Rapid structural change in synaptosomal-associated protein 25 (SNAP25) precedes the fusion of single vesicles with the plasma membrane in live chromaffin cells

Abstract

The SNARE complex consists of the three proteins synaptobrevin-2, syntaxin, and synaptosomal-associated protein 25 (SNAP25) and is thought to execute a large conformational change as it drives membrane fusion and exocytosis. The relation between changes in the SNARE complex and fusion pore opening is, however, still unknown. We report here a direct measurement relating a change in the SNARE complex to vesicle fusion on the millisecond time scale. In individual chromaffin cells, we tracked conformational changes in SNAP25 by total internal reflection fluorescence resonance energy transfer (FRET) microscopy while exocytotic catecholamine release from single vesicles was simultaneously recorded using a microfabricated electrochemical detector array. A local rapid and transient FRET change occurred precisely where individual vesicles released catecholamine. To overcome the low time resolution of the imaging frames needed to collect sufficient signal intensity, a method named event correlation microscopy was developed, which revealed that the FRET change was abrupt and preceded the opening of an exocytotic fusion pore by ∼90 ms. The FRET change correlated temporally with the opening of the fusion pore and not with its dilation.

Keywords: TIR-FRET imaging; electrochemical imaging; image analysis; time superresolution microscopy; transmitter release.

Fig. 1.

Spatiotemporal correlation of exocytic events and fluorescence changes in bovine chromaffin cells expressing SCORE. (A, I) Bright-field image of ECD array. The four Pt conductors (black) are covered by a translucent (and invisible) insulating layer that was removed in the region outlined by the red circle. Within the red circle, the tips of four electrodes are exposed for the detection of fusion events. The yellow dots on a 500-nm grid indicate the release locations that were assumed for a set of RWSs providing the expected fractions of molecules detected by the four individual electrodes for these locations. (A, II) Fluorescence footprint of a cell expressing SCORE placed on the ECD array and excited in TIR mode at 436 nm (average of 1,000 exposures without background subtraction). The ECD electrodes located between glass surface and cell membrane (labeled A–D) are dark. The average fluorescence in a circular area between the electrodes (dashed cyan) with ∼2.5-µm radius served as control for measurements at release sites. (A, III) Schematic cross-sectional view of the ECD array with the cell along the green dotted line in A, II. The heights of the ECD electrode (black) and insulation (brown) layers are 150 nm and 300 nm, respectively. (B, I–III) Amperometric currents from the cell shown in A, II recorded by the four ECD electrodes as indicated by colors. One adjacent AFS spike (B, I) and non-AFS spike (B, II) from the recording (B, III) (blue and red arrows) are shown on expanded scales. Spike starting times (upward arrows in B, I and II) are based on linear fits of the rising phase (black dashed lines, see Materials and Methods). In B, I a short upward arrow marks the foot start time and the horizontal arrow the foot duration. The blue dotted line shows the foot signal from electrode “D” on expanded scale. The fractions of molecules detected by the different electrodes for the B, II event are best matched by the RWS results for the release site marked by the small red dot in A, II. (C) Image sequence of excised six-by-six-pixel region centered at the release site. For clarity, each panel shows a 2.19-s average of 10 consecutive frames. Emission in CFP channel (Top), Venus channel (Middle), and fluorescence ratio of Venus/CFP (Bottom). (D) Time courses of CFP (blue) and Venus (yellow) channels (after background subtraction) averaged over the two-by-two-pixel region framed by the red square in C. (E) Time course of the Venus/CFP ratio (black) suggests a possible transient FRET change (red dashed lines) at the spike starting time (vertical dashed line).

Fig. 2.

FRET change in SCORE precedes fusion. (A, I) Sequence of excised FRET ratio images averaged from 903 non-AFS events like that in Fig.1C. (A, II) SCORE fluorescence (sum of CFP and Venus) averaged as in A, I. (B) Average amperometric spike obtained after temporal alignment of the spikes at their starting times. Average time course of (C) Venus and CFP emission and (D) FRET ratio measured at release sites as the red square in Fig. 1 (bold traces) and from control footprint areas (as the cyan circle in Fig. 1 A, II) over the same time periods (faint traces). (E) FRET time course from the data of Fig. 2D analyzed with the ECOM method on expanded time scale (black), fits of the FRET rising phase (−0.6 s to +0.4 s) with the ECOM step response function (red), and with convolution of step response with exponential distribution of 128-ms time constant (blue); averaged ECD spike (green). (F) Squared deviations of the two fits, colors as in E.

Fig. 3.

Relation of FRET change to fusion pore opening and dilation. (A, I and II) Image sequence of averaged FRET ratio (A, I) and averaged total fluorescence CFP+Venus (A, II) from 581 AFS events. (B) Time courses of FRET change from AFS (solid black line) and non-AFS (dotted black line, redrawn from Fig. 2E) events relative to spike starting times (time 0) and fit of AFS FRET trace with ECOM step response function; averaged amperometric spike of AFS (solid green line) and non-AFS (dotted green line). The horizontal dashed line indicates the 50% FRET change levels. (C) Frequency of foot durations plotted as survival curve on logarithmic scale (black line) and power-law fit (cyan line). (D) Expanded ECD traces (from B) show average foot signal (arrow) in AFS (solid green line) but not in non-AFS (dotted green line) events. Vertical dashed lines indicate spike start time.

Fig. 4.

ECOM method recovers time correlation with 2-ms precision from 219-ms imaging frames. (A–C) Shutter opening (arrows) produces step intensity change (red line) and initiates an ECD photocurrent (green line). Image frame times are indicated by vertical gray lines and include 200-ms exposure time (black horizontal bars) and 19-ms interframe interval. Traces were temporally aligned at onset of photocurrent. Brightness of transition frame depends on time of shutter opening during the frame. (D) Averaged time course of photocurrent (green line) and normalized image brightness (black line) from 260 shutter openings shows 50% rise time at time 0 ± 2 ms. The black curve was fitted well with the step response function of image brightness (dashed red line).

Fig. 5.

SD of timing precision obtained with ECOM analysis is inversely proportional to signal-to-noise ratio. Datasets were averages of 100 (filled symbols) or 1,000 (open symbols) simulated intensity steps with an SNR of 1/10 or 1/31.6, respectively, of final SNR in the average signals.