Biophysical Insights into the Phase Separation of a Nucleolar Protein Nucleophosmin 1

Abstract

Eukaryotic cells have been known to regulate thousands of reactions via compartmentalization of cellular components in membrane-bound organelles like the nucleus, mitochondria, etc. However, recent studies have shown that some of these compartments can further be organized into membrane-less organelles like the nucleolus.3 These membrane-less organelles comprise heterogeneous mixtures of proteins and nucleic acids which assemble through the mechanism of liquid-liquid phase separation. Phase separation is a form of phase transition where one solution spontaneously separates into two or more distinct immiscible phases, the dense and dilute phases. These phase-separated condensates are thought to be formed via transient, weak intermolecular forces that sequester intrinsically disordered proteins (IDPs) and several biomolecules and are responsible for the regulation of cellular functions.21 Biomolecular condensation has emerged as a paradigm for modulating the spatiotemporal coherence of the crowded cellular milieu. This control depends on the localization of reaction components as concentrating certain components together can increase the reaction kinetics and vice-versa. The first membrane-less organelle to be studied is the nucleolus which is an essential component of cellular physiology, consisting of three sub-compartments: - The fibrillar center (FC), the dense fibrillar component (DFC), and the outermost Granular component (GC). vii Nucleophosmin 1 (Npm1) is the scaffold protein responsible for the liquid-like nature of the GC region. It is an RNA-binding protein, belonging to the Nucleophosmin family and is involved in the vectorial assembly of ribosomes, DNA damage repair, centrosome duplication, etc. Npm1 consists of a central intrinsically disordered region (IDR) rich in acidic and basic amino acid stretches, allowing the protein to undergo intra-molecular phase separation. However, mutations in the C-terminal domain of the protein are associated with almost one third of acute myeloid leukemia (AML) cases. Despite the relevance of this protein, the conformational dynamics underlying the liquid-like nature of the GC region are not very well known. In our study, we have worked on the full-length Npm1 as well as its biologically relevant mutants to understand how the biophysical properties of the protein vary for its different mutants. We further ventured into single-molecule studies to comment on the inter domain interactions in the case of monomeric full-length protein via smFRET. Our work can potentially provide a molecular description of the highly dynamic properties of nuclear assemblies and enhance our understanding of the role of biomolecular condensation in nuclear homeostasis.