Function of Cyclin-B3 in oocytes

• Applicant: Professor Dr. Thomas Mayer
• Subject Area: Cell Biology
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 456916924

Cyclins are key regulator of the cell cycle because they associate with and activate cyclin-dependent kinases (Cdks). Female mice genetically ablated for cyclin-B3 (Ccnb3-/-) are viable, but sterile. The cause for sterility is that Ccnb3-/- oocytes arrest at metaphase of the first meiotic division (MI) due to inefficient activation of the ubiquitin ligase APC/C. Notably, this defect can be rescued by the expression of not only mouse cyclin-B3, but also cyclin-B3 from Xenopus laevis indicating that the function of cyclin-B3 is evolutionary conserved. The overarching objective of this project is to dissect the molecular function of cyclin-B3 in female meiosis I. To accomplish this, one approach will be to combine Xenopus egg extract studies with in vitro reconstitution experiments. Xenopus egg extract is an ideal system because it readily allows the expression and removal of proteins by the addition of mRNA and immunodepletion, respectively, as well as detailed time-resolved cell cycle analyses. In vitro assays will reveal which cell cycle proteins are directly regulated by Cdk/cyclin-B3. Structure determination of mammalian cyclin-B3 by our collaboration partner Andreas Boland will be key to understand substrate specificity of Cdk/cyclin-B3. In a parallel, second approach we will translate the mechanistic insights to the situation in intact Xenopus oocytes. These efforts will be accompanied by studies in mouse oocytes, performed by our collaboration partner Katja Wassmann. Based on our extensive preliminary results, we postulate that cyclin-B3 is required for full APC/C activation in meiosis by mediating the degradation of the APC/C inhibitor XErp1/Emi2. To validate and refine this model, we will generate methods to prevent the expression of cyclin-B3 and/or XErp1 and express mutant variants of these proteins in Xenopus oocytes. The resulting functional consequences will be investigated by detailed biochemical as well as microscopic analyses. Due to our longstanding interest in cell cycle research using Xenopus as a model system, all reagents and expertise to perform these experiments are available. The combination of a panel of complementary experimental approaches and different model organisms will enable us to dissect the molecular function of cyclin-B3 during female meiosis. This knowledge will be key to understand not only the different function of the various B-type cyclins in meiosis, but also for our general understanding of the regulatory mechanisms underlying cell cycle progression.

• DFG Programme: Research Grants