Research Projects

Cellular compartmentalization, traditionally attributed to membrane-bounded organelles, has expanded to include membraneless biomolecular condensates formed through phase separation. These condensates, comprising proteins and nucleic acids, are critical for spatiotemporal regulation of cellular activities. However, their aberrant phase transitions, often induced by mutations or prolonged physiological stresses, result in pathological aggregates associated with neurodegeneration. Tau, a neuronal intrinsically disordered protein, exemplifies such behavior. While primarily responsible for stabilizing microtubules, tau also undergoes phase separation to control tubulin polymerization. Stress-induced alterations can convert tau condensates into amyloid-like aggregates, a hallmark of tauopathies. Recent studies reveal that molecular chaperones prevent pathological tau aggregation, while the nucleolus acts as a protein quality control compartment during proteotoxic stress, sequestering misfolded proteins for refolding or degradation. In the proposed study, we will focus on elucidating the role of nucleolin (NCL), found in the dense fibrillar component (DFC) of the nucleolus, in modulating the phase behavior of tau. NCL can form liquid-like condensates in the presence of rRNA and is believed to be the main driver for the formation of the DFC compartment, contributing to the multiphasic nature of the nucleolus. Given the ability of tau to undergo phase separation and its colocalization with NCL in the nucleolus, we hypothesize that, under conditions of cellular stress, tau translocates from the cytoplasm to the nucleolus. Within the nucleolus, NCL, in the presence of rRNA, modulates the phase behavior of tau, potentially influencing aggregation propensity. For the abovementioned, we aim to express and purify both the wild-type and disease-associated variants of tau and NCL using bacterial expression systems and will study their heterotypic condensation in vitro. Subsequently, single-molecule studies on tau-NCL droplets will uncover the molecular details, including conformational and rotational chain dynamics of tau in the presence of NCL and rRNA. Furthermore, using fluorescently-tagged tau and NCL, we will examine in-cell condensate formation and concurrently conduct fluorescence-based studies on live cells to ascertain the material properties and cellular dynamics of the resulting condensates, which will elucidate the physiological factors influencing the condensation and aggregation of tau. By integrating cell-free and in-cellulo approaches, our research will shed light on the physiological significance of the nucleocytoplasmic shuttling of tau under stress and the role of a DFC nucleolar protein, NCL in preventing the formation of its irreversible aggregates. These molecular insights will enhance our understanding of the potential role of nucleolar DFC in mitigating neurodegenerative diseases, offering avenues for therapeutic intervention.