Fig. 1
Tau phosphorylation at 14 residues increases the mobility of Tau near the plasma membrane. A, Schematic of 0N4R Tau tagged with mEos3.2 at the C-terminus with 14 critical Ser/Thr residues replaced by glutamic acid (TauE14-mEos3.2) or alanine (TauA14-mEos3.2). B, C, Representative diffraction-limited TIRF image of N2a cells expressing TauE14-mEos3.2 (B) or TauA14-mEos3.2 (C) acquired in the green channel before sptPALM imaging. D, E, Maps of trajectories of TauE14-mEos3.2 and TauA14-mEos3.2 molecules corresponding to the boxed region shown in panels B and C, respectively. F-I, Comparison of TauWT-mEos3.2, TauE14-mEos3.2 and TauA14-mEos3.2 mobility parameters in live N2a cells. F, G, Average MSD as a function of time (F) and the corresponding area under the curve (AUC) (G). H, J, Distribution of the diffusion coefficients and the corresponding fraction of mobile pool (I). The dashed line indicates the threshold (Log10 [D] ≤ − 1.6) demarcating the immobile and the mobile pools of Tau molecules. Error bars, s.e.m. J, Examples of trajectories of TauA14-mEos3.2 molecules associated with filament-like structures. K, L, Average MSD of TauA14-mEos3.2 trajectories associated with filament-like structures (K) and the corresponding average diffusion coefficient (L). M, Distribution of duration of TauA14-mEos3.2 tracks associated with filament-like structures. N, Distribution of the MSS slope, SMSS, of individual TauA14-mEos3.2 tracks associated with filament-like structures. O, Examples of TauA14-mEos3.2 trajectories associated with filament-like structures classified as immobile or confined pools by MSS analysis. P, Distribution of the confinement radius of immobile and confined pools of TauA14-mEos3.2 molecules from filament-like structures. N = 56 cells expressing TauWT-mEos3.2, 49 cells expressing TauA14-mEos3.2, and 55 cells expressing TauE14-mEos3.2 from 5 independent experiments each. In G and I, statistical analysis was performed using one-way ANOVA with the Tukey’s multiple comparison correction