Computational protocol: The microtubule plus-end-tracking protein TACC3 promotes persistent axon outgrowth and mediates responses to axon guidance signals during development

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Protocol publication

[…] MT dynamics were analyzed from GFP-MACF43 movies using plusTipTracker [, ]. Imaging conditions and tracking parameters were previously validated and same parameters were used: maximum gap length is 8 frames; minimum track length is 3 frames; search radius range 5–12 pixels; maximum forward angle, 50°, maximum backward angle, 10°; maximum shrinkage factor, 0.8; fluctuation radius, 2.5 pixels; time interval, 2 s. MT growth lifetime is the measure of persistent outgrowth till MT undergoes catastrophe. MT growth length is the total growth over a movie and MT growth velocity is the average of each MT growth event. MT dynamics parameters were compiled from multiple individual experiments and to avoid day-to-day fluctuations the final complied data were normalized to the mean of the control data for each experiment. [...] For axon outgrowth imaging, phase contrast images of axons were collected on a Zeiss Axio Observer inverted motorized microscope with a Zeiss 20×/0.5 Plan Apo phase objective and analyzed using ImageJ []. Time-lapse images for axon outgrowth velocity was collected for 4 h with 20 min intervals and images were analyzed using plusTipTracker QFSM plugin and velocity was measured as the average of instantaneous velocity per axon as described []. Axon retraction events were analyzed from the same data set used to assess axon growth velocity. Frame to frame axon growth was tracked manually and retraction events were recorded over a movie. Ratio of the number of retracting frames over total frames for each axon was scored.Axon outgrowth and MT dynamics data were normalized to controls, to account for day-to-day fluctuations in room temperature. Image acquisition and quantitation of fluorescence intensity of fixed and labeled explants were described previously []. Experiments were performed multiple times to ensure reproducibility. Graphs were made in GraphPad Prism. Statistical differences were determined using unpaired two tailed t-tests when comparing two conditions and one-way analysis of variance with Tukey’s post-hoc analysis when multiple conditions were compared. […]

Pipeline specifications

Software tools plusTipTracker, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Xenopus laevis
Chemicals Nocodazole