Conformational Expansion of Tau in Condensates Promotes Irreversible Aggregation

Abstract

Liquid−liquid phase separation (LLPS) of proteins into biomolecular condensates has emerged as a fundamental principle underpinning cellular function and malfunction. Indeed, many human pathologies, including protein misfolding diseases, are linked to aberrant liquid-to-solid phase transitions, and diseaseassociated protein aggregates often nucleate through phase separation. The molecular level determinants that promote pathological phase transitions remain, however, poorly understood.Here we study LLPS of the microtubule-associated protein Tau, whose aberrant aggregation is associated with a number of neurodegenerative diseases, including Alzheimer's disease. Using single molecule spectroscopy, we probe directly the conformational changes that the protein undergoes as a result of LLPS. We perform single-molecule FRET and fluorescence correlation spectroscopy experiments to monitor the intra-and intermolecular changes and demonstrate that the N-and C-terminal regions of Tau become extended, thus exposing the microtubule-binding region. These changes facilitate intermolecular interactions and allow for the formation of nanoscale clusters of Tau. Our results suggest that these clusters can promote the fibrillization of Tau, which can be dramatically accelerated by disease-related mutations P301L and P301S. Our findings thus provide important molecular insights into the mechanism of protein phase separation and the conversion of protein condensates from functional liquid assemblies to pathological aggregates.

Spatiotemporal modulations in heterotypic condensates of prion and α-synuclein control phase transitions and amyloid conversion

“…Nevertheless, our fluorescence depolarization kinetics indicated electrostatic clustering of C-terminal α-Syn and N-terminal PrP into relatively ordered nano-blobs acting as primary units of liquid droplets. The existence of such intermolecular clusters has recently been shown in tau condensates 66 . We posit that these clusters that are detected on the nanosecond timescale can undergo breakings and makings of transient crosslinks on a much slower timescale resulting in a liquid-like behavior on a longer length and timescales as observed by FRAP.…”

Distinct lipid membrane-mediated pathways of Tau assembly revealed by single-molecule analysis

“…Recently, we studied the conformational state of full-length Tau under liquid-liquid phase separation (LLPS) conditions and its relationship with the irreversible aggregation of Tau. 43 Although the LLPS is driven by electrostatic interactions between different domains of Tau with opposite charge, similar conformational changes have been found in the LLPS state in the presence of the crowding reagent PEG, where the N-terminal and C-terminal regions of Tau move away from the central MTBR. The exposure of MTBR that includes the crucial sequence for initiating amyloid aggregation of Tau as well as increased intermolecular interactions facilitate amyloid fibrillization of Tau.…”

“…The exposure of MTBR that includes the crucial sequence for initiating amyloid aggregation of Tau as well as increased intermolecular interactions facilitate amyloid fibrillization of Tau. 43 These studies together suggest a common mechanism where the extension of N-and C-terminal domains and the exposure of the MTBR may rep-Fig. 8 The proposed model for membrane-induced conformational changes of Tau and different effects on Tau aggregation.…”

“…Wild-type Tau and its variants were expressed and purified as previously described. 43,58 Briefly, the plasmid with the Tau gene was transformed into Escherichia coli cells. After induction, the cells were harvested.…”

“…Single-molecule FRET (smFRET) experiments were carried out on a home-built confocal microscope as described previously. 43,59,60 An inverted fluorescence microscope (Ti-U, Nikon) was equipped with a 100× objective (N.A. = 1.4, Nikon).…”