The distinctive feature of several devastating neurological diseases (tauopathies), including Alzheimer’s disease and frontotemporal dementia, consists of the anomalous aggregation of the tau protein in the brain. Broadly speaking, the pathological modifications of the tau protein (path-Tau) — point mutations and hyperphosphorylation — alter its interaction with microtubules (MTs) and promote its pathological aggregation (with loss- and gain-of-function, respectively). Recent studies suggest that the biomolecular condensation of the tau protein via liquid-liquid phase separation (LLPS) is a critical step in its function and pathology. The subsequent aberrant phase transitions of the droplets formed into gel- or solid-type aggregates may play a pivotal role in the progression of tauopathies. Since the initial studies of tau LLPS dating from 2017, there are several works indicating that the tau protein in the presence of crowding agents or of RNA and RNA-binding proteins (RBPs) has a high tendency to undergo condensation, forming liquid droplets. The tau protein is intrinsically disordered (IDP), possessing an asymmetric charge distribution (it presents charged regions in different domains), and the LLPS process is mainly conditioned by the establishment of electrostatic interactions. The hyperphosphorylation of tau also appears to be a determining factor of tau LLPS. Most studies have focused on the post-translational modifications (PTMs) of the tau protein, and so the role played by the point mutations associated with primary tauopathies is still enigmatic and controversial. Overall, the pathological mutations of the tau protein appear to reduce its dynamics within the droplets and increase its transition into insoluble aggregates. Another topic that remains to be clarified is whether the tau protein acts as an inducer of its complex coacervation or whether it has only a passive role, being sequestered in the biomolecular condensates formed by RNA and RBPs. A detailed understanding of the main determinants of the complex coacervation of the tau protein and of how the WT and path-tau variants interact with RNA and RBPs is thus crucial in order to clarify the initial phase of the heterotypic LLPS associated with the tau protein. However, the characterization of the dynamic/heterogeneous features of these interactions remains a challenge due to the intrinsic tendency of the tau protein to self-associate. In order to overcome these problems, in this project advanced fluorescence microscopy techniques (mainly at the single-molecule level) will be used to characterize the initial steps involved in the tau-RNA/RBPs interactions, the complex coacervation of path-tau, and the key structural alterations of path-tau that lead to its phase separation. The innovative character of our proposal involves characterizing the initial steps of interaction of the WT tau protein and path-tau with RNAs (of various sequences and lengths) and with the T-cell intracellular antigen 1 (TIA1) and the GRBP1 protein, using fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) (Tasks 1 and 2, respectively). In Task 3, the complex coacervation process of path-tau with RNA and RBPs will be studied, as well as the dynamics of this protein in the droplets formed, by FCS and fluorescence anisotropy measurements. The results obtained will be correlated with possible transitions of the tau protein into amyloid-type fibrils (Thioflavin T assays). The application of the single-molecule FRET technique in the characterization of the dynamics/conformational properties of the complex coacervates established between the tau protein and RNA and TIA1/G3BP1 will be carried out in Task 4, reinforcing the innovative character of this project. This project brings together the comprehensive experience of a multidisciplinary team in the areas of IDPs, RNA production, and single-molecule fluorescence techniques. The project benefits from the prior experience of the PI in the application of single-molecule methodologies in the study of the function and dysfunction of aggregation-prone proteins (including the tau protein). The assembled team also masters RNA production strategies, which will allow the exploration of a wide variety of RNA sequences and chain lengths in a systematic way. This project will contribute to obtaining a complete description of the initial steps involved in the tau-RNA, tau-TIA1/G3BP1 interactions, which are an ideal clinical target for preventing the progression of tauopathies. The detailed characterization of the physical and structural features of the path-Tau variants in the droplets and of their transition into toxic aggregates may drive the future development of new therapeutic approaches aimed at delaying or reversing the transition of the droplets into toxic aggregates/oligomers.