An autoinhibitory regulatory switch governs heterotypic phase separation and liquid-to-solid phase transition of TDP-43 and tau into cytotoxic amyloids

Biomolecular condensates formed via macromolecular phase separation of proteins and nucleic acids control a myriad of essential cellular processes, whereas abnormal phase transitions into solid-like aggregates are associated with a range of fatal neurodegenerative diseases. Here, we present a unique case to demonstrate that two neuronal proteins, TDP-43 and tau, undergo heterotypic phase separation via domain-specific interactions regulated by an autoinhibitory conformational switch. Using single-molecule FRET (Förster resonance energy transfer), in combination with multi-color high-resolution imaging, fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, single-droplet fluorescence anisotropy imaging, homoFRET microscopy, fluorescence lifetime-FRET imaging, vibrational Raman spectroscopy, and electron microscopy, we unmask the interplay of molecular drivers and dissect the sequence of events associated with the formation of TDP-43:tau co-condensates that undergo liquid-to-solid phase transitions into cytotoxic amyloids. Our cellular studies show that a disease-associated cytosolic fragment of TDP-43 recruits tau into oxidative stress-induced cytoplasmic granules, eliciting cellular toxicity. Our findings provide mechanistic underpinnings of co-condensation-mediated aberrant phase transitions associated with exacerbated neuropathological outcomes.