Analysis of the mitotic exit control system using locked levels of stable mitotic cyclin

Cyclin‐dependent kinase (Cdk) both promotes mitotic entry (spindle assembly and anaphase) and inhibits mitotic exit (spindle disassembly and cytokinesis), leading to an elegant quantitative hypothesis that a single cyclin oscillation can function as a ratchet to order these events. This ratchet is at the core of a published ODE model for the yeast cell cycle. However, the ratchet model requires appropriate cyclin dose–response thresholds. Here, we test the inhibition of mitotic exit in budding yeast using graded levels of stable mitotic cyclin (Clb2). In opposition to the ratchet model, stable levels of Clb2 introduced dose‐dependent delays, rather than hard thresholds, that varied by mitotic exit event. The ensuing cell cycle was highly abnormal, suggesting a novel reason for cyclin degradation. Cdc14 phosphatase antagonizes Clb2–Cdk, and Cdc14 is released from inhibitory nucleolar sequestration independently of stable Clb2. Thus, Cdc14/Clb2 balance may be the appropriate variable for mitotic regulation. Although our results are inconsistent with the aforementioned ODE model, revision of the model to allow Cdc14/Clb2 balance to control mitotic exit corrects these discrepancies, providing theoretical support for our conclusions.

[1]  Katherine C. Chen,et al.  Integrative analysis of cell cycle control in budding yeast. , 2004, Molecular biology of the cell.

[2]  D. Koshland,et al.  The CDC20 gene product of Saccharomyces cerevisiae, a beta-transducin homolog, is required for a subset of microtubule-dependent cellular processes , 1991, Molecular and cellular biology.

[3]  F. Cross,et al.  Two redundant oscillatory mechanisms in the yeast cell cycle. , 2003, Developmental cell.

[4]  D. Barford,et al.  The structure of the cell cycle protein Cdc14 reveals a proline‐directed protein phosphatase , 2003, The EMBO journal.

[5]  Frederick R. Cross,et al.  Multiple levels of cyclin specificity in cell-cycle control , 2007, Nature Reviews Molecular Cell Biology.

[6]  Stephen E. Kearsey,et al.  G1-phase and B-type cyclins exclude the DNA-replication factor Mcm4 from the nucleus , 1999, Nature Cell Biology.

[7]  L. Breeden,et al.  Nascent transcription of MCM2-7 is important for nuclear localization of the minichromosome maintenance complex in G1. , 2007, Molecular biology of the cell.

[8]  M. Crosby,et al.  Cell Cycle: Principles of Control , 2007, The Yale Journal of Biology and Medicine.

[9]  T. Giddings,et al.  Saccharomyces cerevisiae Ndc1p Is a Shared Component of Nuclear Pore Complexes and Spindle Pole Bodies , 1998, The Journal of cell biology.

[10]  David P. Toczyski,et al.  Securin and B-cyclin/CDK are the only essential targets of the APC , 2003, Nature Cell Biology.

[11]  L. Hartwell,et al.  Genetic control of the cell division cycle in yeast. , 1974, Science.

[12]  D. Picard.  Posttranslational regulation of proteins by fusions to steroid-binding domains. , 2000, Methods in enzymology.

[13]  F. M. Yeong,et al.  Early Expressed Clb Proteins Allow Accumulation of Mitotic Cyclin by Inactivating Proteolytic Machinery during S Phase , 2001, Molecular and Cellular Biology.

[14]  M. Kirschner,et al.  Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p. , 1996, Genes & development.

[15]  K Nasmyth,et al.  Destruction of the CDC28/CLB mitotic kinase is not required for the metaphase to anaphase transition in budding yeast. , 1993, The EMBO journal.

[16]  S. Irniger.  Cyclin destruction in mitosis: a crucial task of Cdc20 , 2002, FEBS letters.

[17]  T. Giddings,et al.  Requirement for Bbp1p in the proper mitotic functions of Cdc5p in Saccharomyces cerevisiae. , 2004, Molecular biology of the cell.

[18]  David O. Morgan,et al.  The Cell Cycle: Principles of Control , 2014 .

[19]  L. Hartwell,et al.  Altered fidelity of mitotic chromosome transmission in cell cycle mutants of S. cerevisiae. , 1985, Genetics.

[20]  K. Nasmyth,et al.  Whose end is destruction: cell division and the anaphase-promoting complex. , 1999, Genes & development.

[21]  F. Cross,et al.  Phosphorylation of the Sic1 Inhibitor of B-Type Cyclins in Saccharomyces cerevisiae Is Not Essential but Contributes to Cell Cycle Robustness , 2007, Genetics.

[22]  F. Cross,et al.  Coherence and timing of cell cycle start examined at single-cell resolution. , 2006, Molecular cell.

[23]  M. Tyers,et al.  The phosphatase Cdc14 triggers mitotic exit by reversal of Cdk-dependent phosphorylation. , 1998, Molecular cell.

[24]  Michael Schwab,et al.  Yeast Hct1 Is a Regulator of Clb2 Cyclin Proteolysis , 1997, Cell.

[25]  K Nasmyth,et al.  Characterization of four B-type cyclin genes of the budding yeast Saccharomyces cerevisiae. , 1992, Molecular biology of the cell.

[26]  Joshua E. S. Socolar,et al.  Global control of cell-cycle transcription by coupled CDK and network oscillators , 2008, Nature.

[27]  M. Mendenhall,et al.  An inhibitor of yeast cyclin-dependent protein kinase plays an important role in ensuring the genomic integrity of daughter cells. , 1994, Molecular and cellular biology.

[28]  P. O’Farrell,et al.  RNAi of Mitotic Cyclins in Drosophila Uncouples the Nuclear and Centrosome Cycle , 2008, Current Biology.

[29]  Katherine C. Chen,et al.  Cycling without the Cyclosome: Modeling a Yeast Strain Lacking the APC , 2004, Cell cycle.

[30]  F. Cross,et al.  Testing a mathematical model of the yeast cell cycle. , 2002, Molecular biology of the cell.

[31]  G. Braus,et al.  Two different modes of cyclin Clb2 proteolysis during mitosis in Saccharomyces cerevisiae , 2000, FEBS letters.

[32]  F. Cross,et al.  A potential positive feedback loop controlling CLN1 and CLN2 gene expression at the start of the yeast cell cycle , 1991, Cell.

[33]  A. Shevchenko,et al.  The Bbp1p–Mps2p complex connects the SPB to the nuclear envelope and is essential for SPB duplication , 2000, The EMBO journal.

[34]  S. Jaspersen,et al.  The Saccharomyces cerevisiae spindle pole body (SPB) component Nbp1p is required for SPB membrane insertion and interacts with the integral membrane proteins Ndc1p and Mps2p. , 2006, Molecular biology of the cell.

[35]  Béla Novák,et al.  Supplementary Fig. 1 , 2021 .

[36]  M. Kirschner,et al.  Properties of Saccharomyces cerevisiae wee1 and its differential regulation of p34CDC28 in response to G1 and G2 cyclins. , 1993, The EMBO journal.

[37]  Alan J Tackett,et al.  Genetic and biochemical evaluation of the importance of Cdc6 in regulating mitotic exit. , 2003, Molecular biology of the cell.

[38]  F. Cross,et al.  Mitotic exit in the absence of separase activity. , 2009, Molecular biology of the cell.

[39]  D. Morgan,et al.  Finishing mitosis, one step at a time , 2007, Nature Reviews Molecular Cell Biology.

[40]  P. Nurse,et al.  A quantitative model for the cdc2 control of S phase and mitosis in fission yeast. , 1996, Trends in genetics : TIG.

[41]  F. Cross,et al.  Saccharomyces cerevisiae G1 cyclins differ in their intrinsic functional specificities , 1996, Molecular and cellular biology.

[42]  Kim Nasmyth,et al.  The B-type cyclin kinase inhibitor p40 SIC1 controls the G1 to S transition in S. cerevisiae , 1994, Cell.

[43]  M J Hartmann,et al.  Applications of video mixing and digital overlay to neuroethology. , 2000, Methods.

[44]  A. Murray,et al.  Dominoes and clocks: the union of two views of the cell cycle. , 1989, Science.

[45]  U. Surana,et al.  Cdc20 is essential for the cyclosome-mediated proteolysis of both Pds1 and Clb2 during M phase in budding yeast , 1998, Current Biology.

[46]  Kaoru Irie,et al.  Clb/Cdc28 kinases promote nuclear export of the replication initiator proteins Mcm2–7 , 2000, Current Biology.

[47]  Bela Novak,et al.  Downregulation of PP2ACdc55 Phosphatase by Separase Initiates Mitotic Exit in Budding Yeast , 2006, Cell.

[48]  윤호주.  전사인자(transcription factor)와 기관지천식 , 1999 .

[49]  S. Prinz,et al.  CDC20 and CDH1: a family of substrate-specific activators of APC-dependent proteolysis. , 1997, Science.

[50]  F. Cross,et al.  CLB5: a novel B cyclin from budding yeast with a role in S phase. , 1992, Genes & development.

[51]  K Nasmyth,et al.  Control of cyclin ubiquitination by CDK-regulated binding of Hct1 to the anaphase promoting complex. , 1998, Science.

[52]  M. Kirschner,et al.  Mitosis in transition , 1994, Cell.

[53]  F. M. Yeong,et al.  Exit from mitosis in budding yeast: biphasic inactivation of the Cdc28-Clb2 mitotic kinase and the role of Cdc20. , 2000, Molecular cell.

[54]  L. Hartwell,et al.  Checkpoints: controls that ensure the order of cell cycle events. , 1989, Science.

[55]  Frederick R. Cross,et al.  APC-dependent proteolysis of the mitotic cyclin Clb2 is essential for mitotic exit , 2002, Nature.

[56]  James M. Roberts,et al.  Cell cycle: Oscillation sensation , 2002, Nature.

[57]  Anna Shevchenko,et al.  Exit from Mitosis Is Triggered by Tem1-Dependent Release of the Protein Phosphatase Cdc14 from Nucleolar RENT Complex , 1999, Cell.

[58]  Katherine C. Chen,et al.  Quantitative characterization of a mitotic cyclin threshold regulating exit from mitosis. , 2005, Molecular biology of the cell.

[59]  S. Carr,et al.  Phosphorylation of Sic1p by G1 Cdk required for its degradation and entry into S phase. , 1997, Science.

[60]  K Nasmyth,et al.  The transcription factor Swi5 regulates expression of the cyclin kinase inhibitor p40SIC1 , 1996, Molecular and cellular biology.

[61]  D. Lew,et al.  Control of Swe1p degradation by the morphogenesis checkpoint , 1998, The EMBO journal.

[62]  Angelika Amon,et al.  Separase, Polo Kinase, the Kinetochore Protein Slk19, and Spo12 Function in a Network that Controls Cdc14 Localization during Early Anaphase , 2002, Cell.

[63]  Kim Nasmyth,et al.  Genes involved in sister chromatid separation are needed for b-type cyclin proteolysis in budding yeast , 1995, Cell.