The kinetics of H1 histone kinase activation during the cell cycle of wild-type and wee mutants of the fission yeast Schizosaccharomyces pombe

1994 ◽  
Vol 107 (5) ◽  
pp. 1197-1204 ◽  
Author(s):  
J. Creanor ◽  
J.M. Mitchison
Keyword(s):  
Fission Yeast ◽  
Kinase Activity ◽  
Wild Type ◽  
Kinase Activation ◽  
H1 Histone ◽  
Synchronous Cultures ◽  
Novel Method ◽  
Histone Kinase ◽  
Mutant Cells ◽  
Kinetics Of

H1 histone kinase activity has been followed in selection-synchronised cultures of fission yeast wild-type and wee1 mutant cells, and in induction-synchronised cells of the mutant cdc2-33. The main conclusions are: (1) in all three cases, the peak of activity is near mitosis. (2) The rise in activity is relatively slow starting in wild type at 0.4 of the cycle before mitosis. It is proposed that the beginning of the rise is the first identified event in the mitotic control. (3) The rise is twice as fast in wee and starts nearer to mitosis. (4) In all cases the beginning of the rise is in G2. (5) The fall in activity is also slow, lasting for 0.25 of the cycle, in wild type. Exit from mitosis happens well before activity has fallen to baseline. (6) In a range of size mutants, activity is roughly proportional to cell size. It is suggested that the kinase may have a cytoplasmic function. (7) Estimates have been made of the timing of mitosis in the mutants. In wee, mitosis is 0.14 of the cycle earlier than in wild type because the cells have a longer septated period at the end of the cycle. (8) A novel method has been developed for eliminating the effects of the partial asynchrony in synchronous cultures, without which the kinetic analysis would have been inaccurate.

The size control of fission yeast revisited

1996 ◽  
Vol 109 (12) ◽  
pp. 2947-2957 ◽  
Author(s):  
A. Sveiczer ◽  
B. Novak ◽  
J.M. Mitchison
Keyword(s):  
Fission Yeast ◽  
Kinase Activity ◽  
Size Control ◽  
Time Lapse ◽  
Critical Threshold ◽  
Wild Type ◽  
Linear Rate ◽  
Cycle Times ◽  
Threshold Size ◽  
Histone Kinase

An analysis was made of cell length and cycle time in time-lapse films of the fission yeast Schizosaccharomyces pombe using wild-type (WT) cells and those of various mutants. The more important conclusions about ‘size controls’ are: (1) there is a marker in G2 in WT cells provided by a rate change point (RCP) where the linear rate of length growth increases by approximately 30%. The period before this RCP is dependent on size and can be called a ‘sizer’. The period after the RCP is nearly independent of size and can be called a ‘timer’. The achievement of a critical threshold size is at or near the RCP which is on average at about 0.3 of the cycle (halfway through G2). This is much earlier than was previously believed. (2) The RCP is at about the time when H1 histone kinase activity and the B type cyclin cdc13 start to rise in preparation for mitosis. The RCP is also associated with other metabolic changes. (3) In wee1 mutants, the mitotic size control is replaced by a G1/S size control which is as strong as the mitotic control. As in WT cells, there is a sizer which precedes the RCP followed by a timer but the RCP is at about the G1/S boundary and has a larger increase (approximately 100%) in rate. (4) cdc25 is not an essential part of the size control at mitosis or at the G1/S boundary. (5) Three further situations have been examined in which the mitotic size control has been abolished. First, induction synchronisation by block and release of cdc2 and cdc10. In the largest oversize-cells which are produced, the RCP is pushed back to the beginning of the cycle. There is no sizer period but only a timer. Second, when both the antagonists wee1 and cdc25 are absent in the double mutant wee1-50 cdc25 delta. In this interesting situation there is apparently no mitotic size control and the cycle times are quantised. Third, in rum1 delta wee1-50 where the normal long G1 in wee1 is much reduced, there is probably no size control either in G1 or in G2 causing a continuous shortening of division length from cycle to cycle.

scholarly journals Fission Yeast Rad26 Is a Regulatory Subunit of the Rad3 Checkpoint Kinase

2002 ◽  
Vol 13 (2) ◽  
pp. 480-492 ◽  
Author(s):  
Tom D. Wolkow ◽  
Tamar Enoch
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Fission Yeast ◽  
Complex Analysis ◽  
Kinase Activity ◽  
Regulatory Subunit ◽  
Kinase Activation ◽  
Checkpoint Proteins ◽  
Cycle Arrest ◽  
Phosphoinositide 3 Kinase

Fission yeast Rad3 is a member of a family of phosphoinositide 3-kinase -related kinases required for the maintenance of genomic stability in all eukaryotic cells. In fission yeast, Rad3 regulates the cell cycle arrest and recovery activities associated with the G2/M checkpoint. We have developed an assay that directly measures Rad3 kinase activity in cells expressing physiological levels of the protein. Using the assay, we demonstrate directly that Rad3 kinase activity is stimulated by checkpoint signals. Of the five other G2/M checkpoint proteins (Hus1, Rad1, Rad9, Rad17, and Rad26), only Rad26 was required for Rad3 kinase activity. Because Rad26 has previously been shown to interact constitutively with Rad3, our results demonstrate that Rad26 is a regulatory subunit, and Rad3 is the catalytic subunit, of the Rad3/Rad26 kinase complex. Analysis of Rad26/Rad3 kinase activation in rad26.T12, a mutant that is proficient for cell cycle arrest, but defective in recovery, suggests that these two responses to checkpoint signals require quantitatively different levels of kinase activity from the Rad3/Rad26 complex.

The kinetics of the B cyclin p56cdc13 and the phosphatase p80cdc25 during the cell cycle of the fission yeast Schizosaccharomyces pombe

1996 ◽  
Vol 109 (6) ◽  
pp. 1647-1653 ◽  
Author(s):  
J. Creanor ◽  
J.M. Mitchison
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Mammalian Cells ◽  
Size Control ◽  
Base Line ◽  
Wild Type ◽  
Main Band ◽  
The Times ◽  
G1 Cells ◽  
Mutant Cells ◽  
Kinetics Of

The levels of the B cyclin p56cdc13 and the phosphatase p80cdc25 have been followed in selection-synchronised cultures of Schizosaccharomyces pombe wild-type and wee1 mutant cells. p56cdc13 has also been followed in induction-synchronised cells of the mutant cdc2-33. The main conclusions are: (1) cdc13 levels in wild-type cells start to rise from base line at about mid-G2, reach a peak before mitosis and then fall slowly through G1. Cells exit mitosis with appreciable levels of cdc13. (2) cdc13 levels in wee1 cells fall to zero in interphase. They also start to rise at the beginning of G2, which may be related to the absence of a mitotic size control. (3) cdc25 starts to rise later and reaches a peak after mitosis. This is not what would be expected from a simple mitotic inducer and suggests that cdc25 has an important function at the end of mitosis. (4) An upper (heavier) band of cdc25 peaks at the same time as the main band but rises and falls more rapidly. If this is a hyperphosphorylated form, its timing shows that it is most unlikely to function in the ways shown for such a form in eggs and mammalian cells. (5) Experiments with the mutant cdc10-129 and with hydroxyurea show that the initial signal to begin synthesis of cdc13 originates at Start. (6) In induction synchrony, where G2 spans across cell division, there is evidence that some events in one cycle cannot start in the previous one. (7) Revised timings are given for the times of mitosis in these cultures.

scholarly journals The number of cytokinesis nodes in mitotic fission yeast scales with cell volume

2021 ◽  
Author(s):  
Wasim A Sayyad ◽  
Thomas D Pollard
Keyword(s):  
Plasma Membrane ◽  
Fission Yeast ◽  
Large Population ◽  
Yeast Cells ◽  
Wild Type ◽  
Contractile Ring ◽  
Node Number ◽  
Nmt1 Promoter ◽  
Wide Range ◽  
Mutant Cells

Cytokinesis nodes are assemblies of stoichiometric ratios of proteins associated with the plasma membrane, which serve as precursors for the contractile ring during cytokinesis by fission yeast. The total number of nodes is uncertain, because of the limitations of the methods used previously. Here we used the ~140 nm resolution of Airyscan confocal microscopy to resolve a large population of dim, unitary cytokinesis nodes in 3D reconstructions of whole fission yeast cells. Wild-type fission yeast cells make about 200 unitary cytokinesis nodes. Most, but not all of these nodes condense into a contractile ring. The number of cytokinesis nodes scales with cell size in four strains tested, although wide rga4Δ mutant cells form somewhat fewer cytokinesis nodes than expected from the overall trend. The surface density of Pom1 kinase on the plasma membrane around the equators of cells is similar with a wide range of node numbers, so Pom1 does not control cytokinesis node number. However, varying protein concentrations with the nmt1 promoter showed that the numbers of nodes increase above a baseline of about 200 with the total cellular concentration of either Pom1 or the kinase Cdr2.

scholarly journals A mitotic role for a novel fission yeast protein kinase dsk1 with cell cycle stage dependent phosphorylation and localization.

1993 ◽  
Vol 4 (3) ◽  
pp. 247-260 ◽  
Author(s):  
M Takeuchi ◽  
M Yanagida
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Fission Yeast ◽  
Cell Cycle Progression ◽  
Kinase Activity ◽  
Specific Protein ◽  
Wild Type ◽  
Mobility Shift ◽  
Multicopy Suppressor ◽  
Phosphorylation Pattern

The fission yeast dsk1+ gene, a multicopy suppressor for cold-sensitive dis1 mutants, encodes a novel 61-kd protein kinase. It is a phosphoprotein, and phosphoserine is the major phosphorylated amino acid. Hyperphosphorylation of dsk1 causes a mobility shift, resulting in two dsk1-specific protein bands. The phosphorylation pattern is strikingly altered when cell cycle progression is delayed or arrested. The slowly migrating phosphorylated form is prominent in mitotically arrested cells, and the fast migrating form is enriched in interphase-arrested cells. dsk1 is a protein kinase. It auto-phosphorylates as well as phosphorylates myelin basic protein (MBP). Phosphotyrosine as well as phosphoserine/threonine were found in autophosphorylation, but no tyrosine phosphorylation occurs when MBP was used as the substrate. The dsk1 immunoprecipitates from mitotically arrested cells have a several-fold higher kinase activity than that from wild type. The haploid gene disruptant is viable, indicating that the dsk1+ gene is non-essential for viability. High dosage of dsk1+, however, strongly delays the G2/M progression. Immunofluorescence microscopy using anti-dsk1 antibody shows that localization pattern of dsk1 protein strikingly alters depending on cell cycle stages. In G2-arrested cells, dsk1 locates in the cytoplasm, whereas in mitotically arrested cells, nuclear stain is intense. In wild-type cells, nuclear stain is seen only in mitotic cells. Hence dsk1 protein may play an important role in mitotic control by altering cellular location, degree of phosphorylation and kinase activity. We discuss possible roles of dsk1 kinase as an add-on regulator in mitosis.

scholarly journals Novel alterations in CDK1/cyclin B1 kinase complex formation occur during the acquisition of a polyploid DNA content.

1996 ◽  
Vol 7 (2) ◽  
pp. 209-223 ◽  
Author(s):  
N S Datta ◽  
J L Williams ◽  
J Caldwell ◽  
A M Curry ◽  
E K Ashcraft ◽  
...  
Keyword(s):  
Dna Content ◽  
Kinase Activity ◽  
Cell Cycle Control ◽  
Cyclin B1 ◽  
S Phase ◽  
H1 Histone ◽  
Protein Levels ◽  
M Phase ◽  
Hel Cells ◽  
Histone Kinase

The pathways that regulate the S-phase events associated with the control of DNA replication are poorly understood. The bone marrow megakaryocytes are unique in that they leave the diploid (2C) state to differentiate, synthesizing 4 to 64 times the normal DNA content within a single nucleus, a process known as endomitosis. Human erythroleukemia (HEL) cells model this process, becoming polyploid during phorbol diester-induced megakaryocyte differentiation. The mitotic arrest occurring in these polyploid cells involves novel alterations in the cdk1/cyclin B1 complex: a marked reduction in cdk1 protein levels, and an elevated and sustained expression of cyclin B1. Endomitotic cells thus lack cdk1/cyclin B1-associated H1-histone kinase activity. Constitutive over-expression of cdk1 in endomitotic cells failed to re-initiate normal mitotic events even though cdk1 was present in a 10-fold excess. This was due to an inability of cyclin-B1 to physically associate with cdk1. Nonetheless, endomitotic cyclin B1 possesses immunoprecipitable H1-histone kinase activity, and specifically translocates to the nucleus. We conclude that mitosis is abrogated during endomitosis due to the absence of cdk1 and the failure to form M-phase promoting factor, resulting in a disassociation of mitosis from the completion of S-phase. Further studies on cyclin and its interacting proteins should be informative in understanding endomitosis and cell cycle control.

scholarly journals Glc7/Protein Phosphatase 1 Regulatory Subunits Can Oppose the Ipl1/Aurora Protein Kinase by Redistributing Glc7

2006 ◽  
Vol 26 (7) ◽  
pp. 2648-2660 ◽  
Author(s):  
Benjamin A. Pinsky ◽  
Chitra V. Kotwaliwale ◽  
Sean Y. Tatsutani ◽  
Christopher A. Breed ◽  
Sue Biggins
Keyword(s):  
Protein Kinase ◽  
Chromosome Segregation ◽  
Protein Phosphatase ◽  
Kinase Activity ◽  
Budding Yeast ◽  
Protein Phosphatase 1 ◽  
Wild Type ◽  
Regulatory Subunits ◽  
The Relationship ◽  
Mutant Cells

ABSTRACT Faithful chromosome segregation depends on the opposing activities of the budding yeast Glc7/PP1 protein phosphatase and Ipl1/Aurora protein kinase. We explored the relationship between Glc7 and Ipl1 and found that the phosphorylation of the Ipl1 substrate, Dam1, was altered by decreased Glc7 activity, whereas Ipl1 levels, localization, and kinase activity were not. These data strongly suggest that Glc7 ensures accurate chromosome segregation by dephosphorylating Ipl1 targets rather than regulating the Ipl1 kinase. To identify potential Glc7 and Ipl1 substrates, we isolated ipl1-321 dosage suppressors. Seven genes (SDS22, BUD14, GIP3, GIP4, SOL1, SOL2, and PEX31) encode newly identified ipl1 dosage suppressors, and all 10 suppressors encode proteins that physically interact with Glc7. The overexpression of the Gip3 and Gip4 suppressors altered Glc7 localization, indicating they are previously unidentified Glc7 regulatory subunits. In addition, the overexpression of Gip3 and Gip4 from the galactose promoter restored Dam1 phosphorylation in ipl1-321 mutant cells and caused wild-type cells to arrest in metaphase with unsegregated chromosomes, suggesting that Gip3 and Gip4 overexpression impairs Glc7's mitotic functions. We therefore propose that the overexpression of Glc7 regulatory subunits can titrate Glc7 away from relevant Ipl1 targets and thereby suppress ipl1-321 cells by restoring the balance of phosphatase/kinase activity.

scholarly journals Phosphorylation of p21 in G2/M Promotes Cyclin B-Cdc2 Kinase Activity

2005 ◽  
Vol 25 (8) ◽  
pp. 3364-3387 ◽  
Author(s):  
Bipin C. Dash ◽  
Wafik S. El-Deiry
Keyword(s):  
Kinase Activity ◽  
Cyclin B1 ◽  
S Phase ◽  
Cdk Inhibitor ◽  
Wild Type ◽  
Cdc2 Kinase ◽  
Dependent Protein Kinase ◽  
Kinase Activation ◽  
M Phase ◽  
Dependent Protein

ABSTRACT Little is known about the posttranslational control of the cyclin-dependent protein kinase (CDK) inhibitor p21. We describe here a transient phosphorylation of p21 in the G2/M phase. G2/M-phosphorylated p21 is short-lived relative to hypophosphorylated p21. p21 becomes nuclear during S phase, prior to its phosphorylation by CDK2. S126-phosphorylated cyclin B1 binds to T57-phosphorylated p21. Cdc2 kinase activation is delayed in p21-deficient cells due to delayed association between Cdc2 and cyclin B1. Cyclin B1-Cdc2 kinase activity and G2/M progression in p21−/− cells are restored after reexpression of wild-type but not T57A mutant p21. The cyclin B1 S126A mutant exhibits reduced Cdc2 binding and has low kinase activity. Phosphorylated p21 binds to cyclin B1 when Cdc2 is phosphorylated on Y15 and associates poorly with the complex. Dephosphorylation on Y15 and phosphorylation on T161 promotes Cdc2 binding to the p21-cyclin B1 complex, which becomes activated as a kinase. Thus, hyperphosphorylated p21 activates the Cdc2 kinase in the G2/M transition.

scholarly journals The CDK Pef1 and protein phosphatase 4 oppose each other for regulating cohesin binding to fission yeast chromosomes

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Adrien Birot ◽  
Marta Tormos-Pérez ◽  
Sabine Vaur ◽  
Amélie Feytout ◽  
Julien Jaegy ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Protein Phosphatase ◽  
Opposite Effect ◽  
Kinase Activity ◽  
Chromosome Structure ◽  
Wild Type ◽  
Human Homologue ◽  
Negative Regulators ◽  
Phosphorylation State ◽  
Fine Tune

Cohesin has essential roles in chromosome structure, segregation and repair. Cohesin binding to chromosomes is catalyzed by the cohesin loader, Mis4 in fission yeast. How cells fine tune cohesin deposition is largely unknown. Here, we provide evidence that Mis4 activity is regulated by phosphorylation of its cohesin substrate. A genetic screen for negative regulators of Mis4 yielded a CDK called Pef1, whose closest human homologue is CDK5. Inhibition of Pef1 kinase activity rescued cohesin loader deficiencies. In an otherwise wild-type background, Pef1 ablation stimulated cohesin binding to its regular sites along chromosomes while ablating Protein Phosphatase 4 had the opposite effect. Pef1 and PP4 control the phosphorylation state of the cohesin kleisin Rad21. The CDK phosphorylates Rad21 on Threonine 262. Pef1 ablation, non-phosphorylatable Rad21-T262 or mutations within a Rad21 binding domain of Mis4 alleviated the effect of PP4 deficiency. Such a CDK/PP4-based regulation of cohesin loader activity could provide an efficient mechanism for translating cellular cues into a fast and accurate cohesin response.

scholarly journals A cell cycle-independent, conditional gene inactivation strategy for differentially tagging wild-type and mutant cells

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Sonal Nagarkar-Jaiswal ◽  
Sathiya N Manivannan ◽  
Zhongyuan Zuo ◽  
Hugo J Bellen
Keyword(s):  
Cell Cycle ◽  
Gene Inactivation ◽  
Inversion Method ◽  
Wild Type ◽  
Flip Flop ◽  
Temporal Cues ◽  
A Cell ◽  
Novel Method ◽  
Mutant Cells

Here, we describe a novel method based on intronic MiMIC insertions described in Nagarkar-Jaiswal et al. (2015) to perform conditional gene inactivation in Drosophila. Mosaic analysis in Drosophila cannot be easily performed in post-mitotic cells. We therefore, therefore, developed Flip-Flop, a flippase-dependent in vivo cassette-inversion method that marks wild-type cells with the endogenous EGFP-tagged protein, whereas mutant cells are marked with mCherry upon inversion. We document the ease and usefulness of this strategy in differential tagging of wild-type and mutant cells in mosaics. We use this approach to phenotypically characterize the loss of SNF4Aγ, encoding the γ subunit of the AMP Kinase complex. The Flip-Flop method is efficient and reliable, and permits conditional gene inactivation based on both spatial and temporal cues, in a cell cycle-, and developmental stage-independent fashion, creating a platform for systematic screens of gene function in developing and adult flies with unprecedented detail.

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