Figure 5: Liquid phase transitions for Fluoppi punctate structures.

(a–c) Liquid-like state inside Fluoppi punctate structures in CHO-K1 cells stably co-expressing PB1-p53 and AG-MDM2. (a) Cells were treated with 20 μM nutlin-3 to cause punctum disappearance. After the drug was washed away, formation and growth of puncta were observed. Upon touching (0 s), two round puncta (~2 μm in diameter) fused and relaxed their shape in 2 minutes. The change in aspect ratio was plotted over time (rightmost). Similar results were obtained using 4 other puncta with diameters of <3 μm. (b) Application of FRAP to a round punctum (5 μm in diameter). The change in fluorescence intensity in the bleached region was plotted over time (rightmost). Similar results were obtained from 4 other puncta with diameters of <10 μm. (c) Cells were time-lapse imaged. PC, phase-contrast images. AG + PC, fluorescence images merged with phase-contrast images. right, A montage of a time-lapse imaging (AG + PC) of proliferating cells. One cell-division event is marked by black arrows. See also Supplementary Video 7. Most large puncta (>10 μm) were irregular in shape. Similar images were obtained using another CHO-K1 clone. (d,e) TIRF microscopy images. 2D phase separation (spinodal decomposition) was seen on the surface of a Cos-7 cell co-expressing PB1-HRas and AG-cRaf after stimulation with EGF (C). The same pattern was observed in 5 other cells. This pattern was not seen when monomeric PB1 (mPB1) was used (See Figure S2B). See also Supplementary Video 8. (a–d) Domain structures of transfected constructs are illustrated (leftmost). Scale bars, 1 μm (a,b); 10 μm (c–e). Scale bar in magnified box (d), 1 μm.