Extended Data Fig. 1: EB1 comets exhibit liquid-like property.
From: Phase separation of EB1 guides microtubule plus-end dynamics
(a) GI-SIM image of a typical GFP-EB1 comet fusion at the microtubule plus-ends. COS-7 cells were transfected with EB1-GFP and mCherry-tubulin. The boxed area is magnified in the right panels, which shows time-lapse imaging of EB1 comets over 4 s. White arrows highlight the fusion process of two adjacent comets. Scale bar, 10 μm in original image and 5 μm in magnified montages. (b) Time-lapse analysis of EB1-GFP in HeLa cells by Lattice-SIM. A representative cell with EB1 comet fusion (arrowheads) at different z-plane was shown. Scale bar, 10 μm. (c) Schematic representation of engineering design of the endogenous EB1 gene with an mEGFP tag via CRISPR-Cas9 mediated gene editing to generate EB1-GFP knock-in (KI) in HeLa cells. The mEGFP were inserted before the TAA stop codon of EB1. (d) The EB1-GFP KI HeLa cells were verified by Western blotting using an anti-EB1 antibody. (e) Time-lapse analysis of EB1-GFP KI HeLa cells by Lattice-SIM. The arrows indicate EB1 comets before and after fusion. Scale bar: in original images, 10 μm; in zoom-in images, 5 μm. (f) Schematic of the OptoEB1 construct, which contains mCherry and Cry2 (blue light-induced oligomerization). (g) Representative snapshots of mCherry-Cry2 (mCh-Cry2) in living COS-7 cells upon blue light activation. Note that Cry2 alone does not exhibit comet-like structure after blue-light activation. Scale bar, 10 μm.
