Figure 1. In vivo signalling endosome axonal transport dynamics are unchanged up to over a year in wild-type mice.
(A) Representative series of images (i to iv) acquired by time-lapse confocal microscopy of HcT-555 (pseudocoloured cyan) being retrogradely transported (left to right) in sciatic nerve axons from a live wild-type mouse (postnatal day 408). Four individual endosomes are tracked by coloured triangles (iv), the displacement profiles of which have been plotted (v). Scale bars = 10 μm.
(B) The speed distribution curves of signalling endosome are similar for 1 month and ≥13 month old wild-type animals. The same holds true for the 3 and 8 month data, which are not plotted.
(C) Speed distribution curve produced by combining data from 4 different time points (1, 3, 8 and ≥13 months; 19 mice). A smoothened curve is fitted to the data, which shows that approximately 50% of individual endosome steps are faster than 1.88 μm/s. A value of 1.89 μm/s was calculated from the raw data.
(D) The mean average speed of individual signalling endosomes across the four ages are plotted. Each data point represents an individual endosome.
(E) There is no significant difference between ages in the mean average (×, P =0.851, one-way ANOVA) and the maximum (O, P =0.757, one-way ANOVA) signalling endosome speeds calculated individually for each mouse.
(F-G) No difference is also detected between ages in the percentage of endosomes pausing for at least one frame (F, P =0.876, one-way ANOVA) or the percentage of time paused (G, P =0.221, one-way ANOVA). Pairwise statistical comparisons of the data from different ages in panels E-G were also not significant (P >0.05, Tukey’s multiple comparisons test). NS, not significant. Means ± standard error of the mean (S.E.M.) are plotted for all graphs. n =3 (1 month), 3 (2 months), 4 (8 months), and 9 (≥13 month).
Supplementary Video 1. In vivo imaging of axonal transport.
Representative video constructed from images acquired by time-lapse confocal microscopy of HcT-555 (pseudocoloured cyan) being retrogradely transported (left to right) in exposed sciatic nerve axons of a live, anaesthetised, wild-type mouse (P408). HcT was injected into the gastrocnemius and tibialis anterior muscles of the right leg, and is being transported towards the spinal cord. The video consists of 199 frames, covering 546 s, and is being played at a rate of 5 frames/s. The video is 66.4 mm × 18.6 mm.