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Conformational dynamics of Tau in the cell quantified by an intramolecular FRET biosensor in physiological and pathological context

Impaired interactions of Tau protein with microtubules (MT) and Tau misfolding play a key role in Alzheimer disease (AD) and other neurodegenerative diseases collectively named Tauopathies. However, little is known about the molecular conformational changes that underlie Tau misfolding and aggregation in pathological conditions, due to the difficulty of studying structural aspects of this intrinsically unfolded protein, particularly in the context of living cells. Here we developed a new Conformational-Sensitive Tau sensor (CST), based on human Tau full length protein, to investigate the changes in 3D conformation and aggregation state of Tau upon modulation of its interactions with MTs in living cells, in physiological and pathological conditions. After showing that the CST fully preserves functional Tau activities in living cells, we demonstrated that MT-bound Tau displays a loop-like conformation, while soluble Tau assumes a relaxed conformation. The imaging readout based on CST allowed to discover new conformational properties of full length Tau in living cells, when challenged with Alzheimer-relevant seeds from different sources, and to learn about different ways to induce the self-aggregation of full length Tau in cells. Furthermore, it allowed to investigate the contribution to the pathology of point mutations known to alter Tau/MTs interaction. SIGNIFICANCE The microtubule-associated protein Tau regulates the stability of microtubules (MT) and its alteration and misfolding plays an important role in Alzheimer disease (AD) and in several other neurodegenerative diseases collectively named Tauopathies. However, little is known about the molecular mechanism that modulate the conformational changes of Tau in pathological conditions. Here, we developed a conformational sensitive Tau biosensor providing insights into the 3D conformation of Tau in living cells. This new tool showed that MT-bound Tau displays a loop-like conformation, while soluble Tau assume a relaxed conformation, which might be more prone to aggregation. Furthermore the CST was used to develop an assay to screen molecules and mutations involved in Tau self-aggregation in living cells. Remarkably, the imaging tool developed allowed to demonstrate that Aβ oligomers induced Tau aggregation in living cells.