Elutriation for Cell Cycle Synchronization in Fission Yeast

Abstract In the fission yeast Schizosaccharomyces pombe, as in other eukaryotic cells, Cdc2/cyclin B complex is the key regulator of mitosis. Perhaps the most important regulation of Cdc2 is the inhibitory phosphorylation of tyrosine-15 that is catalyzed by Wee1 and Mik1. Cdc25 and Pyp3 phosphatases dephosphorylate tyrosine-15 and activate Cdc2. To isolate novel activators of Cdc2 kinase, we screened synthetic lethal mutants in a cdc25-22 background at the permissive temperature (25°). One of the genes, slm9, encodes a novel protein of 807 amino acids. Slm9 is most similar to Hir2, the histone gene regulator in budding yeast. Slm9 protein level is constant and Slm9 is localized to the nucleus throughout the cell cycle. The slm9 disruptant is delayed at the G2-M transition as indicated by cell elongation and analysis of DNA content. Inactivation of Wee1 fully suppressed the cell elongation phenotype caused by the slm9 mutation. The slm9 mutant is defective in recovery from G1 arrest after nitrogen starvation. The slm9 mutant is also UV sensitive, showing a defect in recovery from the cell cycle arrest after UV irradiation.

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Cell Cycle Synchronization of the Murine EO771 Cell Line Using Double Thymidine Block Treatment

BioEssays ◽

10.1002/bies.201900116 ◽

2020 ◽

Vol 42 (9) ◽

pp. 1900116

Author(s):

Marie Goepp ◽

Delphine Le Guennec ◽

Adrien Rossary ◽

Marie‐Paule Vasson

Keyword(s):

Cell Cycle ◽

Cell Line ◽

Cell Cycle Synchronization

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Cell Cycle Synchronization at the G2/M Phase Border by Reversible Inhibition of CDK1

Cell Cycle ◽

10.4161/cc.5.22.3463 ◽

2006 ◽

Vol 5 (22) ◽

pp. 2555-2556 ◽

Cited By ~ 84

Author(s):

Lyubomir T Vassilev

Keyword(s):

Cell Cycle ◽

Reversible Inhibition ◽

Cell Cycle Synchronization ◽

M Phase

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Monocyte-induced cell cycle synchronization of a leukemic cell linein vitro

Cytometry ◽

10.1002/cyto.990020309 ◽

1981 ◽

Vol 2 (3) ◽

pp. 179-184

Author(s):

Bjørn Magne Eggen

Keyword(s):

Cell Cycle ◽

Leukemic Cell ◽

Cell Cycle Synchronization

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

Molecular Biology of the Cell ◽

10.1091/mbc.01-03-0104 ◽

2002 ◽

Vol 13 (2) ◽

pp. 480-492 ◽

Cited By ~ 24

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.

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Asymmetry of the spindle pole bodies and spg1p GAP segregation during mitosis in fission yeast

Journal of Cell Science ◽

10.1242/jcs.112.14.2313 ◽

1999 ◽

Vol 112 (14) ◽

pp. 2313-2321 ◽

Cited By ~ 22

Author(s):

L. Cerutti ◽

V. Simanis

Keyword(s):

Cell Cycle ◽

Fission Yeast ◽

Spindle Pole Body ◽

Mitotic Cell ◽

Spindle Pole ◽

The Other ◽

Septum Formation ◽

Spindle Pole Bodies ◽

Interphase Cells ◽

Early Mitosis

In the fission yeast Schizosaccharomyces pombe, the onset of septum formation is induced by a signal transduction network involving several protein kinases and a GTPase switch. One of the roles of the spg1p GTPase is to localise the cdc7p protein kinase to the poles of the mitotic spindle, from where the onset of septation is thought to be signalled at the end of mitosis. Immunofluorescence studies have shown that cdc7p is located on both spindle pole bodies early in mitosis, but only on one during the later stages of anaphase. This is mediated by inactivation of spg1p on one pole before the other. The GAP for spg1p is a complex of two proteins, cdc16p and byr4p. Localisation of cdc16p and byr4p by indirect immunofluorescence during the mitotic cell cycle showed that both proteins are present on the spindle pole body in interphase cells. During mitosis, byr4p is seen first on both poles of the spindle, then on only one. This occurs prior to cdc7p becoming asymmetric. In contrast, the signal due to cdc16p decreases to a low level during early mitosis, before being seen strongly on the same pole as byr4p. Double staining indicates that this is the opposite pole to that which retains cdc7p in late anaphase. Examination of the effect of inactivating cdc16p at various stages of the cell cycle suggests that cdc16p, together with cdc2p plays a role in restraining septum formation during interphase. The asymmetric inactivation of spg1p is mediated by recruitment of the cdc16p-byr4p GAP to one of the poles of the spindle before the other, and the asymmetry of the spindle pole bodies may be established early during mitosis. Moreover, the spindle pole bodies appear to be non-equivalent even after division has been completed.

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