Reconstitution of Dam1 plus end tracking in vitro using TIRF microscopy. (A) Schematic illustration of the in vitro imaging setup. (B, left) Still image of the Alexa Fluor 488–labeled Dam1 complex (100 nM) on dynamic rhodamine-labeled microtubules (MT). The complex shows preferred association with the plus end and the GMPCPP seed. (middle) A time sequence of Dam1 plus end tracking. (right) Kymographs (time/space plot) of Dam1 plus end tracking are shown (Video 4). (C) Still image and kymograph of a time-lapse video showing Dam1 decoration on unlabeled tubulin extensions. Overlay and individual channels are shown. The Dam1 complex tracks plus ends during phases of growth and shrinkage. The microtubule lattice is decorated by additional Dam1 complexes, which are collected during microtubule disassembly (Videos 5 and 6). (B and C) Arrowheads indicate Dam1 localization at the tip of the microtubule. (D) Kymograph from a streaming video recording continous Dam1 tip tracking. Error bars indicate averaged Dam1 outward displacement at the tip and lattice compared with the mean growth speed of microtubules (n ≥ 20). (E) Still series of a spike experiment depicting an individual Dam1 dot moving away from the stable seed. The dashed line indicates the position of the Dam1 signal at time 0. Bars: (B–D) 2 µm; (E) 1 µm.