Toward a unified picture of the exocytotic fusion pore

Abstract

Neurotransmitter and hormone release involve calcium-triggered fusion of a cargo-loaded vesicle with the plasma membrane. The initial connection between the fusing membranes, called the fusion pore, can evolve in various ways, including rapid dilation to allow full cargo release, slow expansion, repeated opening-closing and resealing. Pore dynamics determine the kinetics of cargo release and the mode of vesicle recycling, but how these processes are controlled is poorly understood. Previous reconstitutions could not monitor single pores, limiting mechanistic insight they could provide. Recently developed nanodisc-based fusion assays allow reconstitution and monitoring of single pores with unprecedented detail and hold great promise for future discoveries. They recapitulate various aspects of exocytotic fusion pores, but comparison is difficult because different approaches suggested very different exocytotic fusion pore properties, even for the same cell type. In this Review, I discuss how most of the data can be reconciled, by recognizing how different methods probe different aspects of the same fusion process. The resulting picture is that fusion pores have broadly distributed properties arising from stochastic processes which can be modulated by physical constraints imposed by proteins, lipids and membranes.

Figure 1.

Major players in calcium-triggered exocytosis. Formation of a complex between vesicular v- (red) and plasma membrane t-SNAREs (red) drives membrane fusion. Complex assembly is arrested at an intermediate stage by the synergistic action of Synaptotagmin (light orange) and complexin (black). Membrane depolarization opens voltage-gated calcium channels and allows rapid calcium influx. Tandem C2 domains of Synaptotagmin bind 2–3 calcium ions each and bury their hydrophobic residues at the tips of the calcium-binding loops into the bilayer. Other rearrangements likely allow further SNARE assembly and contribute to fusion pore opening.

Figure 2.

Different types of nanodiscs reconstituted with the neuronal v-SNARE protein VAMP2/Synaptobrevin-2, and various nanodisc-based fusion assays. A. Left: MSP nanodiscs utilize an Apolipoprotein A1 derivative (membrane scaffold protein, MSP) to stabilize the edges of a flat phospholipid bilayer disc [47,155]. These discs are typically 13–17 nm in diameter and can accommodate 4–5 v-SNAREs per face [36,38,156]. Upon fusion of an MSP ND with a target membrane, the maximum size of the fusion pore that appears is limited to ~3–4 nm by the protein scaffold that forms a belt around the disc [36,38]. Right: Larger discs can be made using alternative scaffolds. ApoE-based scaffolds afford ~25 nm discs that can accommodate ~15 v-SNAREs per face [35,37]. With these discs, the fusion pore can expand to ≳ 10 nm diameter with little obstruction from the scaffold ring [35,37]. B. Nanodiscs reconstituted with neuronal v-SNAREs are mixed with small liposomes reconstituted with complementary neuronal t-SNAREs. Bulk cargo release is monitored by an increase in the fluorescence of a cargo-sensitive dye present in the bath. Liposomes were loaded with calcium [36] or glutamate [40] and release was monitored using Mag-Fluo-4 or iGluSnFR, respectively. Release of sulforhodamine B from single t-SNARE liposomes upon fusion with v-SNARE NDs has also been monitored in a dye efflux assay using surface-tethered liposomes [41]. C. ND-cell fusion can be monitored in various manners [37,38,62]. Top: “flipped” t-SNARE cells [56] expressing complementary neuronal t-SNAREs ectopically with the SNARE domain facing the extracellular space, were fused with v-SNARE NDs [37,38]. The cells were pre-loaded with a calcium-sensitive fluorophore, Fluo-4. ND-cell fusion leads to calcium influx into the cytoplasm reported by an increase in Fluo-4 fluorescence. Middle: flipped t-SNARE cell under whole-cell voltage-clamp [38]. The patch pipette is depicted in gray. Perifusion of vNDs (grey bar) leads to a large whole-cell current (red trace) indicating opening of fusion pores on the cell surface. Application of vNDs treated with tetanus neurotoxin (TeNT) do not lead to currents (black trace). Figure modified from [38]. Bottom: single-pore conductance measurements using a flipped t-SNARE cell in the cell-attached patch configuration [37,38]. vNDs are included in the patch pipette. When a vND fuses with the cell surface, a fusion pore opens and allows direct-currents to be measured under voltage clamp. A representative trace is shown on the left (~16 nm diameter MSP vNDs, ~3–4 copies per ND face, transmembrane potential, −16 mV). D. Black-lipid membranes (BLMs) are single bilayer membranes that span a ~100–500 μm hole in a Teflon partition [72]. Recently they have been reconstituted with t-SNAREs and used as target membranes for fusion with ~13 nm v-SNARE NDs [41]. Current from a single fusion event is shown schematically. Currents in the ND-BLM assay have well-defined levels and flicker open-closed like ion channels, at least when pore size is confined by small NDs. When 50 nm diameter vNDs were used, fluctuating, larger currents with no evident stable levels were detected (bottom) [41].

Figure 3.

Properties of single fusion pores. A. Examples of fusion pores that appear during ND-cell fusion, measured in cell-attached recordings. Large, ~25 nm NLPs loaded with ~15 copies per NLP face were used [37]. Most pores have fluctuating currents and no clear transitions between stable states. Some display preferred current levels, but such levels are not consistent across a given sample. The green example is replotted on a larger scale in black to show the threshold (red dashed lines) and a minimum threshold crossing time that are imposed to define open states (colored bars above traces). B. Lifetime distribution of ND-cell fusion pores, for NLPs loaded with 15 v-SNAREs per face. The exponential fit (red curve) resulted in a characteristic time of 15 s [37]. Pore lifetimes are ~6 s for smaller, MSP NDs loaded with 3–4 v-SNAREs per face [38]. C. Top: distribution of conductance values from 99 pores as in A. All points were concatenated to construct the distribution, so pores lasting longer contributed more. Using a different averaging giving equal weight to every pore blunts the peak at ~700 pS. Bottom: distribution of pore sizes estimated from conductance data, assuming the pore is a 15 nm long cylinder [37,38].

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.