Microtubules control Buc Phase separation and Balbiani body condensation in zebrafish oocyte polarity

R. Deis, Y.M. Elkouby

Research output: Working paperPreprint


Molecular condensates provide new paradigms in biology, but their cellular regulation is unclear. Condensates undergo phase separation, decreasing their solubility and compartmentalizing their content. In vertebrate oocytes, RNA-protein (RNP) granules form condensates by phase separation, but the underlying mechanisms are unknown. RNP granules localize to the Balbiani body (Bb), a conserved membraneless organelle, that establishes oocyte polarity. Bb loss results in symmetrical eggs and embryonic lethality. Bb granules aggregate around the centrosome in a nuclear cleft, prior to assembling the mature structure. The Bucky ball (Buc) protein nucleates Bb granules and is essential for Bb formation. Howe ver, the dynamics, mechanisms, and regulation of Bb granule nucleation are unclear. While the mature Bb structure was shown to be a rigid, amyloid-like, phase-separated structure in Xenopus, the early phase separation dynamics prior to maturation are completely unknown. Here, by live, genetic, super-resolution microscopy, and FRAP analyses in zebrafish ovaries, we establish that Buc phase-separates Bb granules and that microtubules play multiple stepwise roles in controlling Buc phase separation and Bb formation specifically at the early nuclear cleft stages. We show that Buc first phase-separates into dynamic liquid droplet-like granules that fuse to form the main Bb aggregate. We demonstrate that early aggregated Buc exhibited dynamic turnover and that this turnover requires dynein-mediated trafficking of Buc on a transient lattice of microtubules that we identified. At later stages, microtubules encapsulated the Bb, indicating a structural role. Thus, microtubules organize multiple steps in Bb condensation. Moreover, in the mature Bb, Buc was stable and required for Bb amyloid formation, finalizing Bb condensation. We found for the first time by live imaging, ThT-positive presumptive amyloid ß-sheets in the mature zebrafish Bb, that we re absent in buc-/- oocytes. Thus, providing the first genetic evidence for Buc dependent formation of presumptive amyloid fibrils in the Bb. Molecular condensation is often viewe d as a self-assembly process. Here, we propose a novel paradigm for the cellular control over condensation mechanisms by microtubules in development. The regulation of Bb assembly and disassembly in the context of phase separation allows studying these mechanisms in physiological conditions, advancing their understanding in neurodegenerative disease and female reproduction.
Original languageEnglish
StatePublished - 13 Mar 2022


  • Balbiani body
  • Xenopus
  • adult
  • amyloid
  • animal experiment
  • animal model
  • cell organelle
  • centrosome
  • controlled study
  • degenerative disease
  • dynein adenosine triphosphatase
  • embryo
  • endogenous compound
  • female
  • lethality
  • microscopy
  • microtubule
  • nonhuman
  • oocyte
  • phase separation
  • protein function
  • reproduction
  • turnover rate
  • vertebrate
  • zebra fish


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