Figure 1.

Photoconversion of Tau^Dendra2 in transfected neurons. Top: Bar diagram of human tau40 with domains, phosphorylation sites, and antibody epitopes. The C-terminal half contains the repeat domain (repeats R1–R4, red, R2 alternatively spliced) plus flanking regions (green), responsible for microtubule binding. The N-terminal half (‘projection domain’) does not bind to microtubules and contains alternatively spliced inserts I1 and I2 (blue). Major target sites of kinases MARK (KXGS motifs with S262, S293, S324, and S356) and PKA (S214) that control binding of Tau to MT are labelled in red. Seventeen major target sites of proline-directed kinases (in the flanking regions of the repeats and projection domain) and some of the responsible kinases are labelled in green. Epitopes of some phosphorylation-dependent antibodies (12E8, AT8, and PHF1) are indicated. Dendra2 is linked to the N-terminus of Tau as indicated. Bottom: (A) Cortical neuron transfected with Dendra2 for 2 days, showing the green fluorescence before photoconversion. Arrow indicates the axon and arrowhead indicates the somatodendritic compartment. (B) Photoconversion of Dendra2 by UV illumination of the soma (ROI1, boxed in B–D). Images were taken in the red fluorescent channel (B–D) at the indicated time points (0–8 min). (C, D) Time-lapse images trace the rapid redistribution of photoconverted Dendra2 from the soma (ROI1) into the dendrites (arrowhead in D) and the axon (arrow in D). (E) Distribution of Tau^D2 in a cortical neuron transfected for 2 days before photoconversion. Tau^D2 appears both in the somatodendritic compartment (arrowheads, top) and in the axon (arrow). (F) Photoconversion of Tau^D2 by UV illumination of the soma (ROI1, boxed in F–H). (G, H) Time-lapse images in red fluorescent channel trace the redistribution of photoconverted Tau^D2 from the soma into the dendrites (arrowhead in H) and the axon (arrow in H). Compared with Dendra2 (B–D), the spreading of Tau^D2 is much slower (images at 0–95 min). The asterisk indicates the non-photoconverted neuron. (I) Intensity versus time in ROI2 in axon (∼150 μm from the soma, red circle in D or H). The signal of Dendra2 reaches a half-maximal level at ∼300 s, whereas Tau^D2 takes much longer (∼5300 s). Data shown here are representations of at least three independent experiments.