Fig. 5

Synaptic vesicle exocytosis reported by pHluorin occurs at a distance from the plasma membrane. (A) Example of x-t images showing a pHluorin event from SypHy (green) at a ribbon shortly after onset of depolarization from -60 mV to -15 mV, shown by the trace to the right. Ribbon fluorescence from deep-red CF633-RBP is pseudocolored red. (B) Example of x-t images showing a pHluorin event from SypHy (green) on the membrane-distal side of a ribbon (red) in a different cell during depolarization to -15 mV. (C) (D) Fluorescence intensity profiles along the x-axis for RBP (red) and pHluorin (green) for the examples shown in A and B. Black lines are fits as described in Figure 3A-D and in Materials and methods. The x-axis position of the pHluorin event was taken to be the peak of the Gaussian from the fit (x₀, shown by the dotted green line), and the position of the plasma membrane was taken to be the parameter x_1/2 (dotted black line) from the fit to the fluorescence profile of the ribbon (red traces). (E) Histogram of the x-axis position of 127 pHluorin events from SypHy and Vglut1-pHluorin during sustained depolarization. The solid arrow shows the average relative position of the plasma membrane, estimated as described in Materials and methods (error bar: ± 1 sem). The dashed arrow shows the estimated position of the membrane at the top and bottom of the optical section, arrived at by assuming that membrane curvature observed along the y-axis for each x-y image also applied in the z-axis. Most of the pHluorin events fell outside the range of membrane positions between the two arrows.