LLPS of Cry2WT-mCherry-Tau 151–254 (CWT 151–254). (A) CWT 151–254 overexpressed in SH SY5Y cells activated by shallow blue light (488 nm, 5% power) rapidly induced condensates (blue arrowheads). Representative fluorescent images at 0 and 5 s after activation are shown. Occasional preactivation foci are sometimes present and do not participate in light-activated phase separation (blue arrow). (B) Temporal evolution of phase separation of CWT 151–254 (black points) and Cry2WT-mCherry (orange points) was monitored on a single confocal plane, quantified by CV across the cell and plotted over time. n = 7 for CWT 151–254 and n = 5 for Cry2WT-mCherry; error bar in SE. (C and D) FRAP of CWT 151–254. (C) Representative time-lapse images of FRAP indicated by the 3 × 3-µm bleach site is marked by the white square. See Video 1. (D) Integrated intensity density for the bleached areas was monitored over time during FRAP. 55% recovery was achieved after bleaching. n = 3. (E and F) CWT 151–254 phase separation is reversible. (E) Time-lapse images of maximum intensity Z-projection of CWT 151–254 show phase separation is reversible after the withdrawal of blue light (0–14 min) and reassembled when subject to blue light reactivation (14–33 min). (F) Quantification of CWT 151–254 from disassembly to reassembly as light power changes. CWT 151–254 distribution quantified by CV (black) and light activation indicated by the blue light power (blue) were plotted over time. n = 8; error bar in SE. (G) Time-lapse images of local shallow light activation of CWT 151–254 demonstrate localized progression toward phase separation. A rectangular area of 3 × 3-µm (white square) was stimulated with blue light. See also Video 2 for local activation and the field activation followed. Scale bar, 10 µm. t, time.