Fig. 7. TNR recycling is mediated by integrins.

a, b Assessment of colocalization between recycling TNR molecules and β1-integrin. a Left: newly-emerged TNR epitopes were labeled 12 h post-blocking concurrently with a labeling of surface-bound β1-integrins, by applying fluorophore-conjugated antibodies directed against the extracellular domain of the receptors. The neurons were fixed and imaged with 2-color-STED. Right: newly-emerged TNR epitopes were labeled 4 h post-blocking, concurrently with β1-integrin. Neurons were incubated a further 12 h to allow for internalization, and remaining surface-bound molecules were stripped with proteinase K. The neurons were fixed and imaged with confocal microscopy. Images on the right of each panel show zoomed views of the dashed boxed. Scale bars = 2 µm (full images), 500 nm (zoomed images). b Quantification of % colocalizing TNR signal (for a) shows newly-emerged TNR epitopes colocalize with both cell surface-bound and internalized β1-integrins. The values are significantly higher than negatives controls, relying on non-specific primary antibodies. Controls were imaged in STED/confocal for comparison to images in the left/right panels, respectively. N = 3 (‘surface β1-integrin’ experiments and negative controls), and 4 (‘internalized β1-integrin’) independent experiments, ≥10 neurons per datapoint. Two-sided Student’s t-test (‘surface integrin’ vs. ‘neg ctrl’: t = 11.61, ***p = 0.0003; ‘internalized integrin’ vs. ‘neg ctrl’: t = 3.177, *p = 0.025. Data represent mean ± SEM, dots indicate individual experiments. c To assess whether β1-integrin receptors are required for TNR endocytosis, newly-emerged TNR epitopes were labeled 12 h post-blocking, after which the neurons were immediately incubated with function-blocking anti-β1-integrin antibodies³³ for 6 h. Neurons were then incubated with proteinase K, to remove remaining surface-bound TNR, and imaged with epifluorescence microscopy. A reduction in fluorescence signal is evident in integrin-blocked cultures. Scale bar = 5 μm. d Quantification of the fluorescence intensity confirms that the amount of internalized TNR is significantly reduced following the blocking of β1-integrin receptors. N = 3 independent experiments, ≥15 neurons per datapoint. Two-sided Student’s t-test (t = 3.343, *p = 0.029). Data represent mean ± SEM, dots indicate individual experiments. Source data are provided in Source Data file.