Regulation of microtubule radial structure by competition between Tau and paclitaxel: Binding and x-ray scattering...

Microtubules (MTs) are a major component of the eukaryotic cytoskeleton. MT architecture is highly regulated by MT-associated proteins such as Tau as well as a number of MT-targeted chemotherapeutic agents such as paclitaxel (PTX). In this study, we examined the ability of each of the six different alternatively spliced isoforms of human wild-type (WT) Tau (4R2N, 4R1N, 4R0N, 3R2N, 3R1N, and 3R0N) and PTX to bind to MTs as well as their effects upon MT structure. MTs were assembled in the physiologically relevant experimental regime of mixing WT Tau protein with unpolymerized tubulin and then treating the resulting MTs with PTX (i.e., Tau-coassembled MTs). The extent of Tau and PTX binding to MTs was assayed by co-sedimentation/Western blotting and high-performance liquid chromatography, respectively. Radial size of MTs was determined by synchrotron small-angle x-ray scattering. We observed that 4R Tau and PTX compete for binding to MTs, whereas 3R Tau and PTX exhibit only limited competition. These observations suggest that both 4R and 3R Tau bind initially to the well-studied binding sites on the outer surface of MTs, followed by binding to the less-well-understood binding site within the MT lumen in an isoform-specific manner. These binding events also lead to distinct effects on MT radial structure compared with MTs formed by PTX and then treated with Tau (i.e., PTX-stabilized MTs). Specifically, the inner radius of MTs first increased and then markedly decreased with increasing Tau concentrations. In addition to providing fundamental insights in the basic biochemistry of MTs, our results have implications regarding the onset and progression of chemotherapy-induced peripheral neuropathy, a consequence of many MT-targeted anticancer therapeutics including PTX. The differential use of the luminal Tau binding site in 4R versus 3R further raises the possibility of differential Tau isoform action in fetal versus adult nervous systems.