Thomas Tischer - Publications

Calcineurin promotes APC/C activation at meiotic exit by acting on both XErp1 and Cdc20.

Andreas Heim, Thomas Tischer, Thomas U. Mayer

Vertebrate oocytes await fertilization arrested at metaphase of the second meiotic division. Fertilization triggers a transient calcium wave, which induces the activation of the anaphase-promoting complex/cyclosome (APC/C) and its co-activator Cdc20 resulting in the destruction of cyclin B and hence meiotic exit. Two calcium-dependent enzymes are implicated in fertilization-induced APC/CCdc20 activation: calcium-/calmodulin-dependent kinase type II (CaMKII) and calcineurin (CaN). While the role of CaMKII in targeting the APC/C inhibitor XErp1/Emi2 for destruction is well-established, it remained elusive how CaN affects APC/CCdc20 activation. Here, we discover that CaN contributes to APC/CCdc20 activation in Xenopus laevis oocytes by two independent but interrelated mechanisms. First, it facilitates the degradation of XErp1 by dephosphorylating it at a site that is part of a phosphorylation-dependent recruiting motif for PP2A-B'56, which antagonizes inhibitory phosphorylation of XErp1. Second, it dephosphorylates Cdc20 at an inhibitory site, thereby supporting its APC/C-activating function. Thus, our comprehensive analysis reveals that CaN contributes to timely APC/C activation at fertilization by both negatively regulating the APC/C inhibitory activity of XErp1 and positively regulating the APC/C-activating function of Cdc20.

EMBO Reports. 2018 December 19, doi:10.15252/embr.201846433

 

Xenopus laevis Kif18A is a highly processive kinesin required for meiotic spindle integrity.

Martin M. Möckel, Andreas Heim, Thomas Tischer, Thomas U. Mayer

The assembly and functionality of the mitotic spindle depends on the coordinated activities of microtubule-associated motor-proteins of the dynein and kinesin superfamily. Our current understanding of the function of motor-proteins is significantly shaped by studies using Xenopus laevis egg extract as its open structure allows complex experimental manipulations hardly feasible in other model systems. Yet, the Kinesin-8 orthologue of human Kif18A has not been described in Xenopus laevis so far. Here, we report the cloning and characterization of Xenopus laevis (Xl) Kif18A. Xenopus Kif18A is expressed during oocyte maturation and its depletion from meiotic egg extract results in severe spindle defects. These defects can be rescued by wildtype Kif18A, but not Kif18A lacking motor-activity or the C-terminus. Single molecule microscopy assays revealed that Xl_Kif18A possesses high processivity, which depends on an additional C-terminal microtubule-binding site. Human tissue culture cells depleted of endogenous Kif18A display mitotic defects, which can be rescued by wildtype, but not tail-less Xl_Kif18A. Thus, Xl_Kif18A is the functional orthologue of human Kif18A whose activity is essential for the correct function of meiotic spindles in Xenopus oocytes.

Biology Open. 2017 Feb 22. doi: 10.1242/bio.023952

 

The Phosphatase Dusp7 Drives Meiotic Resumption and Chromosome Alignment in Mouse Oocytes.

Thomas Tischer and Melina Schuh

Mammalian oocytes are stored in the ovary, where they are arrested in prophase for prolonged periods. The mechanisms that abrogate the prophase arrest in mammalian oocytes and reinitiate meiosis are not well understood. Here, we identify and characterize an essential pathway for the resumption of meiosis that relies on the protein phosphatase DUSP7. DUSP7-depleted oocytes either fail to resume meiosis or resume meiosis with a significant delay. In the absence of DUSP7, Cdk1/CycB activity drops below the critical level required to reinitiate meiosis, precluding or delaying nuclear envelope breakdown. Our data suggest that DUSP7 drives meiotic resumption by dephosphorylating and thereby inactivating cPKC isoforms. In addition to controlling meiotic resumption, DUSP7 has a second function in chromosome segregation: DUSP7-depleted oocytes that enter meiosis show severe chromosome alignment defects and progress into anaphase prematurely. Altogether, these findings establish the phosphatase DUSP7 as an essential regulator of multiple steps in oocyte meiosis.

Cell Reports. 2016 Oct 25;17(5):1426-1437. doi: 10.1016/j.celrep.2016.10.007

 

Live imaging RNAi screen reveals genes essential for meiosis in mammalian oocytes

Sybille Pfender*, Vitaliy Kuznetsov*, Micha? Pasternak*, Thomas Tischer, Balaji Santhanam, Melina Schuh

During fertilization, an egg and a sperm fuse to form a new embryo. Eggs develop from oocytes in a process called meiosis. Meiosis in human oocytes is highly error-prone and defective eggs are the leading cause of pregnancy loss and several genetic disorders such as Down’s syndrome. Which genes safeguard accurate progression through meiosis is largely unclear. Here we develop high-content phenotypic screening methods for the systematic identification of mammalian meiotic genes. We targeted 774 genes by RNA interference within follicle-enclosed mouse oocytes to block protein expression from an early stage of oocyte development onwards. We then analysed the function of several genes simultaneously by high-resolution imaging of chromosomes and microtubules in live oocytes and scored each oocyte quantitatively for 50 phenotypes, generating a comprehensive resource of meiotic gene function. The screen generated an unprecedented annotated data set of meiotic progression in 2,241 mammalian oocytes, which allowed us to analyse systematically which defects are linked to abnormal chromosome segregation during meiosis, identifying progression into anaphase with misaligned chromosomes as well as defects in spindle organization as risk factors. This study demonstrates how high-content screens can be performed in oocytes, and allows systematic studies of meiosis in mammals.

Nature. 2015 Aug 13;524(7564):239-42. doi: 10.1038/nature14568.

 

Fluorogenic ATP analogues for online monitoring of ATP consumption: observing ubiquitin activation in real time.

Hacker SM, Pagliarini D, Thomas Tischer, Hardt N, Schneider D, Mex M, Mayer TU, Scheffner M, Marx A.

Many enzymes use ATP in signal-transducing processes or as an energy source. New fluorogenic ATP analogues signal ATP consumption by ubiquitin-like protein-activating enzymes in real time. Thus the inhibition and stimulation of these ATP-processing enzymes can be studied without auxiliary enzymes and reagents. β-Lapachone was identified as an inhibitor of the ubiquitin-activating enzyme UBA1.

Angew Chem Int Ed Engl. 2013 Nov 4;52(45):11916-9. doi: 10.1002/anie.201304723.

 

Modulation of cell cycle control during oocyte-to-embryo transitions.

Eva Hörmanseder*, Thomas Tischer, Thomas U. Mayer.

Ex ovo omnia - all animals come from eggs - this statement made in 1651 by the English physician William Harvey marks a seminal break with the doctrine that all essential characteristics of offspring are contributed by their fathers, while mothers contribute only a material substrate. More than 360 years later, we now have a comprehensive understanding of how haploid gametes are generated during meiosis to allow the formation of diploid offspring when sperm and egg cells fuse. In most species, immature oocytes are arrested in prophase I and this arrest is maintained for few days (fruit flies) or for decades (humans). After completion of the first meiotic division, most vertebrate eggs arrest again at metaphase of meiosis II. Upon fertilization, this second meiotic arrest point is released and embryos enter highly specialized early embryonic divisions. In this review, we discuss how the standard somatic cell cycle is modulated to meet the specific requirements of different developmental stages. Specifically, we focus on cell cycle regulation in mature vertebrate eggs arrested at metaphase II (MII-arrest), the first mitotic cell cycle, and early embryonic divisions.

EMBO J. 2013 Aug 14;32(16):2191-203. doi: 10.1038/emboj.2013.164.

 

The APC/C inhibitor XErp1/Emi2 is essential for Xenopus early embryonic divisions.

Thomas Tischer*, Eva Hörmanseder, Thomas U. Mayer.

Mitotic divisions result from the oscillating activity of cyclin-dependent kinase 1 (Cdk1). Cdk1 activity is terminated by the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets cyclin B for destruction. In somatic divisions, the early mitotic inhibitor 1 (Emi1) and the spindle assembly checkpoint (SAC) regulate cell cycle progression by inhibiting the APC/C. Early embryonic divisions lack these APC/C-inhibitory components, which raises the question of how those cycles are controlled. We found that the APC/C-inhibitory activity of XErp1 (also known as Emi2) was essential for early divisions in Xenopus embryos. Loss of XErp1 resulted in untimely destruction of APC/C substrates and embryonic lethality. XErp1's APC/C-inhibitory function was negatively regulated by Cdk1 and positively by protein phosphatase 2A (PP2A). Thus, Cdk1 and PP2A operate at the core of early mitotic cell cycles by antagonistically controlling XErp1 activity, which results in oscillating APC/C activity.

Science. 2012 Oct 26;338(6106):520-4. doi: 10.1126/science.1228394.

 

Non-proteolytic ubiquitylation counteracts the APC/C-inhibitory function of XErp1.

Eva Hörmanseder*, Thomas Tischer*, Simone Heubes, Olaf Stemmann, Thomas U. Mayer.

Mature Xenopus oocytes are arrested in meiosis by the activity of XErp1/Emi2, an inhibitor of the ubiquitin-ligase anaphase-promoting complex/cyclosome (APC/C). On fertilization, XErp1 is degraded, resulting in APC/C activation and the consequent degradation of cell-cycle regulators and exit from meiosis. In this study, we show that a modest increase in the activity of the ubiquitin-conjugating enzyme UbcX overrides the meiotic arrest in an APC/C-dependent reaction. Intriguingly, XErp1 remains stable in these conditions. We found that UbcX causes the ubiquitylation of XErp1, followed by its dissociation from the APC/C. Our data support the idea that ubiquitylation regulates the APC/C-inhibitory activity of XErp1.

EMBO Rep. 2011 May;12(5):436-43. doi: 10.1038/embor.2011.32.

*equal contribution