Our lab aims to understand the interaction behaviors of intrinsically disordered protein regions and how they play roles in transcriptional regulation in healthy and diseased human cells. In our lab, we develop and use novel single molecule microscopy techniques in combination with molecular biology, biochemical, and proteomic approaches to study biomolecular condensates. This protocol enables the quantification of the dynamics by which a specific protein binds to a particular type of condensate in live human cells and is broadly applicable to measuring the interaction dynamics of any protein that participates in liquid liquid phase separation.
After creating the cell lines expressing the Halo-tagged protein of interest and Halo-H2B, prepare the Halo ligand staining reagent separately for both of them. Rinse the cells with two milliliters of PBS. Add Halo ligand containing media and incubate them at 37 degrees Celsius with 5%carbon dioxide for one hour.
After removing the staining media, rinse the cells four times with PBS and incubate them with fresh media devoid of Halo ligands. After four such rinse incubation cycles, transfer the cover slip with cells to a microscope stage compatible chamber with sterile forceps. Add phenol red free media to the chamber before imaging.
To begin, obtain ligand stain cells expressing Halo-tagged protein of interest and Halo-H2B. After turning on the microscope system, set the live cell incubation parameters and let the system equilibrate. Put a drop of the oil onto a 100x TIRF objective on the microscope and load the cell sample onto the microscope stage.
To identify a morphologically healthy cell, adjust the Z position and image the cell sample under bright field illumination. Crop the field of view to capture the target cell nucleus. Using laser illumination, adjust the TIRF angle to achieve Hilo illumination with an optimal signal to noise ratio of a single molecule.
Then set the exposure time to 500 milliseconds. During the dead time between frames, photo activate the molecules with a 111 micro watt 405 nanometer beam. And during each exposure, excite H2B-Halo molecules with a 9.1 milliwatt 640 nanometer beam.
Initiate image capturing for 2000 frames continuously and gradually increase the power of the 405 nanometer beam upon reduction of photo activated molecules. For a Halo-tagged protein of interest, split emission wavelengths between two cameras using a long pass dichroic mirror. To the same acquisition parameters demonstrated earlier, add JFX549 channel to acquire one frame every 10 seconds and capture 2000 frames continuously.
To begin, perform live cell single molecule imaging of Halo-tagged protein condensates. Using Image J, convert each channel from the raw imaging data file into an independent TIFF file. If necessary, run pre tracking comb.
text and follow the prompts to replace any frames with the most recent ones. To load the single molecule movie file in slim fast, click on load, followed by image stack. Set the parameters for localization and acquisition under respective option sheets.
Visualize the localizations of all the molecules, and using lock all, generate a file containing the localizations of all the molecules in every frame. Next, go to load, particle data, and select slim fast to load the file with localization in acquisition settings. Using option and sheet tracking, adjust the parameters for trajectory generation.
Click on gen traj to generate a file containing the trajectories of all the molecules. Load the sheet tracked file into eval SPT and set the parameters to filter out the trajectories shorter than 2.5 seconds. Using export data, generate a file with all the filtered trajectories.
Running the imageJmacronucleusnclustermaskv2.0. txt, threshold all the frames of the movie acquired in the JFX549 channel to generate a time-lapse movie populated with the time evolving binary mask highlighting condensate locations. Using convert ASCII slow tracking CSS 3.3, reformat the trajectories and run categorization version 4.
m to sort them based on the lifetime a molecule spends in a condensate. Next, using plotresidencehistcss. m, extract the observed dissociation rate of specifically bound molecules and the photobleaching rate from the in condensate protein of interest and the H2B trajectories.
Finally, calculate the corrected mean residence time of the protein of interest, specifically bound to its condensates. A frame from two color single molecule movie of TAF15-IDR-Halo-FTH1 shows signals from the nucleus in both PA-JF646 and JFX549 channels. Upon assembling and sorting, a clear distinction was observed for the trajectories of PA-JF646 detected molecules bound to the condensates.
The calculated mean residence times after correction for photobleaching were more for TAF15-Halo-FTH1 than Halo-TAF15.
