In Vitro Reconstitutions and Functional Characterizations of Fibrillar Centers of Nucleoli

Abstract

The nucleolus is the largest membraneless biomolecular condensate in cells. It plays a central role in the synthesis of ribosomal subunits. Nucleoli form via spontaneous phase transitions that are driven by key nucleolar protein and RNA molecules. Each nucleolus has a core-shell architecture that is organized around an inner fibrillar center (FC) and an outer granular component (GC). The most abundant nucleolar proteins are nucleophosmin 1 (NPM1) in the GC and nucleolin (NCL) in the FC. We quantified the spatial distribution of NCL and NPM1 by co-expressing these proteins in Xenopus oocytes. As expected, we find that NCL is concentrated in the FC of the nucleolus and depleted in the GC, with the converse being true of NPM1. We used in vitro reconstitutions to show that GFP-tagged NCL forms condensates in the presence of a key binding partner protein Ly-1 Antibody Reactive (LYAR) and RNA. NCL has the curious feature of undergoing auto-proteolysis and LYAR inhibits this auto-proteolysis of NCL. The condensates formed using the minimal ternary system show an intriguing layered structure with NCL at the core of the condensate. Therefore, the minimal system recapitulates the structural organization of bona fide FCs. We report results from a detailed analysis of the driving forces that control the assembly of minimal FCs. Further, we query the impact of designed changes to intrinsically disordered RG-rich domains in NCL on the phase behavior, material properties, and functions of reconstituted FCs. Our results pave the way for understanding how condensates form, how they are regulated, and how material properties impact functions controlled by condensates such as nucleoli.