eb3 mscarlet i  (Addgene inc)

Journal: Nature

Article Title: In situ structural mechanism of epothilone-B-induced CNS axon regeneration

doi: 10.1038/s41586-025-09654-z

Figure Lengend Snippet: a) Live imaging of EpoB-induced regenerating thalamus axon tip transduced with EB3-mScarlet encoding lentiviruses. EB3 signals were found at the regenerating axon tip, suggesting active growth of plus-end-out microtubules. The axotomy site is shown with a white dotted line. Corresponding movies are available in Supplementary Video . b) Scatter dot plots showing counts of tubulin tracker puncta in thalamus control axons in the presence of different concentrations of EpoB. Mean is indicated as horizontal line. One-Way Anova was used for statistical testing, and p-values are 0.0259 (0.1 nM vs 1 nM), <0.0001 (0.1 nM vs 10 nM) and 0.0020 (1 nM vs 10 nM). N = 19 (0.1 nM EpoB), N = 24 (1 nM EpoB) and N = 27 (10 nM EpoB) puncta. c) Tubulin Tracker binding affinity to tubulin monomers, polymerized microtubules and tubulin spirals. Tubulin Tracker alone does not show any fluorescence, but it can bind and fluoresce in the presence of tubulin spirals and microtubules. The data is shown as bar graphs overlaid with data points, in which the fold change in fluorescence between different experimental conditions is plotted. Tubulin spirals were induced by p150 protein. d) Distances traveled by tubulin clusters away from the axotomy site within 30 min of observation. The violin plots represent median and interquartile range. Ax−EpoB+: n = 39 particles (16 axons), and Ax−EpoB−: n = 66 particles (29 axons). Significance was tested using the two-tailed Mann-Whitney test. P-values are reported at the corresponding comparisons on the graphs. e) Distances traveled by tubulin clusters toward regenerating sites within 30 min of observation. The violin plots represent median with interquartile range. Ax−EpoB+: n = 39 particles (16 axons), and Ax−EpoB−: n = 66 particles (29 axons) from at least three biologically independent experiments. Significance was tested using the two-tailed Mann-Whitney test. p-values are reported at the corresponding comparisons on the graphs. f) Net distance (outward vs inward) traveled by tubulin clusters within 30 min of observation. The violin plots represent median with interquartile range. Ax−EpoB+: n = 39 particles (16 axons), and Ax−EpoB−: n = 66 particles (29 axons) from at least three independent experiments. Significance was tested using the two-tailed Mann-Whitney test. p-values are reported at the corresponding comparisons on the graphs. g) Distances traveled by tubulin clusters away from the axotomy site within 10 min of observation. The violin plots represent median with interquartile range. Ax−EpoB+Monas−: n = 60 particles (6 axons), and Ax−EpoB+Monas+: n = 32 particles (7 axons) from at least three independent experiments. Significance has been tested using the two-tailed Mann-Whitney test. p-values are reported at the corresponding comparisons on the graphs. h) Distances traveled by tubulin clusters towards the axotomy site within 10 min of observation. The violin plots represent median with interquartile range. Ax−EpoB+Monas−: n = 60 particles (6 axons), and Ax−EpoB+Monas+: n = 32 particles (7 axons) from at least three independent experiments. Significance has been tested using two-tailed Mann-Whitney test. p-values are reported at the corresponding comparisons on the graphs. i) Distribution of distances between neighboring MIPs at different times after axotomy in the presence of EpoB. The dot plots show median with interquartile range. n = 634 (control), 256 (t = 15 min), 2025 (t = 1 h), 401 (t = 3 h), 336 (t = 6 h), 912 (t = 24 h) particles. Median: 18.5 nm (control) 17.6 nm (15 min) 24.2 nm (1 h), 22.6 nm (3 h), 24.3 nm (6 h), 18.3 nm (24 h). At t = 15 min, the observed microtubules are located at the pre-cut site as no regeneration of microtubules are observed at this time point. At t > 1 h, all the microtubules are located at the post-cut sites. One-Way Anova was used for statistical testing and p-values are >0.9999 (control vs 0.25 h), <0.0001 (control vs 1 h), <0.0001 (control vs 3 h), <0.0001 (control vs 6 h) and >0.9999 (control vs 24 h).

Article Snippet: Lentiviral plasmids containing genes of interest and tagged with fluorescent proteins for labelling neuronal organelles were obtained as follows. pTRIP-CMV-EB3-mScarlet-I was constructed via Gibson assembly of EB3-mScarlet into the pTRIP-CMV-GFP-2A vector opened at the NheI and KpnI sites. eGFP-KIF5A was a gift from J. Bonifacino (Addgene plasmid 172201) . pBa.GFP-KIF5B was a gift from M. Bentley (Addgene plasmid #134625) . pGFP-Kif5c was a gift from M. Peckham (Addgene plasmid #71853) .

Techniques: Imaging, Transduction, Control, Binding Assay, Fluorescence, Two Tailed Test, MANN-WHITNEY