a division septum without completing a normal mitosis. dma1-D1 cells also show an increased rate of chromosome loss during exponential growth. Upon ectopic expression from an inducible promoter, dma1p delays progress through mitosis and inhibits septum formation, giving rise to elongated, multinucleate cells. We propose that dma1 is a component of the spindle assembly checkpoint, required to prevent septum formation and premature exit from mitosis if spindle function is impaired.

| S-EPMC452485 | biostudies-other

Kinetic framework of spindle assembly checkpoint signalling.

Project description:The mitotic spindle assembly checkpoint (SAC) delays anaphase onset until all chromosomes have attached to both spindle poles. Here, we investigated SAC signalling kinetics in response to acute detachment of individual chromosomes using laser microsurgery. Most detached chromosomes delayed anaphase until they had realigned to the metaphase plate. A substantial fraction of cells, however, entered anaphase in the presence of unaligned chromosomes. We identify two mechanisms by which cells can bypass the SAC: first, single unattached chromosomes inhibit the anaphase-promoting complex/cyclosome (APC/C) less efficiently than a full complement of unattached chromosomes; second, because of the relatively slow kinetics of re-imposing APC/C inhibition during metaphase, cells were unresponsive to chromosome detachment up to several minutes before anaphase onset. Our study defines when cells irreversibly commit to enter anaphase and shows that the SAC signal strength correlates with the number of unattached chromosomes. Detailed knowledge about SAC signalling kinetics is important for understanding the emergence of aneuploidy and the response of cancer cells to chemotherapeutics targeting the mitotic spindle.

| S-EPMC4067996 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data