Fig. 3. The YxxC motif regulates LRRTM2 membrane turnover.

a Representative images of DIV15 hippocampal neurons expressing Cre-mCherry, Homer1c-BFP and either SEP-LRRTM2-WT or SEP-LRRTM2-YACA. On the right, Cre-mCherry (blue) is overlaid with Homer (red) and LRRTM2 (green). b Pseudocolor images of SEP fluorescence recovery after photobleaching in spines from neurons expressing Cre-mCherry and SEP-LRRTM2-WT or -YACA. c Corresponding normalized fluorescence recovery curves in spines and d slow pool fraction in both conditions. Data are presented as mean values ± SEM. Data acquired from three independent experiments (SEP-LRRTM2-WT: n = 37 regions, SEP-LRRTM2-YACA: n = 32 regions; ****p < 0.0001)). Data were compared by non-parametric Mann–Whitney test. e Representative images of DIV15 hippocampal neurons expressing Cre-mCherry (blue), Homer1c-BFP (red), and SEP-LRRTM2-WT or -YACA (green). Dashed squares indicate the positions of panel i. f Schematics of the pH change protocol used to visualize intracellular and extracellular protein pools. At pH 7.4, the SEP-tag is fluorescent, whereas at acidic pH (pH 5.5), its fluorescence is quenched. Bath application of pH 5.5 renders surface SEP-tagged proteins non-fluorescent, and NH₄Cl de-acidifies intracellular vesicles, rendering intracellular SEP-tagged proteins fluorescent. g Normalized mean average intensity of SEP-LRRTM2-WT and SEP-LRRTM2-YACA overtime during the pH change protocol illustrated in f. Data are presented as mean values ± SEM. h Percentage of SEP-LRRTM2-WT and SEP-LRRTM2-YACA protein fluorescence signal localized in the intracellular compartments of dendrites. Data are presented as mean values ± SEM. Data were compared by non-parametric Mann–Whitney test. i Pseudocolor images of insets from (e) during the pH change protocol, revealing intracellular protein pools by subtraction of the “baseline” signal (extracellular) from the “NH₄Cl” signal (total) in the shaft and spines. j Percentage of SEP-LRRTM2-WT and SEP-LRRTM2-YACA intracellular pool distribution in spines and dendritic shaft. g–i Data acquired from three independent experiments (SEP-WT: n = 9 and SEP-YACA: n = 11 cells).