Liquid-liquid Phase Separation Modulates the Structural Heterogeneity of Tau Amyloid Fibrils

The protein tau can undergo two types of phase transitions, amyloid fibrillar aggregation leading to neurodegenerative disease, as well as liquid-liquid phase separation (LLPS) leading to the formation of protein condensates. The link between these two processes has yet to be understood fully. In this study, the tau construct, tau (243-386), was found to undergo LLPS only below a NaCl concentration of 135 mM. Hence, fibril formation was studied in 100 and 150 mM NaCl, on either side of the phase boundary established by the salt. Protein molecules inside the condensates lost their dynamicity, as measured by the extent of fluorescence recovery after photobleaching, with a characteristic time similar to that for the formation of amyloid aggregates. Thioflavin T fluorescence intensity increased homogeneously throughout the condensate interior, indicating that amyloid aggregate formation was not restricted to the interface, and with kinetics identical to that observed in a bulk measurement of amyloid fibril formation. Fibrils were seen to be emerging from aged condensates. Hydrogen-deuterium exchange studies coupled to mass spectrometry showed that tau undergoes heterogeneous fibril formation, with structurally different populations of fibrils forming under the same conditions. The structures and local stabilities of the protein molecules assembled inside the fibrils differed when formed under the LLPS and non-LLPS conditions. Consequently, the structural heterogeneity of fibrils formed under LLPS conditions was distinct from that of fibrils formed under non-LLPS conditions. The results indicate that LLPS might facilitate the selective formation of a particular structural polymorph in a heterogeneous fibril population.