Reconstitution of Bim1p MT plus end tracking in vitro using TIRF microscopy. (A) Scheme of the experimental setup. (B) Table summarizing the ability of different Bim1 constructs for MT plus end tacking or lattice association. (C) Full-length Bim1-EGFP autonomously tracks a growing MT plus end in vitro. A kymograph representation demonstrating 70 nM Bim1p tip tracking is shown. Note that Bim1p is not found on the tips of depolymerizing MTs (left; Videos 1 and 2). Association of 1 µM of a Bim1p variant lacking the dimerization domain (Bim1p^1–210) with dynamic MTs. Note that this variant faintly stains the MT lattice but fails to accumulate at growing ends (right; Video 3). Arrows show enrichment of Bim1 on the growing MT end. (D) In a microscopy flow cell, Alexa Fluor 488–labeled Bim1p (green) was bound to rhodamine-labeled, taxol-stabilized MTs. At the indicated time, ATP was added into the flow cell. In the kymograph representation, the Bim1p signal disappears rapidly from the stable MTs; in contrast, it persists in a buffer control (Video 4). (E) In vitro tracking of Bim1p in the presence of the Ipl1–Sli15 complex. Bim1p is removed from growing MT ends when the Ipl1–Sli15 complex and ATP are present (Video 5). Bar, 5 µm.