Figure 2.

Ca²⁺-binding site mutants Doc2b^DN and Doc2b^6A show increased plasma membrane binding at rest but different activity during stimulation. (A) Live confocal microscopy of hippocampal neurons cultured in low density networks co-expressing Doc2b^WT tagged with eGFP (green) and either Doc2b^DN or Doc2b^6A fused to mCherry (red). Both channels were simultaneously imaged for 15 s at rest and then stimulated with 60 mM KCl in the extracellular medium. Representative images were obtained by averaging 4 sequential images each for the resting (naïve) and stimulated state (KCl). (B,C) Representative line scans from 2 of 5 ROIs per cell with a thickness of 10 pixels placed on neurites (see white rectangles in A) showing the fluorescence intensity distribution at rest (light-colored profiles) and after stimulation (dark-colored) for Doc2b^WT-eGFP (B) and Doc2b^DN-mCherry (C). Arrows indicate the plasma membrane (PM); curly brackets indicate cytosol (C). (D) Average PM/C intensity ratio from 5 ROIs of the representative neuron, normalized to the starting value. Both Doc2b^WT–eGFP and Doc2b^DN-mCherry translocated upon KCl stimulation. (E) The PM/C fluorescence ratio was averaged from 6 neurons (N = 6) each containing 2 to 6 ROIs (n = 26 ROIs in total). (F-J) The same methodology was applied to neurons co-expressing Doc2b^WT-eGFP and Doc2b^6A-mCherry. (F) Representative example showing WT and 6A behavior at rest and upon stimulation. (G,H) Line scan profiles for Doc2b^WT-eGFP (G) and Doc2b^6A-mCherry (H). (I) Normalized PM/C intensity ratio averaged from 5 ROIs in the representative neuron. (J) PM/C ratio quantification from n = 36 ROIs from N = 11 neurons, showing a strong translocation of Doc2b^WT-eGFP while in the same cell, Doc2b^6A-mCherry appears insensitive to stimulation. For (D–I) and (E–J) data are represented as mean ± SEM. Friedman ANOVA, paired repeated Post-hoc tests (see Supplementary Table 1).