Fig. 4.

The T198I mutation selectively impacts on activity-dependent sybII retrieval during SV endocytosis. (A, B) Primary cultures of synaptophysin knockout hippocampal neurons were transfected with sybII-pHluorin and either wild-type mCerulean-tagged human synaptophysin (WT), the T198I mutant or empty mCer vector (KO) and challenged with a train of 300 action potentials (10 Hz) before being allowed to recover and exposed to a NH₄Cl containing buffer. (A) Pseudo-coloured images from a representative time-lapse are shown prior to the stimulus (t = 0 s), during the stimulus (t = 44 s), following endocytosis (t = 200 s) and during perfusion of NH₄Cl. Scale bar equivalent to 10 μm. (B) Traces (WT, black; T198I, red; KO, blue) display the average fluorescent sybII-pHluorin response normalised to the peak of stimulation (ΔF/F₀ ± SEM). Stimulation is indicated by the bar. WT n = 9, T198I n = 14, mCer n = 13, * = p < 0.05 for both T198I and KO compared to WT, two-way ANOVA. (C) Wild-type hippocampal neurons transfected with sybII-pHluorin and either WT (black) or T198I (red) mCerulean-tagged human synaptophysin were challenged with a train of 300 action potentials (10 Hz) as indicated by the bar. Traces display the average fluorescent sybII-pHluorin response normalised to the peak of stimulation (ΔF/F₀ ± SEM). WT n = 11, T198I n = 10, ns, two-way ANOVA.