Single-molecule tracking dataset for Ipl1-HaloTag-JF646 in live cells of S. cerevisiae
Description
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.
Files
Steps to reproduce
To visualize the single-molecules of Ipl1, we fused -HaloTag at the c-terminus of the IPL1 gene endogenously and sparsely labeled with the JF646 HaloTag ligand (30 nM). The cells were placed in a LabTek II imaging chamber, covered with a nutrient agarose pad. Single-molecule imaging was performed on a Leica DMi8 infinity TIRF inverted fluorescence microscope equipped with a 100X 1.47 NA oil immersion objective lens, a Photometric Prime95B sCMOS camera, and a 638 nm 150 mW laser. Cells were focused using the FITC channel under the wide field illumination, and the best field was selected based on the fluorescence from CloverGFP-Tub1 or Ndc10-3xGFP. Single-focal plane time-lapse movies were acquired with two imaging regimes (200 ms time interval (slow imaging regime), and 15 ms time interval (fast imaging regime). To quantify residence time, slow imaging movies were tracked using TrackRecord for quantifying the residence time. For the diffusion analysis, the fast imaging movies were tracked with DiaTrack and processed with Sojoiurner to generate .csv file. LogD histogram was used to identify the states of the bound population. Spot-On based kinetic modeling was performed for the robust quantification of diffusion coefficient and bound fraction. Please refer Podh et al. 2022 (STAR Protocols) for detailed methodology.
Institutions
Categories
Funding
Department of Biotechnology
BT/13/IYBA/2020/10
Japan International Cooperation Agency
AC2023-2
Department of Biotechnology
BT/RLF/Re-entry/53/2020