The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.
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Method Article
Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones
In This Article
Summary
Abstract
Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. 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 MTs1-3. 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 types6-8.
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 MTs9,10. 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-ends1,12. EB1 is known to recruit several other proteins to growing MT plus-ends13,14, and has recently been established a....
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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
- 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 nec....
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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.......
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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.......
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Disclosures
The authors declare that they have no competing financial interests.
Acknowledgements
We thank Dr. Gaudenz Danuser and members of his lab for creating the plusTipTracker software and for helpful discussion regarding using the software, in particular Maria Bagonis and Sebastien Besson. We especially thank the Boston College Media Center for their assistance and support in the creation and editing of the video. We also thank members of the Lowery Lab for useful discussions and constructive criticism, and Abigail Antoine for proof-reading the manuscript. This work was funded by an NIH R00 MH095768 award to LAL.
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Materials
| 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/ |
References
- Stepanova, T., et al. Visualization of microtubule growth in cultured neurons via the use of EB3-GFP (end-binding protein 3-green fluorescent protein). The Journal of neuroscience : the official journal of the Society for Neuroscience. 23, 2655-2664 (2003).
- Lee, H., et al.
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