Journal: bioRxiv

Article Title: Gαi2-Mediated Regulation of Microtubules Dynamics and Rac1 Activity Orchestrates Cranial Neural Crest Cell Migration in Xenopus

doi: 10.1101/2023.09.07.556733

Figure Lengend Snippet: A-B. Immunofluorescence against α-tubulin. Pseudocolor scale shows that Gαi2MO increases microtubules distribution towards the leading edge. Magnification: 63X. C-D. Tubulin 3D reconstruction in control and Gαi2 knockdown conditions. E. Quantification of tubulin fluorescence intensity. Gαi2 dramatically increased tubulin signal towards the leading edge. Error bars: s.e.m. *** P≤0.001. n= 3 cells were analyzed per condition for each of three independent experiments. F. Schematic representation of the tubulin distribution measurements from the edge of the nucleus to the cell cortex. T: tubulin. G-H. Images were obtained every 1.5 seconds for a total of 5 minutes using Leica SP8 confocal microscopy. Videos of EB3GFP-labeled cells were cropped to a region of interest, and they were analyzed using plusTipTracker software to detect microtubule growing tips via +TIP tracking proteins . Magnification: 63X. I. Cells were separated into four groups based on growth rate (slow < 11 µm/min < fast) and duration (short < 23 sec < long). Cells injected with Gαi2MO present a higher percentage of slow microtubules, and a lower percentage of fast microtubules compared to control cells. J. The average growth rate of Gαi2 morphant microtubules is 8.52 µm/min ± 0.301 and is significantly lower compared to control cells, whose growth rate is on average 10.53 µm/min ± 0.297. ANOVA statistical analysis for multiple conditions and Student’s T-Test (two-tailed) were performed. N = 3 experiments per condition. K. The quantification of the arrests (pauses) of the comets did not show significant differences between control cells and morphant cells for Gαi2. L. Using TrackMate plugin of ImageJ, the trajectory of comets was tracked in time, specifically those that enter or leave a pre-established region of interest at the cell-cell contact and at the leading edge. At the end of the 2.5 minutes, a relationship was made between the comets that remain in the area versus the total comets that entered the area. Control cells show a 53.8% of catastrophes at the leading edge and 75.1% at cell-cell contact. On the other hand, Gαi2 morphant cells show 42.9% of catastrophes at the leading edge versus only 47.3% at the cell-cell contact.

Article Snippet: We conducted time-lapse tracking of EB3-GFP comets at both 3-second and 1.5-second intervals and quantified various parameters, including growth speed, growth length, comet lifetime, and pauses, using automated analysis with the PlusTipTracker software for Matlab ( ; ).

Techniques: Immunofluorescence, Control, Knockdown, Fluorescence, Confocal Microscopy, Labeling, Software, Injection, Two Tailed Test