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. 2020 Sep 30;219(11):e202006054. doi: 10.1083/jcb.202006054

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© 2020 Zhang et al.

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Figure 5. — Cry2WT-mCherry-Tau 151–254 (CWT 151–254) tracks with EB1 after blue-light activation. (A and B) CWT 151–254 overexpressed with EB1-EGFP in SH SY5Y cells and activated by shallow blue light (488 nm, 5% power) induced colocalization. (A) Kymograph of CWT 151–254 (red) and EB1-EGFP (green) is generated from time-lapse imaging for 66 s at 2-s imaging intervals, with the x axis the selected line crossing the image and the y axis the time of recording. Fluorescence imaging of two channels shows the formed CWT 151–254 (red) condensate colocalized with moving EB1-EGFP (green). Scale bar, 2 µm. (B) Representative time-lapse images at the times of 00:00:00, 00:01:02, and 00:01:06 are shown in the top panel, where the line used to generate the kymograph is shown in white. The kymograph in A moves from left to right. Selected moving foci on the line are indicated with white or blue arrows on both the kymograph and the corresponding time-lapse images. Two fluorescent channels of the middle image at 00:01:02, CWT 151–254 (red, left), EB1-EGFP (green, right), are presented at the bottom panel. See also Video 3. (C and D) CWT 151–254 with I171A and P172A double mutant and EB1-EGFP overexpressed in SH SY5Y cells activated by shallow blue light (488 nm, 5% power). The induced double mutant CWT 151–254 (red) condensate does not colocalize with EB1-EGFP. (C) Kymograph of CWT 151–254 with I171A and P172A double mutant (red) and EB1-EGFP (green) generated from a time-lapse imaging for 3 min and 47 s at 2-s imaging intervals, with the x axis being the selected line crossing the image and the y axis the time of recording. Fluorescence imaging of two channels shows the formed CWT 151–254 with I171A and P172A double mutant (red) condensate does not colocalize with moving EB1-EGFP (green). Scale bar, 2 µm. (D) Representative time-lapse images at the times of 00:00:00, 00:01:16, and 00:01:29 are shown in the top panel, where the line used to generate the kymograph is shown in white. Selected moving foci on the line are indicated with white or blue arrows on both the kymograph and the corresponding time-lapse images. Two fluorescent channels the middle image at 00:01:16, CWT 151–254 I171A and P172A (red, left), EB1-EGFP (green, right), are presented in the bottom panel. (E) Quantification of EB1-EGFP distribution after light activation. EB1 was expressed in SH SY5Y cells with CWT 151–254, or with CWT 151–254 I171A/P172A double mutant, or with no co-overexpression. Distribution of EB1 is evaluated by the change in the CV after 60 s of light activation shown along the y axis. n = 11, 11, and 8 for the groups from left to right along the x axis. Mean for each group is indicated by a purple square. Statistical significance by pairwise t test of each independent group against EB1 without CWT 151–254 is indicated at the top of the plot. (F) CWT 151–254 (red) and EB1-EGFP (green) overexpressed in SH SY5Y cell, treated with 20 µM nocodazole for 2 h, activated by blue light. EB1-EGFP appeared diffuse in the cytoplasm and not colocalized with the CWT 151–254 light-induced spherical condensates. Representative montage image is shown. See also Video 4. Montage of two fluorescent channels is presented from the left, the mCherry channel, the GFP channel separate and the merged. Scale bar, 10 µm unless specifically mentioned. ns, not significant. ***, P < 0.001.