Fig. 5.

CLIP-170 and EB3 droplets concentrate tubulin. (A) Cartoon schematic of domain interactions between EB3, CLIP-170, and tubulin based primarily on (52–54). Interaction sites between EB3 and CLIP-170, EB3 and tubulin, and CLIP-170 and tubulin are shown with green, orange, and blue arrows, respectively. For simplification, monomers of EB3 and CLIP-170 are shown. (B) Representative confocal images with the same contrast settings, of Atto565–tubulin (400 nM) in the presence of purified EB3 (1 µM), GFP-H1 (200 nM) + EB3 (1 µM), GFP-H2 (200 nM) + EB3 (1 µM), GFP-FL-CLIP (200 nM) + EB3 (1 µM), and GFP-FL-CLIP (200 nM and 400 nM) alone. (Scale bar: 20 µm.) Note that the treated plastic surface used for this experiment did not allow for a tubulin shell around the FL-CLIP droplet as shown in (E) for details see Materials and Methods. (C) Quantification of the droplet size from (B) under denoted conditions. Mean with SD from three independent experiments with a total of 27 fields of view. Statistics: two-tailed Student’s t test. (D) Quantification of the integrated density of tubulin fluorescence under denoted conditions with zoom in for the first three conditions. Mean with SD from three independent experiments with a total of 27 fields of view. Statistics: two-tailed Student’s t test. (E) Representative images of EB3/mCherry-EB3/tubulin-647 droplets (8 µM/2 µM/4 µM), GFP-FL-CLIP/tubulin-647 droplets (200 nM/400 nM), and EB3/mCherry-EB3/GFP-FL-CLIP/tubulin-647 droplets (900 nM/100 nM/200 nM/400 nM), with the corresponding line scans. (Scale bar: 5 µm.)