Abstract
Investigating spatial and temporal control of microtubule dynamics in live cells is critical to understanding cell morphogenesis in development and disease. Tracking fluorescently labeled plus-end-tracking proteins over time has become a widely used method to study microtubule assembly. Here, we report a complementary approach that uses only two images of these labels to visualize and analyze microtubule dynamics at any given time. Using a simple color-coding scheme, labeled plus-ends from two sequential images are pseudocolored with different colors and then merged to display color-coded ends. Based on object recognition algorithms, these colored ends can be identified and segregated into dynamic groups corresponding to four events, including growth, rescue, catastrophe, and pause. Further analysis yields not only their spatial distribution throughout the cell but also provides measurements such as growth rate and direction for each labeled end. We have validated the method by comparing our results with ground-truth data derived from manual analysis as well as with data obtained using the tracking method. In addition, we have confirmed color-coded representation of different dynamic events by analyzing their history and fate. Finally, we have demonstrated the use of the method to investigate microtubule assembly in cells and provided guidance in selecting optimal image acquisition conditions. Thus, this simple computer vision method offers a unique and quantitative approach to study spatial regulation of microtubule dynamics in cells.
Highlights
Microtubule assembly in cells is characterized by stochastic conversion between phases of growth and shrinkage at the plus ends, a property known as dynamic instability [1]
The dual color-coded display (dCCD) method is based on the three possible events that can happen to a microtubule plus-end: growth, pause, and catastrophe for a growing microtubule, and shrinkage, pause, and rescue for a shrinking one (Figure 1A) [1]
Building upon recent studies using fluorescently tagged +TIPs to label microtubule plus ends [10], we have developed a computer vision strategy (Figure 1) to create snapshots of microtubule dynamics in space
Summary
Microtubule assembly in cells is characterized by stochastic conversion between phases of growth and shrinkage at the plus ends, a property known as dynamic instability [1] These two phases and the transitions between them, including rescue, catastrophe and pause (Figure 1A), are targets for regulation in many cellular processes, such as mitosis, cell polarization, directed movement, and nerve guidance [1,2,3,4]. Fluorescently tagged plus-end-tracking proteins (+TIPs) have provided a new probe to overcome this limitation [10]. These proteins preferentially associate with growing plus-ends with fast on- and off-rates, allowing the visualization of microtubule plus-ends in the entire cell [10,11]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
