Cell tirf module

Nonfluorescent kin1 motors and GFP-labeled scaffolds (Fig. 2 A) in lysates containing 2 mM ATP were diluted in P12 buffer (12 mM Pipes/KOH, 1 mM EGTA, and 2 mM MgCl₂, pH 6.8). 5 µl of each lysate was added to flow chambers containing taxol-stabilized MTs (Cytoskeleton, Inc.) and 45 µl of oxygen scavenger buffer (1 mM DTT, 1 mM MgCl₂, 2 mM ATP, 10 mM glucose, 0.1 mg/ml glucose oxidase, 0.08 mg/ml catalase, 10 mg/ml BSA, and 10 µM taxol in P12). Linker screening assays (Fig. 2) were performed at the Single Molecule Analysis in Real Time (SMART) Center at the University of Michigan (Ann Arbor, MI). Images were acquired at room temperature using a microscope (IX-81; Olympus) with a 60× 1.49 NA oil immersion TIRF objective with a 4× tube lens (Olympus), equipped with five fiber-coupled lasers (405 nm, 488 nm, 532 nm, 561 nm, and 640 nm) and independently focused via Cell^TIRF module (Olympus). Individual mCitrine-labeled motors (Fig. 2 B) or GFP-labeled scaffolds (Fig. 2, C–F) were excited at 488 nm with 100-ms exposure, and images were collected via an EM CCD detector (iXon 897, 512 × 512, 16 µM array; Andor Technology). For linker screening assays, the SpotTracker plugin for ImageJ (Sage et al., 2005 (link); http://bigwww.epfl.ch/sage/soft/spottracker/) was modified to batch-process motility data (Cai et al., 2009 (link)) and used to calculate the speed and run length.