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

Summary

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

Abstract

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics^1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization¹. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs^1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker⁴, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones⁵. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types^6-8.

Introduction

The goal of this method is to obtain quantitative information regarding microtubule (MT) plus-end-tracking protein (+TIP) dynamics in living growth cones. MT +TIPs are a group of proteins that localize to the plus-ends of MTs^9,10. They perform a range of functions to regulate parameters of MT dynamic instability¹¹, including rates of polymerization, catastrophe, and rescue. One well-used method for analyzing MT dynamics is to track the behavior of the +TIP EB1, which binds specifically to growing MT plus-ends^1,12. EB1 is known to recruit several other proteins to growing MT plus-ends^13,14, and has recently been established as a MT maturation factor¹⁵, promoting both MT growth and catastrophe frequency^15,16.

Many studies of MT dynamics within growth cones have utilized hand-tracking methods to measure changes in EB1-GFP dynamics over time^1-3, as EB1 localization to MT plus-ends can be used as a marker for MT polymerization. A key benefit for examining EB1-GFP comets as a proxy for MT growth is that MT dynamics can be measured even in regions of significant MT overlap. While the method of hand-tracking EB1-GFP comets has provided useful insights into MT behaviors^1-3, it is time-consuming and can be biased. Additionally, as aberrant growth cone behaviors are likely the result of minute shifts in cytoskeletal dynamics, analyzing only a small subset of MTs (usually necessary when hand-tracking) may miss significant information.

Thus, we measure global MT dynamics parameters using the software package, plusTipTracker⁴, after the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones⁵. This software, developed in the Danuser Lab, has been used in several studies characterizing MT dynamics in various cell types^6-8. It is an open-source, user-friendly, MATLAB-based package that includes automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. A long list of specific parameters of MT dynamics are calculated by this software (see Reference 4 for details), but for analysis of MT dynamics in growth cones, the most useful parameters are MT growth track velocity (in microns/minute), growth track lifetime (in seconds), and growth track length (in microns). The software can be downloaded directly from the Danuser Lab website (under “Software”). While the Danuser Lab currently supports a newer interface for +TIP tracking analysis, which is incorporated into a software package called u-track 2.0, the original, stand-alone software will remain available. The underlying algorithms between the two programs are the same (at least as of 2014), with only a difference of interface and analysis outputs. For the novice user with little MATLAB and/or computational analysis experience, plusTipTracker has more user-friendly features, including automated statistical parameter outputs.

Here, we describe the steps for analyzing images of EB1-GFP dynamics in cultured Xenopus laevis growth cones. This protocol was utilized in a recent paper examining MT dynamics¹⁷. See also Lowery et al. 2012⁵ for detailed instructions regarding culturing growth cones expressing EB1-GFP. While this paper primarily focused on examining EB1-GFP dynamics in growth cones, the same protocol can be used for other cell types¹⁷. For all cell types, the time interval between frames should be between 0.5-2 sec for optimal +TIP tracking. A time interval of up to 4 seconds between frames is possible, but this increased interval time results in additional tracking errors.

Protocol

This protocol and video are meant to serve as a companion to the original paper describing the software package in more detail⁴, as well as the Technical Report that comes with the software download on the Danuser Lab website. Readers are encouraged to review these documents carefully if there are additional questions regarding using the software.

1. Prior to Image Analysis

1. Convert each time-lapse movie into a sequence of TIFF (Tagged Image File Format) image files. If there are multiple growth cones/cells in a given movie, first crop each growth cone/cell to create its own image sequence.
   Note: This is not necessary, as individual regions-of-interest (ROI) can be selected within plusTipTracker. However, using smaller image dimensions increases the speed of the computational processing, so this step is recommended if there is significant blank space in the image.
2. Save each TIFF series in its own folder called “images” within a path that MATLAB is set to access (note that “images” is case-sensitive). To add a new path, navigate to the relevant file directory in the “Current Folder” window, right-click on the directory icon, and select “Add to Path – Selected Folders and Sub-Folders”. It is important that the plusTipTracker software folder be added to the Path, as well.

2. plusTipGetTracks

Note: The first step in image analysis is to detect the EB1-GFP comets, link the comets into tracks, and determine the parameters of microtubule dynamics. This is obtained with the command “plusTipGetTracks”⁴.

1. To begin analysis, open MATLAB application and type “plusTipGetTracks” into the command window. This will cause a new dialog box to appear.
2. Click on “Set Up New Projects” and select one (or more) of the previous TIFF image series by selecting the appropriate “images” folder (or directories containing “images” folders). Upon completion of this step, a file directory (roi_1) will be created (in the same folder that holds “images”) that will contain the future data files. Note: the “Set Up New Projects” step can be completed ahead of time, during a separate session.
3. A new window will appear: “Select a polygon, right-click on last point, and click on ‘Create Mask’”. Click on “OK”. The first image of the selected image series will then be displayed. Use the mouse to click and create a polygon that encompasses the entirety of the growth cone. Double click the mouse to close the polygon.
4. Once the polygon has been closed, a dialog box will appear: “Do you want to select another ROI?” If the image has another growth cone to analyze, select “Yes”; otherwise select “No”.
5. Select the projects that will be immediately analyzed. Click on “Select Projects” and select the folder (roi_X) to analyze.
6. A listSelectGUI screen will appear. Select the project(s) from the left side of the screen and move them over to the right side of the screen. Click on “OK”. Choose a location to save the project list and click on “Save”.
7. Select “Detection”, “Tracking”, and “Post-Processing”. Once these selections have been made, the right side of the dialog box will become configurable. Configure each option.
   1. These parameters are used to link detected comets into MT tracks. Details for choosing these control parameters for tracking are included on pages 9-10 of the Technical Report PDF which accompanies the software package download; read this report carefully if problems are encountered. For the purposes of tracking EB1-GFP comets in Xenopus laevis growth cones, use the following Tracking Settings: Search Radius Range (pixels) 5-12, Minimum Sub-Track Length (frames) 3; Max Gap Length (frames) 8; Max Shrinkage Factor 0.8, Max angle Forward 50, Max angle Backward 10, Fluctuation Radius 2.5. These settings are shown in Figure 1.
   
   Note: Max Shrinkage Factor is set to reduce the number of “backward gaps” detected, as “backward gaps” are not useful to analyze in the context of growth cones, given the crowded conditions and likely errors in track linkages. Additionally, both Max Forward angle as well as Fluctuation Radius are set relatively high, as growth cone MTs exhibit small frequent translocations in addition to growths and shrinkages, and increasing these control settings allows for this increased movement during the linkage step.
   1. Fill in the Post-Processing Settings depending upon the desired specific image acquisition settings.
8. Once the settings have been configured, click on “Start”. The software will run whichever settings have been selected. This may take minutes to hours, depending on the number of projects selected and their sizes. The Command Window displays the estimated time remaining for each function. When the plusTipGetTracks step is completed, the Command Window will display “Finished!”
   Note: A long list of specific parameters of MT dynamics have now been calculated by this software (see Reference 4 for details), but for analysis of MT dynamics in growth cones, the most useful parameters to examine are MT growth track velocity (in microns/minute), growth track lifetime (in seconds), and growth track length (in microns).

3. plusTipSeeTracks

Note: Now that the microtubule tracks have been defined, the function “plustipSeeTracks” is used for track visualization⁴. This function can provide multiple outputs for visualization, including spatial MT dynamics maps and speed movies, but here, the focus is solely on using “Track Movies” to display MT tracks superimposed on the growth cone images. While plusTipGetTracks can analyze multiple movies at a time, plusTipSeeTracks can only analyze one movie at a time.

1. Type “plusTipSeeTracks” into the command window.
2. After the dialog box loads, click on “Select Project”. Select the parent directory containing the project to visualize and click on “Select Folder”. A new window will appear: “Select the project you want to visualize”. Choose the file to visualize and click on “OK”.
3. Next, click on “Select Saved ROI”. Navigate to the same roi_X folder as the one selected in the previous step and select the file named “roiYX”.
4. Click on “Select Output Directory” to designate where MATLAB will save the track visualization files. Note: We recommend using the same folder that contains the rest of the data.
5. Select “Make Track Movie” and a screen will appear displaying all of the tracks plusTipGetTracks calculated from the +TIP comets. This step saves the tracked time series in a movie format, in the file “allTracks_X_X_X”. There is an option for saving the movie as an AVI, otherwise the default format is as a Quicktime.mov file.

4. plusTipGroupAnalysis

Note: This final function is used to create groups of movies for analysis and comparison of their MT track parameters.

1. Type “plusTipGroupAnalysis” into the command window. To manually select the groups to compare, first de-select “Auto group from hierarchy”. Then, click on “Select projects”. Navigate to the parent directories containing all the roi_X folders to analyze.
2. A listSelectGUI screen 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 of the screen. Click on “OK”. Choose a location to save the project list and click on “Save”.
3. A window will appear: “Please select first group from the list”. Click on “OK”. The listSelectGUI window will display again. This time, select only those files that correspond to the first group that should be pooled together. Click on “OK”.
4. Then, enter the group name, and click on “OK”. A window will appear: “Select another group?” Answer accordingly and continue selecting groups. A window will appear: “Select a location to save your group list”. Navigate to the location and click on “Save”.
5. Click on “Select Output Directory” to choose where the output folders will be stored.
6. Select which type of group analysis to conduct – whether the MT tracks should be pooled for each group or per cell analysis should be performed. The recommended statistical tests are already designated. To include all of the tracks in the analysis, de-select “Remove tracks at beginning/end of movie”. Otherwise, having this box selected removes any MT growth tracks that are in process as the movie begins or ends.
7. After the Group Analysis selection is made, select “Compare groups”.

Results

Using this software as described here will provide several files of information that quantify +TIP dynamics in living cells.

The function plusTipGetTracks identifies the tracks (using example settings shown in Figure 1), and then provides parameters regarding the +TIP tracks. To view the information that the software has obtained, go into the roi_X directory that was created in step 2.2. The “feat” folder contains “overlayImages”, which is a series of images showing the detect...

Discussion

PlusTipTracker provides a straightforward, graphical user interface to quickly and automatically detect virtually all visible EB1-GFP comets in a cell or growth cone, link the comets into tracks, and calculate MT parameters. Other publications have reported the design of similar types of software (for example, Marx et al. also utilized quantitative analysis of tagged EB1 dynamics in growth cones¹⁸). But, this software appears to be unique in its ease of access, as it is freely downloadable from the we...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
plusTipTracker software	Danuser Lab	http://lccb.hms.harvard.edu/software.html	This software may be hosted by another website in the future.  If the listed site does not exist, search "Danuser Lab Software" on a web search engine to find the site.
MATLAB software	Mathworks	http://www.mathworks.com/products/matlab/