CLIP-170 recognizes composite EB1/tubulin-binding sites at the microtubule end. (A) Western blot of mock-treated and detyrosinated tubulin (tubulinΔY) either Ponceau-stained or probed with an anti–Tyr-tubulin or anti–Glu-tubulin antibody. (B) Kymographs of a growing TAMRA-labeled microtubule (red) in the presence of 75 nM EB1Y→A-GFP (left, green) or EB1-GFP (right, green) in buffer. (C) The peak signal of the EB1 comets obtained from averaged intensity profiles at the indicated conditions. Error bars indicate standard error.(D) Kymographs of a growing TAMRA-labeled microtubule (red) in the presence of 35 nM H2-GFP (green) growing with either mock-treated (left) or detyrosinated tubulin (right) in the presence of unlabeled EB1 (top) or EB1Y→A (bottom). Bars, 5 μm. (E) The peak signal of the H2 comets (top) obtained from averaged intensity profiles at the indicated conditions. Signal of H2-GFP bound to the microtubule lattice (bottom) as averaged from intensity line scans. Error bars indicate the standard error (top) or the standard deviation of the mean lattice intensity from the line scans (bottom). (F) Schematic illustration of the mechanisms of end tracking by vertebrate (left) and fission yeast (right) +TIPs. See text for details.
