Rafael L. Giner-Arroyo

Nucleophosmin (NPM1) is a multifunctional nucleolar protein whose integration into the nucleolus relies on multivalent interactions that drive liquid-liquid phase separation (LLPS). Acetylation of Lys27 and Lys54 within the N-terminal oligomerization domain of NPM1 has been implicated in its nucleolar redistribution during the DNA damage response, but the molecular mechanisms underlying this redistribution remain unclear. Here, we show that Lys27/Lys54 acetylation facilitates NPM1 anchoring in the nucleolus under basal conditions by promoting LLPS. Despite preserving NPM1 structural integrity and oligomerization equilibrium, lysine acetylation reshapes the electrostatic surface potential of the N-terminal domain, neutralizing a predominantly basic region and exposing a previously cryptic acidic surface. This increased charge blockiness enhances the ability of NPM1 to undergo both homotypic and heterotypic LLPS. Thus, lysine acetylation acts as a modulator of NPM1 interaction landscape, promoting its integration into nucleolar condensates despite not constituting the primary determinant of its localization.

Nucleophosmin (NPM1) is a multifunctional nucleolar protein essential for ribosome biogenesis, genome stability, and stress responses. Its integration into the nucleolus depends on its oligomerization and multivalent interactions that enable liquid–liquid phase separation (LLPS). Here, we investigate how tyrosine phosphorylation at Tyr17, Tyr29, and Tyr67 located within the interface between monomers at the N-terminal oligomerization domain regulates NPM1 structure and function. Replacing tyrosines with p- carboxymethyl-L-phenylalanine as phosphomimetic substitutions, we show that phosphorylation at Tyr17 and Tyr67 disrupts key intermonomer interactions and destabilizes the NPM1 pentameric assembly, which drives an order-to-disorder transition and impairs binding to nuclear partners. Consequently, dual phosphorylation at Tyr17 and Tyr67 disturbs both homotypic and heterotypic LLPS, thereby impairing incorporation of NPM1 into the nucleolus. This altered localization of NPM1 serves as a hallmark of p53 activation, driven both by nucleoplasmic NPM1 and by the release of ARF from NPM1-dependent sequestration in the nucleolus. Altogether, our results provide a molecular mechanistic explanation on how phosphorylation-induced structural and dynamic changes drive the release of NPM1 from the nucleolus under genotoxic stress.

Metals play an essential role in cellular homeostasis and are key components of several formulations currently used in the clinic. Synchrotron‐based X‐ray microscopy at submicron resolution is a powerful approach to map intracellular elemental distributions and to monitor how these patterns change upon genetic or pharmacological perturbations. However, existing sample‐preparation protocols often rely on costly and highly specialized equipment for vitrification and dehydration, limiting their widespread adoption. Here, we present an adapted plunge‐freezing and freeze‐drying workflow that enables the preparation of mammalian cell samples for X‐ray fluorescence (XRF) and X‐ray absorption spectroscopy (XAS) studies with submicron resolution in a cost‐effective and versatile manner. Furthermore, we define acquisition parameters optimized for the reliable detection of low‐abundance metals, such as endogenous iron. We anticipate that this accessible protocol will facilitate the broader implementation of synchrotron‐based inner‐shell spectromicroscopy in cell biology.

Nucleophosmin (NPM1) is a nucleolar protein commonly mutated in ~30% of newly diagnosed acute myeloid leukemia (AML) cases. These mutations occur in the terminal exon of the NPM1 gene, affecting the C-terminal DNA-binding domain of the protein and causing its delocalization to the cytoplasm—a hallmark of NPM1-mutated AML. NPM1 shuttling to the nucleoplasm is tightly regulated by posttranslational modifications, such as phosphorylation of Ser254, Ser260, and Tyr271 of the DNA-binding domain. However, the structural mechanisms underlying this process remain unclear. In this work, we show that Ser-to-Asp (S254D–S260D) and Tyr-to-pCMF (para-carboxymethyl phenylalanine) (Y271pCMF) phosphomimetic mutations induce significant structural and dynamical rearrangements, as well as drastic modifications in electrostatic surface potential. These changes compromise recognition of a G-quadruplex sequence from the c-MYC promoter by reducing DNA-binding affinity, reshape histone capturing dynamics, and fade charge segregation in the histone-binding domain. Combination of such substitutions in a triple phosphomimetic variant (S254D–S260D–Y271pCMF) further destabilizes the domain’s structure and triggers protein aggregation. Altogether, these findings suggest that phosphorylation of Ser254, Ser260, and Tyr271 of the C-end DNA-binding domain weakens both DNA affinity and charge block-driven liquid–liquid phase separation, offering a molecular explanation for the delocalization of NPM1 outside of the nucleolus.