Single-molecule tracking dataset for Ipl1-HaloTag-JF646 in live cells of S. cerevisiae

Single-molecule Imaging and Tracking (SMIT) provides direct real-time measurements of protein dynamics in live cells. This dataset contains tracking files (generated through TrackRecord) with raw time-lapse movies acquired for the SMIT of Aurora kinase B (Ipl1) in Saccharomyces cerevisiae. The IPL1 gene was C-terminally fused with -HaloTag and sparsely labeled with HaloTag ligand (JF646-HTL). To identify cell cycle stages, Clover GFP-Tub1 or Ndc10-3xGFP was expressed. The cells were grown to log phase in CSM media, labeled with JF646-HTL, and imaged under the Highly Inclined and Laminated Optical Sheet (HILO) microscope (equipped with 100X 1.47 NA oil immersion TIRF objective lens, Prime95B sCMOS camera, 638 nm 150 mW laser) with a pixel size of 110 nm. Time-lapse movies were acquired with two imaging regimes: 1) Slow imaging regime (200 ms interval, 50 ms exposure, 30% laser power), 2) Fast imaging regime (15 ms interval, 10 ms exposure, 100% laser power). Before acquiring each movie, a single-focal plane image was acquired in the FITC channel to visualize the posotion of spindles or kinetochores (by CloverGFP-Tub1 or Ndc10-3xGFP respectively). For residence time quantification, the slow imaging movies were tracked with TrackRecord, and for the diffusion analysis, the fast imaging movies were tracked with DiaTrack and Sojoiurner. Due to space constrain, 15 time-lapse movies (.mat) and their corresponding mask files (,tiff, from FITC channel) have been uploaded for each dataset.