Stress-Dependent Regulation of Nuclear Compartmentalization via Biomolecular Condensates

The complexity of living cells is characterized by numerous biochemical pathways organized into strictly coordinated networks occurring simultaneously to sustain homeostasis. This spatiotemporal organization is achieved thanks to the inhomogeneous intracellular milieu divided in well-defined subcellular compartments where specific cellular functions occur. The most recognizable and stable modality of compartmentalization are membrane-bound compartments, such as nucleus, endoplasmic reticulum, and mitochondria, each offering designated space and optimal biochemical conditions for crucial events. In addition to organelles, a different and more dynamic modality of compartmentalization is offered by specific macromolecular assemblies that despite lacking lipid membranes, can delimit subcellular domains with distinct biochemical characteristics. These domains are called membrane-less organelles(MLOs) or biomolecular condensates. At the basis of MLOs is the ability of proteins to dynamically aggregate likely due to intrinsically disordered regions, which provide structural plasticity. Biomolecular condensates rapidly concentrate and/or sequester proteins and other macromolecules(nucleic acids) and are involved in the regulation of a wide variety of cellular events in cytosol and nucleus. MLOs are critical hubs organizing rapid nuclear responses to stimuli including stress. Despite their dynamic nature, the modalities regulating MLO formation and plasticity remain elusive. Recent evidence identified a link between MLOs and cellular signalling by two distinct proteins primed to participate in MLO formation in response to increases in the second messenger cAMP. These findings opened the possibility that signalling contributes to spatial compartmentalization of the cell for a new level of subcellular regulation. As a ubiquitous and essential signal, the other major second messenger, calcium(Ca2+) exerts potent control over cellular homoeostasis and stress signaling. Its spatial and temporal fluctuations orchestrate essential adaptive responses and can be sensed by hundreds of calcium-binding proteins suggesting its role in driving or regulating biomolecular condensate plasticity. Some Ca2+-driven condensation events have been observed in cytoplasm; however, whether Ca2+ regulates nuclear compartmentalization via biomolecular condensates remains largely unexplored. In this thesis, we aimed to understand whether calcium acts as a nuclear stress sensor and elicit responses by altering nuclear function through biomolecular condensates. For this, we conceived and employed a multiparametric in silico analysis to identify proteins containing calcium-sensing and condensation-prone domains. Our screening identified 39 putative targets from which we characterized the Penta-EF-hand protein 1(PEF1). To experimentally validate Pef1's ability to respond to Ca2+ signals by forming condensates, we designed a workflow using biochemical and fluorescently tagged approaches to monitor behavior in response to calcium-mobilizing agents and nuclear stress drugs. To determine calcium-dependency we used chemical and genetically encoded calcium chelators. Results indicate PEF1 is present in the nucleus where, in response to Ca2+ increases, is able to form well defined structures. The dynamic nature of Pef1-based nuclear puncta was confirmed by FRAP experiments and hexanediol sensitivity tested liquid-like properties. Low throughput screening with antibodies against nuclear membraneless organelles indicated PEF1 participates in paraspeckles remodeling, a stress-responsive nuclear condensate, in response to DNA transcriptional stress. Overexpression of paraspeckle component PSPC1 caused dramatic nuclear accumulation of PEF1. Ultimately, our findings depict PEF1 as a stress-recruited, calcium-tunable participant of paraspeckle-associated nuclear bodies.