Multi-Step Cellular Control of Molecular Condensation by Microtubules in Early Oogenesis

Abstract

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 b-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.

Tools to analyze the organization and formation of the germline cyst in zebrafish oogenesis

“…Additional advantages of the zebrafish model include the high accessibility of developing ovaries that are present in swimming juvenile fish, and the flat and transparent anatomy of the ovary, which is ideal for advanced quantitative and live microscopy, as we demonstrated previously ( Elkouby and Mullins, 2017a , b ; Elkouby et al, 2016 ; Mytlis and Elkouby, 2021 ; Deis and Elkouby, 2022 preprint; Mytlis et al, 2022 ), and in this work. Furthermore, in zebrafish germline stem cells are maintained and actively produce oocytes throughout life and during regeneration ( Beer and Draper, 2013 ; Cao, et al, 2019 ).…”

“…Owing to multiple experimental advantages, the zebrafish ovary is as an excellent model for the study of oogenesis ( Elkouby and Mullins, 2017b ; Li and Ge, 2020 ), and the toolbox for the investigation of zebrafish oogenesis has been significantly expanded. Advances in genetics ( Jamieson-Lucy et al, 2022 ; Leerberg et al, 2019 ; Beer and Draper, 2013 ), quantitative and live ovarian imaging ( Mytlis et al, 2022 ; Mytlis and Elkouby, 2021 ), live manipulations of cultured ovaries ( Deis and Elkouby, 2022 preprint), and various proteomic and genomic approaches ( Jamieson-Lucy et al, 2022 ; Liu et al, 2022 ; Bogoch et al, 2022 ) have impacted the field greatly. Nevertheless, a fundamental understanding of the cyst is still needed.…”