Fig. 2
Syph Ct undergoes phase separation among themselves when assisted by additional interactions in living cells. a Schematic diagram of Syph Ct-mCh-CRY2PHR consisting of the N-terminal Syph Ct (blue-gray) fused to mCherry (red) and the CRY2PHR domain (gray indicating inactive state). Blue light activation of Syph Ct-mCh-CRY2PHR leads to rapid clustering (blue indicating active CRY2PHR). b Representative time-lapse fluorescence images of light-activated clustering of Syph Ct-mCh-CRY2PHR and CRY2PHR-mCh stimulated with a 488 nm laser for 2500 ms. Middle: Magnified images of the region enclosed by a red rectangle in the top panel. Scale bars; 20 μm (top and bottom), 2 μm (middle). c Schematic diagram of Syph (Ct)2-mCer-MP. Two Syph Cts were linked by a short linker (gray) and fused to mCerulean fluorescent protein and the multimeric protein (MP) of CaMKIIα (pale mint). 12 identical MP subunits are assembled into a circular oligomer, exposing 24 copies of Syph Cts. d Representative fluorescence image of droplets formed by Syph (Ct)2-mCer-MP expressed in living cells. e Representative fluorescence image of droplets formed by purified Syph (Ct)2-mCer-MP (5 μM) in vitro in the presence of 3% PEG-8000. Scale bars; d = 20 μm, e = 10 μm. f Time-lapse images showed fusion of two Syph (Ct)2-mCer-MP droplets in living cells. g Representative fluorescence images of Syph (Ct)2-mCer-MP droplets treated with 3% 1,6-Hexanediol (3% 1,6-HD). Droplets disperse reversibly upon 3% 1,6-HD. Scale bars; f = 2 μm, g = 20 μm. h Representative time-lapse images showing fluorescence recovery of Syph (Ct)2-mCer-MP droplet after photobleaching. i Plot of the average fluorescence intensities after photobleaching of multiple spots. N = 10 cells from 5 coverslips. Scale bars; 2 μm