Publications by authors named "Wolfgang Zachariae"

16 Publications

  • Page 1 of 1

Deprotection of centromeric cohesin at meiosis II requires APC/C activity but not kinetochore tension.

EMBO J 2021 Apr 1;40(7):e106812. Epub 2021 Mar 1.

Laboratory of Chromosome Biology, Max Planck Institute of Biochemistry, Martinsried, Germany.

Genome haploidization involves sequential loss of cohesin from chromosome arms and centromeres during two meiotic divisions. At centromeres, cohesin's Rec8 subunit is protected from separase cleavage at meiosis I and then deprotected to allow its cleavage at meiosis II. Protection of centromeric cohesin by shugoshin-PP2A seems evolutionarily conserved. However, deprotection has been proposed to rely on spindle forces separating the Rec8 protector from cohesin at metaphase II in mammalian oocytes and on APC/C-dependent destruction of the protector at anaphase II in yeast. Here, we have activated APC/C in the absence of sister kinetochore biorientation at meiosis II in yeast and mouse oocytes, and find that bipolar spindle forces are dispensable for sister centromere separation in both systems. Furthermore, we show that at least in yeast, protection of Rec8 by shugoshin and inhibition of separase by securin are both required for the stability of centromeric cohesin at metaphase II. Our data imply that related mechanisms preserve the integrity of dyad chromosomes during the short metaphase II of yeast and the prolonged metaphase II arrest of mammalian oocytes.
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http://dx.doi.org/10.15252/embj.2020106812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8013787PMC
April 2021

APC/C-Cdc20 mediates deprotection of centromeric cohesin at meiosis II in yeast.

Cell Cycle 2017 Jun 17;16(12):1145-1152. Epub 2017 May 17.

a Laboratory of Chromosome Biology , Max Planck Institute of Biochemistry , Martinsried , Germany.

Cells undergoing meiosis produce haploid gametes through one round of DNA replication followed by 2 rounds of chromosome segregation. This requires that cohesin complexes, which establish sister chromatid cohesion during S phase, are removed in a stepwise manner. At meiosis I, the separase protease triggers the segregation of homologous chromosomes by cleaving cohesin's Rec8 subunit on chromosome arms. Cohesin persists at centromeres because the PP2A phosphatase, recruited by the shugoshin protein, dephosphorylates Rec8 and thereby protects it from cleavage. While chromatids disjoin upon cleavage of centromeric Rec8 at meiosis II, it was unclear how and when centromeric Rec8 is liberated from its protector PP2A. One proposal is that bipolar spindle forces separate PP2A from Rec8 as cells enter metaphase II. We show here that sister centromere biorientation is not sufficient to "deprotect" Rec8 at meiosis II in yeast. Instead, our data suggest that the ubiquitin-ligase APC/C removes PP2A from centromeres by targeting for degradation the shugoshin Sgo1 and the kinase Mps1. This implies that Rec8 remains protected until entry into anaphase II when it is phosphorylated concurrently with the activation of separase. Here, we provide further support for this model and speculate on its relevance to mammalian oocytes.
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http://dx.doi.org/10.1080/15384101.2017.1320628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499901PMC
June 2017

Casein Kinase 1 Coordinates Cohesin Cleavage, Gametogenesis, and Exit from M Phase in Meiosis II.

Dev Cell 2017 01 22;40(1):37-52. Epub 2016 Dec 22.

Laboratory of Chromosome Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany. Electronic address:

Meiosis consists of DNA replication followed by two consecutive nuclear divisions and gametogenesis or spore formation. While meiosis I has been studied extensively, less is known about the regulation of meiosis II. Here we show that Hrr25, the conserved casein kinase 1δ of budding yeast, links three mutually independent key processes of meiosis II. First, Hrr25 induces nuclear division by priming centromeric cohesin for cleavage by separase. Hrr25 simultaneously phosphorylates Rec8, the cleavable subunit of cohesin, and removes from centromeres the cohesin protector composed of shugoshin and the phosphatase PP2A. Second, Hrr25 initiates the sporulation program by inducing the synthesis of membranes that engulf the emerging nuclei at anaphase II. Third, Hrr25 mediates exit from meiosis II by activating pathways that trigger the destruction of M-phase-promoting kinases. Thus, Hrr25 synchronizes formation of the single-copy genome with gamete differentiation and termination of meiosis.
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http://dx.doi.org/10.1016/j.devcel.2016.11.021DOI Listing
January 2017

Cell Division: Flipping the Mitotic Switches.

Curr Biol 2016 12;26(24):R1272-R1274

Department of Biological Sciences, Virginia Tech, Blacksburg, VA, USA. Electronic address:

Alternation between DNA replication in the mother cell (S phase) and equal partitioning of the replicated chromosomes to the daughter cells (M phase) during eukaryotic cell division is governed by switches that flip protein kinases on and off. New work reveals that the M-phase promoting kinase is opposed by a phosphatase that also participates in a bistable switching mechanism.
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http://dx.doi.org/10.1016/j.cub.2016.11.007DOI Listing
December 2016

Meiotic prophase requires proteolysis of M phase regulators mediated by the meiosis-specific APC/CAma1.

Cell 2012 Oct;151(3):603-18

Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.

Whereas proliferating cells enter M phase shortly after DNA replication, the first M phase of meiosis is preceded by an extended prophase in which homologous chromosomes undergo recombination. Exit from prophase I is controlled by the recombination checkpoint (RC), which, in yeast, represses the meiosis-specific transcription factor Ndt80 required for the expression of B-type cyclins and other M phase regulators. We show that an extended prophase I additionally requires the suppression of latent, mitotic cell-cycle controls by the anaphase-promoting complex (APC/C) and its meiosis-specific activator Ama1, which trigger the degradation of M phase regulators and Ndd1, a subunit of a mitotic transcription factor. ama1Δ mutants exit from prophase I prematurely and independently of the RC, which results in recombination defects and chromosome missegregation. Thus, control of prophase I by meiotic mechanisms depends on the suppression of the alternative, mitotic mechanisms by a meiosis-specific form of the APC/C.
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http://dx.doi.org/10.1016/j.cell.2012.08.044DOI Listing
October 2012

Limiting amounts of centrosome material set centrosome size in C. elegans embryos.

Curr Biol 2011 Aug 28;21(15):1259-67. Epub 2011 Jul 28.

Max Planck Institute for Cell Biology and Genetics, Dresden 01307 Germany.

Background: The ways in which cells set the size of intracellular structures is an important but largely unsolved problem [1]. Early embryonic divisions pose special problems in this regard. Many checkpoints common in somatic cells are missing from these divisions, which are characterized by rapid reductions in cell size and short cell cycles [2]. Embryonic cells must therefore possess simple and robust mechanisms that allow the size of many of their intracellular structures to rapidly scale with cell size.

Results: Here, we study the mechanism by which one structure, the centrosome, scales in size during the early embryonic divisions of C. elegans. We show that centrosome size is directly related to cell size and is independent of lineage. Two findings suggest that the total amount of maternally supplied centrosome proteins could limit centrosome size. First, the combined volume of all centrosomes formed at any one time in the developing embryo is constant. Second, the total volume of centrosomes in any one cell is independent of centrosome number. By increasing the amount of centrosome proteins in the cell, we provide evidence that one component that limits centrosome size is the conserved pericentriolar material protein SPD-2 [3], which we show binds to and targets polo-like kinase 1 [3, 4] to centrosomes.

Conclusions: We propose a limiting component hypothesis, in which the volume of the cell sets centrosome size by limiting the total amount of centrosome components. This idea could be a general mechanism for setting the size of intracellular organelles during development.
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http://dx.doi.org/10.1016/j.cub.2011.06.002DOI Listing
August 2011

Rec8 phosphorylation by casein kinase 1 and Cdc7-Dbf4 kinase regulates cohesin cleavage by separase during meiosis.

Dev Cell 2010 Mar;18(3):397-409

Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.

During meiosis, two rounds of chromosome segregation after a single round of DNA replication produce haploid gametes from diploid precursors. At meiosis I, maternal and paternal kinetochores are pulled toward opposite poles, and chiasmata holding bivalent chromosomes together are resolved by cleavage of cohesin's alpha-kleisin subunit (Rec8) along chromosome arms. This creates dyad chromosomes containing a pair of chromatids joined solely by cohesin at centromeres that had resisted cleavage. The discovery that centromeric Rec8 is protected from separase during meiosis I by shugoshin/MEI-S332 proteins that bind PP2A phosphatase suggests that phosphorylation either of separase or cohesin may be necessary for Rec8 cleavage. We show here that multiple phosphorylation sites within Rec8 as well as two different kinases, casein kinase 1delta/epsilon (CK1delta/epsilon) and Dbf4-dependent Cdc7 kinase (DDK), are required for Rec8 cleavage and meiosis I nuclear division. Rec8 with phosphomimetic mutations is no longer protected from separase at centromeres and is cleaved even when the two kinases are inhibited. Our data suggest that PP2A protects centromeric cohesion by opposing CK1delta/epsilon- and DDK-dependent phosphorylation of Rec8.
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http://dx.doi.org/10.1016/j.devcel.2010.01.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2994640PMC
March 2010

Dbf4-dependent CDC7 kinase links DNA replication to the segregation of homologous chromosomes in meiosis I.

Cell 2008 Nov;135(4):662-78

Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.

Meiosis differs from mitosis in that DNA replication is followed by the segregation of homologous chromosomes but not sister chromatids. This depends on the formation of interhomolog connections through crossover recombination and on the attachment of sister kinetochores to microtubules emanating from the same spindle pole. We show that in yeast, the Dbf4-dependent Cdc7 kinase (DDK) provides a link between premeiotic S phase, recombination, and monopolar attachment. Independently from its established role in initiating DNA replication, DDK promotes double-strand break formation, the first step of recombination, and the recruitment of the monopolin complex to kinetochores, which is essential for monopolar attachment. DDK regulates monopolin localization together with the polo-kinase Cdc5 bound to Spo13, probably through phosphorylation of the monopolin subunit Lrs4. Thus, activation of DDK both initiates DNA replication and commits meiotic cells to reductional chromosome segregation in the first division of meiosis.
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http://dx.doi.org/10.1016/j.cell.2008.10.026DOI Listing
November 2008

Identification of the C. elegans anaphase promoting complex subunit Cdc26 by phenotypic profiling and functional rescue in yeast.

BMC Dev Biol 2007 Mar 20;7:19. Epub 2007 Mar 20.

The Gurdon Institute and Department of Genetics, University of Cambridge, Cambridge, UK.

Background: RNA interference coupled with videorecording of C. elegans embryos is a powerful method for identifying genes involved in cell division processes. Here we present a functional analysis of the gene B0511.9, previously identified as a candidate cell polarity gene in an RNAi videorecording screen of chromosome I embryonic lethal genes.

Results: Whereas weak RNAi inhibition of B0511.9 causes embryonic cell polarity defects, strong inhibition causes embryos to arrest in metaphase of meiosis I. The range of defects induced by RNAi of B0511.9 is strikingly similar to those displayed by mutants of anaphase-promoting complex/cyclosome (APC/C) components. Although similarity searches did not reveal any obvious homologue of B0511.9 in the non-redundant protein database, we found that the N-terminus shares a conserved sequence pattern with the N-terminus of the small budding yeast APC/C subunit Cdc26 and its orthologues from a variety of other organisms. Furthermore, we show that B0511.9 robustly complements the temperature-sensitive growth defect of a yeast cdc26Delta mutant.

Conclusion: These data demonstrate that B0511.9 encodes the C. elegans APC/C subunit CDC-26.
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http://dx.doi.org/10.1186/1471-213X-7-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1847674PMC
March 2007

Monopolar attachment of sister kinetochores at meiosis I requires casein kinase 1.

Cell 2006 Sep;126(6):1049-64

Research Institute of Molecular Pathology, Dr. Bohrgasse 7, A-1030 Vienna, Austria.

In meiosis, a single round of DNA replication is followed by two consecutive rounds of chromosome segregation, called meiosis I and II. Disjunction of maternal from paternal centromeres during meiosis I depends on the attachment of sister kinetochores to microtubules emanating from the same pole. In budding yeast, monopolar attachment requires recruitment to kinetochores of the monopolin complex. How monopolin promotes monopolar attachment was unclear, as its subunits are poorly conserved and lack similarities to proteins with known functions. We show here that the monopolin subunit Mam1 binds tightly to Hrr25, a highly conserved casein kinase 1 delta/epsilon (CK1delta/epsilon), and recruits it to meiosis I centromeres. Hrr25 kinase activity and Mam1 binding are both essential for monopolar attachment. Since CK1delta/epsilon activity is important for accurate chromosome segregation during meiosis I also in fission yeast, phosphorylation of kinetochore proteins by CK1delta/epsilon might be an evolutionary conserved process required for monopolar attachment.
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http://dx.doi.org/10.1016/j.cell.2006.07.029DOI Listing
September 2006

The yeast APC/C subunit Mnd2 prevents premature sister chromatid separation triggered by the meiosis-specific APC/C-Ama1.

Cell 2005 Mar;120(6):773-88

Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.

Cohesion established between sister chromatids during pre-meiotic DNA replication mediates two rounds of chromosome segregation. The first division is preceded by an extended prophase wherein homologous chromosomes undergo recombination. The persistence of cohesion during prophase is essential for recombination and both meiotic divisions. Here we show that Mnd2, a subunit of the anaphase-promoting complex (APC/C) from budding yeast, is essential to prevent premature destruction of cohesion in meiosis. During S- and prophase, Mnd2 prevents activation of the APC/C by a meiosis-specific activator called Ama1. In cells lacking Mnd2 the APC/C-Ama1 enzyme triggers degradation of Pds1, which causes premature sister chromatid separation due to unrestrained separase activity. In vitro, Mnd2 inhibits ubiquitination of Pds1 by APC/C-Ama1 but not by other APC/C holo-enzymes. We conclude that chromosome segregation in meiosis depends on the selective inhibition of a meiosis-specific form of the APC/C.
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http://dx.doi.org/10.1016/j.cell.2005.01.032DOI Listing
March 2005

Emi1 and Erp1: who can stop these eggs?

Dev Cell 2005 Mar;8(3):301-3

Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.

Vertebrate eggs prevent parthenogenetic development by producing cytostatic factor (CSF), which blocks exit from metaphase of meiosis II until fertilization. CSF was never purified but recently suspected to inhibit the anaphase-promoting complex (APC), an ubiquitin ligase required for entry into anaphase. In a recent paper in Genes & Development, Schmidt et al. describe the Xenopus APC inhibitor Erp1, which seems to be the best candidate yet for the downstream effector of CSF activity.
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http://dx.doi.org/10.1016/j.devcel.2005.02.004DOI Listing
March 2005

Spo13 facilitates monopolin recruitment to kinetochores and regulates maintenance of centromeric cohesion during yeast meiosis.

Curr Biol 2004 Dec;14(24):2183-96

Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7, A-1030 Vienna, Austria.

Background: Cells undergoing meiosis perform two consecutive divisions after a single round of DNA replication. During the first meiotic division, homologous chromosomes segregate to opposite poles. This is achieved by (1) the pairing of maternal and paternal chromosomes via recombination producing chiasmata, (2) coorientation of homologous chromosomes such that sister chromatids attach to the same spindle pole, and (3) resolution of chiasmata by proteolytic cleavage by separase of the meiotic-specific cohesin Rec8 along chromosome arms. Crucially, cohesin at centromeres is retained to allow sister centromeres to biorient at the second division. Little is known about how these meiosis I-specific events are regulated.

Results: Here, we show that Spo13, a centromere-associated protein produced exclusively during meiosis I, is required to prevent sister kinetochore biorientation by facilitating the recruitment of the monopolin complex to kinetochores. Spo13 is also required for the reaccumulation of securin, the persistence of centromeric cohesin during meiosis II, and the maintenance of a metaphase I arrest induced by downregulation of the APC/C activator CDC20.

Conclusion: Spo13 is a key regulator of several meiosis I events. The presence of Spo13 at centromere-surrounding regions is consistent with the notion that it plays a direct role in both monopolin recruitment to centromeres during meiosis I and maintenance of centromeric cohesion between the meiotic divisions. Spo13 may also limit separase activity after the first division by ensuring securin reaccumulation and, in doing so, preventing precocious removal from chromatin of centromeric cohesin.
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http://dx.doi.org/10.1016/j.cub.2004.12.020DOI Listing
December 2004

Swm1/Apc13 is an evolutionarily conserved subunit of the anaphase-promoting complex stabilizing the association of Cdc16 and Cdc27.

Mol Cell Biol 2004 Apr;24(8):3562-76

Max Planck Institute of Molecular Cell Biology and Genetics. Scionics Computer Innovation GmbH, Dresden, Germany.

The anaphase-promoting complex (APC/C) is a large ubiquitin-protein ligase which controls progression through anaphase by triggering the degradation of cell cycle regulators such as securin and B-type cyclins. The APC/C is an unusually complex ligase containing at least 10 different, evolutionarily conserved components. In contrast to APC/C's role in cell cycle regulation little is known about the functions of individual subunits and how they might interact with each other. Here, we have analyzed Swm1/Apc13, a small subunit recently identified in the budding yeast complex. Database searches revealed proteins related to Swm1/Apc13 in various organisms including humans. Both the human and the fission yeast homologues are associated with APC/C subunits, and they complement the phenotype of an SWM1 deletion mutant of budding yeast. Swm1/Apc13 promotes the stable association with the APC/C of the essential subunits Cdc16 and Cdc27. Accordingly, Swm1/Apc13 is required for ubiquitin ligase activity in vitro and for the timely execution of APC/C-dependent cell cycle events in vivo.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC381669PMC
http://dx.doi.org/10.1128/mcb.24.8.3562-3576.2004DOI Listing
April 2004

Destruction with a box: substrate recognition by the anaphase-promoting complex.

Mol Cell 2004 Jan;13(1):2-3

Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.

Destruction boxes mark cyclin B and other proteins degraded in mitosis for ubiquitination by the anaphase-promoting complex (APC/C). In a paper in this issue of Molecular Cell, Yamano et al. show that destruction boxes directly bind to the APC/C in a cell cycle-regulated manner. Interestingly, this interaction does not require APC/C activators of the Cdc20 family, which were thought to be essential for recruiting substrates to the APC/C.
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http://dx.doi.org/10.1016/s1097-2765(03)00530-6DOI Listing
January 2004

The CCT chaperonin promotes activation of the anaphase-promoting complex through the generation of functional Cdc20.

Mol Cell 2003 Jul;12(1):87-100

Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany.

The WD repeat protein Cdc20 is essential for progression through mitosis because it is required to activate ubiquitin ligation by the anaphase-promoting complex (APC/C). Here we show in yeast that Cdc20 binds to the CCT chaperonin, which is known as a folding machine for actin and tubulin. The CCT is required for Cdc20's ability to bind and activate the APC/C. In vivo, CCT is essential for Cdc20-dependent cell cycle events such as sister chromatid separation and exit from mitosis. The chaperonin is also required for the function of the Cdc20-related protein Cdh1, which activates the APC/C during G1. We propose that folding of the Cdc20 family of APC/C activators is an essential and evolutionary conserved function of the CCT chaperonin.
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http://dx.doi.org/10.1016/s1097-2765(03)00244-2DOI Listing
July 2003