What we present here is a robust and simple method to analyze microtubule dynamics using live-cell spinning disk confocal imaging in cells synchronized in prometaphase, and images are afterwards analyzed on a MATLAB-based platform. The use of red-shifted fluorescent protein in combination with spinning disk microscopy reduces phototoxicity. Therefore, a larger number of cells can be imaged within the same preparation.
Microtubule dynamicity is altered in a large number of pathological conditions. Therefore, understanding the regulation and the behavior of microtubule dynamics in those processes can help us understanding the mechanism of disease, and eventually, developing therapies to compensate for them. And the method is also upscaleable, so it could potentially be applied in a drug discovery effort.
The method can be modified by changing fluorescence desynchronization protocol and obtaining cells from different phases of the cell cycle. This can be useful when screening drugs against microtubules and identifying defect on dividing and non-dividing cells. It is necessary to optimize the transfection protocol and seeding density for each cell type.
The expression level of EB3 should be low, in order to detect single growing microtubules. Start by rinsing asynchronously growing HeLa cells in DPBS, then incubate them with trypsin-EDTA for five minutes, at 37 degrees Celsius. Stop the trypsinization by adding RPMI-1640 medium supplemented with 10%heat inactivated FCS at a three to one ratio, the added trypsin-EDTA.
Calculate the concentration of the cells according to manuscript directions, and seed 50, 000 cells into each well of a prepared, chambered coverslip. Return the coverslip to the incubator, and grow cells for 24 hours at 37 degrees Celsius and 5%carbon dioxide. On the next day, remove the cells from the incubator, and drop-wise add 100 microliters of transfection mixture into each well.
Return the cells to the incubator, and after four hours of incubation supplement the cells with fresh medium and additionally incubate for 20 hours. Prepare a 2.5 micromolar solution of dimethylenastron, or DME, in phenol red free DMEM, supplemented with 10%FCS and two millimolar L-glutamine. Replace the growth medium in the chambered coverslip with the DME growth medium, and return the cells to the incubator.
After 3.5 hours of incubation, transfer the cells to the microscope by mounting the chambered coverslip into an environmental chamber set to 37 degrees Celsius and 5%carbon dioxide, with dark panels for imaging. Then continue the incubation for a total of four hours. Perform the time lapse imaging on an inverted microscope with a 100X, 1.49 numerical aperture, oil immersion objective, a dual disk confocal system, and a reliable auto focus system.
Define the imaging parameters as described in the text manuscript. Find a cell in prophase, and focus in the Z-plane corresponding to the center of the monopolar mitotic spindle. Then acquire images every 5 seconds, over a total of one minute, with no binning and no illumination between exposures.
Start by loading the numerical analysis software and adding the u-track V2.2.0 folder into the software search path, called movie selector GUI, from the command window. Then import the raw files generated by the image acquisition software. Manually enter the numerical aperture of the objective and the time interval used for imaging.
Once all the images are loaded, save the entered time lapse series as a movie by selecting save as movie list, and the u-track option on the right side of the dialogue window. Select microtubule plus-ends from the pop-up menu, and click okay, which opens a new dialogue window to determine the parameters of the three steps of the analysis. For step one, click settings and select comet detection from the drop down menu.
Define the parameters for the difference of Gaussian filter and the watershed segmentation, as described in the manuscript. Then select apply settings to all movies and click apply. For step two, select the microtubule plus-end dynamics, and use the setting options to define the values for linking, gap closing, merging, and splitting, and Kalman filter functions, according to the text manuscript.
For problems with dimensionality, choose two from the drop down menu, and use five frames for maximum gap to close, and three frames for minimum length of track segments from first step. As before, select apply settings to all movies and click apply. For step three, choose microtubule dynamics classification as a track analysis method, and define the parameters through the setting button.
Select the boxes for remove tracks at the beginning and end of the movie, and make statistics histograms. From the drop down list, select using two to three frames before forward gap, and 95th percentile of forward gap speed distribution, for forward and backward reclassification, respectively. Once all parameters are defined, select apply check/uncheck to all movies, and run all movies boxes from the control panes u-track window, then press run.
A message is displayed once the movie processing is complete. The pEB3-tdTomato plasmid was transiently expressed in HeLa cells. The cells were synchronized with DME treatment.
Time lapse movies of microtubule growth were analyzed, and the resulting growth speed and dynamicity were plotted. The parameters described to affect the analysis, such as maximum gap length and maximum shrinkage factor, were modified for the same set of time lapse movies. The corresponding values of growth speed and dynamicity were calculated.
While the resulting growth speed was not significantly affected, the dynamicity was different when the maximum gap length was modified. In all three cases, the detection of microtubule subtracks was similar, yet the reconstruction of the full MT trajectories was mostly affected when maximum gap length was set to 15. In order to assess whether imaging conditions interfered with microtubule behavior, the first and second halves of the time lapse series were analyzed separately, and the corresponding growth speed and dynamicity were compared.
As expected, no significant differences were detected. It is important to keep in mind, that because the cells are synchronized in prometaphase, the density of microtubule is very high. Therefore, one has to set the parameters of the analysis very carefully, not to misidentify different microtubule.
The measurement of microtubule dynamics with this method can be compared with studies of other biological targets directly, indirectly, regulating microtubules.
