Fig. 2: The residence time of individual TauC3 molecules is increased on axonal microtubules without modifying axonal microtubule polymerization.
A Single-molecule tracking of a single TauC3 molecule in an axon-like process of a differentiated PC12 cell. The position of tau at selected times is displayed; dashed lines indicate the limit of the neurite. TauC3 was fused N-terminally with a HaloTag to enable substoichiometric labeling with TMR-HTL as indicated below. Scale bar, 0.5 μm. B Pseudotrajectory generated from the time series shown in A (3.4 s = 156 frames) indicating fast and undirected movement in the longitudinal and transverse direction of the axonal process. For comparison, the thickness and density of MTs are indicated schematically by gray bars. The starting point is indicated by a black circle and the time is color-coded as shown on the right. Representative histograms of the residence times of Halo-TauC3 on MTs in PC12 cells (C) and in primary neurons prepared from tau knockout mice (D). The monoexponential fit is indicated by a red line. The time constant (residence time) is given above (mean ± SEM for n = 8 (PC12 cells) and n = 5 (primary neurons) from 2413 and 1410 temporally immobile molecules, respectively). The residence time of full-length tau was previously determined (Janning et al. [18]) and is shown in gray. E Representative micrographs of FDAP time-lapse recordings of PAGFP-α-tubulin in a process of a PC12 cell. mCherry-tagged Tau or TauC3 was co-expressed by lentiviral (LV) expression. The photoactivated segment is indicated by a white box. Scale bar, 5 µm. F FDAP diagrams of PAGFP-α-tubulin in cells co-expressing mCherry-Tau (Tau) or mCherry-TauC3 (TauC3). Mean ± SEM of 42 cells for Tau and 34 cells for TauC3 is shown. G Scatter plots of average association (avg k*on), average dissociation (avg koff) rate constants with mean ± SEM and percent of polymerized tubulin (n = 42 and 34 cells for Tau and TauC3, respectively). A schematic representation is shown on the left which gives avg k*on and avg koff of the tubulin-MT equilibrium and the calculation of polymerized tubulin. All transfected tau constructs are based on the human tau sequence. Statistically significant differences determined by unpaired Student’s t test are indicated. *p < 0.05; **p < 0.01.
