Tau is a microtubule (MT) binding protein expressed in neurons that is directly linked to Alzheimer’s disease (AD). Tau has been widely studied both in its normal interactions with MTs and in its formation of Tau filaments that lead to hallmark disease-related aggregation. Though there are many functions for which Tau binds to MTs, the mechanisms are poorly understood and reports are controversial. For example, the reported Tau–MT binding affinity values vary over an order of magnitude, and Tau has been reported to bind MT protofilaments either longitudinally or laterally. Through various experiments including MT-binding assays, fluorescence spectroscopy, and transmission electron microscopy (TEM), we have gained a better understanding for Tau–MT binding behavior.
Firstly, we tested the hypothesis of a secondary tubulin binding site for Tau on MTs. Tau is known to polymerize tubulin into MTs and is believed to incorporate itself into the inner MT wall. Using Dolastatin-10 tubulin rings, we reveal through TEM and cosedimentation analysis that Tau does bind to the molecular surface of the inner MT wall with a higher affinity than its exterior MT-binding site. This also provides supporting evidence that Tau binds MTs longitudinally (versus laterally) to stabilize the MT structure.
Secondly, given that Tau polymerizes tubulin in vitro, we questioned its potential binding activity with tubulin at the growing MT end and involvement with the protein network (called +TIPs) that regulates MT growth. We measured a moderate binding affinity of Tau for GTP-like tubulin polymer, which models the MT end, and a strong affinity for GDP-tubulin polymer, which models the MT body. Additionally, since MT growth is regulated by EB1 protein, we ran competition assays with EB1 and Tau to test for synergistic or competitive MT-binding. For binding to GDP-tubulin polymer, we saw that Tau competes EB1 off of MTs and EB1 activity decreases. The sum of these results indicate that Tau actively avoids the MT end and the network of +TIPs. Using fluorescence microscopy with labeled chimeric MTs, we then confirmed that Tau preferentially binds to GDP-tubulin versus GTP-tubulin, the first MT-associated protein discovered to do so. Thirdly, we sought out the possibility that MTs could induce Tau–Tau interactions and even Tau filament formation. Recent studies have shown support for the formation of Tau oligomers on the MT surface, which would explain why traditional techniques have produced inconsistent reports on the Tau–MT binding affinity. Therefore, we hypothesized that using different experimental techniques would produce different apparent results. We tested this by measuring the Tau–MT KD using different experimental procedures and corresponding analysis calculations as well as varied protein concentrations. However, the results were still inconsistent, indicating that yet another interaction was occurring in addition to the expected Tau–MT and Tau oligomerization interactions. Through fluorescence microscopy, we reveal the novel presence of Tau filaments forming within the context of MTs during a cosedimentation assay. This explains the inconsistencies of Tau–MT binding behavior in previously reported binding studies and may contribute to the current understanding of the initial formation of Tau filaments found in AD. Our TEM analyses show the Tau filaments are similar to heparin-induced Tau filaments, which are commonly used to model AD filaments.