Figure 8.

Expression of open-syntaxin increases the number of dead-end vesicles by 70%. A, Quantitative immunocytochemistry to assess the difference in SNAP-25 and syntaxin insertion in the PM upon open-syntaxin overexpression. Left, TIRFM images of anti-syntaxin and anti-SNAP-25 labeled cells either overexpressing open-syntaxin (top) or not (bottom). Right, average fluorescence intensity of syntaxin and SNAP-25 immunolabeling in the PM. Overexpressed open-syntaxin was clearly transported to the PM as the syntaxin immunofluorescence was significantly increased compared with control (***p < 0.001, n = 10 and n = 14 for control and open-syntaxin overexpressing cells, respectively). B, C, Cells transfected with NPY-mCherry and eGFP alone (B) or together with open-syntaxin (C) were patched with 6 μm free [Ca²⁺] in the pipette solution. Shown are the average membrane capacitance increase (stippled line) and the cumulative number of secreted LDCVs visualized in TIRFM normalized to the footprint area (solid line). D, Analysis of TIRFM recording in cells expressing open-syntaxin (red) or control cells (black). Represented are the average density of LDCVs near the PM at the beginning and the end of the experiment and the average total secretion. LDCVs visible in TIRFM at the end of the experiment were separated between newcomer and dead-end vesicles (n = 16 and n = 15 for open-syntaxin expressing and control cells, respectively; *p < 0.05; **p < 0.01). See Materials and Methods for details on the normalization procedure.