Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The aim of this procedure is to obtain quantitative information regarding Microtubule plus end tracking protein or plus tip dynamics and living growth cones using the software package plus tip tracker. Here we describe the steps for analyzing images of EB one GFP dynamics and cultured XUS lavis growth cones, but a similar protocol can be used for other cell types following the acquisition of high resolution live images of tagged EB one and cultured embryonic growth cones. The first step in the procedure is to use the command plus tip, get tracks to detect the EB one GFP comets, link the comets into tracks, and determine the parameters of microtubule dynamics.

The second step is to use the command plus tip C tracks to visualize the microtubule tracks superimposed on the growth cone images. The final step is to use plus tip group analysis to analyze and compare the microtubule track parameters of groups of movies. The main advantage of this technique over existing methods like using hand tracking to analyze individual plus tip tracks, is that this method allows for rapid and automated detection, tracking, visualization, and analysis.

The first step in image analysis is detect the EB one GFP comets, link the comets into tracks and determine the parameters of microtubule dynamics. This is obtained with the command plus tip. Get tracks prior to image analysis.

Convert each time-lapse movie into a sequence of TIFF image files and save each series in its own folder called images. Note that images is case sensitive. To begin analysis, open MATLAB application and type plus tip get tracks into the command window.

This will cause a new dialogue box to appear. Click on, set up new projects and select one or more of the previous TIFF image series by selecting the appropriate images folder. Upon completion of this step, a File directory will be created.

Click on, okay. Once the First image has been displayed, use the mouse to click and create a polygon. Then encompasses The entirety of the growth cone.

Double click the mouse to close the polygon. A dialogue box will appear if the image has another growth cone. To analyze, select yes.

Otherwise, select no. The next step is to select the projects that will be immediately analyzed. Click on select projects and select the folder.

To analyze a list, select gooey screen will appear. Select the projects from the left side of the screen and move them over to the right side. Click on, okay.

Choose a location to save the project list and click on save, select detection, tracking, and post-processing. Once these selections are made, the right side of the dialogue box will become configurable. Fill in the post-processing settings.

Depending upon the desired specific image acquisition settings, click on start. The software will then run whichever steps have been inputted. The command window displays the estimated time remaining for each function and Will indicate when the step is done running.

Now that micro tubial tracks have Been defined, the function plus tip C tracks is used for track visualization and to display the micro tubial tracks superimposed on growth cone images. This is obtained with the command plus tip C tracks type plus tip C tracks into the command window. After the dialogue box loads, click on select project.

Select the parent directory containing the project to visualize and click on select folder. Once the window appears, choose the file to visualize and click on. Okay, next, click on select saved ROI navigate to the same region of interest folder as the one selected in the previous step and select the file named R-O-I-Y-X.

Click on select output directory to designate where MATLAB will save the track visualization files and then select make track movie. A screen will appear displaying all of the tracks. Plus tip get tracks calculated from the plus tip comments.

The step saves the track time series in a movie format. The final function is used to create groups of movies for analysis and comparison of the micro tubial track parameters. This is obtained with the command plus tip group analysis.

Once plus tip get tracks has been run for multiple movies and different data subsets are ready for comparison. Type plus tip group analysis into the command window To manually select the groups to compare first deselect auto group from hierarchy. Then click on select projects.

Navigate to the parent directories containing all Of the ROI folders. To analyze a list, select GUI Window will appear. Select all of the projects to include in the groups from the left side of the screen and move them over to the right side.

Click on, okay. Then choose a location to save the project list and click on save a window will appear. Click on.

Okay, and the list select gooey window will display. Again, select only the files that correspond to the first group that should be pulled together and click on.Okay. Enter the group name and click on, okay.

A window will Appear, answer accordingly and continue selecting groups. Then choose a location to save the group list and click on save. Click on select output directory to choose where the output folders will be stored.

Finally, select which type of group analysis to conduct. Here we select pool data from all cells and groups. Click on compare groups.

This image displays the parameters of Micro tubial dynamics obtained from the function plus tip git tracks. This movie demonstrates the microtubule tracks obtained from the function plus tip C tracks. Finally, this image shows the microtubule parameters obtained from the function plus tip group analysis.

Using a spreadsheet application. These parameters acquired from different groups of movies can also be examined as box plots using A processing program. After watching this video, you Should have a good understanding of how to use plus tip tracker to obtain quantitative information regarding microtubule plus tip dynamics and living growth cones by utilizing the functions plus tip git tracks plus tip C tracks, and plus tip Group analysis.