Despite decades of research in Alzheimer’s disease (AD), no treatment has been found to efficiently prevent or cure its progression. The microtubule binding protein Tau plays central roles in the pathogenesis of AD and is found, in the form of paired helical filaments (PHFs), as the main component of the neurofibrillary tangles, a major pathological hallmark of AD. Therefore, a better understanding of the molecular and structural determinants of Tau normal and pathological functions is crucial to elucidate the role of Tau in AD. The work presented in this thesis represents our efforts to contribute to addressing this knowledge gap by investigating the sequence, molecular and structural determinants of Tau aggregation, membrane binding, and toxicity and by exploring the interplay between these different properties. In Chapter 1, we assess the effect of manipulating the aggregation conditions on the fibril polymorphism of 4R Tau, the K18 fragment and the four individual repeat peptides within the microtubule binding domain, namely R1, R2, R3 and R4. The fibrilar structures formed by Tau, K18, R2 and R3 are diverse, polymorphic and depend upon the aggregation conditions. This work paves the way to a better understanding of molecular basis underlying Tau fibrils formation and demonstrates the importance of interplay between the different sequence motifs in Tau. In Chapter 2, we describe the discovery of a Tau-derived peptide that forms fibrils with a unique capacity to seed the aggregation of full-length Tau and templates its aggregation into highly ordered twisted fibrils that resemble native PHFs. This has tremendous potential applications in the understanding the biophysical properties of the PHFs, but also provide a powerful platform for designing Tau-specific imaging probes or the screening for small molecule modulators of fibrillization and clearance. In Chapter 3, we investigate the sequence determinants and structural consequences of the interactions of Tau with membranes, and describe for the first time the formation of stable protein/lipid complexes. Using NMR, we determined that the core of these complexes is comprised of short motifs localized in R2 and R3. We designed novel mutants that disrupt Tau interactions with lipids, thus providing powerful tools for investigating the role of membrane interactions in regulating the functions of Tau. Our findings point toward a novel form of Tau complexes that might be part of a membrane-dependent mechanism that regulates Tau oligomerization and toxicity. In Chapter 4, we explore the effect of tyrosine phosphorylation in regulating Tau properties. We report that phosphorylation at Y310, which is located within R3, results in the greatest inhibition of Tau fibril formation. We also identified two novel Tau kinases, Fes and Btk that phosphorylate Tau at positions Y310 and Y394, and developed antibodies specific for pY310 and pY394, thereby providing novel targets and tools that could lead to the discovery of novel mechanisms and pathways involved in regulating Tau functions. In addition to providing several tools to allow further understanding of Tau functions and implications in AD, our work underscores the importance of investigating the individual domains of Tau, especially R2 and R3, as structural determinant of Tau self-assembly and binding to membranes and demonstrates the critical involvement of phosphorylation of the Y310 residue in Tau biophysical and cellular properties.