Identifying Novel Regulators of Bucky ball Localization and Prion-like Nucleation During Balbani Body Phase-Separation in Zebrafish Oocytes Polarity

Vertebrate oocyte polarity along the animal-vegetal (AV) axis is essential for embryonic development. Oocyte polarity is established by the Balbiani body (Bb), a large phase-separated aggregate of mRNA-protein (mRNP) granules that is conserved from insects to humans. Loss of the Bb results in radially symmetrical eggs and embryonic lethality. Our lab identified the symmetry breaking events leading to Bb formation in zebrafish. Bb granules first localize to the centrosome at the onset of meiosis, where they subsequently nucleate to assemble the mature Bb. The only known essential protein for Bb formation is Bucky ball (Buc), an intrinsically disordered protein (IDP) with a prion-like domain. IDPs self-aggregate, triggering mRNP granule phase-separation, an emerging theme in biology that compartmentalizes components into membrane-less condensates. Our genetic analysis revealed that upon Buc localization to the centrosome, it specifically functions to nucleate Bb granules. In the mature Bb, we detected presumptive amyloid b-sheets that were absent in buc^-/- oocytes, providing the first genetic evidence for prion-like mechanisms in oocytes polarity. However, how Buc localizes to the centrosome and nucleates granules is unclear. Buc protein partners, targets, and upstream regulators are largely unknown. To understand Buc regulation, we are identifying regulators by immunoprecipitation (IP) and mass spectrometry (MS) analysis, using a fully functional transgenic Buc-GFP as bait. We developed a method for oocyte stage specific isolation, yielding hundreds of oocytes at Bb formation stages, all with Buc-GFP-positive Bbs. Our analysis will reveal regulators of physiological prion-like aggregation and mRNP phase-separation for the first time in vertebrates.