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. Author manuscript; available in PMC: 2019 Nov 1.

Published in final edited form as: FEBS Lett. 2018 Oct 26;592(21):3563–3585. doi: 10.1002/1873-3468.13270

Figure 4. — Possible fates of the fusion pore and the fused vesicle, and how various methods would report them. A. Possible pathways that can be taken by the fusion pore and fused vesicle ghost. B. How the different pore/vesicle states in A (a-i) would appear in admittance measurements. ΔC_m and G_p refer to membrane capacitance (proportional to membrane area) and pore conductance (only detectable within a window, typically corresponding to pores ≲ 3 – 5 nm diameter), respectively. For small pores G_p may not be detected, depending on experimental parameters and vesicle size. Multiple states (e.g. “c/d/f/h”) indicate they would all produce the same signal, i.e. they could not be discriminated. Typical time resolution is 1–10 ms. C. Left: schematic of the detection principle. Released cargo such as catecholamines are oxidized as soon as they reach the surface of a carbon-fibre electrode, generating an oxidation current. Right: How the states depicted in A would appear in amperometric recordings of release, which have 0.1–1 ms time resolution. D. TIRFM detection of the states in A. Left: lumenal cargo is fused to a fluorescent protein (e.g. NPY-pHluorin). Upon fusion, the fluorescence of the granule rapidly increases (due to pH neutralization which enhances GFP fluorescence and release of the probe toward the glass surface where the evanescent field intensity is highest), then decreases due to diffusion of the labeled probes away from the fusion site. Right: if a slowly releasable cargo is fused to a pH sensitive fluorescent protein, signals increase due to pH neutralization after fusion, then return to baseline due to pore resealing and re-acidification. If the cargo is labeled with a pH-insensitive probe, or if the fluorescent probe is placed at the cytoplasmic end of a membrane cargo, then no signal is produced upon fusion up to >1 min [120]. Retention of cargo does not simply scale with cargo size; it can also be due to interactions with the dense-core matrix or membrane. To test how much, if any, of the cargo was lost during fusion, ammonium chloride is applied to collapse pH gradients. E. How dye influx measurements would report the states depicted in A. A mixture of dyes are placed in the extracellular bath. Exocytosis allows both dyes to enter a granule, increasing the fluorescence intensity at the fusion site. One of the dyes (red) is excited at low power and probes the vesicle’s size. The other (green) is excited at high power and probes when the fusion pore reseals. Pore resealing (arrowhead) prevents exchange of bleached dye with unbleached dyes in the bath and leads to a drop in the fluorescence intensity at the fusion site.

Figure 4. — Possible fates of the fusion pore and the fused vesicle, and how various methods would report them. A. Possible pathways that can be taken by the fusion pore and fused vesicle ghost. B. How the different pore/vesicle states in A (a-i) would appear in admittance measurements. ΔC_m and G_p refer to membrane capacitance (proportional to membrane area) and pore conductance (only detectable within a window, typically corresponding to pores ≲ 3 – 5 nm diameter), respectively. For small pores G_p may not be detected, depending on experimental parameters and vesicle size. Multiple states (e.g. “c/d/f/h”) indicate they would all produce the same signal, i.e. they could not be discriminated. Typical time resolution is 1–10 ms. C. Left: schematic of the detection principle. Released cargo such as catecholamines are oxidized as soon as they reach the surface of a carbon-fibre electrode, generating an oxidation current. Right: How the states depicted in A would appear in amperometric recordings of release, which have 0.1–1 ms time resolution. D. TIRFM detection of the states in A. Left: lumenal cargo is fused to a fluorescent protein (e.g. NPY-pHluorin). Upon fusion, the fluorescence of the granule rapidly increases (due to pH neutralization which enhances GFP fluorescence and release of the probe toward the glass surface where the evanescent field intensity is highest), then decreases due to diffusion of the labeled probes away from the fusion site. Right: if a slowly releasable cargo is fused to a pH sensitive fluorescent protein, signals increase due to pH neutralization after fusion, then return to baseline due to pore resealing and re-acidification. If the cargo is labeled with a pH-insensitive probe, or if the fluorescent probe is placed at the cytoplasmic end of a membrane cargo, then no signal is produced upon fusion up to >1 min [120]. Retention of cargo does not simply scale with cargo size; it can also be due to interactions with the dense-core matrix or membrane. To test how much, if any, of the cargo was lost during fusion, ammonium chloride is applied to collapse pH gradients. E. How dye influx measurements would report the states depicted in A. A mixture of dyes are placed in the extracellular bath. Exocytosis allows both dyes to enter a granule, increasing the fluorescence intensity at the fusion site. One of the dyes (red) is excited at low power and probes the vesicle’s size. The other (green) is excited at high power and probes when the fusion pore reseals. Pore resealing (arrowhead) prevents exchange of bleached dye with unbleached dyes in the bath and leads to a drop in the fluorescence intensity at the fusion site.