An Investigation of the Phase Separation and Interaction of Oskar and Vasa through Cellular Reconstitution of Drosophila Protein Condensates

Abstract

Germ granules are essential to species throughout the animal kingdom to sequester and target mRNA to the future germline. In doing so, they ensure proper germ cell specification and suppression of somatic gene expression in primordial germ cells. In Drosophila, germ granule formation begins with the localization of Oskar expression to the posterior pole of the oocyte. Oskar recruits other essential germ granule proteins, such as Vasa, and RNAs to form phase-transitioned condensates. Oskar and Vasa are fundamental components of germ granules, but it is not well understood how these proteins contribute to the formation of phase-separated condensates and, conversely, what is required for these proteins to phase separate. Here, I describe the reconstitution of protein condensates of Oskar and Vasa in Drosophila S2R+ cells. Mutagenic analyses were then conducted in this cellular reconstitution system to probe the roles of the canonical domains of Oskar and Vasa in their interaction and condensation ability. Confirming published data, the Oskar-LOTUS and Vasa-CTD domains were required for Oskar and Vasa colocalization. The RGG-rich region of Vasa was also required for the interaction of these two proteins, which was previously not known. Condensation ability was governed by Oskar’s LOTUS, IDR, and SGNH domains and the RGG-rich domain and amino acids 636-646 of Vasa. Interestingly, Vasa with a deletion of amino acids 636-646 consistently formed condensates in the absence of Oskar, suggesting that this 11-amino-acid sequence restricts ectopic condensation of Vasa. This thesis confirms the role of Oskar as the primary germ granule organizer but also suggests that other germ granule components could exert substantial effects on the dynamics of condensation. Further study of the condensation of germ granules, particularly through addition of RNA to this cellular reconstitution system, could reveal broader strategies that are employed in other types of condensates, such as disease-associated condensates.