Fig. 4. Dynamic TNR molecules are endocytosed in neurons over hours, and recycle with a periodicity of ~3 days.

a Newly-emerged TNR epitopes were labeled 4 h post-blocking, and monitored by live epifluorescence imaging. Arrowheads indicate neuronal somas. Scale bar = 10 µm. Quantification of the intensity in somas (normalized to t₀ timepoint) indicates significant internalization. N = 5 independent experiments, 1-4 neurons per datapoint. Friedman test (χ²₆ = 25.46, ***p < 0.001), followed by two-sided Dunn’s multiple comparisons test (‘6 h’: *p = 0.033; ‘8 h’: **p = 0.005; ‘10 h’: **p = 0.005; ‘12 h’: **p = 0.002). Data represent mean ± SEM, dots indicate individual experiments. b Internalized TNR in axons vs. dendrites. Epitopes were allowed to internalize for 12 h, followed by surface stripping with proteinase K. The remaining signal was imaged with confocal microscopy in neurites visualized using DiO (green), with axons identified by immunostaining AnkyrinG (blue). Scale bar = 4 µm (1 µm zoom). Quantification of the signal density reveals no differences between dendrites and axons. N = 4 experiments, ≥10 neurons per datapoint. Two-sided paired t-test (t = 0.741, p = 0.513). Data represent mean ± SEM, dots indicate individual experiments. c Newly-emerged TNR epitopes were labeled 6 h post-blocking. The fraction present on the surface of neurites was measured at different intervals by imaging neurons in epifluorescence before and after stripping with proteinase K. Quantification of the fluorescence ratio before/after stripping (normalized to the ‘0d’ timepoint) reveals peaks of TNR resurfacing at 3 and 6 days post-labeling (~3 day periodicity). Amounts stripped at ‘3d’ and ‘6d’ are significantly higher than at ‘1d’ and ‘2d’, or ‘5d’ and ‘7d’. N = 4 independent experiments, 5 before/after images per datapoint. Kruskal-Wallis followed by Fisher’s LSD (Days 2, 3, 4: H₂ = 8.29, *p = 0.016, ‘3d’/‘2d’; *p = 0.046; ‘3d’/‘4d’: **p = 0.005; Days 4, 5, 6: H₂ = 6.74, *p = 0.036, ‘6d’/‘5d’: *p = 0.022, ‘6d’/‘7d’: *p = 0.028). Scale bar = 20 µm. Data represent the mean (lines) ± SEM (shaded regions); dots indicate individual experiments. Source data are provided in Source Data file.