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Tau is an intrinsically disordered protein that binds and stabilizes axonal microtubules (MTs) in neurons of the central nervous system. The binding of Tau to MTs is mediated by its repeat domain and flanking proline-rich domains. The positively charged (basic) C-terminal half of Tau also mediates the assembly Tau into fibrillar aggregates in Alzheimer's disease (AD) and tauopathy brains. In recent years, another assembly form of Tau has been identified: Tau can undergo liquid-liquid phase separation (LLPS), which leads to its condensation into liquid-dense phases, either by complex coacervation with polyanions like heparin or RNA or through "self-coacervation" at high Tau concentrations. Condensation of Tau in the absence of polyanions can be enhanced by the presence of molecular crowding agents in a dilute Tau solution. In vitro experiments using recombinant purified Tau are helpful to study the physicochemical determinants of Tau LLPS, which can then be extrapolated into the cellular context. Tau condensation is a new aspect of Tau biology that may play a role for the initiation of Tau aggregation, but also for its physiological function(s), for example, the binding to microtubules. Here we describe how to study the condensation of Tau in vitro using light microscopy, including fluorescence recovery after photobleaching (FRAP), to assess the shape and molecular diffusion in the condensates, a proxy for the degree of condensate percolation. We also describe turbidity measurements of condensate-containing solutions to assess the overall amount of LLPS and time-resolved dynamic light scattering (trDLS) to study the formation and size of Tau condensates.

Biomolecular condensation of the neuronal microtubule-associated protein Tau (MAPT) can be induced by coacervation with polyanions like RNA, or by molecular crowding. Tau condensates have been linked to both functional microtubule binding and pathological aggregation in neurodegenerative diseases. We find that molecular crowding and coacervation with RNA, likely coexisting in the cytosol, synergize to enable Tau condensation at physiological buffer conditions and produce condensates with a strong affinity to charged surfaces. During condensate-mediated microtubule polymerization, this synergy enhances bundling and spatially arranges microtubules. We further show that different Tau condensates efficiently induce pathological Tau in cells, including small accumulations at the nuclear envelope that correlate with nucleocytoplasmic transport deficits. Fluorescent lifetime imaging reveals different molecular packing densities of Tau in cellular accumulations, and a condensate-like density for nuclear envelope Tau. These findings suggest that a complex interplay between interaction partners, post-translational modifications, and molecular crowding regulates the formation and function of Tau condensates. Conditions leading to prolonged existence of Tau condensates may induce the formation of seeding-competent Tau and lead to distinct cellular Tau accumulations.

Biomolecular condensation of the neuronal microtubule-associated protein Tau (MAPT) can be induced by coacervation with polyanions like RNA, or by molecular crowding. Tau condensates have been linked to both functional microtubule binding and pathological aggregation in neurodegenerative diseases. We find that molecular crowding and coacervation with RNA, two conditions likely coexisting in the cytosol, synergize to enable Tau condensation at physiological buffer conditions and to produce condensates with a strong affinity to charged surfaces. During condensate-mediated microtubule polymerization, their synergy enhances bundling and spatial arrangement of microtubules. We further show that different Tau condensates efficiently induce pathological Tau aggregates in cells, including accumulations at the nuclear envelope that correlate with nucleocytoplasmic transport deficits. Fluorescent lifetime imaging reveals different molecular packing densities of Tau in cellular accumulations and a condensate-like density for nuclear-envelope Tau. These findings suggest that a complex interplay between interaction partners, post-translational modifications, and molecular crowding regulates the formation and function of Tau condensates. Conditions leading to prolonged existence of Tau condensates may induce the formation of seeding-competent Tau and lead to distinct cellular Tau accumulations.

Tau is a neuronal protein that stabilizes axonal microtubules (MTs) in the central nervous system. In Alzheimer’s disease (AD) and other tauopathies, phosphorylated Tau accumulates in intracellular aggregates, a pathological hallmark of these diseases. However, the chronological order of pathological changes in Tau prior to its cytosolic aggregation remains unresolved. These include its phosphorylation and detachment from MTs, mislocalization into the somatodendritic compartment, and oligomerization in the cytosol. Recently, we showed that Tau can interact with phenylalanine-glycine (FG)-rich nucleoporins (Nups), including Nup98, that form a diffusion barrier inside nuclear pore complexes (NPCs), leading to defects in nucleocytoplasmic transport. Here, we used surface plasmon resonance (SPR) and bio-layer interferometry (BLI) to investigate the molecular details of Tau:Nup98 interactions and determined how Tau phosphorylation and oligomerization impact the interactions. Importantly, phosphorylation, but not acetylation, strongly facilitates the accumulation of Tau with Nup98. Oligomerization, however, seems to inhibit Tau:Nup98 interactions, suggesting that Tau-FG Nup interactions occur prior to oligomerization. Overall, these results provide fundamental insights into the molecular mechanisms of Tau-FG Nup interactions within NPCs, which might explain how stress-and disease-associated posttranslational modifications (PTMs) may lead to Tau-induced nucleocytoplasmic transport (NCT) failure. Intervention strategies that could rescue Tau-induced NCT failure in AD and tauopathies will be further discussed.

BiP is the endoplasmic member of the Hsp70 family. BiP is regulated by several co-chaperones including the nucleotide-exchange factor (NEF) Bap (Sil1 in yeast). Bap is a two-domain protein. The interaction of the Bap C-terminal domain with the BiP ATPase domain is sufficient for its weak NEF activity. However, stimulation of the BiP ATPase activity requires full-length Bap, suggesting a complex interplay of these two factors. Here, single-molecule FRET experiments with mammalian proteins reveal that Bap affects the conformation of both BiP domains, including the lid subdomain, which is important for substrate binding. The largely unstructured Bap N-terminal domain promotes the substrate release from BiP. Thus, Bap is a conformational regulator affecting both nucleotide and substrate interactions. The preferential interaction with BiP in its ADP state places Bap at a late stage of the chaperone cycle, in which it coordinates release of substrate and ADP, thereby resetting BiP for ATP and substrate binding.