Fig. 1.

CLIP-170 condenses into droplets in cells and in vitro. (A) Representative confocal images of RPE-1 cells transfected with GFP-CLIP-170 at three different overexpression levels (1: low, 2: high, and 3: very high overexpression). Zoom-in shows +TIP networks with adjusted contrast for visualization; for images with the same contrast, see (SI Appendix, Fig. S1A). For each cell, the whole cell fluorescence intensity (WCFI) and peak of +TIP network fluorescence intensity (TFI) are indicated; the blue outlining of the image corresponds to colored data points in (B). (Scale bar: 10 µm.) (B) Analysis of A showing the correlation between peak +TIP network fluorescence intensity and WCFI in RPE-1 cells expressing GFP-CLIP-170. The dashed line shows exponential plateau curve fit. Each dot represents 5 analyzed +TIP networks from one cell, data from two independent experiments with a total of 42 cells. (C) Percentage of +TIP networks with trailing foci in fixed cells expressing the indicated CLIP constructs or stained with antibodies to analysis endogenous CLIP-170 or EB3. Mean with SD from three independent experiments. Statistics: one-way ANOVA test. (D) Representative time-lapse TIRF images (Top) and kymograph (Bottom) of +TIP network from GFP-CLIP-170 expressing RPE-1 cell. Cyan and white arrowheads denote trailing foci formation and dissolving, respectively, in both time-lapse images and kymograph. (Scale bar: 2 µm.) (E) Phase diagram of GFP-FL-CLIP in vitro at increasing KCl and protein concentration. The blue shaded dot denotes where phase separation occurred, results of three independent experiments. The red dotted square represents the physiological cell concentration of CLIP-170 where we observe phase separation. (F, Top) representative confocal images of purified GFP-FL-CLIP at indicated concentrations. (Scale bar: 20 µm.) Bottom: quantification of the coverslip surface coverage (Left) and droplet size (Right) for GFP-FL-CLIP condensates at 100, 200, or 400 nM in the absence of PEG. Statistics: two-tailed Student’s t test. Mean with SD from three independent experiments with a total of 27 fields of view per condition. (G) Time-lapse TIRF images of purified GFP-FL-CLIP (1 µM) undergoing fusion. Representative of three experimental replicates. (Scale bar: 10 µm.) (H) Representative TIRF images and recovery curve of purified GFP-FL-CLIP (2 µM) droplets after photobleaching (dashed box). The blue curve shows mean with SD of three individual experiments with a total of 47 condensates. The gray curve shows the FRAP curve for GFP-CLIP droplets in cells. (Scale bar: 5 µm.)