Figure 1.
Synaptotagmin-7 staining in cultured hippocampal neurons overlaps with both synaptotagmin-1 and overexpressed SNAP-23/SNAP-25.
A: Hippocampal neuron overexpressing EGFP-SNAP25 (green channel, upper right) and stained for syt-1 (red, upper left) and syt-7 (blue, lower left). Scale bar = 10 µm. The lower right panel shows the combined image. B: Close-up image shows that syt-1 is concentrated in spots, which represent presynapses, whereas both EGFP-SNAP25 and syt-7 have a more wide-spread distribution. Scale bar = 2 µm. C: Hippocampal neuron overexpressing EGFP-SNAP23 (green channel, upper right) and stained for syt-1 (red, upper left) and syt-7 (blue, lower left). Scale bar = 10 µm. The combined picture is shown in the lower right panel. D: Close-up image shows a widespread distribution of both EGFP-SNAP-23 and syt-7, which overlaps with, but is not restricted to, the areas stained for syt-1. Scale bar = 2 µm.
Figure 2.
Expression of SNAP-23 in SNAP-25 KO neurons leads to asynchronous release, which is exacerbated by deletion of Synaptotagmin-7.
A, top: Representative EPSC traces of SNAP-25 KO neurons rescued with SNAP-25 (black) or SNAP-23 (blue) and SNAP-25/Syt-7 DKO neurons rescued with SNAP-25 (red), or SNAP-23 (green). Stimulation is indicated by an arrow. A, bottom: Representative mEPSC traces of SNAP-25 KO neurons rescued with SNAP-25 (black) or SNAP-23 (blue) and SNAP-25/Syt-7 DKO neurons rescued with SNAP-25 (red), or SNAP-23 (green). B: Expression of SNAP-23 in SNAP-25 KO neurons (blue) led to reduced peak EPSC amplitudes in SNAP-25 KO neurons, which were further significantly reduced in the absence of Syt-7 (green). In contrast, syt-7 deletion (red: SNAP-25 expression in SNAP-25/Syt-7 DKO neuron) was without consequence in the presence of SNAP-25 (black: SNAP-25 expression in SNAP-25 KO neuron). C: SNAP-23 rescued neurons displayed a 3–4 fold reduction in the charge transferred upon a stimulus; this reduction was independent of Syt-7 expression. D: The time-to-peak (EPSC) was prolonged in SNAP-23-rescued SNAP-25 KO neurons. Additional deletion of Syt-7 leads to a total of 50-fold increase in the time-to-peak. E: Spontaneous mEPSC amplitudes were unchanged in all four conditions. F: The frequency of mEPSCs was increased when SNAP-23 was expressed in SNAP-25 KO neurons, and further significantly increased when SNAP-23 was expressed in the SNAP-25/Syt-7 double KO background. G: Explanation of abbreviated labeling, used in Fig. 2–5. * = p<0.05; ** = p<0.01; *** = p<0.001.
Figure 3.
Pool sizes and release probabilities of neurons expression SNAP-25 or SNAP-23 in the presence and absence of synaptotagmin-7.
A: Pool sizes estimated by back-extrapolation of cumulative release during a train [41]. The lines are linear fits to the steady state. B: Pool Sizes. Solid Bars: Estimates from train stimulation and back extrapolation Cross-hatched bars: Estimates from sucrose stimulation. The expression of SNAP-23 reduced the pool estimate using back-extrapolation, and syt-7 deletion led to a further decrease. C: Release Probabilities, derived by dividing EPSC amplitudes by the pool size identified by back extrapolation (solid bars), or by dividing the EPSC charge by the charge identified by sucrose application (squared bars). The expression of SNAP-23 reduced the release probability, but it was not further reduced by deleting syt-7.
Figure 4.
SNAP-23 expressing neurons display asynchronous release, which is shifted to later times by the lack of synaptotagmin-7.
A: Example traces (stimulus artifacts and action potential associated currents have been blanked); arrows indicate the five stimuli. Color coding as in Figure 2. B: Charge transfer plotted versus stimuli at 50 Hz. The expression of SNAP-23 reduced the charge, which was further exacerbated by the deletion of syt-7. C: Charge transferred as a result of 50 Hz stimulation (5 stimuli). “During STP”: Integrated release during the interval from the beginning of the train until 20 ms after the train. “Post STP”: Release after the 5th EPSC (+20 ms) until current has relaxed to baseline. “Cumulative”: “During STP”+“Post STP”. Deletion of syt-7 in the presence of SNAP-23 shifted release to after the short train of action potentials. D: As (B), 5 Hz instead of 50 Hz. E: As (C), 5 Hz instead of 50 Hz. *: p<0.05; **: p<0.01; ***:p<0.001.
Figure 5.
Syt-7 limits the build-up of release during prolonged stimulation in the presence of SNAP-23.
A: Examples of 40 Hz, 100 AP trains in the four groups. Color coding as in Figure 2. B: Cumulative release during and after a 40 Hz, 100 AP train. Train stimulation is indicated by a horizontal bar. Shown are SNAP-25 KO neurons rescued with SNAP-25 (black), or SNAP-23 (blue), compared to SNAP-25/Syt-7 DKO neurons rescued with SNAP-25 (red) and SNAP-23 (green). The thick lines are means of all experiments; thin lines indicate the mean ± SEM (number of cells: SNAP-25+syt7, n = 25 cells; SNAP-25−syt7, n = 31 cells; SNAP-23+syt7, n = 19 cells; SNAP-23−syt7, 26 cells). C: Zoom-in of the first 0.5 s of (B), showing that in the presence of SNAP-23, but in the absence of syt-7, release becomes stronger than in the presence of syt-7 at ∼0.4 s (train starts at 0.1 s). **: p<0.01.
Figure 6.
Vesicles fusing asynchronously in the presence of SNAP-23 carry synaptotagmin-1, not synaptotagmin-7.
A: pHluorin constructs. pHluorin was fused to the N-terminal of syt-1 or syt-7, preceded by a signal peptide from preprotachykinin (ss-ppt), to ensure proper orientation. For syt-7, two different splice variants were used, a long, and a short. Both have the calcium-binding C2-domains, but they vary in the length of the linker between the Trans Membrane Region (TMR) and the first C2-domain. B. Experimental paradigm. pHluorin-syt situated in acidified intracellular compartments will not be fluorescent, but will gain fluorescence upon fusion with the plasma membrane. C. Example images of pH-Syt1, pH-Syt7S (short isoform), pH-Syt-7L (long isoform) in SNAP-25 KO neurons co-expressing CFP-SNAP-25. At 0 s, the neurons are exposed to high-K solution (45 mM K+) to induce vesicular release. “Base” is the normal extracellular solution. D. Same as C, but cells co-express CFP-SNAP-23. E. Quantification of pHluorin fluorescence in SNAP-25 expressing SNAP-25 KO neurons, for all three pHluorin constructs. High-K exposure has been marked “KCl”. The experiment ended with exposure to a solution with pH 5.5, followed by exposure to an ammonium solution to unquench all pHluorins and reveal the total fluorescence. The fluorescence values have been subtracted by the fluorescence in pH 5.5 solution. ‘N’ gives the number of cells (number of synapses analyzed: 414 for CFP-SNAP-25/pH-Syt-1, 591 for CFP-SNAP-25/pH-Syt-7S, 511 for CFP-SNAP-25/pH-Syt-7L). F. Same as E, but neurons co-express EGFP-SNAP-23. ‘N’ gives the number of cells (number of synapses analyzed: 473 for CFP-SNAP-23/pH-Syt-1, 561 for CFP-SNAP-23/pH-Syt-7S, 439 for CFP-SNAP-23/pH-Syt-7L).
Figure 7.
Schematic drawings of possible arrangements of synaptotagmins and Q-SNAREs result in different energy landscapes of fusion.
In the control situation, syt-1 and SNAP-25 results in a relatively high energy barrier at rest, and an efficient removal of the fusion barrier under stimulation conditions, leading to fast evoked release and a large difference between evoked and spontaneous fusion rates. In the presence of SNAP-23, syt-7 creates a lower fusion barrier (leading to a higher frequency of spontaneous release than syt-1), which cannot be as efficiently removed, leading to slower (asynchronous) evoked fusion. In the presence of SNAP-23 and in the absence of syt-7 the energy barrier for fusion becomes even more ‘squeezed’ between the calcium-independent and calcium-dependent case, leading to high rates of spontaneous fusion and very asynchronous evoked fusion. The calcium sensor for secretion remains unknown in this situation.