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.

Zinc finger proteins targeted to MAPT safely repress tau mRNA in the adult mouse brain and protect against Aβ toxicity.

Information on protein–protein interactions (PPIs) is of critical importance for studying complex biological systems and developing therapeutic strategies. Here, we present a double‐readout bioluminescence‐based two‐hybrid technology, termed LuTHy, which provides two quantitative scores in one experimental procedure when testing binary interactions. PPIs are first monitored in cells by quantification of bioluminescence resonance energy transfer (BRET) and, following cell lysis, are again quantitatively assessed by luminescence‐based co‐precipitation (LuC). The double‐readout procedure detects interactions with higher sensitivity than traditional single‐readout methods and is broadly applicable, for example, for detecting the effects of small molecules or disease‐causing mutations on PPIs. Applying LuTHy in a focused screen, we identified 42 interactions for the presynaptic chaperone CSPα, causative to adult‐onset neuronal ceroid lipofuscinosis (ANCL), a progressive neurodegenerative disease. Nearly 50% of PPIs were found to be affected when studying the effect of the disease‐causing missense mutations L115R and ∆L116 in CSPα with LuTHy. Our study presents a robust, sensitive research tool with high utility for investigating the molecular mechanisms by which disease‐associated mutations impair protein activity in biological systems.

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.

The microtubule (MT) association of protein Tau is decreased upon phosphorylation. Increased levels of phosphorylated Tau in the cytosol pose the risk of pathological aggregation, as observed in neurodegenerative diseases. We show that binding of 14-3-3ζ enhances cytosolic Tau solubility by promoting phosphorylated Tau removal from MTs, while simultaneously inhibiting Tau aggregation both directly and indirectly via suppression of condensate formation. These 14-3-3ζ activities depend on site-specific binding of 14-3-3 to Tau phosphorylated at S214 and S324. At sub-stoichiometric 14-3-3ζ concentrations, or in the presence of other 14-3-3ζ binding partners, multivalent electrostatic interactions promote Tau:14-3-3ζ co-condensation, offering a phosphorylation-independent mode of Tau-14-3-3ζ interactions. Given the high abundance of 14-3-3 proteins in the brain, 14-3-3 binding could provide efficient multi-modal chaperoning activity for Tau in the healthy brain and be important for preventing Tau aggregation in disease.