Fig. 4: Quantitative live cell imaging.
A Schematic representation of the live cell imaging approach to determine Tau dynamics in axon-like processes of neuronally differentiated PC12 cells. The schematic shows that calculation of the effective diffusion constant (Deff) using a one-dimensional diffusion model allows determination of the fraction of microtubule-bound Tau75. Schematic representations of the expressed Tau constructs are shown on the right. The MT-binding repeat regions (RR1–RR4) are indicated by yellow boxes, the N-terminal PAGFP fusion by green. The lysine deletion of the aggregation-prone construct is located in the second repeat. B Effect of β-Hsp90 on the dynamics of aggregation-prone Tau in axon-like processes. FDAP diagrams, scatterplots of effective diffusion constants (Deff), bar graphs of the fraction of Tau bound to microtubules, and scatterplots of k*on and koff rate constants are shown. Mean ± SE of 12 (control), 16 (3.12 µM β-Hsp90), 17 (6.25 µM β-Hsp90), 18 (12.5 µM β-Hsp90) and 12 (25 µM β-Hsp90) cells are shown. Deff β-Hsp90 25 µM vs control p-value = 0.0074. MT bound Tau β-Hsp90 25 µM vs control p-value = 0.0340. C Effect of β-Hsp90 on wild-type Tau dynamics in axon-like processes. In contrast to aggregation-prone Tau, β-Hsp90 does not affect fluorescence decay, Deff, MT-bound fraction and k*on and koff values. This indicates that the increased binding of aggregation-prone Tau is caused by the inhibition of soluble Tau aggregate formation without affecting the physiological Tau-microtubule interaction. Shown are mean ± SE of n = 14 (control), n = 16 (6.25 µM β-Hsp90), n = 15 (12.5 µM β-Hsp90), n = 11 (25 µM β-Hsp90) cells. D Effect of β-Tau on the dynamics of aggregation-prone Tau in axon-like processes. β-Tau does not affect Deff and the MT-bound fraction of aggregation-prone Tau. However, β-Tau leads to a significant increase in the k*on and koff rates, suggesting that it increases the dynamics of the Tau-microtubule interaction. k*on β-Tau 25 µM vs control p-value = 0.0010. koff β-Tau 25 µM vs control p-value = 0.0285. Mean ± SE of 19 (control) and 16 (25 µM β-Tau) cells are shown. Statistically significant differences from control determined by one-way ANOVA with Dunnett’s post hoc test (B, C) or unpaired two-tailed Student’s t-tests (D) are indicated. *p < 0.05, ** p < 0.01. In all experiments, incubation with the compound lasted 24 h, and the final concentration of DMSO was 0.125%.
