Our protocol can be used to determine spacial and temporal dynamic events during mitosis, and can also be used to visualize the effects of mytotic regulators in real time. Using dual color imaging allows a simultaneous analysis of two molecular markers. For example, we can visualize the microtubules of the mytotic spindle and the kinetic core structure of replicated chromosomes.
To induce fluorescent protein expression, four days after RNA interference treatment, expose inducible metallo-thyanin promoter transfected cells to a final concentration of 500 micromolar copper sulfate for 24 to 36 hours. To prepare the fluorescent protein expressing cells for imaging, transfer the cells to a sterile 15 milliliter tube, and, after counting, sediment the cells by centrifugation. Resuspend the pellet in fresh, 25 degree Celsius warmed SIM, supplemented with 10%FBS, at a two times 10 to the 6 cells per milliliter concentration, and treat the cells with additional 500 micromolar copper sulfate.
Then add 200 to 500 microliters of cells to one well of a multi-well live cell chamber, and place the chamber on an inverted fluorescent microscope stage. While the cells are settling in the chamber, open the live cell imaging software and click new and experiment. To insert a time lapse loop over which the images will be taken, click the time lapse loop icon.
Set the interval to seconds, and divide the desired overall experiment length in seconds by the interval to set the number of cycles. Allow the loop to repeat over the desired total time period. To insert an infrared focus check to maintain the focus of the objective, click the move XY icon and select Z drift compensation to add a Z drift compensation step within the time lapse loop layer.
To allow the acquisition of a multi-channel image, click the multi-channel group icon to add a multi-channel group layer, and click the add a Z stack loop icon to add a Z stack loop layer. Then set the desired step size and number of slices, and set the exposure of each channel to as low as possible to minimize photo bleaching. To image a cell division, select a 40 or 60 times oil immersion objective, and the mCherry florescence channel, and align the objective along the top or bottom of the well.
Then move away from the vertical well divider to avoid interference with the Z drift compensation step. Locate a cell or cells in late G2 or early M phase, and click the live view button to begin viewing cells in the software screen. Finding appropriate cells at the G2 to M transition is key to imaging a cell division.
Locate a cell with an intact nucleus and exactly two centrosomes for best results. Using the fine focus knob of the microscope, focus on the cells of interest and click find offset to set the infrared focus check. Click start to initiate the time lapse imaging program, and select the desired channel and adjust the mean pixel intensities to adjust the histograms as necessary, to clearly visualize the cells of interest.
Check the cells after 15 to 20 minutes to confirm that the nuclear envelope breakdown has occurred, as determined by the disappearance of the round, dark refracted spot near the cell's center. After another 15 to 20 minutes, check to determine whether anaphase onset has occurred. Then stop the program and save the file.
To determine the nuclear envelope breakdown to anaphase onset timing, click the frame up button to determine the time of nuclear envelope breakdown and the time of initial chromosome separation in minutes, and subtract the breakdown time from the onset time to obtain the nuclear envelope breakdown to anaphase onset time for a given cell. Then continue scanning for pre-dividing cells to obtain multiple ends for a given condition for up to 12 hours from the initial settling. Cells that are about to divide can be targeted by the presence of two centrosomes and an intact nucleus, as indicated by refracted light and a darker spot within the cell when viewed in the alpha tubulin channel.
Nuclear envelope breakdown can be visualized through the disappearance of this dark spot, resulting in the uniform coloring of the cytoplasm. After nuclear envelope breakdown, the time each cell takes to form the spindle and to congress and segregate the chromosomes can be measured by noting the time points of these events relative to nuclear envelope breakdown. Treatment with double stranded RNA targeted against shortstop, an actin microtubule crosslinking protein suspected to affect cell cycle dynamics results in a significant mytotic delay.
This treatment results in a significant delay, with many cells arresting at the metaphase and never transitioning to anaphase during the entire two to three-hour imaging experiment. Co-treatment with double-stranded RNA against shot and rough deal, an important component of this checkpoint, leads to a suppression of the arrest phenotype, resulting in nuclear envelope breakdown to anaphase times similar to controlled, untreated cells. Be sure to select appropriate cells, and don't waste time imaging cells that do not begin division.
If a cell doesn't begin to divide within 20 minutes, find another cell. Researchers can follow this procedure to help identify new mytotic regulators, or possibly novel dry compounds that affect specific events in cell division.
