Regulation and cellular functions of the Arp2/3 nucleation complex in actin-driven chromosome transport in oocyte meiosis

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Oocyte meiosis is a highly specialized form of cell division that produces the fertilizable egg, and is therefore essential for sexual reproduction of all animal species and is highly relevant to human health. Indeed, aneuploidy resulting from errors in oocyte meiosis is the leading cause of pregnancy loss and birth defects in humans that is developing into a major social issue with increasing maternal age in Western countries.Oocyte meiosis is specialized, because oocytes are very large and divide with extreme asymmetry in order to preserve accumulated nutrients to support early embryonic development. As it recently emerged from studies in various animal model species, the actin cytoskeleton has major, conserved functions in transporting and positioning chromosomes in the exceptionally large cytoplasm of oocytes – functions that are mediated by microtubules in small somatic cells. Recent studies in mouse oocytes and Xenopus egg extracts, as well as our preliminary data in starfish oocytes showed that the Arp2/3 complex has important and conserved roles in these processes. As shown in mouse oocytes by others and as indicated by our preliminary data in starfish oocytes, in this cellular context the Arp2/3 complex is recruited and activated by RanGTP produced locally on chromosomes. This is the first known mechanism that links actin nucleation to chromatin, and is therefore likely to be key to actin-driven transport and positioning of chromosomes in oocytes. How RanGTP recruits and activates the Arp2/3 complex is not at all understood.In the course of the first funding period of the Priority Programme SPP 1464 we established starfish oocytes as an experimental system for purification and biochemical and cellular characterization of F-actin regulators. In this project we will build on this experimental system to purify and identify the molecules involved in RanGTP-mediated activation of Arp2/3. To this end, we will combine assays that had been developed to identify microtubule regulators activated by RanGTP with purification of F-actin interactors. We will validate these newly identified candidates in live cell assays in starfish oocytes and further characterize their cellular functions. Simultaneously, we will biochemically characterize these newly identified regulators of Arp2/3 in in vitro nucleation assays.It is widely accepted that RanGTP has key functions in organizing the microtubule spindle around chromosomes. The major aim of the proposed project is to establish at the molecular and cellular level that RanGTP has equally important functions in regulating the Arp2/3 complex to nucleate actin on chromatin and thereby driving chromosome transport and positioning in large oocytes, a mechanism essential to prevent aneuploidy of eggs.