The general aim of our research projects is to gain a better understanding of cell cycle regulation in meiosis. We want to elucidate the mechanisms of chromosome segregation during female meiosis to understand why it goes wrong so frequently. Chromosomes are held together through a protein complex named Cohesin, which has to be removed in a step-wise manner during the two meiotic divisions. We study how arm cohesin is removed in meiosis I, and the centromere associated pool of Cohesin only in meiosis II. Chromosome segregation in meiosis also requires a specific orientation of chromosomes in meiosis I, and sister chromatids in meiosis II. The second aim of our projects is to elucidate how the so-named spindle assembly checkpoint, which prevents precocious Cohesin removal, recognizes and promotes correct attachments in meiosis. A third aim is to gain a better understanding of cell cycle regulation in oocyte meiosis, by studying oocyte-specific roles of A- and B-type cyclins. Global analysis of changes in the phospho-proteome is used to gain insights into how progression from meiosis I into meiosis II without intervening S-phase is achieved. The model system we are mainly using are mouse oocytes, but we also use complementary systems such as Xenopus laevis oocytes for biochemical approaches, and budding yeast for phospho-proteome analysis.
- 1) Protection and Deprotection of Centromeric Cohesin
Chromatid pairs are segregated in meiosis I, and single chromatids in meiosis II. To maintain chromatid pairs together throughout meiosis I, centromeric Cohesin has to be protected from proteolytic cleavage by Separase through Shugoshin 2 (Sgo)- dependent recruitment of PP2A. PP2A is a phosphatase that dephosphorylates a meiosis-specific subunit of the Cohesin complex and thereby prevents Cohesin removal by Separase. PP2A and Sgo2 are localised to the centromeres in meiosis I , but strikingly, they also co-localize in meiosis II. Therefore, localisation of Sgo2 and PP2A alone cannot explain why centromeric Cohesin is protected in meiosis I, and why this protection is lost in meiosis II ("deprotected"). We have found that the kinase Mps1 is required for Sgo2 dependent protection f centromeric cohesin. Furthermore we have identified I2PP2A/Set and Cyclin A2 as being essential for deprotection of centromeric Cohesin in oocytes. Very recently we discovered that Separase brings about sister kinetochore individualization at exit from meiosis I, and this kinetochore individualization is a prerequisite for sister chromatid segregation in the following meiosis II. Our current projects aim at identifying the signaling pathways required for protection and deprotection of Cohesin in female meiosis.
- 2) Spindle Assembly Checkpoint Control
Chromosome segregation in mitosis and meiosis depends on the activity of the APC/C (anaphase promoting complex/cyclosome), an E3 ubiquitin ligase that targets key cell cycle regulators for degradation. Degradation of Cyclin B and Securin is required for full activation of the protease Separase. Separase cleaves one subunit of the Cohesin complex (Scc1 in mitosis, Rec8 in meiosis) to allow the separation of paired sister chromatids in mitosis, and paired chromosomes in meiosis I. Activation of Separase depends on the inactivation of the Spindle Assembly Checkpoint or SAC. The SAC verifies that correct tension-generating attachments to the bipolar spindle are present on kinetochores. In the presence of erroneous attachments, metaphase to anaphase transition is prevented through the inhibition of the APC/C.
Chromosome attachment in meiosis I is fundamentally different from mitosis: two sister chromatids are oriented side-by-side towards the same pole (monopolar attachment), and not towards opposite poles (bipolar attachment). Nevertheless, we and others have been able to show that the SAC is active in meiosis I and components of the mitotic SAC are essential for generating oocytes of the correct ploidy. Our projects aim at elucidating the role of the SAC as oocytes progress from meiosis I into meiosis II. We address the role of SAC and the molecular link between error correction and SAC control in meiosis I and II.
- 3) Cell Cycle Control in Oocyte Meiosis
In meiosis, two cell divisions without intervening S-phase take place. Using mouse and X. laevis oocytes we are interested in understanding how cell cycle progression is regulated during the two divisions in oocytes, and how Separase is inactivated between meiosis I and II. We use both mouse and frog oocytes to determine the role of A-and B-type cyclins for meiotic progression. Furthermore we study how the meiotic cohesin Rec8 is recognized and cleaved by Separase in mouse oocyte meiosis I and II. To gain a more global understanding of cell cycle regulation at the transition from meiosis I into meiosis II we recently started to analyse the meiotic phosphoproteome using budding yeast. Our data will allow us to compare meiotic and mitotic datasets and dissect how cell division kinases and phosphatases bring about two cell divisions without intermediate S-phase in meiosis.