Graded regulation of microtubule-binding of Tau by the phosphorylation state of the proline-rich region in living neurons.

Tau protein is a microtubule-associated protein that plays a crucial role in maintaining neuronal morphology and axonal transport. While phosphorylation is known to regulate Tau-microtubule interactions, the contribution of specific phosphorylation patterns in situ remains poorly understood due to the complexity of the intracellular environment. In this study, we combined fluorescence recovery after photobleaching (FRAP) in primary cultured rat hippocampal neurons with dephosphorylation-mimetic mutations and computational modeling to analyze the effects of phosphorylation on Tau-microtubule interaction. We particularly focused on the proline-rich region, of which phosphorylation has been studied in physiological and pathological perspectives, and generated a dephosphorylation-mimetic Tau mutant by substituting key phosphorylation sites with alanine residues and compared its microtubule-binding dynamics to those of WT-Tau in FRAP experiments. Experimental data, together with simulation-based parameter exploration, revealed that the overall number of phosphorylated sites plays a more dominant role than their specific locations in modulating Tau-microtubule affinity. These findings provide new insights into the post-translational regulation of Tau and establish a computational-experimental framework for interrogating intracellular protein dynamics.