(a) Representative super-resolution radial fluctuations (SRRF) microscopy of an axon stained for endogenous Rab5 (purple), Rab7 (green), and Rab10 (orange). The inset at ×6 higher magnification shows examples of partial co-localisation of Rab10 with Rab5 and Rab7. Scale bar = 5 µm. (b) Intensity along the same axonal segment shown in (a) was plotted to show the correlation between the three markers. (c) Co-localisation of endogenous Rab10 and Rab5 was analysed comparing axons from starved neurons versus incubated 30 min with brain-derived neurotrophic factor (BDNF). Confined-displacement algorithm (CDA) has been used to compute Manders coefficients (Welch’s corrected unpaired Student’s t-test; M1: t(46.27), p-value = 0.0244; M2: t(48.94), p-value = 0.4794). Data points showing significant co-localisation compared to random distribution are marked with coloured dots. No significant differences were found between starved and BDNF-stimulated neurons. (d) Co-localisation of endogenous Rab10 and Rab7 was analysed in a similar experimental set-up (Welch’s corrected unpaired Student’s t-test; M1: t(72.32), p-value = 0.0621; M2: t(62.33), p-value = 0.1043). (e) Labelled HCT and anti-TrkB were co-internalised in the presence of BDNF for 30, 60, and 90 min, and their level of overlap with endogenous Rab10 in axons was evaluated using confocal microscopy. Relative areas positive for HCT and TrkB are shown in cyan (normalised to 30 min) and the fraction of the normalised area that was triple positive for HCT, TrkB and Rab10 is plotted in white. Whereas the double TrkB/HCT-positive area significantly increased by 90 min (one-way ANOVA, F(2,72), p-value <0.0001; Bonferroni multiple comparison test p value is shown in the plot), the triple TrkB/HCT/Rab10 surface remained low and fairly constant at all time points (one-way ANOVA, F(2,72), p-value = 0.2730; Bonferroni multiple comparisons test for 60 vs. 90 min, t(72), p-value >0.9999). (f) Representative SRRF microscopy from the same three time points. Double-positive puncta for TrkB (green) and HCT (purple) is indicated with cyan arrowheads. Triple-positive puncta of TrkB/HCT (cyan) and Rab10 (orange) are indicated with white empty triangles. Scale bar = 5 µm. Source data of the plots have been included in Figure 3—source data 1.